Antibacterial and DNA interaction studies of zinc(II) complexes with quinolone family member, ciprofloxacin

Article abstract of DOI:10.1016/j.ejmech.2009.10.024

DNA binding and cleavage characteristics of Zn(II) complexes have been investigated. The DNA interaction property of the complexes has been investigated using absorption spectra, viscosity measurements, as well as gel electrophoresis studies. Intrinsic bi

Full text of DOI:10.1016/j.ejmech.2009.10.024

European Journal of Medicinal Chemistry 45 (2010) 439–446
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Antibacterial and DNA interaction studies of zinc(II) complexes
with quinolone family member, ciprofloxacin
*
Mohan Patel , Mehul Chhasatia, Pradhuman Parmar
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:
DNA binding and cleavage characteristics of Zn(II) complexes have been investigated. The DNA inter-
action property of the complexes has been investigated using absorption spectra, viscosity measure-
ments, as well as gel electrophoresis studies. Intrinsic binding constant (Kb) has been estimated under
similar set of experimental conditions. Absorption spectral study indicate that the Zn(II) complexes
intercalate between the base pairs of the DNA tightly with intrinsic binding constant in the range of
1.0 ꢀ 104–4.0 ꢀ 104 Mꢁ1 in phosphate buffer. The proposed DNA binding mode supports the large
enhancement in the relative viscosity of DNA on binding. The antimicrobial activity of all the ligands and
metal complexes has been examined by minimum inhibitory concentration method (MIC).
Ó 2009 Elsevier Masson SAS. All rights reserved.
Received 13 March 2009
Received in revised form
23 July 2009
Accepted 15 October 2009
Available online 29 October 2009
Keywords:
Zn(II) complexes
Ciprofloxacin
DNA interaction
Intrinsic binding constant (Kb)
MIC
1. Introduction
spectroscopic techniques (UV spectroscopy), viscosity measure-
ments and gel electrophoresis technique. The antimicrobial effi-
Fluoroquinolones represent an important group of chemother-
apeutic compounds, which exhibit high antibacterial activities.
An efficient representative of this group, ciprofloxacin (Cip ¼
C17H18FN3O3, 1-cyclopropyl-6-fluoro-4-oxo-7-(1-piperazinyl)-1,4-
dihydroquinoline-3-carboxylic acid) [1] is widely used in clinical
practice as a broad spectrum antimicrobial agent [2]. Quinolones
comprise a group of well-known antibacterial agents and the first
members being in clinical practice over 40 years [3,4]. They can
act as antibacterial drugs that effectively inhibit DNA replication
and are commonly used in treatment of many infections [5,6].
Studies on the biological properties of quinolone-metal complexes
have been focused on the interaction with DNA, antibacterial
activity tests on diverse microorganisms, cytotoxicity and poten-
tial antitumor activity [7–15]. In this context, we have studied the
interaction of Zn(II) with ciprofloxacin, in the presence of
nitrogen-donor ligands such as bpdmed(A1)/mtma(A2)/apq(A3)/
bpeed(A4)/dcnd(A5)/dpeda(A6). The coordination behaviors of the
ligands towards transition metal salts have been investigated and
the data have been correlated with their elemental analysis,
thermal properties, magnetic measurements, IR and their DNA
binding and cleavage behavior were also being examined using
ciency (MIC) of the compounds has been screened against five
different microorganisms.
2. Results and discussion
2.1. 1H NMR and 13C NMR spectra of Schiff bases
The 1H and 13C NMR spectra of the ligands were carried out in
CDCl3/MeOD and reported along with the possible assignment. The
1H NMR spectra of ligands exhibiting peaks at about 6.5–8.19 ppm
were assigned to the aromatic protons. The singlet peak which
appeared at 7.5 ppm was assigned to the azomethine proton
(–CH]N–). In the 13C NMR spectra, the peaks observed at about
105.9–135.4 and 128.5–143.0 ppm were assigned to aromatic and
thiophene carbons respectively. Peaks observed at about
140.0– 150.9, 165.3–167.3 ppm were assigned to C–N and C]N
carbons respectively.
2.2. Spectroscopic studies of the mixed-ligand complexes
2.2.1. Infrared spectroscopy
The prominent IR spectral data of the complexes are shown in
Table 1. The
n(C]O) stretching vibration band appears at
1708 cmꢁ1 for ciprofloxacin, where as for complexes it appear at
1615–1629 cmꢁ1; this shift towards lower energy suggests that
* Corresponding author. Tel.: þ91 2692226858x220.
