Interactions with herring sperm DNA and biological studies of sparfloxacin drug-based copper(II) compounds

Article abstract of DOI:10.1002/aoc.2919

Copper(II) complexes of the type [Cu(SPF)(Lⁿ)Cl] (where SPF is sparfloxacin and Lⁿ = substituted terpyridines) were synthesized and found to have a distorted octahedral geometry. Superoxide dismutase-like activity of the complexes wa

Full text of DOI:10.1002/aoc.2919

Full Paper
Received: 12 May 2012
Revised: 3 August 2012
Accepted: 6 August 2012
Published online in Wiley Online Library
(wileyonlinelibrary.com) DOI 10.1002/aoc.2919
Interactions with herring sperm DNA and
biological studies of sparfloxacin drug-based
copper(II) compounds
Mohan N. Patel*, Hardik N. Joshi and Chintan R. Patel
Copper(II) complexes of the type [Cu(SPF)(Ln)Cl] (where SPF is sparfloxacin and Ln = substituted terpyridines) were synthesized
and found to have a distorted octahedral geometry. Superoxide dismutase-like activity of the complexes was measured using
a nitroblue tetrazolium/reduced nicotinamide adenine dinucleotide/phenazine methosulfate system and expressed in terms
of the concentration of complex which terminates the formation of formazan by 50% (IC50 value), which was found to range
from 0.572 to 1.522 mM. Interactions of the complexes with herring sperm DNA were studied by absorption titration, viscosity
measurement and gel electrophoresis under physiological conditions. The antimicrobial efficiency of the complexes was
tested against five different microorganisms and showed good biological activity. All the complexes showed good cytotoxic
activity, with LC50 values ranging from 4.01 to 9.64 mg mlꢀ1. Copyright © 2012 John Wiley & Sons, Ltd.
Supporting information may be found in the online version of this article.
Keywords: sparfloxacin; intercalation; IC50; LC50; bactericidal activity
bronchitis and community-acquired pneumonia.[16,17] DNA
interaction with sparfloxacin drug-based complexes of copper(II),
Introduction
Studies on the interaction of transition metal complexes with
DNA are a subject of great interest in recent years. Interest in this
area is a fundamental requirement, not only for gaining insight
into the reactive models for protein–nucleic acid interactions
and probes of DNA structure, but also for obtaining information
about drug design and tools of molecular biology.[1–7] It is well
established that transition metal complexes can interact with
DNA through a non-covalent mechanism, such as electrostatic
interaction, groove binding or intercalation. Considerable work
needs to be done to develop applications for binding these
complexes to DNA through an intercalation mode with the
ligands containing fully planar intercalating into adjacent base
pairs of DNA.[3,8] The intercalating ability not only correlates with
the planarity of ligand, but also relates to the coordination geometry
of the metal ion and type of donor atom in the ligand.[9,10] In
addition, both the metal ion type and its valency play important roles
in deciding the binding extent of complexes to DNA.[11] This
provides valuable information to explore the potential chemothera-
peutical agents.
Fluoroquinolones belong to a group of synthetic antimicrobial
agents which have been used clinically used for the past 30 years.
They are derived from the basic structure of nalidixic acid and
have substituents at N-1, C-5, C-7 at position 8 and a fluorine
atom at position 6. Fluoroquinolones inhibit bacterial DNA gyrase
and topoisomerase IV enzymes, resulting \in inhibition of DNA
replication and transcription.[12–15] The fluorine atom at position
6 enhances gyrase inhibition and cell penetration. Piperazinyl
substituents provide activity against Gram-negative bacteria
and the pyrrolidinyl moiety is active against Gram-positive cocci.
The function substituted at position 8 controls anaerobe activity.[15]
Sparfloxacin, as a third-generation quinolone antimicrobial drug, is
mainly used for the treatment of acute exacerbations of chronic
nickel(II) and zinc(II) ions have been studied by various
groups.[18–24] Over the last several years, ternary copper–quinolone
complexes with biomolecules have been thoroughly studied.[25–30]
Superoxide dismutases (SOD) are metalloenzymes which
catalyze the dismutation of superoxide anion (Oꢀ2 ) into hydrogen
peroxide (H2O2) and dioxygen.[31] Numerous diseases have been
shown to involve overproduction of the superoxide radical,
which results in growing interest in the search for mimics.[32]
The direct utilization of enzymes as pharmaceutical agents poses
many problems.[33] Considerable efforts have been directed to
obtaining stable, nontoxic and inexpensive low-molecular
biomimetic molecules, which are able to catalyze superoxide
dismutation and to provide important therapeutic applications.
In continuation of our previous work,[34] herein we synthesized
copper(II) complexes with sparfloxacin and terpyridine derivatives.
The interaction of complexes with DNA has also been investigated
using electronic absorption spectroscopy, viscosity measurement
and gel electrophoresis. The potency of synthesized compounds
against five bacteria was evaluated by determining the minimum
inhibitory concentration (MIC). Electron scavenging capacity was
determined using a non-enzymatic nitroblue tetrazolium/reduced
nicotinamide adenine dinucleotide/phenazine methosulfate
(NBT/NADH/PMS) system. Cytotoxic activity of the complexes
has been performed to measure the LC50 value.
*
Correspondence to: M. N. Patel, Department of Chemistry, Sardar Patel University,
Vallabh Vidyanagar-388120, Gujarat, India. E-mail: jeenen@gmail.com
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar-388120,
Gujarat, India
Appl. Organometal. Chem. 2012, 26, 641–649
Copyright © 2012 John Wiley & Sons, Ltd.

