Synthesis, characterization and biological studies of some homodinuclear complexes of zinc with second-generation quinolone drug and neutral bidentate ligands

Article abstract of DOI:10.1016/j.poly.2010.03.007

The novel homodinuclear zinc(II) complexes with the quinolone antibacterial drugs ciprofloxacine and neutral bidentate ligands have been synthesized and characterized by elemental analysis, TG analyses and various spectroscopic techniques. The metal ion exhibits octahedral geometry with two water molecule in the inner sphere cavity environment. The interaction of complexes with DNA was determined using absorption titration, viscosity measurements and electrophoresis technique. The intrinsic binding constants (K_b) of complexes were determined, which were ranging from 1.0 × 10⁴ to 3.5 × 10⁴ per mole. Suggesting that complexes bind more strongly to DNA. Effect on viscosity has also been checked to authenticate the binding of metal complexes with DNA. An antimicrobial activity of all the ligands and metal complexes has been examined by minimum inhibitory concentration method (MIC).

Full text of DOI:10.1016/j.poly.2010.03.007

Polyhedron 29 (2010) 1918–1924
Contents lists available at ScienceDirect
Polyhedron
journal homepage: www.elsevier.com/locate/poly
Synthesis, characterization and biological studies of some homodinuclear
complexes of zinc with second-generation quinolone drug and neutral bidentate
ligands
a
a
b
b
M.N. Patel a, , M.R. Chhasatia , P.A. Dosi , H.S. Bariya , V.R. Thakkar
*
a Department of Chemistry, Sardar Patel University, Vallabh, Vidyanagar 388 120, Gujarat, India
b BRD School of Bioscience, 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 homodinuclear zinc(II) complexes with the quinolone antibacterial drugs ciprofloxacine and
neutral bidentate ligands have been synthesized and characterized by elemental analysis, TG analyses
and various spectroscopic techniques. The metal ion exhibits octahedral geometry with two water mol-
ecule in the inner sphere cavity environment. The interaction of complexes with DNA was determined
using absorption titration, viscosity measurements and electrophoresis technique. The intrinsic binding
constants (Kb) of complexes were determined, which were ranging from 1.0 ꢀ 104 to 3.5 ꢀ 104 per mole.
Suggesting that complexes bind more strongly to DNA. Effect on viscosity has also been checked to
authenticate the binding of metal complexes with DNA. An antimicrobial activity of all the ligands and
metal complexes has been examined by minimum inhibitory concentration method (MIC).
Ó 2010 Elsevier Ltd. All rights reserved.
Received 23 August 2009
Accepted 4 March 2010
Available online 15 March 2010
Keywords:
Ciprofloxacin
DNA-binding
Cleavage
Minimum inhibitory concentration
1. Introduction
The role of zinc in various biological systems is important, since
it is crucial for numerous cell processes and is a major regulatory
Quinolones are a large and constantly expanding group of syn-
thetic antibacterial agents [1,2], and ciprofloxacin (CPFL) is one of
the most popular members of this family (Fig. 1). It is a synthetic,
broad-spectrum antibacterial drug that is used to treat pneumonia,
bronchitis and some types of gonorrhea, diarrhea caused by bacte-
ria, typhoid fever, prostate, sinus and urinary tract infections [3,4].
It is active against DNA gyrase enzyme. DNA gyrase introduces
negative supercoils in DNA [5] by wrapping the DNA around the
enzyme. The enzyme then catalyzes the breakage of a segment of
the wrapped DNA, the passage of a segment of the same DNA
through the break and finally the relegation of the break [6]. In this
way, a knot of DNA is resolved and is exposed for replication.
The design of drug based metal complexes is of particular inter-
est in pharmacological research. Metal combinations with pharma-
ceutical agents are known to improve drug activity and to decrease
their toxicity. Quinolone antibiotics are chelating agents for a vari-
ety of metal ions. The coordinated metal ions play an important
role in maintaining proper structure and/or function. The interac-
tion of quinolone with metal ions and the biologically active com-
ion in the metabolism of cells [17,18]. In the literature, different
zinc complexes with biological activity have been reported, but
only zinc complexes with drugs are used for the treatment of Alz-
heimers disease [19], while others show antibacterial [20], anti-
convulsant [21], antidiabetic [22], anti-inflammatory [23] and
antimicrobial activity [24].
Here, we present the interaction of Zn2+ with a second-genera-
tion quinolone, ciprofloxacin, and bidentate ligands in an attempt
to examine the mode of coordination and biological properties of
the prepared complexes. More specifically, the complexes have
been synthesized and characterized using diverse spectroscopic
techniques (IR and mass spectroscopy). The biological activity of
the complexes has been evaluated by determining the minimum
inhibitory concentration (MIC) against five microorganisms. To this
aim, we have also investigated the DNA binding assay of the Zn(II)
complexes with ciprofloxacin and some neutral bidentate ligands
using a combination of spectroscopic (UV–Vis), hydrodynamic (vis-
cometric) and gel electrophoresis techniques.
plexes formed as
a result of this interaction are especially
2. Experimental
important [7,8]. Although reports indicate that the coordination
of quinolones to metal ions appears to be important for the activity
of quinolone antibiotics [9–11], it has detrimental effect on their
absorption [12–16].
