Interaction of drug based binuclear mixed-ligand complexes with DNA

Article abstract of DOI:10.1016/j.bmc.2009.06.023

Here in we tried to increase an antibacterial activity of ciprofloxacin drug due to formation of mixed-ligand complexes. Synthesized compounds were found to be more potent compare to drugs, ligands and metal salt against selective gram^(+ve) and

Full text of DOI:10.1016/j.bmc.2009.06.023

Bioorganic & Medicinal Chemistry 17 (2009) 5648–5655
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Interaction of drug based binuclear mixed-ligand complexes with DNA
*
Mohan N. Patel , Mehulsinh R. Chhasatia, Deepen S. Gandhi
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:
Here in we tried to increase an antibacterial activity of ciprofloxacin drug due to formation of mixed-
ligand complexes. Synthesized compounds were found to be more potent compare to drugs, ligands
and metal salt against selective gram(+ve) and gram(ꢀve) organisms. Interaction of the complexes with
nucleic acid (DNA) were investigated using spectroscopic technique, viscosity measurement and gel elec-
trophoresis and it was found that the complexes bind to DNA via intercalative mode.
Ó 2009 Elsevier Ltd. All rights reserved.
Received 18 April 2009
Revised 7 June 2009
Accepted 11 June 2009
Available online 17 June 2009
Keywords:
Mixed-ligand complex
Oxidative DNA cleavage
FAB-MS spectra
MIC
1. Introduction
assayed against five different microorganism namely Escherichia
coli, Pseudomonas aeruginosa, Staphylococcus aureus, Bacillus subtilis
Quinolones are an important group of antibiotics and several
quinolones are in common clinical use. The first quinolone antibi-
otic, nalidixic acid, was synthesized in 1962 by Lesher et al.1 Cipro-
floxacin is a typical second-generation fluoroquinolone, has been
in clinical use for more than a decade and sold for $US 1.5 billion
in 1996.2 The drug has been the center of great interest and success
and more than 15,000 articles have been published about cipro-
floxacin. In connection with the outbreak of suspected anthrax bio-
logical warfare, many experts consider ciprofloxacin the drug of
choice for treating victims. Binding of flouroquinolone to DNA is
relatively weak, thus it is unlikely that their binding to DNA trig-
gers the formation of gyrase–DNA complex.3–6 Likewise, binding
of fluoroquinolone to gyrase is weak, even though the presence
of mutated gyrase alleles in resistant bacteria implicates gyrase
in the interactions.7–11 Numerous studies have shown that drug
binding to DNA12–15 and gyrase is enhanced in the presence of me-
tal ions and metal ions are essential for antibacterial efficiency of
drug–DNA interaction. Understanding the interactions between
fluoroquinolone and DNA may help to elucidate the mechanism
of action of this important class of antibacterial agents, and may
ultimately lead to the design of better, more potent antibacterial
agents with fewer side effects.
and Serratia merscences.
2. Result and discussion
The structural characterizations of ligands have been done
using IR spectra, 1H and 13C NMR spectra, and elemental analyses.
The complexes under investigation have been characterized using
IR spectra, magnetic measurements, TG analysis, FAB-mass spectra
and electronic spectra. All the complexes were insoluble in ether,
hexane, chloroform, water and methanol but were completely sol-
uble in dimethyl sulphoxide.
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 are reported along with the possible assignment
as below.
2.1.1. N,N0-Bis-(4-methoxy-phenyl)-1,2-dimethyl-ethane-1,2-
diimine (A1ꢁbmpdme)
1H NMR (ppm): 6.67–6.86 (8H, m, Ar-H), 2.07 (6H, s, Al-H), 3.73
(6H, s, OCH3); 13C NMR (ppm): 114.2–120.6 (Ar-C), 158.6 (Ar, C-O),
168.9 (C@N), 144.1 (C–N), 15.4 (Al–C), 55.4 (OCH3).
To this aim, we have investigated the DNA-binding assay of
Co(II) mixed-ligand complexes with ciprofloxacin and some neu-
tral bidentate ligands(NN) using a combination of spectroscopic
(UV-spectroscopy), hydrodynamic techniques (viscometric) and
gel electrophoresis technique. Antibacterial activity (MIC) has been
2.1.2. N,N0-Bis-(4-methoxy-benzylidene)-ethane-1,2-diamine
(A2ꢁbmbed)
1H NMR (ppm): 6.65–7.91 (8H, m, Ar-H), 3.8 (4H, t, Al-H), 8.3
(2H, s, CH@N), 3.4 (6H, s, OCH3); 13C NMR (ppm): 113.9–134.4
(Ar-C), 157.3 (C–O), 165.5 (C@N), 130.4 (C–N), 55.3 (OCH3).