M. N. Patel et al.
Experimental
R=4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
4-Methylphenyl
2-Pyridyl
2
R-CHO
Materials and Reagents
N
All reagents, chemicals and solvents used were of analytical reagent
grade and used as purchased. Double-distilled water was used
throughout. Sparfloxacin was generously supplied by Bayer
AG (Wuppertal, Germany). 2-Acetylpyridine, p-fluorobenzaldehyde,
p-chlorobenzaldehyde, p-bromobenzaldehyde, p-methylbenzaldehyde,
pyridine-2-carbaldehyde, thiophene-2-carbaldehyde, p-benzyloxyben-
zaldehyde, NADH, NBT and PMS were purchased from Loba
Chemie Pvt. Ltd (India). Cupric chloride dihydrate was purchased
rom E. Merck (India) Ltd, Mumbai. Ethidium bromide, bromophenol
blue, agarose and Luria Broth (LB) were purchased from Himedia
(India).
Thiophene-2-yl
4-Benzyloxyphenyl
O
2-Acetylpyridine
aq. NaOH
EtOH
NH3
R
N
Physical Measurement
N
N
An elemental analysis (C, H and N) of the synthesized complexes
was performed with a model 240 Perkin Elmer elemental
analyzer. The metallic content of the complex was determined
after decomposing it in acid mixture on heating and titrating
against ethylenediaminetetraacetic acid (EDTA) solution volumet-
rically. Magnetic moments were measured by Gouy’s method
using mercury tetrathiocyanatocobaltate(II) as the calibrant
(wg = 16.44 ꢁ 10ꢀ6 cgs units at 20ꢂC), on a Citizen balance.
Diamagnetic correction was made using Pascal’s constant.
Electronic spectra for solution state were recorded on a
UV-160A UV–visible spectrophotometer (Shimadzu, Japan).
Electronic spectra for solid state were recorded on a Lambda
19 spectrophotometer (PerkinElmer, USA). Infrared spectra
were recorded on an FT-IR ABB Bomen MB 3000 spectropho-
tometer as KBr pellets in the range 4000–400 cmꢀ1. LC mass
spectra were recorded using a Thermo mass spectrophotometer
(USA). An MIC study was carried out by means of a laminar air flow
cabinet (Toshiba, Delhi, India).
Scheme 1. General synthesis of ligand
washed with ether/hexane and dried in a vacuum desiccator
(Scheme 2). Yield: 64.51%; m.p. 286ꢂC; meff 1.94 BM. Anal. Calcd
for C40H35ClCuF3N7O3 (816.18): C, 58.75; H, 4.31; N, 11.99;
Cu, 7.77%. Found: C, 58.57; H, 4.20; N, 11.81; Cu, 7.68%.
IR: nmax (cmꢀ1) n(C═O)pyridone 1625(vs), n(CO2)asym 1597(vs),
n(CO2)sym 1397(vs), n = n(CO2)asym ꢀ n(CO2)sym = 200, n(M-N) 540,
n(M-O) 518 cmꢀ1. UV–visible l (nm) (e, Mꢀ1 cmꢀ1), as solid: 723,
344, 296; as solution: 720 (360), 340 (15429), 291(36222),
ΛM = 21 mho cm2 molꢀ1
.
[Cu(SPF)(L2)Cl] (2)
Complex 2 was synthesized using 40-(4-chlorophenyl)-2,20:60,200-
terpyridine as ligand (L2) (1.5 mmol). Yield 62.15%; m.p. >
300ꢂC; meff 1.89 BM. Anal. Calcd for C40H35Cl2CuF2N7O3 (834.20):
C, 57.59; H, 4.23; N, 11.75; Cu, 7.62%. Found: C, 57.41; H, 4.03; N,
Synthesis of Ligands
NH2
O
OH
R
All tridentate ligands were synthesized similarly by following
literature procedure.[35] 2-Acetyl pyridine (20.0 mmol) was added
to an ethanolic solution of various aldehydes (10.0 mmol in 70 ml
EtOH). KOH pellets (26 mmol) and 30 ml aqueous NH3 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 ꢁ 10 ml) and EtOH (2 ꢁ 5 ml).
Crystallization from a CHCl3–MeOH system gave a white crystalline
solid. The proposed reaction is shown in Scheme 1.
F
O
N
N
N
HN
F
N
N
Sparfloxacin
R=4-Fluorophenyl
4-Chlorophenyl
4-Bromophenyl
4-Methylphenyl
2-Pyridyl
CuCl2.2H2O
CH3ONa
pH=6.8
Thiophene-2-yl
4-Benzyloxyphenyl
Synthesis of Complexes
[Cu(SPF)(L1)Cl] (1)
A methanolic solution of CuCl2.2H2O (1.5 mmol) was added to a
methanolic solution of 40-(4-fluorophenyl)-2,20:60,200-terpyridine
(L1) (1.5 mmol), followed by the addition of a previously prepared
methanolic solution of sparfloxacin (1.5 mmol) in the presence
of CH3ONa (1.5 mmol). After mixing the above solutions, the pH
of the solution is in the basic range (>6.8). Therefore, the pH of
the reaction mixture was adjusted to ~6.8 by addition of dilute
hydrochloric acid. The resulting solution was refluxed for 2 h in
a water bath, followed by concentrating to half of its original
volume. A fine, green, amorphous product obtained, which was
O
N
O
N
R
Cu
F
N
O
Cl
N
H2N
N
F
NH
Scheme 2. General synthesis of complex.