E-mail address: jeenen@gmail.com (M. Patel).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.10.024

440
M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
Table 1
Infrared spectral data of the Zn(II)complexes.
Compounds
n
(C]O)
n
(COO)asy
n
(COO)sym
Dn
cmꢁ1
n
(C–Cl)
n
(C]N)
n
(C]N)
n
(M–N)
n(M–O)
cmꢁ1
cmꢁ1
cmꢁ1
cmꢁ1
cmꢁ1
cmꢁ1
ring
cmꢁ1
cmꢁ1
pyridone
azomethine
I
II
III
IV
V
1629
1628
1620
1626
1615
1627
1590
1585
1601
1596
1605
1588
1382
1376
1380
1385
1384
1383
208
209
221
211
221
205
1117
1131
1142
1145
1136
1150
1579
1576
–
1564
1570
1576
–
–
1602
–
–
–
545
546
540
535
540
542
505
509
510
512
508
510
VI
coordination occurs through the carbonyl oxygen of pyridine ring
[16]. The sharp band in ciprofloxacin at 3520 cmꢁ1 [17] is due to
hydrogen bonding; which is attributed to ionic resonance structure
and peak observed because of free hydroxyl stretching vibration.
This band completely vanished in the spectra of the metal
complexes indicating deprotonation of the carboxylic proton. The
band at w31,000 cmꢁ1 and were assigned [32] to a ligand-metal
charge transfer transition. Zn(II) is belongs to d10 system so the
complexes which synthesized are diamagnetic in nature because of
unavailability of unpaired electrons in this system [33].
2.2.3. TG analyses
data were further supported by
at 505–512 cmꢁ1
The strong absorption band obtained at
1624 cmꢁ1 and 1340 cmꢁ1 in ciprofloxacin were assigned to
(COO)asy and (COO)sym respectively, while in the metal
complexes these bands were observed at 1600 and 1380 cmꢁ1
n
(M–O) [18] band which appeared
The water content in the complexes was determined by thermal
analysis, using a 5000/2960 SDTA, TA instrument (USA) differential
thermal analysis apparatus operating at a heating rate of 10 ꢂC per
minute in the range of 20–800 ꢂC in N2 in order to establish their
compositional differences as well as to ascertain the nature of
associated water molecules. It was observed that all the complexes
showed a loss in weight corresponding to three water molecules in
the range 50–130 ꢂC, indicating the water of crystallization. In the
second step weight loss during 130–180 ꢂC corresponding to two
coordinated water molecules. For Zn(II) complexes a loss in weight
was seen corresponding to a piperazine (pip) molecule in the
temperature range 180–250 ꢂC, followed by liberation of Cip.(L) in
the temperature range 250–500 ꢂC. Finally, decomposition of An
occurred in the temperature range 520–800 ꢂC, and the remaining
weight was consistent with metal oxide [34]. Probable structure of
the complexes from the above analytical facts is given in Fig. 1.
.
n
n
,
respectively. The frequency separation (Dn) in the investigated
complexes is greater than 200 cmꢁ1, suggesting an unidentate
bonding nature for the carboxyl group [19–23]. The n(C]N) peak
for the Schiff bases was observed at 1590–1620 cmꢁ1 which on
complexation were shifted to 1564–1579 cmꢁ1, which indicates
N– N coordination of the ligands [24–27]. This data was further
supported by
case of the
n
(M–N) [28] which appeared at 535–546 cmꢁ1.In the
n
(C–S) band of mtma observed at 700 cmꢁ1 is shifted
lower to 670 cmꢁ1 in the spectra of the complex indicating the
participation of the sulfur atom of the thiophene ring. This data was
further supported by a new band observed at 425 cmꢁ1 which can
be assigned to n(M–S) [29–31] mode.
2.2.4. Mass spectroscopy
2.2.2. Electronic spectra and magnetic measurements
The FAB mass spectrum of complex-II shows the molecular ion
peak at m/z 1248 due to [M þ 2Hþ] in absence of three lattice water
molecules. The peak at m/z 1148 is due to removal of two coordi-
nate water and two methoxy group [M-C2H10O4]. The peak
The UV–Vis. spectra of the complexes have been recorded using
UV-160A UV–Vis. spectrophotometer, Shimadzu (Japan). The elec-
tronic spectra of the Zn(II) complexes exhibited only a high-intense
Fig. 1. Probable structure of complex [Zn2(Cip)2(mtma)2(pip)(H2O)2].