* Corresponding author. Tel.: +91 2692 226858x220.
E-mail address: jeenen@gmail.com (M.N. Patel).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.06.023

M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
5649
4
4
2.1.3. N,N0-Bis-(1-phenylethylidene)-benzene-1,2-diamine
(A3ꢁbpebd)
4T1g(F)?4T2g(F)(
m
1), T1g(F)?4A2g(F)(
m
2), T1g(F)?4T1g(P)(
m3), tran-
sitions for a high-spin octahedral geometry.29,30
1H NMR (ppm): 6.5–7.5 (14H, m, Ar-H), 1.74 (6H, s, Al–H); 13C
NMR (ppm): 121.9–130.4 (Ar-C), 18.2 (Al–C), 170.4 (C@N), 148.4
(C–N).
2.2.3. Magnetic behavior
The observed magnetic moments of cobalt(II) complexes are gi-
ven in Table 2. The theory of magnetic susceptibility of cobalt(II)
complex was given originally by Schlapp and Penney and the best
summary of results on the magnetic behavior of cobalt compound
is that of Figgis and Nyholm.31,32 The observed values of magnetic
moment for cobalt(II) complexes are generally diagnostic of the
coordination geometry about the metal ion. The low-spin square-
planar cobalt(II) complexes have magnetic susceptibility value
2.9 BM, arising from one unpaired electron along with contribution
of apparently large orbital.32 Both tetrahedral and high-spin octa-
hedral cobalt(II) complexes possess three unpaired electrons but
may be distinguished by the magnitude of the deviation of leff.
from the spin-only value. The magnetic moment of tetrahedral co-
balt(II) complexes with an orbitally nongenerate ground term is in-
creased above the spin-only value via contribution from higher
orbitally degenerate terms and occurs in the range 4.2–4.7 BM.33
Octahedral cobalt(II) complexes however maintain a large contri-
The 1H NMR spectra of ligands exhibiting peaks at about 6.5–
7.91 ppm were assigned to the aromatic protons. The singlet peak
which appeared at 8.3 ppm was assigned to the azomethine proton
(–CH@N–). In the 13C NMR spectra, the peak observed at about
113.9–134.4 was assigned to aromatic carbons. Peaks observed at
about 130.4–148.4, 165.5–170.4 ppm were assigned to C–N and
C@N carbons, respectively.
2.2. Characterization of the mixed-ligand complexes
2.2.1. Infrared spectral resolution
In Table 1 the most characteristic absorptions of IR spectra of
the complexes are listed. The m(C@O) stretching vibration band ap-
pears at 1708 cmꢀ1 for ciprofloxacin, where as for complexes it ap-
pear at 1615–1630 cmꢀ1; this shift towards lower energy suggests
that coordination occurs through the carbonyl oxygen of pyridine
ring.16 The sharp band in ciprofloxacin at 3520 cmꢀ1 is due to
hydrogen bonding;17 which is attributed to ionic resonance struc-
ture and peak observed because of free hydroxyl stretching vibra-
tion. This band completely vanished in the spectra of the metal
complexes indicating deprotonation of the carboxylic proton. The
4
bution due to T1g ground term and exhibit leff. in the range 4.8–
5.6 BM.34 The magnetic measurements on the complexes reported
herein 4.8–5.2 BM show that all are paramagnetic and have three
unpaired electrons indicating a high-spin octahedral configuration.
data were further supported by
504–512 cmꢀ1 18
The strong absorption band obtained at
1624 cmꢀ1 and 1340 cmꢀ1 in ciprofloxacin were assigned to
(COO)asy and (COO)sym, respectively, while in the metal com-
plexes these bands were observed at 1607 and 1383 cmꢀ1, respec-
tively. The frequency separation in the investigated
complexes is greater than 200 cmꢀ1, suggesting a unidentate bond-
m
(M–O) band which appeared at
2.2.4. Thermal response of synthesized complex
.
