In vitro bacteriostatic and DNA interaction studies of drug-based mixed-ligand complexes of cobalt(II)

Article abstract of DOI:10.1007/s00044-010-9310-9

The dinuclear cobalt(II) complexes with ciprofloxacin and bidentate ligands were synthesized and characterized using infrared spectra, electronic spectra, magnetic measurements, elemental analyses, thermal investigation, and mass spectroscopy. Here in we

Full text of DOI:10.1007/s00044-010-9310-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:220–230
DOI 10.1007/s00044-010-9310-9
ORIGINAL RESEARCH
In vitro bacteriostatic and DNA interaction studies of drug-based
mixed-ligand complexes of cobalt(II)
•
Mohan Patel Mehul Chhasatia Bhupesh Bhatt
•
Received: 21 July 2009 / Accepted: 13 January 2010 / Published online: 3 February 2010
Ó Springer Science+Business Media, LLC 2010
Abstract The dinuclear cobalt(II) complexes with cipro-
floxacin and bidentate ligands were synthesized and char-
acterized using infrared spectra, electronic spectra, magnetic
measurements, elemental analyses, thermal investigation,
and mass spectroscopy. 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 compared to drugs, ligands, and
metal salt against selective gram(?ve) and gram(-ve) organ-
isms. Interaction of the complexes with nucleic acid (DNA)
was investigated using spectroscopic technique, viscosity
measurement, and gel electrophoresis and it was found that
the complexes bind to DNA via intercalative mode.
forming polynuclear complexes (Hernandez-Gil et al.,
2009; Efthimiadou et al., 2007; Chen et al., 2000). The
synthesis and characterization of new metal complexes
with quinolone antibacterial agents are of great importance
for understanding the drug–metal ion interaction and for
their potential pharmacological use. In continuation of our
previous work (Patel et al., 2008a, b), herein we synthe-
sized and characterized the mixed-ligand complexes of
Co(II) with ciprofloxacin and neutral bidentate ligands,
such as bpdmed(A1), mtma(A2), apq(A3), bpeed(A4),
dcnd(A5), and dpeda(A6). In a few preliminary analytical
investigations it was shown that quinolone is coordinated
to the metal ion in the range of 6–7.5 pH. The DNA
binding and cleavage properties of the complexes have
been investigated by ultraviolet spectroscopy, viscosity
measurements, and gel electrophoresis method.
Keywords Dinuclear mixed-ligand complexes of Co(II) ꢀ
Ciprofloxacin ꢀ MIC ꢀ DNA interaction
Introduction
Results and discussion
Ciprofloxacin (Cip) is synthetic, broad-spectrum antibac-
terial drug used to treat pneumonia, bronchitis and some
types of gonorrhea, diarrhea caused by bacteria, typhoid
fever, prostate, sinus, and urinary tract infections. Cipro-
floxacin is a quinolone antibacterial agent, with 4-oxo-3-
carboxylic acid group that is essential for their bactericidal
activity. Due to the ring carbonyl group at position 4 and
one of the oxygen atoms of carboxylato group at position 3,
the compound acts as bidentate ligand. Quinolones can also
act as bridging ligands and hence they are capable of
Chemistry
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 were reported along with the possible
1
assignment. The H NMR spectra of ligands, exhibiting
peaks at about 6.5–8.19 ppm were assigned to the aromatic
protons (Patel et al., 2008a, b). 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.
M. Patel (&) ꢀ M. Chhasatia ꢀ B. Bhatt
Department of Chemistry, Sardar Patel University, Vallabh
Vidyanagar, 388 120 Gujarat, India
e-mail: jeenen@gmail.com
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Med Chem Res (2011) 20:220–230
221
20,500–22,525 cm-1 (Table 2) were assigned (Chohan
Infrared spectroscopy
et al., 2005; Chohan and Supuran, 2005; Sathyanarayana,
4
4
2001) to 4T1g(F) ? A2g(F)(t2) and 4T1g(F) ? T1g(P)(t3)
transition, respectively, for a high-spin octahedral geome-
try. In addition, the magnetic susceptibility measurements
(4.8–5.1 BM) for the Co(II) complexes are also indicative
of three unpaired electrons per Co(II) ion, suggesting
consistency with their octahedral environment. Electronic
spectral data of the complexes of the compounds are pro-
vided in Table 3.