2.1. Materials and methods
Ciprofloxacin hydrochloride (CPFLꢁHCl) was purchased from
Bayer AG (Wuppertal, Germany). Ethylenediamine (A6-en), 2,3-
butanedione, p-anisaldehyde, p-anisidine, 1,8-diaminonaphthaline
* Corresponding author. Tel.: +91 2692226856x220.
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.2010.03.007

M.N. Patel et al. / Polyhedron 29 (2010) 1918–1924
1919
2.4. Synthesis of the coordination compounds
[Zn2(Cip)2(bmpdme)2(pip)(H2O)2]ꢁ3H2O (I). A methanolic solu-
tion (50 mL) of Zn(OAc)2ꢁH2O (2.01 g, 10 mmol), was added to
warm methanolic solution (50 mL) of bmpdme (2.96 g, 10 mmol),
followed by addition of a previously prepared solution of CPFLꢁHCl
(3.67 g, 10 mmol) in water (20 mL); the pH of the solution was ad-
justed to 6.0–7.0 using dilute NaOH solution. During the reaction,
the piperazine ring of ciprofloxacin hydrochloride was substituted
by a chloride ion in the presence of NaOH [27]. The resulting solu-
tion was refluxed for 8–10 h on a steam bath, and then was kept
overnight at room temperature. A fine amorphous product was ob-
tained which was washed with ether and dried in a vacuum
desiccator.
In a similar way, complexes II–VI were prepared with the use of
the corresponding ligands (Supplementary data 2: Proposed struc-
tures of the synthesized complexes). The physical parameters of all
the complexes are shown in Table 1 and the proposed reaction is
shown in Scheme 1.
Fig. 1. 4-(3-Carboxy-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)pip-
erazin hydrochloride [CPFLꢁHCL].
(A4-dan), acetophenone, glycerol and zinc(II) acetate were pur-
chased from E. Merck (India) Ltd., Mumbai. Xylene cyanol FF, ethi-
dium bromide and Luria broth were purchased from Himedia, India.
Agarose was purchased from the Sisco Research Lab., India. Bromo-
phenol blue, o-phenylenediamine (A5-opd), acetic acid and EDTA
were purchased from Sd Fine Chemicals, India. Sperm herring
DNA was purchased from Sigma Chemical Co., India. All the chem-
icals and solvents used were of analytical grade.
2.5. Antibacterial activity – minimum inhibitory concentration
The antibacterial activity of the compounds (ligand and com-
plexes) was studied against Staphylococcus aureus, Bacillus subtilis,
Escherichia coli, Pseudomonas aeruginosa and Serratia merscences.
All bacterial species were incubated and activated at 37 °C for
24 h by inoculating them to 2% Luria Broth (w/v) in double distilled
water. The compounds were dissolved in DMSO and then diluted
using cautiously adjusted Luria broth. The MIC was determined
using twofold serial concentrations in liquid media containing
2.2. Instrumentation
C, H and N elemental analyses were performed with a model
Perkin Elmer 240 elemental analyzer. The metal contents of the
complexes were analyzed by EDTA titration [25] after decompos-
ing the organic matter with a mixture of HClO4, H2SO4 and HNO3
(1:1.5:2.5). IR spectra (400–4000 cmꢂ1) were recorded on a FT-IR
Shimadzu spectrophotometer with the samples prepared as KBr
pellets. Thermogravimetric analyses were obtained with a model
5000/2960 SDTA, TA instrument (USA). The FAB-mass spectra 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.
The electronic spectra were recorded on a UV-160A UV–Vis spec-
trophotometer, Shimadzu (Japan)
12 000–0.1
lM of the compound being tested. Test cultures were
incubated at 37 °C (24 h). The lowest concentrations of antimicro-
bial agents that result in complete inhibition of microorganisms
were represented as the MIC in
lM. In each case triplicate tests
were performed and an average was taken as the final reading [28].
2.6. DNA binding and cleavage experiments
2.6.1. UV–Vis spectroscopy
The concentration of DNA was measured by using its standard
extinction coefficient at 260 nm (6600 Mꢂ1 cmꢂ1) [29]. Absor-
bances at 260 nm (A260) and 280 nm (A280) were measured to
check the purity of the DNA. The ratio of A260 to A280 was found
to be 1.8, indicating that the DNA was satisfactorily free from
protein. Phosphate buffer (1 mM, pH 7.2) was used for the
absorption titration experiment. The absorption titration was car-
2.3. Synthesis of the Schiff bases
The neutral bidentate ligands A1(bmpdme), A2(bmbed) and
A3(bpebd) were synthesized and characterized as per the method-
ology previously reported by us [26] (Supplementary data 1: Struc-
ture of the ligands used).
ried out by varying the DNA concentration (0–20
taining constant concentration of the complex (4
Absorption spectra were recorded after each successive addition
lM) and main-
a
l
M).
Table 1
Experimental and physical parameters of the complexes.