M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
441
Fig. 2. Mass spectrum of complex [Zn2(Cip)2(mtma)2(pip)(H2O)2].
occurring at m/z 615 correspond to fragment produced by cleavage
of Zn–N bond with piperazine [M-C26H26N2O7ClFSZn þ Hþ]. The
appearance of peak at m/z 1077 is due to removal of two chlorine
atom [M-C2H10O4Cl2 þ Hþ]. The peak at m/z 961 is due to removal
of one ligand [M-C13H19NO4S ꢁ Hþ]. The peak arising at m/z 218
corresponds to the ligand [C12H11NOS]. The M þ 2 peak appears due
to the isotopes of Cl and Zn. The mass spectrum is shown in Fig. 2.
MTCC 121. In case of Serratia marcescens MTCC 86 complex III is
more potent than all standard drugs; while for Pseudomonas aer-
uginosa MTCC 1688 complex VI is more potent than all standard
drugs except gatifloxacin. In case of Escherichia coli MTCC 443
complexes II, III, V and VI are more potent than gatifloxacin, nor-
floxacin and pefloxacin. Enhancement in the activity can be
explained on the basis of chelation theory and/or may be due to
Overtone’s concept [37–39].
2.3. Antibacterial screening of Zn(II) complexes
2.4. DNA binding and cleavage study
The susceptibility of certain strains of bacterium towards mixed
ligand–metal complexes was judged by measuring the minimum
inhibition concentration (MIC). A stock solution of 2000 mM was
prepared by dissolving each complex in DMSO solution. As assessed
by colour, the complexes remain intact during biological testing.
The results are shown in Table 2. MIC was determined with the help
of progressive double dilution method [35,36] in liquid media
2.4.1. Absorption spectroscopic studies
In order to quantitatively compare the binding strength of the
complexes, the intrinsic binding constants Kb of the complexes with
sperm herring DNA were calculated using the following function
equation [40]:
containing compound with a varying range of 0.3–3200 mM, in all
½DNAꢃ=ð
where [DNA] is the concentration of DNA in base pairs, the
apparent absorption coefficient 3a 3f and 3b correspond to Aobsd
3
a L 3f Þ [ ½DNAꢃ=ð
3
b L3f Þ D1=Kbð
3
b L 3f
Þ
(1)
cases, from the experimental examination we have found that all
the metal complexes were more active relative to metal salts and
Schiff bases. All the complexes are more potent than all standard
drugs against Staphylococcus aureus MTCC 1430 and Bacillus subtilis
,
/
[Zn], the extinction coefficient for the free zinc complex, for each
Table 2
Minimum inhibitory concentration data (MIC) (
m
M).
Compounds
Gram positive
Gram negative
Staphylococcus
aureus
Bacillus
subtilis
Serratia
marcescens
Pseudomonas
aeruginosa
Escherichia
coli
Zn(OAc)2 H2O
Ciprofloxacin
Gatifloxacin
Norfloxacin
Enrofloxacin
Pefloxacin
Levofloxacin
Sparfloxacin
1240
1.63
5.06
2.51
1.95
2.10
1.66
1.27
1.94
2115
2531
2528
2080
1884
1248
0.75
0.46
0.75
0.72
0.67
0.50
992
1984
1.63
2.93
4.07
1.67
5.10
1.66
1.53
1.66
2539
2761
2739
2080
1884
1525
1.87
2.69
1.49
2.15
2.33
1.89
1736
1.36
1.01
3.76
1.39
5.70
1.66
1.53
2.21
2750
2991
2950
2458
2041
1664
1.49
1.92
1.86
1.79
1.66
1.26
1736
1.36
2.93
2.82
1.39
2.70
0.97
1.27
1.38
2750
2761
3161
2647
2041
1803
2.24
1.92
1.86
2.15
1.66
1.89
1.09
4.00
2.51
3.90
2.40
2.21
2.04
1.38
1904
2301
2318
1891
1570
1387
0.37
0.31
0.60
0.36
0.33
0.50
Ofloxacin
L1
L2
L3
L4
L5
L6
I
II
III
IV
V
VI

442
M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
Fig. 3. Absorption titration spectra of complex [Zn2(Cip)2(bpdmed)2(pip)(H2O)2] (4
mM) with sperm herring DNA; [DNA] ¼ 2–20
m
M. Incubation time 10 min at 37 ꢂC.
addition of DNA to the zinc complex and zinc complex in the fully
effects of the complexes, on the viscosity of rod-like DNA are shown
in Fig. 4. It is observed that as the concentration of complexes
increase, the viscosity of DNA increases steadily, which indicates
that the complexes bind to DNA through a classical intercalation
mode. Compare to all complexes, the binding ability of classical
intercalator ethidium bromide is more. Among all complexes,
complexes III and VI bind more strongly than other. This difference
of DNA-binding mode between complexes should be caused by their
different ancillary ligands.
bound form, respectively. In plots [DNA]/(3a
given by the ratio of slope to the y intercept.