The prominent IR spectral data of the complexes are shown
in Table 1. The m(C=O) stretching vibration band appears
at 1,708 cm-1 for ciprofloxacin, where as for complexes it
appear at 1,609–1,627 cm-1; this shift toward lower
energy suggests that coordination occurs through the car-
bonyl oxygen of pyridine ring (Turel et al., 1997). The
sharp band in ciprofloxacin at 3,520 cm-1 is due to
hydrogen bonding (Silverstein and Webster, 2004); 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 (Panchal
and Patel, 2006). The data were further supported by m(M–O)
Thermal analysis
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°C per minute in the range of 20–800°C
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°C, indicating that these water molecules
were water of crystallization. In the second step weight loss
during 130–180°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 tem-
perature range of 180–250°C, followed by liberation of
Cip.(L) in the temperature range of 250–500°C. Finally,
decomposition of An occurred in the temperature range of
520–800°C, and the remaining weight was consistent with
(Yan et al., 2005) band which appeared at 509–515 cm-1
.
The strong absorption band obtained at 1,624 and
1,340 cm-1 in ciprofloxacin were assigned to m(COO)asy
and m(COO)sym, respectively, while in the metal complexes
these bands were observed at 1,600 and 1,380 cm-1
,
respectively. The frequency separation (Dm) in the inves-
tigated complexes is greater than 200 cm-1, suggesting a
unidentate bonding nature for the carboxyl group (Chohan
et al., 2005; Chohan and Supuran, 2005; Nakamoto, 1986).
The m(C=N) peak for the Schiff bases was observed at
1,590–1,620 cm-1 which on complexation were shifted to
1,558–1,575 cm-1, which indicates the N–S and the N–N
coordination of the ligands. This data was further supported
by m(M–N) (Chandra et al., 2005) which appeared at
532–542 cm-1. In the case of the m(C–S) band of mtma
observed at 700 cm-1 is shifted lower to 680 cm-1 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 420 cm-1 which can
be assigned to m(M–S) (Chandra et al., 2005; Patel et al.,
2008a, b) mode.
Table 2 Electronic spectral data of the complexes
Compounds t2
t3
B0
10Dq
b
% b leff BM
I
18790 21500 646.0 11291 0.665 33.46 4.8
16500 20620 506.4 10770 0.521 47.84 5.0
17577 21725 548.0 11355 0.564 43.56 4.8
18625 21500 633.7 11285 0.652 34.73 4.9
17560 22525 518.2 11745 0.533 46.63 5.1
18471 20500 656.5 10784 0.676 32.38 4.9
II
III
IV
V
Electronic spectroscopy and magnetic measurements
The Co(II) complexes exhibited well-resolved bands at
16,500–18,790 cm-1 and a strong high energy band at
VI
Table 1 Infrared spectral data of the Co(II) complexes
Compounds m(C=O) (cm-1
pyridone
)
m(COO)asy m(COO)sym Dm
m(C–Cl) m(C=N) (cm-1
)
m(C=N)
m(M–N) m(M–O) m(M–S)
(cm-1
)
(cm-1
)
(cm-1) (cm-1
)
azomethine
(cm-1) ring (cm-1
)
(cm-1
)
(cm-1
)
I
1609
1622
1619
1627
1620
1618
1600
1584
1590
1608
1564
1602
1380
1383
1384
1384
1384
1386
205
201
206
224
205
216
1144
1118
1160
1145
1144
1140
1575
1558
–
–
540
542
540
540
540
532
511
510
515
509
511
512
–
II
–
–
III
IV
V
1600
420
–
1560
1575
1571
–
–
–
–
VI
–
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Med Chem Res (2011) 20:220–230
Table 3 Experimental and physical parameters of the complexes
Complexes empirical formula
Elemental analysis % found (required)
m.p. (°C)
% Yield
Formula weight
(gm/mol)
C
H
N
M
C62H66Cl2Co2F2N8O11 (I)
56.12 (56.16)
50.29 (50.28)
52.32 (52.30)
57.39 (57.35)
59.67 (59.64)
62.52 (62.56)
5.08 (5.02)
4.57 (4.53)
4.52 (4.54)
5.41 (5.40)
5.86 (5.82)
4.73 (4.74)
8.44 (8.45)
8.92 (8.89)
9.10 (9.14)
8.87 (8.85)
8.50 (8.53)
7.94 (7.91)
7.46 (7.49)
300 dec.