Complexes empirical formula
Elemental analysis % found (required)
m.p. (°C)
% Yield
Formula weight (g/mol)
C
H
N
M
C66H74Cl2F2N8O15Zn2 (I)
54.39 (54.33)
5.14 (5.11)
7.64 (7.68)
8.95 (8.97)
312
330
280
>360
356
300
68.4
69.0
66.5
67.8
68.2
65.7
1459.06
1459.32
1479.09
1182.73
1082.62
986.53
[Zn2(Cip)2(bmpdme)2(pip)(H2O)2]ꢁ3H2O
C66H74Cl2F2N8O15 Zn2 (II)
54.35 (54.33)
59.58 (59.60)
50.77 (50.78)
46.59 (46.60)
41.35 (41.39)
5.10 (5.11)
4.98 (5.00)
4.58 (4.60)
4.65 (4.66)
5.10 (5.11)
7.65 (7.68)
7.49 (7.51)
9.45 (9.47)
10.34 (10.35)
11.35 (11.36)
8.93 (8.97)
[Zn2(Cip)2(bmbed)2(pip)(H2O)2]ꢁ3H2O
C74H74Cl2F2N8O11Zn2 (III)
8.75 (8.77)
[Zn2(Cip)2(bpebd)2(pip)(H2O)2]ꢁ3H2O
C50H54Cl2F2N8O11Zn2 (IV)
11.04 (11.06)
12.05 (12.08)
13.24 (13.26)
[Zn2(Cip)2(dan)2(pip)(H2O)2]ꢁ3H2O)
C42H50Cl2F2N8O11Zn2(V)
[Zn2(Cip)2(opd)2(pip)(H2O)2]ꢁ3H2O
C34H50Cl2F2N8O11Zn2 (VI)
[Zn2(Cip)2(en)2(pip)(H2O)2]ꢁ3H2O

1920
M.N. Patel et al. / Polyhedron 29 (2010) 1918–1924
Scheme 1. Proposed reaction scheme for synthesis of complex I.
of DNA followed by allowing it to attain equilibrium (approxi-
mately 10 min). For complexes I–VI, the observed data were fur-
ther utilized to obtain the intrinsic binding constant, Kb using the
following equation [30]:
2.6.3. Gel electrophoresis study
pUC19 DNA was prepared by transformation of pUC19 into safe
competent cells (E. coli strain), and amplification of a clone as out-
lined by Sambrook and Russell [32]. After concentrating by ethanol
precipitation, the DNA was stored in TE buffer (pH 8.0) at ꢂ20 °C.
The relative amount of the supercoiled (SC) form was checked by
gel electrophoresis on an agarose bed. Electrophoresis was carried
out in a Submarine Mini-gel Electrophoresis Unit, also the degree
of DNA cleavage activity was expressed in terms of the percentage
of cleavage of the SC-DNA according to the following equation
[33]:
½DNAꢃ=ðea
ꢂ
ef Þ ¼ ½DNAꢃ=ðeb
ꢂ
ef Þ þ 1=Kbðeb
ꢂ
ef
Þ
where [DNA] is the concentration of DNA in terms of nucleotide
phosphate, [NP] is the apparent absorption coefficient ef ea and
,
eb correspond to the extinction coefficient of the free complex,
the extinction coefficient for each addition of DNA to the com-
plex and the extinction coefficient for the complex in the fully
bound form respectively and Kb is the ratio of the slope to y
intercept.
ð% of SCÞcontrol ꢂ ð% of SCÞsample
%DNA cleavage activity ¼
ꢀ 100
ð% of SCÞcontrol
2.6.2. Viscosity measurements
Viscosity measurements were carried out using an Ubbelohde
2.7. Aliquots of complex
viscometer maintained at
a constant temperature of 27.0
( 0.1) °C in a thermostatic bath. DNA samples with an approximate
average length of 200 base pairs were prepared by sonication in or-
der to minimize complexities arising from DNA flexibility. The flow
time was measured with a digital stopwatch with a precision of
DMSO containing the metal complexes (50
l
M) was taken in a
clean eppendroff tube and TE buffer (pH 8.0), pUC19
(0.12
l
g mlꢂ1) was added. The contents were incubated for 1 h
at 37 °C, loaded on 1% agarose gel after mixing of the loading
buffer (0.03% Bromophenol blue + 0.03% xylene cyanol + 60%
glycerol sterilized, distilled), 60 mM EDTA was added and elec-
trophoresis was performed at 100 V for 2 h in TAE buffer using
0.1 s. Each sample was measured thrice and an average flow time
1/3
was calculated. Data are presented as (
tion ratio [Zn]/[DNA] [31], where
presence of complex and 0 is the viscosity of DNA alone. Viscosity
g
/
g0
)
versus concentra-
g
is the viscosity of DNA in the
g
1.0% agarose gel containing 0.5 l
g mlꢂ1 ethidium bromide until
values were calculated from the observed flow time of DNA-con-
taining solutions (t > 100 s) corrected for the flow time of the buf-
the bromophenol blue reached to 3/4 of the gel. A control exper-
iment was done in the presence of the reactive substances Zn(II)
and ciprofloxacin.
fer alone (t0),
g
= t ꢂ t0.

M.N. Patel et al. / Polyhedron 29 (2010) 1918–1924
1921
The plasmid band was visualized by viewing the gel under a
3.3. TG analysis
transilluminator and photographed. The efficiency of the DNA
cleavage was measured by determining the ability of the complex
to form open circular (OC) or nicked circular (NC) DNA from its
supercoiled (SC) form by quantitatively estimating the intensities
of the bands, and the extent of cleavage was determined by using
volume quantization AlphaDigiDoc™ RT. Version V.4.1.0 PC-Image
software.