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Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 641–649

Sparfloxacin drug-based copper(II) compounds
11.56; Cu, 7.52%. IR: nmax (cmꢀ1) n(C═O)pyridone 1605(vs), n(CO2)asym
1561(vs), n(CO2)sym 1360(vs), n = n(CO2)asym ꢀ n(CO2)sym = 201,
n(M-N) 555, n(M-O) 517cmꢀ1. UV–visible l (nm) (e, Mꢀ1 cmꢀ1), as
solid: 728, 342, 295; as solution: 719 (332), 349.5 (15429), 290.5
Antibacterial Activity (MIC)
Synthesized complexes were tested for their impact on micro-
organisms, namely Escherichia coli (MTCC 433), Pseudomonas
aeruginosa (MTCC P-09), Serratia marcescens (MTCC 7103),
Bacillus subtilis (MTCC 7193) and Staphylococcus aureus (MTCC
3160). Impact was tested in terms of MIC using suspended LB in
sterile double-distilled water as a medium. Gram-positive and
Gram-negative cultures were incubated for 24 h. at 37ꢂC. A control
test with no active ingredient was also performed by adding just an
equivalent amount of solvent.[36] MIC was determined using
twofold serial dilution in liquid media containing various concentra-
tions of test compounds. Bacterial growth was measured by the
turbidity of the culture after 18h. If a particular concentration of
compound inhibited bacterial growth, half the concentration
of the compound was tested. This procedure was carried out
until the concentration at which bacteria grew normally was found.
The lowest concentration which inhibited bacterial growth totally
was determined as the MIC value. All equipment and culture media
employed during the process were sterile.
The bactericidal action of all compounds was evaluated
against the same microorganisms. The inoculum was prepared
by diluting an overnight culture of microorganisms grown in
LB, to obtain 106 viable bacteria mlꢀ1. Bacteria were exposed to
various concentrations of compounds. Control tubes without
compound were included in each run. The final volume was
1 ml. Cultures were incubated at 37ꢂC for 2 h. A 100 ml bacterial
culture from each dilution was spread over previously prepared
agar plates. The plates were then incubated for 24 h. The number
of colonies present on the plates were counted. The number of
colonies should be in the range 30–300.
(39008), ΛM = 27 mho cm2 molꢀ1
.
[Cu(SPF)(L3)Cl] (3)
Complex 3 was synthesized using 40-(4-bromophenyl)-2,20:60,
200-terpyridine as ligand (L3) (1.5 mmol). Yield 61.34%; m.p. 296ꢂC;
meff 1.92 BM. Anal. Calcd for C40H35BrClCuF2N7O3 (878.65): C,
54.68; H, 4.02; N, 11.16; Cu, 7.23%. Found: C, 54.46; H, 3.90; N,
10.99; Cu, 7.14%. IR: nmax (cmꢀ1) n(C═O)pyridone 1603(vs), n(CO2)asym
1570(vs), n(CO2)sym 1365(vs), n = n(CO2)asym ꢀ n(CO2)sym = 205,
n(M-N) 540, n(M-O) 510 cmꢀ1. UV–visible l (nm) (e, Mꢀ1 cmꢀ1), as
solid: 719, 339, 288; as solution: 711 (166), 335 (11035), 291
(24451), ΛM = 24 mho cm2 molꢀ1
.
[Cu(SPF)(L4)Cl] (4)
Complex 4 was synthesized using 40-(4-methylphenyl)-2,20:60,
200-terpyridine as ligand (L4) (1.5 mmol). Yield 66.42%, m.p. >
300ꢂC; meff 1.90 BM. Anal. Calcd for C41H38ClCuF2N7O3 (813.78):
C, 60.51; H, 4.71; N, 12.05; Cu, 7.81%. Found: C, 60.33; H, 4.53; N,
11.87; Cu, 7.71%. IR: nmax (cmꢀ1) n(C═O)pyridone 1636(vs), n(CO2)asym
1597(vs), n(CO2)sym 1378(vs), n = n(CO2)asym ꢀ n(CO2)sym = 219,
n(M-N) 556, n(M-O) 514 cmꢀ1. UV–visible l (nm) (e, Mꢀ1 cmꢀ1), as
solid: 710, 335, 292; as solution: 704 (375), 340 (24897), 291
(27392) ΛM = 22 mho cm2 molꢀ1
.
[Cu(SPF)(L5)Cl] (5)
Complex 5 was synthesized using 40-(2-pyridyl)-2,20:60,200-terpyridine
as ligand (L5) (1.5 mmol). Yield 63.50%; m.p. 290ꢂC; meff 1.91 BM. Anal.
Calcd for C39H35ClCuF2N8O3 (800.74): C, 58.50; H, 4.41; N, 13.99; Cu,
DNA Interaction Activity
7.94%. Found: C, 58.33; H, 4.26; N, 13.80; Cu, 7.86%. IR: nmax (cmꢀ1
)
Absorption Titration
n(C═O)pyridone 1628(vs), n(CO2)asym 1558(vs), n(CO2)sym 1355(vs),
Perturbation in spectral behavior of the complex on interacting
with DNA is a tool for determining the interaction of complexes
with DNA. Curves were constructed by increasing the nucleic acid
concentration. After adding an equivalent amount of DNA to a
reference cell, incubation for 10 min at room temperature
was followed by measurement of absorption. DNA-mediated
hypochromism (decrease in absorbance) for test compounds
was calculated. The results of this assay were generated
under the same conditions as the plasmid degradation assay.
It was specifically done to nullify the change in absorbance because
of the increasing amount of DNA. From the absorption data, the
intrinsic binding constant (Kb) of the complexes with DNA was
determined using the following equation:[37]
n = n(CO2)asym ꢀ n(CO2)sym = 203, n(M-N) 556, n(M-O) 520 cmꢀ1
.
UV–visible l (nm) (e,
M
ꢀ1 cmꢀ1), as solid: 735, 349, 290; as
solution: 729 (184), 340 (15251), 291(36589), ΛM = 26 mho
cm2 molꢀ1
.