The thermal behavior of the complexes was determined using a
5000/2960 SDTA, TA instrument (USA) differential thermal analy-
sis apparatus operating at a heating rate of 10 °C per minute in
the range of 20–800 °C in N2. The TG curves of Co(II) complexes
showed in the following decomposition 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 that these
water molecules were water of crystallization. In the second step
weight loss during130–180 °C corresponding to two coordinated
water molecules. For Co(II) complexes a loss in weight was seen
corresponding to a piperazine (pip) molecule in the temperature
range 180–250 °C, followed by liberation of Cip.(L) in the temper-
ature range 250–500 °C. Finally, decomposition of An occurred in
the temperature range 520–800 °C, and the remaining weight
was consistent with metal oxide.35 Probable structure of the
complexes from the above analytical facts is given below in
Figure 1.
m
m
(Dm)
ing nature for the carboxyl group.16,19–23 The
m(C@N) peak for the
ligands A1–A6 was observed at 1600–1629 cmꢀ1 which on com-
plexation were shifted to 1569–1602 cmꢀ1, which indicates the
N–N coordination of the ligands.24–27 This data was further sup-
ported by
m .
(M–N) which appeared at 535–542 cmꢀ1 28
2.2.2. Reflectance spectra
The characteristic bands of the electronic spectra of the com-
plexes are given in Table 2. The Co(II) complexes exhibited well-re-
solved bands at 16,500–18,790 cmꢀ1 and a strong high-energy
band at 20,500–22,525 cmꢀ1 (Table 2) were assigned to
Table 1
Infrared spectral data of the Co(II) complexes
Complexes
m
(C@O) cmꢀ1 pyridone
m
(COO)asy cmꢀ1
m
(COO)sym cmꢀ1
D
m
cmꢀ1
m
(C–Cl) cmꢀ1
m
(C@N) cmꢀ1 azomethine
m
(M–N) cmꢀ1
m
(M–O) cmꢀ1
I
II
III
IV
V
1617
1623
1620
1615
1627
1630
1607
1608
1587
1605
1588
1600
1383
1388
1375
1384
1383
1377
224
220
212
221
205
223
1145
1146
1130
1106
1150
1101
1569
1602
—
—
—
535
538
540
540
542
536
510
509
504
512
508
510
VI
—
Table 2
Electronic spectral data of Co(II) complexes
Complexes
m2
m3
B0
10Dq
b
%b
leff BM
I
II
III
IV
V
16,525
17,430
16,615
16,577
17,125
15,920
21,920
20,525
22,365
21,920
20,620
20,500
461.50
579.07
452.13
465.46
553.12
466.97
11403.40
10760.86
11622.30
11405.92
10795.94
10686.53
0.4752
0.5963
0.4656
0.4793
0.5696
0.4809
52.471
5.0
4.9
5.2
4.8
4.9
4.8
40.362
53.436
52.063
43.035
51.907
VI

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M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
2.2.5. FAB-MS
may represent the molecular ion peak in absence of water of crys-
tallization and presence of four H+ ion that is, [Co2(Cip)2(bmpd-
me)2(pip)(H2O)2] + 4H+. There also exists several triplet at with
peak value 1679(M), 1681(M+2) and 1683(M+4) which corre-
The mass spectrum of the complex(I) [Co2(Cip)2(bmpdme)2(pi-
p)(H2O)2]ꢂ3H2O (Fig. 2) shows triplet peak at 1394(M),
1396(M+2) and 1398(M+4). The most abundance peak at 1394
a
Figure 1. Probable structures of the complexes.

M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
5651
Figure 2. Mass spectrum of complex (I).
sponds to complex bound to matrix (289) that is, [Co2(Cip)2(bmpd-
me)2(pip)(H2O)2] + 289; 1494(M), 1496(M+2) and 1498(M+4)
which corresponds to fragment [Co2(Cip)2(bmpdme)2(pip)] + 136,
these pattern indicates the presence of two Cl atom in this frag-
ments.17 There exist a doublet at 645(M) and 647(M+2) which cor-
responds to fragment [Co(bmpdme)] + 289 + 4H+ consist of single
Cl atom.17 The other fragment was observed at 951(M) related to
[Co2(bmpdme)2(pip)] + 136 + 4H+. Thus the m/z of all the frag-
ments of complex with the relative intensity confirms the stoichi-
ometry of the complex as per Figure 1.
and standard drug are represented in Table 3. The ligands
(A1–A6) exhibit a little antimicrobial activity. The antimicrobial
activity of the complexes against all the five microorganisms is
much higher than metal salt, while in competition with the cipro-
floxacin. The results of our study indicate that the in case of S. aur-
eus the complexes II, III, V and VI (MIC = 0.65, 0.68, 0.28 and
1.03
plexes I, IV (MIC = 2.07, 1.73
cin. In case of B. subtilis except III and IV (MIC = 1.69 and
1.71 M) all the complexes are much potent than the standard
lM) are more potent than all the standard drugs and com-
l
M) are less potent than ciprofloxa-
l
drugs. For S. merscences all the complexes are active compare to
2.3. In vitro biological studies of mixed-ligand complexes
gatifloxacin and norfloxacin, but less active than ciprofloxacin.