ꢁ
3f) vs [DNA], Kb is
Fixed amounts (4 mM) of complexes were titrated with
increasing amounts of DNA. The electronic spectral traces are given
in Fig. 3. For complexes, the absorption spectra show clearly that
the addition of DNA to the complexes yields hyperchromism and
a blue shift to the ratio of [DNA]/[M]. Obviously, these spectral
characteristics suggest that all the complexes interact 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 hypsochromism
of the peak maxima in the UV–visible spectra of the complexes. In
2.4.3. Cleavage of plasmid pUC19 DNA
There has been considerable interest in DNA cleavage reactions
that are activated by transition metal complex [45,46]. The delivery
of metal ion to the helix, in locally generating oxygen or hydroxide
radicals, yields an efficient DNA cleavage reaction. Fig. 5 illustrates
the gel electrophoretic separations showing the cleavage of plasmid
pUC19 DNA induced by the complexes under aerobic conditions and
in presence of H2O2, respectively [47]. When circular plasmid DNA is
conducted byelectrophoresis, the fastest migrationwill be observed
for the supercoiled form-I (SC). If one strand is cleaved, the super-
coiled will relax to produce a slower-moving open circular form-II
(OC). If both strands are cleaved, a nicked form-III (NC) will be
generated that migrates in between. This clearly shows that the
relative binding efficacy of the complexes to DNA is much higher
than the binding efficacy of metal salt itself (Table 4). The different
DNA-cleavage efficiency of the complexes was due to the different
binding affinity of the complexes to DNA, which has been observed
in other cases. One of the most interesting electrophoretic results of
the complexes takes place when experiment is done in presence of
H2O2 in TAE buffer. The DNA þ complex þ H2O2 systems (Fig. 6)
cleave the supercoiled DNA form (I) and convert into nicked form
(III) and open circular form (II) more than complex alone. Therefore,
we concluded that the mixture of complex with H2O2 have been
found to be efficient oxidant.
the plot of [DNA]/(3a
ꢁ
3f) vs. [DNA], the binding constant Kb is given
by the ratio of the slope to the y intercept. The binding constant for
Zn(II) complexes varies in the range of 1.0–4.0 ꢀ 104 Mꢁ1, all data
are given in Table 3. The DNA-binding constant of the title
complexes are comparable to those of some DNA intercalative
polypyridyl Ru(II) complexes 1.0–4.8 ꢀ 104 Mꢁ1 [41,42].
2.4.2. Viscosity measurements
The binding modes of the complexes were further investigated
by viscosity measurements. Photophysical probes generally provide
necessary, but not sufficient, clues to support a binding model.
Hydrodynamic measurements that sensitive to length change (i.e.
viscosity and sedimentation) are regarded as the least ambiguous
and the most critical tests of binding mode in solution in the absence
of crystallographic structural data [43,44]. A classical intercalation
model results in lengthening the DNA helix, as base pairs are
separated to accommodate the binding ligand, leading to the
increase of DNA viscosity. However, a partial and/or non-classical
intercalation of ligand may bend (or kink) DNA helix, resulting in the
decrease of its effective length and, concomitantly, its viscosity. The
3. Conclusion
Table 3
The binding constants (Kb) of Zn(II) complexes with DNA in Phosphate buffer pH 7.2.
Six binuclear Zn(II) complexes using ciprofloxacin and neutral
bidentate ligands, were synthesized and characterized. Antibacterial
study indicates that all the metal complexes were more active
compared to metal salts and Schiff bases. All the complexes were
found to be more potent than all standard drugs against S. aureus and
B. subtilis. For S. marcescens, complex III and for P. aeruginosa, complex
VI is more potent than all standard drugs. The strong antimicrobial
Complexes
Kb (Mꢁ1
)
I
II
III
IV
V
2.5 ꢀ 104
3.0 ꢀ 104
3.0 ꢀ 104
1.0 ꢀ 104
1.0 ꢀ 104
4.0 ꢀ 104
VI

M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
443
were purchased from E. Merck (India) Ltd. Mumbai. Xylene cyanol FF,
ethidium bromide and Luria Broth were from Himedia, India. Agarose
was purchased from Sisco research lab., India. Bromophenol blue,
acetic acid and EDTA were purchased from Sd fine chemicals, India.
Sperm herring DNA was purchased from Sigma Chemical Co., India.
Organic solvents were purified by standard methods [48].