TG curves of the Zn(II) complexes show the following decompo-
sition steps. It was observed that all the complexes showed a loss in
weight corresponding to three water molecules in the range 50–
130 °C, indicating loss of water of crystallization. The second step
weight loss, during 130–180 °C, corresponds to two coordinated
water molecules. Loss in weight in the temperature range 180–
250 °C corresponds to a piperazine (pip) molecule followed by lib-
eration of Cip in the temperature range 250–500 °C. Finally, decom-
position of An occurred in the temperature range 520–800 °C and
the remaining weight was consistent with the metal oxide [49].
3. Results and discussion
All the complexes were insoluble in ether, hexane, chloroform,
water and methanol, partially soluble in dimethyl formamide,
and completely soluble in dimethylsulfoxide. Schiff bases A1–A3
were prepared by condensation of the amine and aldehyde/ketone
in ethanol. The complexes under investigation were characterized
using IR spectra, FAB-mass spectra, TG analysis and electronic
spectra.
3.4. Mass spectrum
The FAB-mass spectrum of complex I (Fig. 2) shows the molec-
ular ion peak at m/z 1406 due to [M+2H+] rid of three lattice water
molecules. The peaks at m/z 1388 and 1370 are due to removal of
coordinated water, one after another. The peaks occurring at m/z
641 and 726 correspond to the fragments [C31H28ClFN3O5Zn + H+]
and [C35H36ClFN5O5Zn + H+], produced by cleavage of one of the
Zn–N bonds with piperazine. The peak arising at m/z 296 corre-
sponds to the ligand [C18H20N2O2], whereas the peak at m/z 344
corresponds to the fragment [C13H8ClFNO3Zn] (Supplementary
data 3: FAB-MS of Complexes I–VI).
3.1. IR spectra
The
m
(C@O) stretching vibration band appears at 1708 cmꢂ1
for ciprofloxacin, whereas for complexes it appeared at 1619–
1630 cmꢂ1; this shift towards lower energy suggests that the
coordination occurs through the carbonyl oxygen of the pyridine
ring [34]. A sharp band in ciprofloxacin at 3520 cmꢂ1 [35] is due
to hydrogen bonding; which is attributed to the ionic resonance
structure and the peak is observed because of a free hydroxyl
stretching vibration. This band completely vanished in the spectra
of metal complexes indicating deprotonation of the carboxylic
3.5. Antimicrobial activity
The efficiencies of the ligands and the complexes have been
tested against three Gram(ꢂve), E. coli, S. merscences, P. aeruginosa,
and two Gram(+ve), S. aureus, B. subtilis, microorganisms. The results
are presented in Table 3. From the experiment we found that all
the metal complexes were more active than the metal salts and li-
gands. Complexes I, II and III have good activity against S. aureus
compared to all standard drugs whereas complexes IV, V and VI
were more active than gatifloxacin. In the case of B. subtilis, except
for complex VI, all the complexes were more active than standard
drugs. In the case of S. merscences, complex V had better activity
than standard drugs whereas complexes I–IV were found to be
more active than gatifloxacin, norfloxacin and pefloxacin. In the
case of P. aeruginosa, complex I was the most potent among the
six synthesized complexes, however the increasing order of their
activity is I < III < II < V < VI < IV. In the case of E. coli the order of
activity is III < II < I < V < IV < VI. The data shows that the metal
complexes exhibit antimicrobial properties and it is important to
note that these complexes exhibit enhanced activity in contrast
to the free ligand. Enhanced antibacterial activity is because the li-
pid membrane that surrounds the cell favors the passage of only li-
pid-soluble materials, so liposolubility is an important factor
controlling the antimicrobial activity [50].
proton. The data were further supported by a m(M–O) [36] band
which appeared at 502–514 cmꢂ1. The strong absorption band
obtained at 1624 and 1340 cmꢂ1 in ciprofloxacin were assigned
to
plexes these bands were observed at 1591 and 1386 cmꢂ1
respectively. The frequency of separation ( ) for the carboxyl
group in the investigated complexes is greater than 200 cmꢂ1
m(COO)as and m(COO)s respectively, while in the metal com-
,
D
m
,
suggesting a unidentate bonding nature [34,37–41]. The m(C@N)
peak foꢂr 1 the Schiff bases A1–A3 was observed at 1610–
1640 cm
which on complexation was shifted to 1564–
1580 cmꢂ1, which indicates the N–N coordinating behavior [42–
45]. This data was further supported by m(M–N) [46] which ap-
peared at 537–548 cmꢂ1. Infrared spectral data of the complexes
are shown in Table 2.
3.2. Electronic spectral measurements
In the electronic spectra of the zinc(II) complexes there are
M ? L charge-transfer bands at ꢄ30 800 cmꢂ1, which were as-
signed to the octahedral structure. Zn(II) being d10 system, with
It may be suggested that these complexes deactivate various
cellular enzymes, which play a vital role in various metabolic path-
ways of these microorganisms. It has also been proposed that the
ultimate action of the toxicant is the denaturation of one or more
no unpaired electron, always exhibits
[47,48].
a diamagnetic nature
Table 2
Infrared spectral data of the Zn(II) complexes.