For P. aeruginosa complex I (MIC = 0.69
the gatifloxacin drugs. In case of E coli complexes I, II, III and V
(MIC = 2.42, 2.27, 2.37 and 1.40 M) are more potent than gatiflox-
acin norfloxacin and pefloxacin. It was observed that all the com-
plexes were more potent bactericides than the ligands. It was
found that the complex V having planer aromatic ancillary ligand
lM) is more potent than
2.3.1. Effect of complexes on the microorganism
The complexes exhibit activities against three Gram(ꢀve) that is,
E. coli, S. merscences and P. aeruginosa and two Gram(+ve) that is, S.
aureus, B. subtilis microorganisms. The results concerning in vitro
antimicrobial activity (MIC) of the metal salt, ligands, complexes
l
Table 3
Antimicrobial activity data (
lM)
Compounds
Gram positive
Gram negative
S. aureus
B. subtilis
S. marcescens
P. aeruginosa
E. coli
Co(NO3)2ꢂ6H2O
515.00
1.63
858.00
1.09
1374.00
1.63
1030.00
1.36
1030.00
1.36
Ciprofloxacin
Gatifloxacin
5.06
4.00
2.93
1.01
2.93
Norfloxacin
2.50
2.51
4.07
3.76
2.82
Enrofloxacin
1.95
3.90
1.67
1.39
1.39
Pefloxacin
2.10
2.40
5.10
5.70
2.70
Levofloxacin
1.66
2.21
1.67
1.66
0.97
Sparfloxacin
1.27
2.04
1.53
1.53
1.27
Ofloxacin
1.94
1.38
1.66
2.21
1.38
A1
A2
A3
A4
A5
A6
I
1855.00
1687.00
1760.00
3160.00
5086.00
8319.00
2.07
1518.00
1518.00
1600.00
3476.00
5548.00
9151.00
0.28
2024.00
1687.00
1760.00
4108.00
6010.00
9983.00
1.73
2193.00
1855.00
1920.00
3792.00
6010.00
10815.00
0.69
2361.00
2193.00
2080.00
4108.00
6473.00
10815.00
2.42
II
0.65
0.19
2.27
1.30
2.27
III
IV
V
0.68
1.71
0.28
1.69
1.71
0.37
2.71
3.42
2.80
2.37
3.42
0.93
2.37
2.99
1.40
VI
1.03
0.41
4.11
3.08
4.11

5652
M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
Figure 4. Viscosity data of the complexes with DNA.
Figure 3. Absorption spectra of the complex I.
2.3.2.2. Viscosity measurement.
Furthermore, the interac-
tions between the complex and DNA were investigated by viscosity
measurements. Optical photophysical probes provided necessary,
but not sufficient, clues to support a binding model. Hydrodynamic
measurements that are sensitive to length change (i.e., viscosity
and sedimentation) of DNA are regarded as the least ambiguous
and the most critical tests of binding mode in solution in absence
of crystallographic structural data.38,39 A classical intercalation
model usually resulted in lengthening the DNA helix, as base pairs
were separated to accommodate the binding ligand leading to the
increase of DNA viscosity, where as non-classical intercalation
mode usually results in static bend or kink in the helix and de-
creases the viscosity of DNA. As seen in Figure 4, the viscosity of
DNA increased on increasing the ratio of Co(II) complexes to
DNA. This result further suggested an intercalative binding mode
of the complex with DNA and also parallel to the above spectro-
scopic results, such as hypochromism and hypsochromism of com-
plexes in the presence of DNA. But the viscosity of DNA decreases
on increasing the ratio of ciprofloxacin to DNA. This result suggests
that ciprofloxacin bind DNA via partial, non-classical intercalation.
was more potent bacteriostatic compare to the one with aliphatic
ancillary ligand (complex VI). It was clearly observed that with in-
crease in aromatic nature of ancillary ligands (complexes III and
IV) potency of complex was not much affected, but the presence
of methoxy group along with increase in aromatic character shows
mark increase in potency against bacteria (complex II) which is
further supported from Kb values from absorption titration and
gel electrophoresis data. The inhibition activity seems to be gov-
erned in certain degree by the facility of coordination at the metal
center. This may support the argument that some type of biomo-
lecular binding to the metal ions or intercalation or electrostatic
interactions causing the inhibition of biological synthesis and pre-
venting the organisms from reproducing. The strong antimicrobial
activities of these complexes against tested organisms suggest fur-
ther investigation on these complexes.