4.2. Instrumentation
Infrared spectra were recorded on a FT-IR Shimadzu spectro-
photometer as KBr pellets in the range 4000–400 cmꢁ1. C, H and N
elemental analyses were performed with a model 240 Perkin Elmer
elemental analyzer. The reflectance spectra of the complexes were
recorded in the range of 1700–350 nm (as MgO discs) on a Beckman
DK-2A spectrophotometer. The metal contents of the complexes
were analyzed by EDTA titration [49] after decomposing the organic
matter with a mixture of HClO4, H2SO4, and HNO3 (1:1.5:2.5). MIC
study was carried out by means of laminar air flow cabinet, Toshiba,
Delhi, India, Thermogravimetric analyseswas obtainedwith a model
5000/2960 SDTA, TA instrument (USA). The 1H NMR and 13C NMR
was recorded on a Bruker Avance (400 MHz). The electronic spectra
were recorded on a UV-160AUV–Vis. spectrophotometer, Shimadzu
(Japan). The magnetic moments were measured by Gouy’s method
using mercury tetrathiocyanatocobaltate(II) as the calibrant
(
cg ¼ 16.44 ꢀ10ꢁ6 cgs units at 20 ꢂC), Citizen Balance. The diamag-
Fig. 4. The effect of the increasing amount of complexes on the relative viscosity of
DNA at 27 ꢄ 0.1 ꢂC.
netic correctionwas made using Pascal’s constant [50]. The FAB mass
spectra were recorded on a Jeol SX 102/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. M-Nitro benzyl alcohol (NBA) was used as the matrix.
The matrix peaks appear at m/z 136, 137, 154, 289, 307.
activitiesof thesecomplexes against testedorganisms suggestfurther
investigation on these complexes. DNA-binding study indicate that
intrinsic binding constant of all complexes varies in the range of
1.0 ꢀ 104–4.0 ꢀ 104 Mꢁ1, which is comparable to those of some DNA
4.3. Synthesis and physical properties of Schiff bases
intercalative polypyridyl Ru(II) complexes1.0 ꢀ 104–4.8 ꢀ 104 Mꢁ1
.
The complexes bind to DNA by classical intercalative mode. Gel
electrophoresis data suggests that presence of H2O2 enhances the
DNA cleavage to a significantextent. Hence, the proposed work seems
to be worth for generating database to develop new effective useful
DNA probes.
4.3.1. N,N0-bis-(phenyl)-1,2-dimethyl-ethane-1,2-diimine
(A1wbpdmed)
An ethanolic solution (100 mL) of aniline (1.86 g, 20 mmol) was
added drop wise to ethanolic solution (100 mL) of 2,3-butanedione
(0.86 g, 10 mmol) and refluxed on water bath for 8 h. The resulting
mixture was filtered. The obtained yellow crystalline product was
washed with n-hexane, recrystallized in ethanol and dried in air.
Yield: 58%, m.p.: 114 ꢂC, Found %: C, 81.49, H, 6.71, N,11.69. C16H16N2
(236.31) requires %: C, 81.32, H, 6.82, N, 11.85. Ir (cmꢁ1): 1613
(C]N), 1572 (C]C); 1H NMR (ppm): 6.81–7.42 (10H, m, Ar–H), 2.19
(6H, s, Al–H); 13C NMR (ppm): 118.8–129.0 (Ar–C), 168.3 (C]N),
150.9 (C–N), 15.4 (Al–C).
4. Experimental
4.1. Materials and methods
All the chemicals used were of analytical grade. Thiophene-
2-carboxaldehyde, 1,8-diaminonaphthalene were purchased from
Lancaster, England. Ciprofloxacin hydrochloride was purchased from
Bayer AG (Wyppertal, Germany). Anthranilic acid, benzoyl chloride,
ethylenediamine, 2,3-butanedione, aniline, hydrazine hydrate, pyri-
dine, cyclohexanone, benzil, p-anisidine, glycerol, and zinc acetate
4.3.2. 4-Methoxy-N-(thiophene-2-ylmethylene)aniline (A2wmtma)
An ethanolic solution (100 mL) of thiophene-2-carbaldehyde
(1.12 g, 10 mmol) was added drop wise to an ethanolic solution
Fig. 5. Agarose gel electrophoresis of pUC19 plasmid DNA (0.12
mg) with different complexes in TE buffer using 1% agarose gel containing 1 mg/mL ethidium bromide: Lane 1: DNA
(control); Lane 2: DNA þ Metal salt; Lane 3: DNA þ cip; Lane 4: DNA þ I; Lane 5: DNA þ II; Lane 6: DNA þ III; Lane 7: DNA þ IV; Lane 8: DNA þV; Lane 9: DNA þVI.