[Cu(SPF)(L6)Cl] (6)
Complex 6 was synthesized using 40-(thiophen-2-yl)-2,20:60,
200-terpyridine as ligand (L6) (1.5 mmol). Yield 60.78%; m.p. > 300ꢂC,
meff: 1.95 BM. Anal. Calcd for C38H34ClCuF2N7O3S (805.78): C, 56.64;
H, 4.25; N, 12.17; Cu, 7.89%. Found: C, 56.45; H, 4.03; N, 11.98;
Cu, 7.80%. IR: nmax (cmꢀ1) n(C═O)pyridone 1605(vs), n(CO2)asym 1565
(vs), n(CO2)sym 1359(vs), n =n(CO2)asym ꢀ n(CO2)sym = 206, n(M-N) 542,
n(M-O) 506 cmꢀ1. UV–visible l (nm) (e, Mꢀ1 cmꢀ1), as solid: 733, 347,
293; as solution: 727 (236), 342 (31291), 288.5 (38162), ΛM =23 mho
cm2 molꢀ1
.
½DNAꢃ=ðea ꢀ efÞ ¼ ½DNAꢃ=ðeb ꢀ efÞ þ 1=Kbðeb ꢀ efÞ
(1)
[Cu(SPF)(L7)Cl] (7)
Complex 7 was synthesized using 40-(4-benzyloxyphenyl)-2,20:60,
200-terpyridine as ligand (L7) (1.5 mmol). Yield 68.63%; m.p. > 300ꢂC;
meff: 1.88 BM. Anal. Calcd for C47H42ClCuF2N7O4 (905.88): C, 62.32;
H, 4.67; N, 10.82; Cu, 7.01%. Found: C, 62.14; H, 4.50; N, 10.59;
Cu, 6.93%. IR: nmax (cmꢀ1) n(C═O)pyridone 1636(vs), n(CO2)asym 1597
(vs), n(CO2)sym 1381(vs), n =n(CO2)asym ꢀ n(CO2)sym =216, n(M-N)
545, n(M-O) 515 cmꢀ1. UV–visible l (nm) (e, Mꢀ1 cmꢀ1), as solid: 713,
337, 284; as solution: 703 (2056), 330 (7730), 279 (9101), ΛM =25
where [DNA] is the concentration of herring sperm (HS) DNA in
base pairs, ea corresponds to the apparent extinction coefficient
obtained by calculating Aobsd/[complex], ef corresponds to the
extinction coefficient of the complex in its free form and eb refers
to the extinction coefficient of the complex in fully bound form.
The data were fitted to the above equation to obtain a straight line
with a slope value corresponding to the term 1/(ea ꢀ ef) and the
y-intercept to 1/Kb(eb ꢀ ef). Kb was determined from the ratio
of slope to the y-intercept.
mho cm2 molꢀ1
.
Appl. Organometal. Chem. 2012, 26, 641–649
Copyright © 2012 John Wiley & Sons, Ltd.
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M. N. Patel et al.
Viscosity Experiments
SOD-Like Activity
In viscosity measurement, DNA samples of approximately 200
base pairs in length were prepared by sonication in order to
minimize complexities arising from DNA flexibility.[38] Flow times
were measured with a digital stopwatch. Each sample was
measured three times and an average flow time was calculated.
Relative viscosities for DNA in the presence and absence of the
complexes were calculated from the following relation:
SOD-like activity of the complexes was determined by using NBT
(75 mM), NADH (79 mM) and PMS (30 mM) containing varying
concentrations of test compounds (0.5–3.0 mM). The percentage
inhibition of NBT reduction was calculated using the following
equation:[42]
ð% inhibition of NBT reductionÞ ¼ ð1 ꢀ k0=kÞ ꢁ 100
(3)
where, k0 and k are the slopes of the straight line of absorbance
as a function of time in the presence and absence of a
compound, respectively. The IC50 value of the complexes was
determined by plotting the graph of percentage inhibition of
NBT reduction against increase in the concentration of complex.
Concentration of the complexes which caused 50% inhibition of
NBT reduction was reported as IC50.
ꢀ / ðt ꢀ t0Þ
(2)
where t is the observed flow time of DNA-containing solution and
t0 is that of phosphate buffer alone. Data were presented as
(ꢀ/ꢀ0)1/3 versus [complex]/[DNA],[39] where ꢀ is the viscosity
of DNA in the presence of copper(II) complexes and ꢀ0 is
the viscosity of DNA alone.
Results and Discussion
DNA Cleavage Study by Gel Electrophoresis
Magnetic and Electronic Behavior
Gel electrophoresis of plasmid DNA (pUC19 DNA) was carried out on
a 1% agarose gel bed submerged in TAE buffer (0.04 M Tris–acetate,
pH 8, 0.001 M EDTA). 15 ml of reaction mixture containing plasmid
DNA in TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0) and 200 mM
complex were allowed to proceed for 24 h at 37ꢂC. All the reactions
were quenched by the addition of 5 ml loading buffer (40% sucrose,
0.25% bromophenol blue, 0.25% xylene cyanole FF, 200 mM EDTA).
The aliquots were loaded directly onto 1% agarose gel and
electrophoresed at 50 V in 1ꢁ TAE buffer. Gel was stained with
0.5 mg mlꢀ1 ethidium bromide and was photographed on a UV
trans-illuminator. The percentage of each form of DNA was
quantified based on illumination using AlphaDigiDocTM RT
Version V.4.0.0 PC-Image software.
The magnetic moment of copper(II) in any of its geometry lies
around 1.8 BM, which is very close to the spin-only value, i.e. 1.73
BM. The found values in our case lie in the range 1.88–1.95 BM.
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 expected spin-only values due to the spin orbit
coupling contribution.[43] The UV–visible spectra of the complexes
in solid and solution states show a similar pattern, suggesting that
the complexes retain their structure in solution. Complexes
exhibited one broad band along with a shoulder at ~14 000 cmꢀ1
and ~11 000 cmꢀ1 respectively, which was attributed to the d–d
transition for Cu(II) atom in a distorted octahedral geometry.[44–48]
All complexes are soluble in DMSO and non-electrolytes
(ΛM ≤ 20–27 mho cm2 molꢀ1).
