DNA-interaction and in vitro antimicrobial studies of some mixed-ligand complexes of cobalt(II) with fluoroquinolone antibacterial agent ciprofloxacin and some neutral bidentate ligands

Article abstract of DOI:10.1016/j.bmcl.2009.03.078

Six new mixed-ligand complexes of Co(II) with ciprofloxacin (Cip) and neutral bidentate ligands have been synthesized and characterized. Binding and cleavage of DNA with the complex were investigated using spectroscopic method, viscosity measurements and gel electrophoresis techniques. Antibacterial activity has been assayed against two Gram^(-ve) and three Gram^(+ve) microorganisms using the doubling dilution technique.

Full text of DOI:10.1016/j.bmcl.2009.03.078

Bioorganic & Medicinal Chemistry Letters 19 (2009) 2870–2873
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
DNA-interaction and in vitro antimicrobial studies of some mixed-ligand
complexes of cobalt(II) with fluoroquinolone antibacterial agent
ciprofloxacin and some neutral bidentate ligands
*
M. N. Patel , M. R. Chhasatia, D. S. Gandhi
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar, Gujarat 388 120, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Six new mixed-ligand complexes of Co(II) with ciprofloxacin (Cip) and neutral bidentate ligands have
been synthesized and characterized. Binding and cleavage of DNA with the complex were investigated
using spectroscopic method, viscosity measurements and gel electrophoresis techniques. Antibacterial
activity has been assayed against two Gram(ꢀve) and three Gram(+ve) microorganisms using the doubling
dilution technique.
Received 27 September 2008
Revised 16 March 2009
Accepted 20 March 2009
Available online 25 March 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
Mixed-ligand complexes of cobalt(II)
MIC
Oxidative cleavage
Binding constant (Kb)
Derivatives of compounds composed of 3-carboxy-4-oxo-1,4-
dihydroquinoline (i.e., 4-quinolones) are active against a wide
range of gram-positive and gram-negative organisms.1 The first
member of the quinolone family kept forward for clinical practice
was nalidixic acid; it is used for the treatment of urinary tract
infections.2 Major increase in the potency was obtained by addi-
tion of fluorine atom on position 6 of the quinoline ring, addition
of piperazinyl group on position 7 to enhance permeability and po-
tency.3 Binding of DNA with the complexes have been studied by
different methods to understand the selectivity and efficiency of
DNA reorganization, cleavage by cobalt complexes, and to develop
new effective useful DNA probes.
proton (–CH@N–). In the 13C NMR spectra, the peaks observed at
about 115.0–136.4 and 113.5–144.5 ppm were assigned to aro-
matic and pyridine carbons, respectively. Peaks observed in range
123.5–140.0 and 160.5–180.5 ppm were assigned to C–N and
C@N carbons, respectively.
The m
(C@O) stretching vibration band appears at 1708 cmꢀ1 for
ciprofloxacin, where as for complexes it appear at 1627–
1633 cmꢀ1; this shift towards lower energy suggests that coordina-
tion occurs through the carbonyl oxygen of pyridine ring.8 The sharp
band in ciprofloxacin at 3520 cmꢀ1 due to hydrogen bonding;9
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 deproto-
nation of the carboxylic proton. The data were furtꢀh1er supported
All the chemicals used in the experiment were of analytical grade
and solventswere purified by standard methods.4 Physical measure-
ments were made as per S-1. The diamagnetic correction was made
using Pascal’s constant.5 The Schiff bases A1–A5 were prepared by
condensation of the amine and aldehyde/ketone in ethanol.6 The
complexes (I)–(VI) were prepared as per reported method.7 Synthe-
sis and physicochemical parameters of the ligands and synthesized
complexes are as Supplementary data S-2. All the complexes were
insoluble in common solvent while partially soluble in dimethyl-
formamide and completely soluble in dimethylsulfoxide.
by m
(M–O)10 band which appeared at 502–514 cm . The strong
absorption band obtained at 1624 cmꢀ1 and 1340 cmꢀ1 in ciproflox-
acin were assigned to (COO)asy and (COO)sym, respectively, while
in the metal complexes these bands were observed about 1590
m
m
and 1375 cmꢀ1, respectively. The frequency separation (
Dm) in the
investigated complexes is greater than 200 cmꢀ1, suggesting a uni-
dentate bonding nature for the carboxyl group.11 In the investigated
compound the
1585 cm.ꢀ1 This shifted to higher frequency at 1612 cmꢀ1 which
m
(C@N) band of 2,20-bipyridylamine appeared at
The 1H NMR spectra of ligands exhibiting peaks at about 6.65–
7.91 ppm were assigned to the aromatic protons. The singlet peak
which appeared at 8.6–8.7 ppm was assigned to the azomethine
points that N–N coordination of the chelating agent.12 The
m(C@N)
peak for the Schiff bases A1–A5 was observed at 1601–1629 cmꢀ1
which on complexation were shifted to 1560–1605 cmꢀ1, which
indicates the N–N coordination of the chelating agent.13 This data
* Corresponding author.
E-mail address: jeenen@gmail.com (M.N. Patel).
was further supported by
m
(M–N)14 which appeared at
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.03.078