Compounds
m
(C@O) (cmꢂ1
)
m
(COO)as
m
(COO)s
D
m
m
(C–Cl)
m
(C@N) (cmꢂ1
)
m
(M–N)
m(M–O)
pyridone
(cmꢂ1
)
(cmꢂ1
)
(cmꢂ1
)
(cmꢂ1
)
azomethine
(cmꢂ1
)
(cmꢂ1
)
I
II
III
IV
V
1619
1625
1630
1621
1625
1622
1591
1600
1610
1587
1598
1596
1386
1380
1381
1376
1385
1372
205
220
229
211
213
224
1130
1141
1125
1128
1126
1124
1567
1564
1580
–
–
–
539
537
548
540
538
541
511
508
514
509
505
502
VI

1922
M.N. Patel et al. / Polyhedron 29 (2010) 1918–1924
Fig. 2. FAB-mass spectrum of complex I.
Table 3
in Fig. 3. For the complexes, the absorption spectra clearly show
MIC data of the compounds (lM).
that addition of DNA to the complexes yields hyperchromism
and a blue shift corresponding to the ratio of [DNA]/[M]. Obviously,
these spectral characteristics suggest that all the complexes inter-
act with DNA, most likely through a mode that involves a stacking
interaction between the aromatic chromophore and the base pairs
of DNA. Addition of increasing amounts of DNA resulted in hypso-
chromism of the peak maxima in the UV–Vis spectra of the binu-
clear zinc(II) complexes.
Compounds
Gram positive
Gram negative
S. aureus
B.
S.
P.
E. coli
subtilis
merscences
aeruginosa
Zn(OAc)2ꢁH2O 1373.4.00 1099.00 2198.00
1923.00
1.36
1.01
3.76
1.39
5.70
1.66
1.53
2.21
1923.00
1.36
2.93
2.82
1.39
2.70
0.97
1.27
1.38
Ciprofloxacin
1.63
5.06
2.51
1.95
2.10
1.66
1.27
1.94
1.09
4.00
2.51
3.90
2.40
2.21
2.04
1.38
1.63
2.93
4.07
1.67
5.10
1.66
1.53
1.66
Gatifloxacin
Norfloxacin
Enrofloxacin
Pefloxacin
Levofloxacin
Sparfloxacin
Ofloxacin
An electronic interaction between the complex chromophore
and DNA bases meets one of the criteria for intercalative binding
[55]. Using the absorption measurements for I, a linear plot of
A1
A2
A3
A4
A5
A6
I
1856.00
1687.00
1761.00
3161.00
5086.00
8319.00
0.55
0.34
0.54
2.54
3.23
1519.00 2025.00
1518.00 1687.00
1600.00 1761.00
3477.00 4109.00
5548.00 6011.00
9151.00 9983.00
2193.00
1856.00
1921.00
3793.00
6011.00
10815.00
1.37
1.71
1.68
2.54
1.85
2362.00
2193.00
2080.00
4109.00
6473.00
10815.00
2.74
2.06
1.68
2.96
2.77
[DNA]/(ef
ꢂ
ea) versus [DNA] (Fig. 3, inset) was obtained. Assuming
all the complexes were bound with DNA, the experimental Kb was
obtained by substituting the absorbance into Beer’s law. The bind-
ing constants (Kb) are given in Table 4. The binding constant Kb for
the Zn(II) complexes varies in the range 1.0–3.5 ꢀ 104 Mꢂ1, which
is in good agreement with that determined experimentally. The
DNA-binding constant of the title complexes are comparable to
0.55
0.34
0.34
0.85
0.92
1.52
2.06
2.06
2.01
2.96
0.92
3.04
II
III
IV
V
VI
3.55
2.53
3.04
proteins of the cell, which as a result, impairs normal cellular pro-
cesses. The following five principal factors [51–54] should be con-
sidered for metal complexes showing antimicrobial activity, (i) the
chelate effect, i.e. ligands that are bound to metal ions in a biden-
tate fashion, like quinolones and NN-donor ligands; (ii) the nature
of the ligands; (iii) the total charge of the complex; generally the
antimicrobial efficiency decreases in the order cationic > neu-
tral > anionic complex; (iv) the nature of the ion neutralizing the
ionic complex; (v) the nuclearity of the metal center in the com-
plex; dinuclear centers are usually more active than mononuclear
ones. Thus, factors (i), (ii) and (v) can be considered to increase in
the antibacterial activity in this study.
3.6. DNA binding studies
Fig. 3. Absorption spectra of [Zn2(Cip)2(bmpdme)2(pip)(H2O)2]ꢁ3H2O (4
absence and in the presence of increasing amounts of DNA, where a to h represents
the increasing amount of DNA, inset: plot of [DNA]/(ef a) vs. [DNA]. Arrow shows
the absorption change upon increasing the DNA concentration.
lM) in the
3.6.1. Absorption titration
Increasing amounts of DNA were titrated with preset amounts
(4 lM) of the complexes. The electronic spectral traces are given
ꢂ
e

M.N. Patel et al. / Polyhedron 29 (2010) 1918–1924
1923
Table 4
The binding constants (Kb) of Zn(II) complexes with DNA in phosphate buffer pH 7.2.