444
M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
Table 4
Gel electrophoresis data.
6 h., and concentrated up to one-third volume after that kept it
overnight over desiccator. Fine crystalline particles obtained on
filtration, washed with 1:1 absolute ether:hexane and dried in air.
Yield: 66%, m.p.: 135 ꢂC, Found %: C, 81.70, H, 7.68, N, 10.67.
C18H20N2 (264.36) requires %: C, 81.78, H, 7.63, N, 10.60. Ir (cmꢁ1):
1630 (C]N), 1565 (C]C); 1H NMR (ppm): 7.40–7.70 (10H, m, Ar–
H), 2.07 (6H, s, Al–H), 3.9 (4H, t, Al–H); 13C NMR (ppm): 125.9–131.4
(Ar–C), 18.2 (Al–C), 169.0 (C]N), 52.5 (C–N).
Compounds
% SC(I)
% NC(III)
% OC(II)
DNA Control
DNA þ Metal salt
DNA þ cip
58
49
44
45
44
43
45
42
48
35
33
34
42
41
42
41
42
41
31
39
32
40
41
44
42
43
39
22
22
25
42
40
39
39
40
38
11
12
24
15
15
13
13
15
13
43
45
41
16
19
19
20
18
21
DNA þ I
DNA þ II
DNA þ III
DNA þ IV
4.3.5. N, N0-dicyclohexylidene-naphthalene-1,8-diamine
(A5wdcnd)
DNA þ V
DNA þ VI
DNA þ H2O2
An ethanolic solution (100 mL) of cyclohexanone (1.96 g,
20 mmol) was added to ethanolic solution (100 mL) of 1,8-dia-
minonaphthalene (1.58 g, 10 mmol). The mixture was stirred
continuously for 4 h to get fine yellow crystalline product. Obtained
crystalline product was washed with n-hexane. The product was
recrystallized in ethanol and dried in air. Yield: 68%, m.p.: 135 ꢂC,
Found %: C, 83.00, H, 8.09, N, 8.74. C22H26N2 (318.45) requires %: C,
82.97, H, 8.23, N, 8.80. Ir (cmꢁ1): 1600 (C]N), 1570 (C]C); 1H NMR
(ppm): 6.50–7.28 (6H, m, Ar-H), 1.31–2.37 (20H, m, Al–H); 13C NMR
(ppm): 105.9–134.7 (Ar–C), 169.0 (C]N), 140.0 (C–N), 22.3 (Al–C),
25.3 (Al–C), 36.9 (Al–C).
DNA þ H2O2 þ Metal salt
DNA þ H2O2 þ cip
DNA þ H2O2 þ I
DNA þ H2O2 þ II
DNA þ H2O2 þ III
DNA þ H2O2 þ IV
DNA þ H2O2 þ V
DNA þ H2O2 þ VI
(100 mL) of 4-methoxyaniline (1.43 g, 10 mmol) and refluxed on
water bath for 8 h. The resulting mixture was filtered. The obtained
crystalline product was washed with n-hexane, recrystallized in
ethanolanddriedin air. Yield:68%, m.p.: 275 ꢂC, Found %: C, 66.38, H,
5.18, N, 6.47. C12H11NOS (217.29) requires %: C, 66.33, H, 5.10, N, 6.45.
Ir (cmꢁ1): 1620 (C]N), 700 (C–S), 1580 (C]C); 1H NMR (ppm):
6.95–7.31 (4H, m, Ar–H), 7.1–7.2 (3H, m, Thio-H), 3.72 (3H, s, OCH3),
7.5 (1H, s, CH]N); 13C NMR (ppm): 113.9–124.4 (Ar–C), 157.3 (C–O),
128.5–130.0 (Thio-C), 165.4 (C]N), 142.4 (C–N), 55.3 (OCH3).
4.3.6. N,N0-(1,2-diphenyl-ethane-1,2-diylidene)dianiline
(A6wdpeda)
An ethanolic solution of benzil (2.10 g, 10 mmol) and aniline
(1.86 g, 20 mmol) were refluxed on water bath for 12 h., and
concentrated up to one-third volume after that kept it overnight
over sulfuric acid in desiccator. Fine crystalline particles
obtained on filtration, washed with 1:1 absolute ether:hexane
and dried in air. Yield: 64%, m.p.: 165 ꢂC, Found %: C, 86.60, H,
5.57, N, 7.70. C26H20N2 (360.45) requires %: C, 86.64, H, 5.59, N,
7.77. Ir (cmꢁ1): 1610 (C]N), 1530 (C]C); 1H NMR (ppm):
6.80–7.60 (20H, m, Ar–H); 13C NMR (ppm): 124.2–135.4 (Ar–C),
167.3 (C]N), 141.5 (C–N).