M. N. Patel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2870–2873
2871
537–542 cmꢀ1. Detail IR Spectral data of complex is as Supplemen-
tary data S-3.
served that all the complexes were more potent bactericides than
the ligands. The inhibition activity seems to be governed in certain
degree by the facility of coordination at the metal centre. This may
support the argument that some type of bimolecular binding to the
metal ions or intercalation or electrostatic interactions causing the
inhibition of biological synthesis and preventing the organisms
from reproducing. The strong antimicrobial activities of these com-
plexes against tested organisms suggest further investigation on
these complexes.18
The magnetic moments of the complexes are in the range 4.7–
5.1 BM show that all are paramagnetic and have three unpaired elec-
trons indicating a high-spin octahedral configuration.15 The Co(II)
complexes exhibited well-resolved bands at 17471–17577 cmꢀ1
and a strong high-energy band at 20202–22122 cmꢀ1 were assigned
to 4T1g(F)?4A2g(F)(
m m3) transitions, respectively,
2), 4T1g(F)?4T1g(P)(
for a high-spin octahedral geometry.16
It was observed that all the complexes showed a loss in weight
corresponding to three water molecules in the range 50–130 °C,
indicating the presence of lattice water molecules. In the second
step weight loss during 130–180 °C corresponding to two coordi-
nated water molecules. For Co(II) complexes a loss in weight corre-
sponding to a piperazine (pip) molecule was found between 180
and 250 °C, followed by liberation of Cip.(L) in the temperature
range 250–500 °C. Finally, decomposition of An occurred in the
temperature range 520–800 °C, and the remaining weight was con-
sistent with metal oxide.17
Complex binding with DNA through intercalation usually
results in hyperchromism and blue shift, because intercalative
mode involving
a strong stacking interaction between an
aromatic chromophore and the base pairs of DNA. The electronic
absorption spectra of complexes mainly consist of two resolved
bands.19 Change in absorbance at peak maximum shows moder-
ate hypsochromism shift (ꢂ4 nm). For each complex, increasing
concentration of DNA has been monitored for an evaluation of
the intrinsic binding constant. Using absorption measurements
for (I), a linear plot of [DNA]/(ef/ea) versus [DNA] was obtained
The ESI mass spectrum of the complex [Co2(Cip)2(dcbd)2(pi-
p)(H2O)2]ꢁ3H2O (I) shows the peak at m/z = 1336 corresponds to
the molecular ion peak of complex (in absence of water of crystal-
lization). For fragments at m/z = 781.3 and 763 there exist a peak at
m, m + 2 and m + 4, which indicates presence of two Cl atoms in
fragments. There exist several other fragments at m/z = 710, 669,
442 and 567 with peak at m and m + 2 only, which indicates pres-
ence of single Cl atom9 S-4.
The antimicrobial activity of all the complexes against all the
five microorganisms is much higher than metal salt while in com-
petition with the ciprofloxacin (S-5). The results of our study indi-
cate that the compounds II, III and IV (MIC = 0.648, 0.223 and
using Eq. 1 (S-6). Assuming all the molecules of complexes were
bound with DNA, the experimental Kb was obtained by
substituting the absorbance into Beer’s law. The Kb value derived
from the plot for all the complexes were in range of
1.0 ꢃ 104 ꢀ 2.5 ꢃ 104 Mꢀ1. These spectral characteristics are con-
sistent with a mode of interaction that involves a stacking inter-
action between the complex and the base pairs of DNA, which
means that the titled complexes can intercalate into the double
helix structure of DNA.
Hydrodynamic measurements that are sensitive to length
change (i.e., viscosity and sedimentation) are regarded as the least
ambiguous and the most critical tests of a binding model in solu-
tion in the absence of crystallographic structural data (S-7). A clas-
sical intercalation model demands that the DNA helix lengthens as
base pairs are separated to accommodate the bound ligand, leading
to the increase of sperm herring DNA viscosity. In contrast, a par-
tial, non-classical intercalation of ligand could bend (or kink) the
DNA helix, reduce its effective length and, concomitantly, its vis-
cosity.20 The effects of complexes on the viscosity of DNA are
shown in Figure 1. For complexes the viscosity of DNA increases
steadily with increasing concentration of the complex. The exper-
imental results suggest that complexes bind to DNA through a clas-
sical intercalation mode and the order of intercalation mode of
complexes to DNA are II > V > IV > VI > III > I. This difference of
DNA-binding mode between complexes should be caused by their
different ancillary ligands.
0.754
against Staphylococcus aureus compare to all tested drugs. Except
VI (MIC = 1.672 M) all the compounds posses higher antimicro-
bial activity against Bacillus subtilis compare to all tested drugs.
In case of Serratia merscences compounds I (MIC = 0.717 M) and
IV (MIC = 1.131 M) exhibit higher antimicrobial activity compare
to all tested drug. In case of Pseudomonas aeruginosa compound II
posses highest activity (MIC = 0.876 M). In case of Escherichia coli
compounds III (MIC = 0.876 M) shows the best activity. Thus the
lM, respectively) exhibit higher antimicrobial activity
l
l
l
l
l
synthesized complexes were found more potent against gram(+ve)
and not much active against gram(ꢀve) species. The result indicates
that the compound III is most potent against all the test species
compares to other synthesized compounds. No doubt it is the most
potent compare to all but weak in case of S. merscences. It was ob-
Figure 1. The effect of the complexes on the viscosity of DNA.