2.3.2. Interaction of the mixed-ligand complexes with DNA
2.3.2.1. Absorption spectroscopy.
The application of elec-
tronic absorption spectroscopy in DNA-binding studies is one of
the most useful techniques.36 Complex binding with DNA through
intercalation usually results in hyperchromism and hypochro-
mism, 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. The low energy absorption band
centered at 455–472 nm is assigned to metal-to-ligand charge
transfer (MLCT) transition and the other band centered at 263–
2.3.2.3. Oxidative and non-oxidative nucleic acid cleav-
age.
There has been considerable interest in DNA cleavage
reactions that are activated by transition metal complex.40,41 The
delivery of metal ion to the helix, in locally generating oxygen or
hydroxide radicals, yields an efficient DNA cleavage reaction. Fig-
ure 5 illustrates the gel electrophoretic separations showing the
cleavage of plasmid pBR322 DNA induced by the complexes under
aerobic conditions and in presence of H2O2, respectively.42 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 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 complexes to
DNA is much higher than the binding efficacy of metal salt itself or
ciprofloxacin (Table 5). The different DNA cleavage efficiency of the
complexes was due to the different binding affinity of the com-
plexes to DNA, which has been observed in other cases. One of
286 nm is attributed to intraligand (IL) p– p
* transition by compar-
ison with the spectrum of other polypyridyl Ru(II) complexes.37
The absorption spectra of the complex (I) in the absence and pres-
ence of increasing amounts of DNA are illustrated in Figure 3.
Change in absorbance at peak maximum shows moderate hypso-
chromism shift (ꢁ5 nm). For each complex, increasing concentra-
tion of DNA has been monitored for an evaluation of the intrinsic
binding constant, which observed in the range of
1.0 ꢃ 104ꢀ4.0 ꢃ 104 Mꢀ1. (Insert Fig. 3 for the plot for the calcula-
tion of intrinsic binding constant). The binding constants are given
in Table 4. These spectral characteristics are consistent with a
mode of interaction that involves a stacking interaction between
the complex and the base pairs of DNA, which means that the titled
complexes can intercalate into the double helix structure of DNA.
Table 4
The binding constants (Kb) of Co(II) complexes with DNA in phosphate buffer pH 7.2
Complexes
Kb (Mꢀ1
)
I
II
III
IV
V
1.0 ꢃ 104
4.0 ꢃ 104
1.5 ꢃ 104
2.0 ꢃ 104
2.0 ꢃ 104
2.5 ꢃ 104
Figure 5. Gel electrophoresis data with pBR322: Lane 1: DNA (control); Lane 2:
DNA + metal salt; Lane 3: DNA + cip; Lane 4: DNA + I; Lane 5: DNA + II; Lane 6:
DNA + III; Lane 7: DNA + IV; Lane 8: DNA + V; Lane 9: DNA + VI; Lane 10:
DNA + H2O2 + I; Lane 11: DNA + H2O2 + II; Lane 12: DNA + H2O2 + III; Lane 13:
DNA + H2O2 + IV; Lane 14: DNA + H2O2 + V; Lane 15: DNA + H2O2 + VI.
VI

M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
5653
Table 5
4.1.2. Instrumentation
Gel electrophoresis data of the compounds
Infrared spectra were recorded on a FT-IR Shimadzu spectro-
photometer as KBr pellets in the range 4000–400 cmꢀ1. C, H and
N elemental analyses were performed with a model 240 Perkin El-
mer elemental analyzer. The reflectance spectra of the complexes
were recorded in the range 1700–350 nm (as MgO discs) on a Beck-
man DK-2A spectrophotometer. The metal contents of the com-
plexes were analyzed by EDTA titration after decomposing the
Compounds
% SC
% NC
% OC
DNA control
DNA + Co(NO3)2ꢂ6H2O
DNA + Cip.