Complexes
Kb (Mꢂ1
)
I
II
III
IV
V
1.0 ꢀ 104
3.5 ꢀ 104
1.0 ꢀ 104
1.0 ꢀ 104
2.0 ꢀ 104
2.0 ꢀ 104
VI
those of some DNA intercalative polypyridyl Ru(II) complexes, 1.0–
Fig. 5. Gel electrophoresis data with pUC19. Lane 1: DNA (control); Lane 2:
DNA + metal salt; Lane 3: DNA + CPFLꢁHCl; Lane 4: DNA + I; Lane 5: DNA + II; Lane
6: DNA + III; Lane 7: DNA + IV; Lane 8: DNA + V; Lane 9: DNA + VI.
4.8 ꢀ 104 Mꢂ1 [56–58]. In the plot of [DNA]/(ea
ꢂ
ef) versus [DNA],
the binding constant Kb is given by the ratio of the slope to the
intercept.
3.6.2. Viscosity measurements
The binding of intercalative drugs to DNA has also been charac-
terized recently through the changes in viscosity of DNA. Fig. 4
summarizes the relative viscosity (go) of DNA in the absence and
presence of the complexes. The viscosity measurements were car-
ried out by varying the concentration of the complexes. The inter-
calation of drugs with DNA usually causes an increase in the
viscosity of DNA [59]. The viscosity experiment displays that the
relative viscosity (go) of DNA increased with an increasing concen-
Fig. 6. Gel electrophoresis data with pUC19. Lane 1: DNA (control); Lane 2:
DNA + H2O2; Lane 3: DNA + H2O2 + metal salt; Lane 4: DNA + H2O2 + CPFL; Lane 5:
DNA + H2O2 + I; Lane 6: DNA + H2O2 + II; Lane 7: DNA + H2O2 + III; Lane 8:
DNA + H2O2 + IV; Lane 9: DNA + H2O2 + V; Lane 10: DNA + H2O2 + VI.
tration of the complexes, but the extent of increase was lesser than
that for the known intercalator EB [60]. For all the complexes, as
the amounts of complexes increased, the viscosity of DNA in-
creased steadily, which is similar to that of the classical intercala-
tive complex [Ru(phen)2(DPPZ)]2ꢂ. From Fig. 4 we can say that
complexes I, II and VI intercalate more strongly than complexes
III, IV and V. This indicates that I, II and VI bind to DNA more
strongly than the III, IV and V. Based on the above experiments,
the results clearly indicate the different ability of the ligands in
the all complexes to stack and overlap with the base pairs.
Table 5
Gel electrophoresis data.
Compounds
% SC
% NC
% OC
% Cleavage
DNA control
58
49
44
48
47
48
47
48
48
35
34
31
42
44
42
45
47
46
31
39
32
35
42
41
39
41
39
24
26
30
47
48
46
43
41
40
11
12
24
24
11
11
15
11
13
41
40
39
11
18
12
12
12
14
0
DNA + metal salt
DNA + CPFLꢁHCl
DNA + I
DNA + II
DNA + III
DNA + IV
DNA + V
DNA + VI
DNA + H2O2
DNA + H2O2 + metal salt
DNA + H2O2 + CPFLꢁHCl
DNA + H2O2 + I
DNA + H2O2 + II
DNA + H2O2 + III
DNA + H2O2 + IV
DNA + H2O2 + V
DNA + H2O2 + VI
15.52
24.14
17.24
18.97
17.24
18.97
17.24
17.24
39.66
41.38
46.55
27.59
24.14
27.59
22.41
18.97
20.69
3.6.3. Gel analysis and quantification of pUC19 DNA
There has been considerable interest in DNA cleavage reactions
that are activated by transition metal complexes [61,62]. The deliv-
ery of the metal ion to the helix, locally generating oxygen or
hydroxide radicals, yields an efficient DNA cleavage reaction. Figs.
5 and 6 illustrate electrophoretic separations presenting the cleav-
age of plasmid pUC19 DNA by the complexes under aerobic condi-
tions in the presence and absence of H2O2, respectively [63]. When
circular plasmid DNA is conducted by electrophoresis, the fastest
migration will be observed for the supercoiled form (SC). If one
strand is cleaved, the supercoiled form will relax to produce a
slower-moving open circular form (OC). If both strands are cleaved,
a nicked form (NC) will be generated that migrates in between.
This clearly shows that the relative binding efficacy of the com-
plexes to DNA is much higher than the binding efficacy of the
metal salt itself (Table 5). The different DNA cleavage efficiencies
of the complexes is due to the different binding affinity of the com-
plexes to DNA, which has been observed in other cases. One of the
most interesting electrophoretic results of the complexes takes
place when the experiment was carried out in the presence of
H2O2 in TAE buffer. The DNA + complex + H2O2 systems (Fig. 6)
cleaved the supercoiled DNA form (I) and converted it into the
nicked form (II) and linear form (III) more than just the complex
alone. It can be concisely seen from the data that the mixture of
complex with H2O2 proves to be an efficient cleaving agent via
the electrostatic mode of interaction [64].
4. Conclusion
The antimicrobial activity shows that the complexes have been
found to be more potent against Gram(+ve) than Gram(ꢂve) species.
The DNA-binding constants of the title complexes varies in the
Fig. 4. Change in viscosity of DNA on addition of the complexes.

Patel et al. / Polyhedron 29 (2010) 1918–1924
M.N.