2872
M. N. Patel et al. / Bioorg. Med. Chem. Lett. 19 (2009) 2870–2873
Table 1
Gel electrophoresis data of the compounds without H2O2
Compound
Conc. of comp. (lM)
% SC
% NC
% OC
Ref.
DNA (control)
DNA + Co(NO3)ꢁ6H2O
DNA + Cip
DNA + I
DNA + II
DNA + III
DNA + IV
DNA + V
DNA + VI
DNA (control)
DNA + [Co(en)2Cl2]+
DNA + [Co(bpy)2Cl2]+
DNA (control)
DNA + [CuL2]ClO4
DNA + [CuL(phe)]ClO4
—
87
58
40
15
28
14
20
20
—
—
13
42
44
40
30
38
67
51
17
14.2
21.9
22.6
—
This work
This work
This work
This work
This work
This work
This work
This work
This work
24
10
10
10
10
10
10
10
10
—
16
45
42
48
12
29
62
—
—
—
7
46
71
21
85.8
78.1
77.4
93
54
29
500
500
24
24
25
25
33
33
—
—
25
Table 2
Gel electrophoresis data of the compounds with H2O2
Compound
Conc. of comp. (
l
M)
Additional reagent
Conc. of reagent
—
% SC
% NC
% OC
Ref.
DNA (control)
DNA
—
—
87
—
—
—
—
20
16
—
11
15
99
91
1
—
13
23
31
59
25
14
11
14
23
13
—
—
—
—
—
—
—
This work
This work
This work
This work
This work
This work
This work
This work
This work
This work
26
26
26
24
24
27
27
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
H2O2
—
NaN3
D2O
—
NaN3
—
100
100
100
100
100
100
100
100
100
—
lM
lM
lM
lM
lM
lM
lM
lM
lM
77
69
41
75
66
73
86
60
72
1
9
99
4
25.7
—
—
DNA + Co(NO3)2ꢁ6H2O
10
10
10
10
10
10
10
10
—
80
80
—
500
—
DNA + Cip
DNA + I
DNA + II
DNA + III
DNA + IV
DNA + V
DNA + VI
DNA (control)
DNA + [CuL2(phe)]ClO4
DNA + [CuL2(phe)]ClO4
DNA (control)
DNA + [Co(phe)2Cl2]+
DNA (control)
DNA + [Cu(qnsa)2(phe)]
100
lM
14
—
lL
96
74.3
—
20 mM
—
15
H2O2
150
lM
—
There has been considerable interest in DNA cleavage reac-
tions that are activated by transition metal complex. The deliv-
ery of metal ion to the helix, in locally generating oxygen or
hydroxide radicals, yields an efficient DNA cleavage reaction.21,22
The cleavage of pUC19 DNA induced by the complexes under
aerobic conditions in absence and presence of H2O2 was carried
out using electrophoresis technique23 (S-8). When circular plas-
mid DNA conducted by electrophoresis, the fastest migration will
observed for the supercoiled form (SC). If one strand cleaved, the
supercoiled will relax to produce a slower-moving open circular
form (OC). If both strands cleaved, a nicked form (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 or ciproflox-
acin. 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 inter-
esting electrophoretic results of the complexes takes place when
experiment done in presence of H2O2. The DNA + complex + H2O2
systems cleave the supercoiled DNA form (I) and convert into
nicked form (III) to open circular form (II) more than complex
alone. Gel electrophoresis data of the complexes with and with-
out H2O2 along with some reported results are presented in
Tables 1 and 2. Therefore, we concluded that the mixture of
complex with H2O2 have been found to be efficient oxidant.
Hence the proposed work seems to be worth for generating
database to develop new effective useful DNA probes.
Acknowledgments
We wish to express our gratitude to Professor J. S. Parmar, Head,
Department of Chemistry, Vallabh Vidyanagar and Dr. V. R. Thak-
kar, BRD School of Bioscience, Vallabh Vidyanagar, Gujarat, India,
for providing us the necessary laboratory facilities. M.R.C. is highly
thankful to the UGC for JRF and financial assistance of UGC grant
32-226/2006(SR).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.03.078.
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