Brine Shrimp Assay
Brine shrimp lethality bioassay is widely used in the bioassay for
bioactive compounds by the Meyer method.[40] Brine shrimp
(Artemia cysts) eggs were hatched in a shallow rectangular plastic
dish (22 ꢁ 32 cm), filled with artificial sea water. Artificial sea
water was prepared using 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 eggs
were sprinkled into the large compartment, which was open to
ordinary light. After hatching, active nauplii free from egg shells
were collected from the brighter portion of the hatching chamber
and used for the assay. 1 ml seawater and 10 shrimps were added
to each vial (30 shrimps per dilution) and the volume was adjusted
with sea water to 2.5 ml per vial. A sample of the test compound
was prepared by dissolving 10 mg of each compound in 10 ml
DMSO. Solutions were transferred from the stock solutions to 18
vials to make final concentrations of 2, 4, 8, 10, 12 and 16 mg mlꢀ1
(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 a control, having the same amount of DMSO only. When
the shrimp nauplii were ready, 1 ml artificial sea water and 10
shrimps were added to each vial (30 shrimps per dilution). The
volume was adjusted to 2.5ml per vial using artificial sea water.
The number of survivors was counted after 24 h. Data were
analyzed by a simple method to determine the LC50 values, in
which the log of concentration of samples was plotted against
percentage mortality of nauplii.[41]
IR Spectra
The IR spectra of quinolones are quite complex, owing to the
presence of numerous functional groups in the molecules.[49] In
the IR spectrum of sparfloxacin the valence vibration of the carbox-
ylic stretch n(C═O)carb was found at 1714 cmꢀ1 and the pyridone
stretch n(C═O)p at 1641 cmꢀ1. Characterization of quinolone metal
complexes can be achieved by studying the most typical vibrations
that are characteristic of the coordination type of quinolones.
In the IR spectra of complexes 1–7 the absorption of the
n(C═O)carb has disappeared. Two very strong characteristic bands
are present in the range 1603–1636 cmꢀ1 and 1355–1397 cmꢀ1
assigned as n(O-C-O) asymmetric and symmetric stretching
vibrations, respectively, whereas n(C═O)p is shifted from 1643 to
1603–1636 cmꢀ1 upon bonding. The difference n = n(CO2)asym
ꢀ n(CO2)sym is a useful characteristic for determining the coordi-
nation mode of the ligands. The Δ values fall in the range
200–219 cmꢀ1, indicating a monodentate coordination mode
of the carboxylato group[49–51] of the sparfloxacin. The overall
changes in the IR spectra suggest that sparfloxacin is coordi-
nated to copper via the pyridone (Op) and one carboxylate
(Oc) oxygen.[50] A table showing the characteristic absorptions
of IR spectra of complexes is available online as supporting
information.
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Appl. Organometal. Chem. 2012, 26, 641–649

Sparfloxacin drug-based copper(II) compounds
Mass Spectra
According to Overtone’s concept of cell permeability, the lipid
membrane that surrounds the cell favors the passage of only the
lipid-soluble materials, owing to which liposolubility is an important
factor, which controls antimicrobial activity. It has been observed
that different compounds exhibit microbial activity of little variation
against bacterial species. This difference in activity depends either
on the impermeability of the cells of the microbes, which is single
layer in the case of Gram-positive and multilayer in the case of
Gram-negative,[57] or differences in the ribosomes of microbial
cells.[58] It can possibly be concluded that chelation increased the
activity of these complexes.
In addition to our study regarding MIC, we performed bacteri-
cidal activity in terms of colony-forming units (CFU) mlꢀ1 of metal
complexes against the same microorganisms (two Gram-positive
and three Gram-negative. Results reveal a decrease in the number
of colonies with increasing concentration of compounds. The
results are shown in supporting information for all complexes
against S. aureus. The number of colonies counted in this technique
is in the range 30–300. From the MIC values and CFU, it is found that
complex 1 is more potent against all five microorganisms.
The mass spectrum of complex 1 shows a molecular ion peak at
m/z = 816.17. The peak observed at m/z = 781.20 is due to loss of
chlorine atom in complex 1. The peaks observed at m/z = 425.08
and 427.21, m/z = 489.06 and 491.16, confirm the presence of
chlorine atom in the case of copper–terpyridine and copper–
sparfloxacin moieties, respectively. The peaks observed at
m/z = 390.06, m/z = 454.09 are due to loss of chlorine atom from
the copper–terpyridine and copper–sparfloxacin moieties, respec-
tively. The peaks observed at m/z = 327.12, m/z = 392.23 are due
to terpyridine moiety and sparfloxacin, respectively. The mass
spectrum and proposed mass fragmentation pattern of complex
1 are available as supporting information.
Antibacterial Activity
Table 1 shows the in vitro antibacterial activity data of the cupric
chloride dihydrate, sparfloxacin and synthesized complexes
(1–7). It is clear from the data that complexes 1 and 2 are
active compared to sparfloxacin against all the bacterial
species employed. Complex 3 is as active as sparfloxacin.