1924
range 1.0–3.5 ꢀ 104 Mꢂ1, which are comparable to the values of
some DNA intercalative polypyridyl Ru(II) complexes, 1.0–
4.8 ꢀ 104 Mꢂ1. All the complexes exhibit good binding and cleav-
age activity. Complexes I, II and VI intercalate more strongly than
complexes III, IV and V. This indicates that complexes I, II and VI
bind to DNA strongly than complexes III, IV and V. The mixture
of the complexes with H2O2 have been found to be efficient oxi-
dants via electrostatic interaction by generating more strand
breaks in plasmid DNA compare to the complexes alone.
[25] A.I. Vogel, Textbook of Quantitative Inorganic Analysis, fourth ed., ELBS and
Longman, London, 1978.
[26] M.N. Patel, M.R. Chhasatia, D.S. Gandhi, Bioorg. Med. Chem. 17 (2009) 5648.
[27] G. Wu, G. Wang, X. Fu, L. Zhu, Molecules 8 (2003) 287.
[28] R.N. Jones, A.L. Barry, T.L. Gaven, J.A. Washington, E.H. Lennette, A. Balows, W.J.
Shadomy, Manual of Clinical Microbiology, fourth ed., vol. 972, American
Society for Microbiology, Washington, DC, 1984.
[29] M.E. Reichmann, S.A. Rice, C.A. Thomas, P. Doty, J. Am. Chem. Soc. 76 (1954)
3047.
[30] A. Wolfe, G.H. Shimer, T. Meehan, Biochemistry 26 (1987) 6392.
[31] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45.
[32] J. Sambrook, D.W. Russell, Preparation of Plasmid DNA by Alkaline Lysis with
SDS: Minipreparation, Molecular Cloning, A Laboratory Manual, third ed., vol.
1, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, p. 1.
[33] J. Yang, R.N.S. Wong, M.S. Yang, Chem.-Biol. Interact. 125 (2000) 221.
[34] I. Leban, I. Turel, N. Bukovec, J. Inorg. Biochem. (1999) 241.
[35] R.M. Silverstein, F.X. Webster, Spectrometric Identification of Organic
Compounds, sixth ed., John Wiley & Sons, Inc., 2004.
Acknowledgments
We wish to express our gratitude to the Head, Department of
Chemistry, Vallabh Vidyanagar, Gujarat, India for providing neces-
sary laboratory facilities. The authors are also thankful to the UGC
for financial assistance of UGC grant 32-226/2006(SR).
[36] C. Yan, Y. Li, J. Lou, C. Zhu, Syn. React. Inorg. Metal–Org. Chem. 34 (5) (2004)
979.
[37] K. Nakamoto, Infrared and Raman Spectra of Inorganic and Coordination
Compounds, fourth ed., Wiley Interscience Publication, 1986.
[38] J.R. Anacona, I. Rodriguez, J. Coord. Chem. 57 (2004) 1263.
[39] G.B. Deacon, R.J. Philips, Coord. Chem. Rev. 23 (1980) 227.
[40] Z.H. Chohan, C.T. Suparan, A. Scozzafava, J. Enz. Inhib. Med. Chem. 20 (3)
(2005) 303.
Appendix A. Supplementary data
[41] N.H. Patel, P.K. Panchal, P.B. Pansuriya, M.N. Patel, J. Macro. Sci.: Part A: Pure
Appl. Chem. 43 (2006) 1083.
[42] P.K. Panchal, P.B. Pansuriya, M.N. Patel, Toxicol. Environ. Chem. 88 (1) (2005)
57.
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.poly.2010.03.007.
[43] H.M. Parekh, P.K. Panchal, M.N. Patel, J. Therm. Anal. Cal. 86 (3) (2006) 803.
[44] N. Raman, A. Kulandaisamy, K. Jayasubramananian, Polish J. Chem. 76 (2002)
1085.
References
[45] H.M. Parekh, P.K. Panchal, P.B. Pansuriya, M.N. Patel, Polish J. Chem. 80 (2006)
989.
[46] S. Chandra, N. Gupta, L.K. Gupta, Synth. React. Inorg. Metal–Org. Chem. 34 (5)
(2004) 919.
[47] A.B.P. Lever, J. Lewis, R.S. Nyholm, J. Chem. Soc. (1963) 2552.
[48] D.N. Sathyanarayana, Electronic Absorption Spectroscopy and Related
Techniques, first ed., University press (India) Ltd., Hyderabad, 2001.
[49] S.H. Patel, P.B. Pansuriya, M.R. Chhasatia, H.M. Parekh, M.N. Patel, J. Therm.
Anal. Cal. 91 (2) (2008) 413.
[50] Z.H. Chaohan, M. Hassan, K.M. Khan, C.T. Supuran, J. Enz. Inhib. Med. Chem. 20
(2005) 183.
[51] G. Psomas, C. Dendrinou-Samara, P. Philippakopoulos, V. Tangoulis, C.P.
Raptopoulou, E. Samaras, D.P. Kessissoglou, Inorg. Chim. Acta 272 (1998)
24.
[1] L.A. Mitscher, Chem. Rev. 105 (2005) 559.
[2] G. Sheehan, N.S.Y. Chew, in: A.R. Ronald, D.E. Low (Eds.), Fluoroquinolone
Antibiotics, Birkhauser, Basel, Switzerland, 2003, p. 1.