4.3.3. 3-Amino-2-phenyl-3H-quinazolin-4-one (A3wapq)
The solution of anthranilic acid (1.37 g, 0.1 mol) was prepared in
pyridine (100 mL) followed by addition of benzoyl chloride
(2.814 g, 0.2 mol). The resulting mixture was stirred for 0.5 h., and
finally treated with 5% NaHCO3 (15 mL). The separated solid was
crystallized in ethanol. Yield: 80%, m.p.: 120 ꢂC, The obtained
2-phenyl-3,1-benzoxazin-4-one (0.557 g, 0.05 mol) in ethanol
(50 mL) was mixed with hydrazine hydrate (0.125 g, 0.05 mol) in
ethanol (50 mL) and refluxed for 3 h. The obtained product was
crystallized in ethanol. Yield: 85%, m.p.: 196 ꢂC, Found %: C,
70.67, H, 4.62, N, 17.59. C14H11N3O (237.26) requires %: C, 70.87,
H, 4.67, N, 17.71. Ir (cmꢁ1): 1680 (C]O), 1590 (C]N), 1545 (C]C);
1H NMR (ppm): 7.28–8.19 (9H, m, Ar–H); 13C NMR (ppm):
127.02–133.02(Ar–C),164.7.6(C]O),165.3 (C]N, ring),145.5 (C–N).
4.4. Synthesis and physical properties of the mixed-ligand
complexes
4.4.1. [Zn2(Cip)2(mtma)2(pip)(H2O)2]$3H2O (II)
A methanolic solution of Zn(OAc)2$H2O (2.01 g, 10 mmol) was
added to methanolic solution of mtma (1.12 g, 10 mmol), followed
by addition of a previously prepared solution of Cip$HCl (3.67 g,
10 mmol) in water; pH was adjusted to 6–7.5 pH using dilute
NaOH solution. During reaction the piperazine ring of cipro-
floxacin was substituted by chloride ion in the presence of NaOH
[51]. The resulting red solution was refluxed for 8–10 h on
a steam bath, and then was kept overnight at room temperature.
4.3.4. N, N0-bis-(1-phenylethylidene)-ethane-1,2-diamine
(A4wbpeed)
An ethanolic solution of ethylenediamine (0.60 g, 10 mmol) and
acetophenone (2.40 g, 20 mmol) were refluxed on water bath for
Fig. 6. Agarose gel electrophoresis of pUC19 plasmid DNA (0.12 mg) with different complexes in TE buffer using 1% agarose gel containing 1 mg/mL ethidium bromide: Lane 1: DNA
(control); Lane 2: DNA þ H2O2; Lane 3: DNA þ H2O2 þ Metal salt; Lane 4: DNA þ H2O2þcip; Lane 5: DNA þ H2O2 þ I; Lane 6: DNA þ H2O2 þ II; Lane 7: DNA þ H2O2 þ III;
Lane 8: DNA þ H2O2 þ IV; Lane 9: DNA þ H2O2 þ V; Lane 10: DNA þ H2O2 þ VI.

M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
445
Table 5
Experimental and physical parameters of the complexes.
Complexes empirical formula
Elemental analysis % found (required)
m.p. ꢂC
% Yield
Formula
weight
(gm/mol)
C
H
N
M
C62H66Cl2F2N8O11Zn2 (I)
C54H56Cl2F2N6O13S2Zn2 (II)
C58H56Cl2F2N10O13Zn2 (III)
55.61 (55.62)
49.87 (49.86)
51.94 (51.96)
56.86 (56.82)
59.10 (59.12)
62.04 (62.05)
4.98 (4.97)
4.37 (4.34)
4.25 (4.21)
5.32 (5.35)
5.76 (5.77)
4.73 (4.74)
8.34 (8.37)
9.75 (9.77)
315
340
220
270
>360
280
69.5
67.2
69.5
67.8
66.4
68.0
1338.96
1300.86
1340.81
1395.06
1503.24
1587.23
6.44 (6.46)
10.43(10.45)
8.01 (8.03)
7.43 (7.45)
7.15(7.12)
10.08 (10.05)
9.73(9.75)
9.33 (9.38)
8.74 (8.70)
7.44(7.49)
C
66H74Cl2F2N8O11Zn2 (IV)
C74H86Cl2F2N8O11Zn2 (V)
C82H74Cl2F2N8O11Zn2 (VI)
A fine amorphous product was obtained which was washed with
ether and dried in a vacuum desiccator. The complexes (I and III–
VI) were prepared by same method. The physical parameters of
the complexes are shown in Table 5 and probable reaction
scheme is shown in Scheme 1.