Complexes 4, 5, 6 and 7 are less active against all the species
than sparfloxacin. Complex 1 is the most active amongst all
the complexes. All the complexes show better activity against
E. coli, S. aureus and P. aeruginosa than those of [Cu(pr-norf)
(bipyam)Cl] and [Cu(PPA)(phen)Cl].[52] All the complexes show
better activity than [Cu(sf)(bipyam)Cl] against S. aureus. Complexes
1, 2, 3 and 4 show better activity than [Cu(sf)(bipyam)Cl] against P.
aeruginosa.[28] Except for complex 7, the rest of the complexes
show better activity than [Cu(sf)(bipyam)Cl] against E. coli.[28] All
the complexes are less active than [Cu(sf)2] against P. aeruginosa.[50]
Complexes 1, 2, 3, 4 and 6 are more active against S. aureus than
[Cu(sf)2]. Except for complex 7, the rest of the complexes show
better activity than [Cu(sf)2] against E. coli.[50] Chelation could
facilitate the ability of a complex to cross the cell membrane[53,54]
and can be explained by Tweedy’s chelation theory.[55] Chelation
considerably reduces the polarity of the metal ion because of partial
sharing of its positive charge with donor groups and possible
p-electron delocalization over the whole chelate ring. Such a
chelation could enhance the lipophilic character of the central
metal atom, which subsequently favors its permeation through
the lipid layers of the cell membrane and blocking the metal
binding sites on enzymes of the microorganism. The other possible
mode of action may be explained by Overtone’s concept.[56]
DNA Interaction Study
Absorption Titration
It is well known that electronic absorption spectroscopy is an
effective method for examining the binding modes and binding
extent of metal complexes with DNA.[59] Hypochromism and
red shift are associated with binding of the metal complexes to
the DNA helix, due to the intercalative mode, involving a strong
stacking interaction between the aromatic chromophore of the
complexes and the base pairs of DNA. The magnitude of the
hypochromism and red shift are dependent on the strength of
intercalation.[60] The spectral curve for diverse complex:DNA
mixing ratios (i.e. constant concentration of complex and varying
concentration of DNA; Fig. 1) suggests that the complexes bind
to DNA by intercalation. The binding constant values (Table 2)
were found in the range 8.090 ꢁ 103 to 9.350 ꢁ 104
M
ꢀ1, which is
lower than those of the classical intercalator (ethidium bromide),
[Mn(sf)2(H2O)2] (1.17ꢁ 105),[61] [Co(sf)2(H2O)2] (2.59 ꢁ 106),[27]
[Ni(sf)2(H2O)2] (7.46 ꢁ 105), [Ni(sf)2(phen)] (1.13 ꢁ 106),[26] [Cu(flmq)
(phen)Cl] (2.39 ꢁ 105) and [Cu(flmq)2(py)2] (8.12 ꢁ 105),[62] but
comparable to those of [Cu(SPF)(phendione)Cl].5H2O (3.7ꢁ 104),
[Cu(SPF)(nitrophen)Cl].5H2O (5.19 ꢁ 104)[63] and [Cu(SPF)(dpa)Cl].5H2O
Table 1. Bacteriostatic concentration of CuCl2.2H2O, sparfloxacin
and complexes (mg mlꢀ1
)
Compound
Gram-positive
Gram-negative
S. B.
S.
P.
E.
aureus subtilis marcescens aeruginosa coli
CuCl2.2H2O
459.95 479.90
469.84
0.55
0.24
0.33
0.53
0.77
1.36
0.97
2.45
409.83 579.97
Sparfloxacin
0.47
0.20
0.25
0.44
0.65
1.12
0.81
2.00
0.74
0.41
0.54
0.79
1.14
1.76
1.37
2.72
0.59
0.29
0.38
0.62
0.90
1.44
1.05
2.26
0.47
0.16
0.21
0.40
0.57
1.20
0.73
2.17
[Cu(SPF)(L1)Cl]
[Cu(SPF)(L2)Cl]
[Cu(SPF)(L3)Cl]
[Cu(SPF)(L4)Cl]
[Cu(SPF)(L5)Cl]
[Cu(SPF)(L6)Cl]
[Cu(SPF)(L7)Cl]
Figure 1. Absorption spectral traces on addition of HS DNA to the solu-
tion of complex 1 after incubating for 10 min at room temperature in
phosphate buffer at 7.2 pH (shown by arrow). Inset: plot of [DNA]/(ea ꢀ ef)
vs. [DNA] for absorption titration of HS DNA with complex 1.
Appl. Organometal. Chem. 2012, 26, 641–649
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

M. N. Patel et al.
increase in the relative viscosity of HS DNA but less so compared
to that of ethidium bromide. This behavior is similar to that of
copper complexes reported by Raman et al.[68] and Song et al.[69]
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.
Table 2. The binding constant (Kb) values of compounds
ꢀ1
)
Compound
Kb (M
Sparfloxacin
2.930 ꢁ 103
9.350 ꢁ 104
8.632 ꢁ 104
7.028 ꢁ 104
4.751 ꢁ 104
9.649 ꢁ 103
2.134 ꢁ 104
8.090 ꢁ 103
[Cu(SPF)(L1)Cl]
[Cu(SPF)(L2)Cl]
[Cu(SPF)(L3)Cl]
[Cu(SPF)(L4)Cl]
[Cu(SPF)(L5)Cl]
[Cu(SPF)(L6)Cl]
[Cu(SPF)(L7)Cl]
DNA Cleavage Study by Gel Electrophoresis
DNA cleavage activity of the complexes was studied by agarose
gel electrophoresis using supercoiled pUC19 plasmid DNA. DNA
cleavage is controlled by relaxation of the supercoiled circular
form of pUC19 DNA into a nicked circular form and a linear form.