[3] B.M. Lomaestro, G.R. Bailie, Ann. Pharmacother. 25 (1991) 1249.
[4] J.H. Paton, D.S. Reeves, Drugs 36 (1988) 193.
[5] M. Gellert, K. Mizuuchi, M.H. O’Dea, H.A. Nash, Proc. Natl. Acad. Sci. USA 73
(1976) 3872.
[6] L.L. Shen, D.T.W. Chu, Curr. Pharm. Des. 2 (1996) 195.
[7] H.T. Yu, L.H. Hurley, S.M. Kerwin, J. Am. Chem. Soc. 118 (1996) 7040.
[8] G.S. Son, J.A. Yeo, M.S. Kim, J. Am. Chem. Soc. 120 (1998) 6451.
[9] M. Gellert, K. Mizuuchi, M.H. O’Dea, Proc. Natl. Acad. Sci. USA. 74 (1977) 4772.
[10] N.R. Cozarelli, Science 207 (1980) 953.
[11] G. Palu, S. Valisena, G. Ciarrocchi, Proc. Natl. Acad. Sci. USA 89 (1992) 9671.
[12] R.W. Frost, J.D. Carlson, A.J. Dietz, J. Clin. Pharmacol. 29 (1989) 953.
[13] D.E. Nix, W.A. Watson, M.E. Lener, Clin. Pharmacol. Ther. 46 (1989) 700.
[14] R.W. Frost, K.C. Lasseter, A.J. Noe, Antimicrob. Agents Chemother. 36 (1992) 830.
[15] M. Kara, B.B. Hasinoff, D.W. McKay, R.C. Campbell, Br. J. Clin. Pharmocol. 31
(1991) 257.
[16] T. Motoya, M. Niyashit, A. Kawachi, K. Yamada, J. Pharm. Pharmacol. 52 (2000)
397.
[17] N. Farrell, Coord. Chem. Rev. 232 (2002) 1.
[18] G.K. Walkup, S.C. Burdette, S.J. Lippard, R.Y. Tsien, J. Am. Chem. Soc. 122 (2000)
5644.
[19] M . Di Vaira, C. Bazzicalupi, P. Orioli, L. Messori, B. Bruni, P. Zatta, Inorg. Chem.
43 (2004) 3795.
[52] C. Dendrinou-Samara, G. Psomas, C.P. Raptopoulou, D.P. Kessissoglou, J. Inorg.
Biochem. 83 (2001) 7.
[53] A.D. Russell, in: S.S. Block (Ed.), Disinfection, Sterilization and Preservation,
fourth ed., Lea and Febinger, Philadelphia, 1991, p. 27.
[54] H.W. Rossmore, in: S.S. Block (Ed.), Disinfection, Sterilization and Preservation,
fourth ed., Lea and Febinger, Philadelphia, 1991, p. 290.
[55] C. Chulvi, R.M. Oritz, L. Perello, M.A. Romero, Thermochim. Acta 1556 (2)
(1989) 393.
[56] A. Juris, V. Balzani, F. Barigelletti, S. Campagna, P. Belser, A. von Zelewsky,
Coord. Chem. Rev. 84 (1988) 85.
[57] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901.
[58] M. Baldini, M. Belicchi-Ferrari, F. Bisceglie, P.P. Dall’ Aglio, G. Pelosi, S. Pinelli, P.
Tarasconi, Inorg. Chem. 43 (2004) 7170.
[20] Z.Q. Li, F.J. Wu, Y. Gong, C.W. Hu, Y.H. Zhang, M.Y. Gan, Chin. J. Chem. 25 (2007)
1809.
[21] J. d’Angelo, G. Morgant, N.E. Ghermani, D. Desmaele, B. Fraisse, F. Bonhomme,
E. Dichi, M. Sghaier, Y. Li, Y. Journaux, J.R.J. Sorenson, Polyhedron 27 (2008)
537.
[22] H. Sakurai, Y. Kojima, Y. Yoshikawa, K. Kawabe, H. Yasui, Coord. Chem. Rev.
226 (2002) 187.
[59] J.B. Leepecq, C. Paoletti, J. Mol. Biol. 27 (1967) 87.
[60] J.M. Veal, R.L. Rill, Biochemistry 30 (1991) 1132.
[61] R.P. Hertzberg, P.B. Dervan, J. Am. Chem. Soc. 104 (1) (1982) 313.
[62] D.S. Sigman, D.R. Graham, L.E. Marshall, K.A. Reich, J. Am. Chem. Soc. 102 (16)
(1980) 5419.
[23] Q. Zhou, T.W. Hambley, B.J. Kennedy, P.A. Lay, P. Turner, B. Warwick, J.R. Biffin,
H.L. Regtop, Inorg. Chem. 39 (2000) 3742.
[63] B.K. Santra, P.A.N. Reddy, G. Neelakanta, S. Mahadevan, M. Nethaji, A.R.
Chakravarty, J. Inorg. Biochem. 89 (2002) 191.
[24] N.C. Kasuga, K. Sekino, M. Ishikawa, A. Honda, M. Yokoyama, S. Nakano, N.
Shimada, C. Koumo, K. Nomiya, J. Inorg. Biochem. 96 (2003) 298.
[64] S. Ramakrishnan, M. Palaniandavar, J. Chem. Soc., Dalton Trans. (2008)
3866.