DNA + I
DNA + II
DNA + III
DNA + IV
DNA + V
DNA + VI
DNA + I + H2O2
DNA + II + H2O2
DNA + III + H2O2
DNA + IV + H2O2
DNA + V + H2O2
DNA + VI + H2O2
100
58
50
19
21
20
21
26
29
17
14
19
18
24
18
—
—
—
42
36
55
46
45
44
44
44
58
52
45
54
49
50
14
26
33
35
35
30
37
25
34
36
28
27
32
organic matter with
a mixture of HClO4, H2SO4, and HNO3
(1:1.5:2.5).44 MIC study was carried out by means of laminar air
flow cabinet, Toshiba, Delhi, India, Thermogravimetric analyses
was obtained with a model 5000/2960 SDTA, TA instrument
(USA). The 1H NMR and 13C NMR were recorded on a Bruker Avance
(400 MHz). The electronic spectra were recorded on a UV-160A
UV–vis. spectrophotometer, Shimadzu (Japan). The magnetic mo-
ments were measured by Gouy’s method using mercury tetrathioc-
yanatocobaltate(II) as the calibrant (
v
g = 16.44 ꢃ 10ꢀ6 cgs units at
20 °C), Citizen Balance. The diamagnetic correction was made
using Pascal’s constant.45 The FAB-mass spectra were recorded
on a Jeol SX 102/Da-600 mass spectrometer/Data System using
Argon/Xenon (6 kv, 10mA) 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.
the most interesting electrophoretic results of the complexes takes
place when experiment is done in presence of H2O2 in TAE buffer.
The DNA + complex (10 lM) + H2O2 (100 lM) systems (Fig. 5, Lane
10–15) cleave the supercoiled DNA form (I) and convert into
nicked form (II) and linear form (III) more than complex alone.
Therefore, we concluded that the mixture of complex with H2O2
have been found to be efficient oxidant.
4.2. Method
4.2.1. Synthesis of ligand
3. Conclusion
4.2.1.1. N,N0-Bis-(4-methoxy-phenyl)-1,2-dimethyl-ethane-1,2-
diimine (A1ꢁbmpdme).
An ethanolic solution (100 mL) of p-
In all the complexes, the quinolone ligand bound to cobalt(II)
via the pyridone oxygen and one carboxylate oxygen. Interaction
of Co(II) with the deprotonated quinolone ligand and neutral
bidentate, results in the formation of the neutral dinuclear com-
plexes. For all the complexes, an octahedral environment around
Co(II) has been suggested. The interaction of complexes I–VI with
DNA revealed that all the complexes can bind to DNA by the inter-
calative mode. The best inhibition among the compounds studied
anisidine (2.46 g, 20 mmol) was added drop wise to ethanolic solu-
tion (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 ob-
tained crystalline yellow product was recrystallized in ethanol,
washed with n-hexane and dried in air (see Scheme 1).
4.2.1.2. N,N0-Bis-(4-methoxy-benzylidene)-ethane-1,2-diamine
(A2ꢁbmbed).
An ethanolic solution of ethylenediamine
in this work is provided by the compound V (MIC = 0.28
against S. aureus while compounds (MIC = 0.28 M), II
(MIC = 0.19 M), V (MIC = 0.37 M) and VI (MIC = 0.41 M) against
B. subtilis. In case of P. aeruginosa I and V (MIC = 0.69 and 0.93 M)
lM)
(0.60 g, 10 mmol) and p-anisaldehyde (2.50 g, 20 mmol) were re-
fluxed on water bath for 8 h. The resulting mixture was filtered.
The obtained crystalline yellow product was recrystallized in eth-
anol, washed with n-hexane and dried in air.
I
l
l
l
l
l
prove to be better active. Hence complexation of ligands with me-
tal is responsible for the better MIC value than ligands itself. From
the gel electrophoresis data it is crystal clear that there occurs a
mark increase in DNA cleavage property of ciprofloxacin on
complexation.
4.2.1.3. N,N0-Bis-(1-phenylethylidene)-benzene-1,2-diamine
(A3ꢁbpebd).
The preparation of N,N0-bis-(1-phenylethyli-
dene)-benzene-1,2-diamine was carried out by refluxing an etha-
nolic solution of acetophenone (2.4 g, 20 mmol) and o-
phenylenediamine (1.08 g, 10 mmol) on water bath for 8 h. Fine
yellow crystalline product obtained on filtration was further crys-
tallized in ethanol, washed with n-hexane and dried in air.
4. Experimental
4.1. Materials and methods
4.2.2. Synthesis and physical properties of the mixed-ligand
complexes
4.1.1. Drug and chemicals
All the chemicals used were of analytical grade. 1,8-Diamino-
naphthalene (danꢁA4) was purchased from Lancaster, England.
Ciprofloxacin hydrochloride was purchased from Bayer AG
(Wuppertal, Germany). Ethylenediamine (enꢁA6), 2,3-butanedi-
one, p-anisaldehyde, p-anisidine, acetophenone, glycerol, and co-
balt nitrate were purchased from E. Merck (India) Ltd, Mumbai.