The principle of this method is that molecules migrate in the gel
as a function of their mass, charge and shape, with supercoiled
DNA migrating faster than open circular molecules of the same
mass and charge. Figure 3 shows the electrophoretic separation
of pUC19 DNA when reacted with complexes under aerobic
conditions. When circular plasmid DNA was subjected to electro-
phoresis, the intact supercoiled DNA (form I) migrates relatively
quickly. If scission occurs on one strand, the supercoiled DNA will
relax to generate a slower-moving open circular form (form II). If
both strands are cleaved, a linear form (form III) will be generated
that migrates between form I and form II.[70] DNA cleavage ability
of the complexes has been quantified by measuring the transfor-
mation of the SC to OC and L forms. Results show that the cleavage
ability of complexes is more comparable to the metal salt and drug
(Table 3). Complex 1 shows the maximum cleavage ability
compared to all synthesized complexes. The similar behaviour of
Cu(II) complexes with plasmid DNA is shown by compounds of type
(7.25 ꢁ 104) and [Cu(SPF)(pyc)Cl].5H2O (7.93 ꢁ 104).[64] Kb values of all
the complexes are higher than that of sparfloxacin (2.930 ꢁ 103
M
)
ꢀ1
and [CuCl2(H2O)(SPAR)], [CuCl(bipy)(SPAR)]Cl and [CuCl2(phen)(SPAR)]
(in 103 order).[65] The reason for Kb values lower than the reported
complexes is the non-planarity of terpyridines. Therefore the above
results indicate that complexes may bind with DNA via the
intercalative mode. The binding mode is further confirmed
by viscosity measurement.
Viscosity Experiments
Although spectral methods are necessary, they are not sufficient
to support a binding mode. To clarify further the interaction
mode of the mononuclear copper(II) complexes with HS DNA,
viscosity measurements were carried out. In classical intercalation
the DNA helix lengthens as base pairs are separated to accom-
modate the bound ligand, leading to increase in DNA viscosity,
whereas a partial, non-classical ligand intercalation causes a bend
in the DNA helix, reducing its effective length and thereby its
viscosity. Therefore viscosity measurement is regarded as the
least ambiguous and the most critical means for studying the
binding mode of metal complexes with DNA in solution and
provides stronger arguments for intercalative binding mode.[66,67]
The effects of copper(II) complexes on the viscosity of HS DNA are
shown in Fig. 2. As illustrated in this figure, on increasing the
amount of the copper(II) complexes the relative viscosity of HS
DNA increases steadily, which confirms that the copper(II) com-
plexes are bound to HS DNA by intercalation. This phenomenon
may be explained by the insertion of the compounds between
the DNA base pairs, leading to an increase in the separation of base
pairs at intercalation sites and thus an increase in overall DNA
length. It is clear from the figure that all these complexes show an
Figure 3. Gel electrophoresis diagram showing the cleavage of SC
pUC19 DNA with series of copper(II) complexes, incubated at 37ꢂC, using
1% agarose gel, at 50 mV for 1.5 h. Lane 1, DNA control; lane 2, DNA +
CuCl2.2H2O; lane 3, DNA + sparfloxacin; lane 4, DNA + [Cu(SPF)(L1)Cl]; lane
5, DNA + [Cu(SPF)(L2)Cl]; lane 6, DNA + [Cu(SPF)(L3)Cl]; lane 7, DNA + [Cu
(SPF)(L4)Cl]; lane 8, DNA + [Cu(SPF)(L5)Cl]; lane 9, DNA + [Cu(SPF)(L6)Cl];
lane 8, DNA + [Cu(SPF)(L7)Cl].
Table 3. Complex-mediated DNA cleavage data by gel
electrophoresis
Lane Compound Form I (SC) Form II (OC) Form III (LC) % Cleavage
1
2
3
4
5
6
7
8
9
Control
89
85
56
15
18
21
25
31
28
34
11
15
26
64
58
57
54
49
49
47
—
—
18
21
24
22
21
20
23
19
—
CuCl2.2H2O
4.5
Sparfloxacin
[Cu(SPF)(L1)Cl]
[Cu(SPF)(L2)Cl]
[Cu(SPF)(L3)Cl]
[Cu(SPF)(L4)Cl]
[Cu(SPF)(L5)Cl]
[Cu(SPF)(L6)Cl]
37.07
83.14
79.77
76.40
71.91
65.17
68.53
61.80
10 [Cu(SPF)(L7)Cl]
Figure 2. Effect of increasing amount of EtBr, sparfloxacin and
complexes on the relative viscosity of HS DNA at 27 ꢄ 0.1ꢂC.
wileyonlinelibrary.com/journal/aoc
Copyright © 2012 John Wiley & Sons, Ltd.
Appl. Organometal. Chem. 2012, 26, 641–649

Sparfloxacin drug-based copper(II) compounds
[Cu(Hpr-norf)(phen)Cl], [Cu(Hpr-norf)(bipy)Cl], [Cu(erx)(phen)Cl]
and [Cu(erx)(bipy)Cl] reported by Katsaros et al.[71] The difference
in DNA cleavage efficiency of complexes was due to the difference
in binding affinity of complexes to DNA.
supporting information. Figure 4 illustrates that the amount of
chromophore required to yield 50% inhibition of the reduction of
NBT (IC50) value of complexes is higher than exhibited by the
copper salt. The copper(II) complexes showed SOD-like activity,
which was evaluated by the scavenger concentration causing
50% inhibition in the detector formation (IC50). The quantitative
Brine Shrimp Assay
ꢀ
•
reduction of NBT to blue Formazan by the O2 was followed
spectrophotometrically at 560nm. Compounds exhibit SOD-like
activity at biological pH with IC50 values ranging from 0.572 to
1.522mM. The results show that complexes exhibit greater scaveng-
ing activity towards superoxide radicals than sparfloxacin. All the
complexes have been compared with those reported by Casanova
et al.[74,75] The superoxide scavenging data suggest that complexes
1, 2, 3 and 4 were found to be more active, and complexes 5, 6 and
7 have similar activity with respect to [Cu(stz)(py)3Cl]. All the com-
plexes were found to be more active than [Cu(Hstz)(MeOH)Cl2]
and [CuL2(4-mHim)2]. The IC50 values of the copper(II) complexes
are better than recently reported Ni(II) complexes.[76] The results
show that complexes exhibit greater scavenging activity towards
superoxide radicals, which may be accredited to the redox potential
All the synthesized compounds were screened for their cytotoxicity
(brine shrimp bioassay) using the protocol of Meyer et al.[40]
A
general bioassay that appears capable of detecting a broad
spectrum of bioactivity present in the complex is the brine shrimp
lethality bioassay. The technique is easily mastered, of little cost
and utilizes a small amount of test material. The aim of this method
is to provide a front-line screen that can be backed up by more
specific and more expensive bioassays after isolating the active
compounds. The brine shrimp lethality bioassay is a development
in the assay procedure of bioactive compounds, which indicates
cytotoxicity as well as a wide range of pharmacological activities
(such as anticancer, antiviral, insecticidal, pesticidal, AIDS) of the
compounds. Results for lethality were noted in term of deaths of
nauplii. The mortality rate of nauplii was found to increase with
increasing concentration of complexes. A plot of log of sample
concentration versus percentage of mortality showed a linear
correlation. From the graph, the LC50 values of the samples were
Table 4. SOD-like activities of sparfloxacin and copper(II) complexes
Complex
IC50
Reference
calculated and were found to be in the range 4.01–9.64 mg mlꢀ1
.