300 dec.
280
69.5
71.2
69.5
67.8
66.4
68.0
1326.00
1289.97
1331.93
1382.11
1490.29
1574.28
C54H58Cl2Co2F2N6O13S2 (II)
C58H60Cl2Co2F2N10O13 (III)
C66H74Cl2Co2F2N8O11 (IV)
C74H86Cl2Co2F2N8O11 (V)
C82H74Cl2Co2F2N8O11 (VI)
6.54 (6.51)
10.57 (10.52)
8.14 (8.11)
7.53 (7.52)
7.15 (7.12)
370
[370
270
of peak at m/z 998 is due to removal of two coordinate
water molecule and one ligand [M-C16H20N2O2]. The
peak occurring at m/z 575 correspond to fragment pro-
duced by cleavage of Co–N bond with piperazine
[M-C33H36N5O6ClFCo]. The peak arising at m/z 236 corre-
spond to the ligand [C16H16N2]. The mass spectrum is shown
in Fig. 2.
Antimicrobial activity
The results concerning in vitro antimicrobial activity
(MIC) of the ligands, standard drugs, metal salt, and
complexes are represented in Table 4. The ligands (A1–A6)
exhibit a little antimicrobial activity. The antimicrobial
activity of all the complexes against all the five microor-
ganisms is much higher than metal salt, while in compe-
tition with the ciprofloxacin. The results of our study
indicate that all the synthesized compounds were found
more potent antimicrobial against Serratia aureus and
Bacillus subtilis compared to all tested drugs. In case of
Serratia merscences all the compounds were more active
compared to pefloxacin drug. In case of Pseudomonas
aeruginosa compound II was found more potent antimi-
crobial compared to all tested drugs. In case of E. coli
compounds I, II, and III (MIC = 1.13, 0.77, 1.12 lM,
respectively) shows higher activity compare to all the tes-
ted drugs. The inhibition activity seems to be governed in
Fig. 1 Structure of the complex (I)
metal oxide (Patel et al., 2008a, b). Structure of the com-
plexes from the above analytical facts is given below in
Fig. 1.
Mass spectroscopy
The FAB mass spectrum of complex-I shows the molecular
ion peak at m/z 1,252 due to [M?] in absence of three
lattice water molecules. The peak at m/z 1,234 is due to
removal of one coordinate water molecule. The appearance
Fig. 2 Mass spectrum of
complex (I)
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Med Chem Res (2011) 20:220–230
223
Table 4 MIC Data of the compounds (lM)
Compounds
Gram positive
Gram negative
S. aureus
B. subtilis
S. merscences
P. aeruginosa
E. coli
Co(NO3)2ꢀ6H2O
Ciprofloxacin
514.75
1.63
857.92
1.09
1372.68
1.63
1029.51
1.36
1029.51
1.36
Gatifloxacin
5.06
4.00
2.93
1.01
2.93
Norfloxacin
2.51
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
1.66
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
2115.86
2531.11
2528.87
2080.49
1884.12
1248.43
0.37
1904.20
2301.07
2318.13
1891.36
1570.10
1387.15
0.3
2539.00
2761.28
2739.61
2080.49
1884.12
1525.87
1.13
2750.62
2991.39
2950.34
2458.76
2041.13
1664.58
1.13
2750.62
2761.28
3161.08
2647.90
2041.13
1803.30
1.13
II
0.31
0.31
2.32
0.77
0.77
III
IV
V
0.75
0.3
2.25
1.12
1.12
0.72
1.08
3.25
1.44
3.25
1.0
1.0
3.0
3.0
2.68
VI
0.63
0.63
1.90
2.22
2.54
certain degree by the facility of coordination at the metal
center. This may support the argument that some type of
biomolecular binding to the metal ions or intercalation or
electrostatic interactions causing the inhibition of biologi-
cal synthesis and preventing the organisms from repro-
ducing. Comparing with our earlier effort cited on
antibacterial activities of Co(II) complexes, ligands A1, A2,
and A6 illustrate superior MIC value than the reported
(Patel et al., 2009) MIC of ligands such as N,N0-dicyclo-
hexylidene-benzene-1,2-diamine (dcbd), N,N0-bis-(4-
methoxybenzylidene)-benzene-1,2-diamine (bmbbd), N,N0-
bis-(3-chlorophenyl)-1,2-diphenylethane-1,2-diimine (bcp-
ded), bis(benzylidene)ethylenediamin (benen), N,N0-bis-(4-
The presence of four unsubstituted phenyl rings (A6) and
presence of thiophene ring (A1), can improve MIC value of
complexes (Patel et al., 2009). The strong antimicrobial
activities of these complexes against tested organisms
suggest further investigation on these complexes.