autoclaved for 20 min at 120 ꢂC and at 15 lb/in2 pressure before
inoculation. Inoculation of 10 L of glycerol stock was done to the
previously prepared corning tubes with the help of a micropipette
using sterilized tip under sterile condition. The bacteria were then
cultured for 24 h at 37 ꢂC in an incubator.
m
4.5. Dilution method
4.6. DNA-binding and cleavage assay
Before inoculation all the bacteria were incubated and acti-
vated at 30 ꢂC for 24 h into Luria Broth. The compounds were
dissolved in DMSO and then diluted using cautiously adjusted
Luria Broth. Two-fold serial concentrations of the compounds
were employed to determine the (MIC) ranging from 0.3 to
4.6.1. UV–Vis. spectroscopy
All experiments involving the interaction of the complexes with
DNA were carried out in buffer (0.2 M Na2HPO4, 0.2 M NaH2PO4, pH
7.2). A solution of DNA in the buffer gave a ratio of UV absorbance at
260 and 280 nm of about 1.68, indicating that the DNA was suffi-
ciently free of protein [53]. The DNA concentration per nucleotide
was determined by absorption spectroscopy using the molar
absorption coefficient (6600 Mꢁ1 cmꢁ1) at 260 nm [54].
3200
concentrations of antimicrobial agents that result in complete
inhibition of microorganisms were represented as (MIC) M. The
m
M. Test cultures were incubated at 37 ꢂC (24 h). The lowest
m
control experiments were performed with only equivalent volume
of solvents without added test compounds. All experiments were
performed in triplicate and the average was taken as the final
reading [52]. The ciprofloxacin, gatifloxacin, norfloxacin, enro-
floxacin, pefloxacin, levofloxacin, sparfloxacin and ofloxacin were
used as a standard drug.
4.6.2. Viscosity study
Relative viscosity measurements were carried out using an
Ubbelohde viscometer maintained at a constant temperature of
27.0 (ꢄ0.1) ꢂC in a thermostatic bath [55]. Flow time was measured
with a digital stopwatch. Each sample was measured three times
and an average flow time was calculated. Data were presented as
4.5.1. Inoculation procedure
The target micro-organism cultures were prepared separately in
15 mL of 2% Luria Broth medium. First the Luria Broth was
(h/h0) h is the relative
1/3 versus binding ratio [M]/[DNA] [56], where
viscosity of DNA in the presence of complex and h0 is the viscosity
of DNA alone. Viscosity values were calculated from the observed
flow time of DNA-containing solutions (t > 100 s) corrected for the
flow time of buffer alone (t0),
h
¼ t ꢁ t0.
4.6.3. Gel electrophoresis
Isolation of plasmid DNA from pure culture of E. coli was carried
out by conventional method [57]. For the gel electrophoresis
experiments, supercoiled pUC19 DNA (0.12 mg) in TE buffer (10 mM
Tris–HCl, pH 8.0 and 1 mM EDTA) was treated with different Zn(II)
complexes; and in the presence of hydrogen peroxide in the reac-
tion solution. The samples were incubated for 30 min at 37 ꢂC, then
a loading buffer containing 10 mM TE (pH 7.5), 0.03% bromophenol
blue, 0.03% xylene cyanol FF, 60% glycerol and 60 mM EDTA was
added and electrophoresis was performed at 100 V for 2 h in 1X
TAE buffer (0.04 M Tris–Acetate, pH 8, 0.001 M EDTA) using 1.0%
agarose gel containing 1.0 mg/mL ethidium bromide. Bands were
visualized by UV light and photographed on a capturing system
(AlphaDigiDocÔ RT. Version V.4.1.0 PC-Image software).
Acknowledgement
We wish to express our gratitude to Prof. (Miss) R. G. Patel, Head,
Department of Chemistry Vallabh Vidyanagar, Gujarat, India, for
providing the necessary laboratory facilities authors also wish to
acknowledge the help of Dr. V.R. Thakkar and Mr. H.S. Bariya,
Department of Biochemistry, Sardar Patel University. M.R.
Scheme 1. Reaction scheme of complex [Zn2(Cip)2(mtma)2(pip)(H2O)2]$3H2O.

446
M. Patel et al. / European Journal of Medicinal Chemistry 45 (2010) 439–446
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