From the data recorded in the table (supporting information),
complex 1 is the most potent amongst all the compounds. The
order of potency of compounds is 1 > 2 > 3 > 4 > 6 > 5 > 7. All
the complexes show good cytotoxic activity compared with
sparfloxacin. The shrimp lethality assay is considered a useful tool
for preliminary assessment of toxicity. Further investigations are
required to explore the exact mechanism of their cytotoxic proper-
ties, which may be helpful in exploring new types of potent
cytotoxic agents with the hope of adding new and alternative
chemotherapeutic agents in clinical implications.
Sparfloxacin
168.7 mM
0.572 mM
This work
This work
This work
This work
This work
This work
This work
This work
[74]
[Cu(SPF)(L1)Cl]
[Cu(SPF)(L2)Cl]
[Cu(SPF)(L3)Cl]
[Cu(SPF)(L4)Cl]
[Cu(SPF)(L5)Cl]
[Cu(SPF)(L6)Cl]
[Cu(SPF)(L7)Cl]
[Cu(stz)(py)3Cl]
[Cu(Hstz)(MeOH)Cl2]
[Cu(stz)2(1,2-dmHim)2]
[CuL2(4-mHim)2]
[Ni(sf)2(bipy)]
0.672 mM
0.787 mM
1.057 mM
1.373 mM
1.212 mM
1.522 mM
1.310 mM
5.170 mM
1.030 mM
5.860 mM
28.30 mM
26.54 mM
27.45 mM
12.69 mM
[75]
[75]
SOD-Like Activity
[75]
[78]
It has been proposed that electron transfer between copper(II)
and superoxide anion radicals occurs through direct binding.
The aꢀxial site of copper(II) complex is the likely attachment point
[Ni(erx)2(bipy)]
[Ni(oxo)2(bipy)]
[Ni(flmq)2(bipy)]
[78]
[78]
[78]
•
for O2 . The copper(II) arrangement facilitates interaction with the
radical and their dissociation to oxygen and hydrogen peroxide.
The fast exchange of axial solvent mꢀolecules and a limited steric
•
hindrance to the approach of the O2 in the complexes give rise
to better SOD mimics.[72]
Superoxide radicals were generated in vitro using a non-enzymatic
(NBT/NADH/PMS) system and determined spectrophotometrically.
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 complexes were
investigated by NBT assay. SOD data for the complexes have
been compiled in Table 4.[73] Copper gives good SOD activity,
although its structure is totally unrelated to native enzyme.
The ping-pong mechanism of SOD activity is given in the
form of equations (4) and (5):
O2ꢅꢀ þ CuII ! O2 þ CuI
(4)
(5)
O2ꢅꢀ þ CuI þ 2Hþ ! H2O2 þ CuII
A plot of absorbance values (Aobs560) against time (t) with varying
concentration of complex 1 from 0.5 to 3 mM is given in the
Figure 4. Plot of percentage of inhibiting NBT reduction with increase in
concentration of complex 1.
Appl. Organometal. Chem. 2012, 26, 641–649
Copyright © 2012 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/aoc

M. N. Patel et al.
[17] J. K. Aronson (Ed), Sparfloxacinin, in Meyler’s Side Effects of Drugs: The
International Encyclopedia of Adverse Drug Reactions and Interactions
Ed., Elsevier, Amsterdam, 2006 pp., pp. 3172.
of Cu(II) complex, which depends on the geometry at the metal
center. The mechanism for scavenging of reactive oxygen species
may move forward via an unstable octahedral adduct under the
influence of the Jahn–Teller effect.[43] Hence there is a possibility
of rapid interconversion of Cu(II) and Cu(I) via electron transfer
between copper and reactive oxygen radical anions following the
principle of electroneutrality.[77,78]
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In this work we have synthesized seven Cu(II) metallointercalators
with different neutral tridentate ligands and sparfloxacin. The
antibacterial activity of sparfloxacin is changed upon coordination
with the metal ion. Hypochromism and bathochromism of the
band in absorption titration and the increase in relative viscosity
of DNA suggest that all complexes bind with DNA via the classical
intercalative mode. Complexation of drug with metal ion enhances
their DNA cleavage ability. Upon determination of the antioxidant
activity in the NBT/NADH/PMS system, complex 1 showed the
highest scavenging ability for the oxygen radical. The tested
copper(II) complexes have strong cytotoxic activity but this investi-
gation 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 is suggested that
the complexes can be used as potent cytotoxic agents with the
hope of adding to the arsenal of weapons used against the fatal
disease of cancer.
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Acknowledgment
The authors thank the Head, Department of Chemistry, Sardar
Patel University, India, for making it convenient to work in
the laboratory. The authors report no conflicts of interest. The
authors alone are responsible for the content and writing of the
paper.
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Supporting information may be found in the online version of
this article.
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