Xylene cyanol FF, ethidium bromide and luria broth were pur-
chased from Himedia, India. Agarose was purchased from Sisco
research Lab., India. Bromophenol blue, o-phenylenediamine
(opdꢁA5), 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.43
4.2.2.1. [Co2(Cip)2(bmpdme)2(pip)(H2O)2]ꢂ3H2O (I).
A meth-
anolic solution of Co(NO3)2ꢂ6H2O (2.91 g, 10 mmol) was added to a
methanolic solution of bmpdme (2.96 g, 10 mmol), followed by
addition of a previously prepared solution of CipꢂHCl (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 ciprofloxacin
was substituted by chloride ion in the presence of NaOH.46 The
resulting red solution was refluxed for 8–10 h. on a steam bath,
and then was kept overnight at room temperature. A fine reddish
brown crystalline product was obtained which was washed with
ether and dried in a vacuum desiccator. The physical parameters
of the complexes are shown in Table 6 and probable reaction
scheme is shown in Scheme 2. Yield 68.5%, mp >360 °C, Calcd for

5654
M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
NH2
Ethanol
+
N
N
O
O
O
O
O
N,N'-Bis-(4-methoxyphenyl)-
1,2-dimethyl-ethane-1,2-diamine
2,3- Butenedion
4-Methoxy Aniline
Scheme 1. Reaction scheme of A1ꢁbmpdme.
C66H74Cl2Co2F2N8O15: C, 54.82; H, 5.16; N, 7.75; Co, 8.15; M+,
1446.28. Found: C, 54.80; H, 5.19; N, 7.72; Co, 8.17; M+, 1446.11.
O
HO
O
O
N
N
O
N
N
4.2.2.2. [Co2(Cip)2(bmbed)2(pip)(H2O)2]ꢂ3H2O (II).
It was
+
prepared by using bmbed (2.96 g, 10 mmol). Yield 69.4%, mp
350 °C, Calcd for C74H74Cl2Co2F2N8O15: C, 57.63; H, 4.84; N, 7.27;
Co, 7.64; M+, 1542.54. Found: C, 57.60; H, 4.85; N, 7.24; Co, 7.61;
M+, 1542.19.
F
Co(NO3)2.6H2O
NaOH
NH2+Cl-
4.2.2.3. [Co2(Cip)2(bpebd)2(pip)(H2O)2]ꢂ3H2O (III).
It was
pH 6-7.5
prepared by using dan (3.12 g, 10 mmol). Yield 67.5%, mp 255 °C,
Calcd for C74H74Cl2Co2F2N8O11: C, 60.13; H, 5.05; N, 7.58; Co,
7.97; M+, 1478.19. Found: C, 60.14; H, 5.04; N, 7.56; Co, 7.95; M+,
1478.20.
O
N
N
O
N
Cl
F
4.2.2.4. [Co2(Cip)2(dan)2(pip)(H2O)2]ꢂ3H2O (IV).
It was pre-
N
pared by using dan (1.58 g, 10 mmol). Yield 68.0%, mp 350 °C, Calcd
for C50H54Cl2Co2F2N8O11: C, 51.34; H, 4.65; N, 9.58; Co, 10.08; M+,
1169.78. Found: C, 51.30; H, 4.66; N, 9.56; Co, 10.06; M+, 1169.78.
H2OCo
O
N
O
O
3H2O
O
N
O
F
Co
O
OH2
4.2.2.5. [Co2(Cip)2(opd)2(pip)(H2O)2]ꢂ3H2O (V).
It was pre-
Cl
N O
N
O
pared by using opd (1.08 g, 10 mmol). Yield 68.0%, mp 360 °C
(dec.), Calcd for C42H50Cl2Co2F2N8O11: C, 47.16; H, 4.71; N, 10.48;
Co, 11.02; M+, 1069.73. Found: C, 47.15; H, 4.72; N, 10.45; Co,
11.01; M+, 1069.66.
Scheme 2. Synthesis of Complex I [Co2(Cip)2(bmpdme)2(pip)(H2O)2]ꢂ3H2O.
4.2.2.6. [Co2(Cip)2(en)2(pip)(H2O)2]ꢂ3H2O (VI).
It was pre-
pared by using en (0.60 g, 10 mmol). Yield 67.9%, mp 360 °C
(dec.), Calcd for C34H50Cl2Co2F2N8O11: C, 41.94; H, 5.18; N, 11.51;
Co, 12.11; M+, 973.58. Found: C, 41.93; H, 5.15; N, 11.49; Co,
12.10; M+, 973.58.
4.2.4. Function of complexes on the DNA
Isolation of plasmid DNA from pure culture of E. coli was carried
out by conventional
Method,48 the basic principle employed for isolation is based on
three steps:
4.2.3. Function of complexes on the microorganism
All the bacteria were incubated and activated at 30 °C by inoc-
ulation into luria broth for 24 h. The compounds were dissolved in
DMSO and then diluted using luria broth. Twofold serial concentra-
tions of the compounds were employed to determine the Mini-
1. Weakening of bacterial cell wall by the action of lysozyme.
2. Cells are lysed by EDTA and detergent at high pH.
3. Insoluble cells debris consisting of genomic DNA and protein is
precipitated with high salt and centrifuged down leaving the
plasmid in solution (supernant).
mum Inhibitory Concentration (MIC) ranging from 11,000
0.1 M. Test cultures were incubated at 37 °C (24 h). The lowest
concentrations of antimicrobial agents that resulted in complete
inhibition of growth were represented as MIC ( M). In each case
lM to
l
The basic principle employed is ‘alkali-lysis’ in which at the alka-
line pH, both the genomic and plasmid DNA are denatured. On
reduction of the pH the plasmid DNA molecule being small in size,
l
triplicate tests were performed and the average was taken as the
final value.47
Table 6
Experimental and physical parameters of the complexes
Complexes empirical formula
Elemental analysis % found (required)
mp (°C)
% Yield
Formula weight (gm/mol)
C
H
N
M
C66H74Cl2Co2F2N8O15 (I)
C74H74Cl2Co2F2N8O15 (II)
C74H74Cl2Co2F2N8O11 (III)
C50H54Cl2Co2F2N8O11 (IV)
C42H50Cl2Co2F2N8O11 (V)
C34H50Cl2Co2F2N8O11 (VI)
54.80 (54.82)
57.60 (57.63)
60.14 (60.13)
51.30 (51.34)
47.15 (47.16)
41.93 (41.94)
5.19 (5.16)
4.85 (4.84)
5.04 (5.05)
4.66 (4.65)
4.72 (4.71)
5.15 (5.18)
7.72 (7.75)
7.24 (7.27)
7.56 (7.58)
9.56 (9.58)
10.45 (10.48)
11.49 (11.51)
8.17 (8.15)
7.61 (7.64)
7.95 (7.97)
10.06 (10.08)
11.01 (11.02)
12.10 (12.11)
>360
350
255
350
360 dec.
360 dec.
68.5
69.4
67.5
68.0
68.0
67.9
1446.11
1542.19
1478.20
1169.78
1069.66
973.58

M. N. Patel et al. / Bioorg. Med. Chem. 17 (2009) 5648–5655
5655
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l
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½DNAꢄ
½DNAꢄ
1
¼
þ
ð1Þ
ð
ꢀa
ꢀ
ꢀf
Þ
ð
ꢀb
ꢀ
ꢀf
Þ
Kbðꢀb
ꢀ
ꢀf
Þ
where, [DNA] is the concentration of DNA in terms of nucleotide
phosphate [NP], the apparent absorption coefficient ef a and b cor-
respond to the extinction coefficient of the free complex, the extinc-
tion coefficient for each addition of DNA to the complex and the
extinction coefficient for the complex in the fully bound form,
respectively and Kb is the ratio of the slope to the y intercept.
Viscosity measurements were carried out using an Ubbelodhe
,
e
e
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1/3
Data were presented as
[DNA],52 where
plex and 0 is the viscosity of DNA alone. Viscosity values were cal-
culated from the observed flow time of DNA-containing solutions
(t >100 s) corrected for the flow time of buffer alone (t0),
= t ꢀ t0.
For the gel electrophoresis experiments, supercoiled pBR322
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(g/g0)
versus binding ratio [Co]/
g
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bromophenol blue, 0.03% xylene cyanol FF, 60% glycerol and
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1.0 mg/mL ethidium bromide. Bands were visualized by UV light
and photographed on a capturing system (AlphaDigiDocTM RT.
Version V.4.1.0 PC-Image software).
Acknowledgments
Authors would like to acknowledge Head, Department of Chem-
istry, Sardar Patel University, Vallabh Vidyanagar, Gujarat, India,
for providing the necessary laboratory facilities. Authors are also
thankful to UGC for financial assistance Grant 32-226/2006(SR).
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