DNA binding
Absorption spectroscopic titrations
The interaction of the complexes with DNA was investi-
gated using absorption spectra. The absorption spectra of
the complexes in the absence and presence of DNA (at a
constant concentration of complexes) are given in Fig. 3.
As the concentration of DNA is increased, the band of
complexes exhibit hyperchromism and hypsochromism
shift (*5 nm). These spectral characteristics may suggest
that there are some interactions between the complex and
DNA. In order to compare quantitatively the binding
strength of the complex, the intrinsic binding constants Kb
of complexes with DNA were determined from the decay
of the absorbance monitored for [Co2(Cip)2(bpdmed)2
(pip)(H2O)2]ꢀ3H2O. An intrinsic binding constant Kb of
[Co2(Cip)2(bpdmed)2(pip)(H2O)2]ꢀ3H2O was obtained at
3.0 9 104 M-1. The data is not as strong as that of
methoxyphenyl)-1,2-diphenylethane-1,2-diimine
(bmp-
ded), and 2,20-bipyridylamine for gram(?ve) microorgan-
isms. While, ligand A6 is more effective antibacterial agent
than all the above ligands for gram(-ve) microorganisms.
The ligands A1 and A2 also add their influence when
coordinated to cobalt metal ion and hence complex-I and -
II show potent antibacterial activity even than complexes
cited in our earlier work (Patel et al., 2009), for S. aureas
and B. subtilis as well as for gram(-ve) microorganisms.
The structural activity relationship for antibacterial prop-
erties reveals scope of structural modification for devel-
opment of antibacterial activity against microorganisms.
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Med Chem Res (2011) 20:220–230
Fig. 3 Absorption spectra of
the complex (I)
Table 5 The binding constants (Kb) of Co(II) complexes with DNA
in phosphate buffer pH 7.2
Complexes
Kb (M-1
)
I
3.0 9 104
2.0 9 104
2.0 9 104
1.0 9 104
3.0 9 104
3.0 9 104
II
III
IV
V
VI
Fig. 4 Viscosity data of the complexes with DNA
[Ru(IP)2dppz]2? (2.1 9 107 M-1) (Liu et al., 1999), but
comparable to those of some DNA intercalative polypyri-
dyl Ru(II) complexes 1.0–4.8 9 104 M-1 (Juris et al.,
1988). The binding constant (Kb) data of all other com-
plexes are shown in Table 5. The binding parameters of
complexes in this work compared with our previous work
confirm the superiority of complexes II, V, and VI as can
bind to DNA with three times greater affinity than
[Co2(Cip)2(bmpded)2(pip)(H2O)2]ꢀ3H2O and two times
greater affinity than [Co2(Cip)2(bmbbd)2(pip)(H2O)2]ꢀ3H2O,
[Co2(Cip)2(bepded)2(pip)(H2O)2]ꢀ3H2O, and [Co2(Cip)2
(benen)2(pip)(H2O)2]ꢀ3H2O (Patel et al., 2009).
DNA helix must lengthen as base pairs are separated to
accommodate the binding ligand, leading to the increase of
DNA viscosity. In contrast, a partial intercalation ligand
could bend (or kink) the DNA helix, reduce its effective
length and, concomitantly, its viscosity (Satyanarayana
et al., 1992, 1993). The effect of the complex [Co2(Cip)2
(bpdmed)2(pip)(H2O)2]ꢀ3H2O on the viscosity of rod-like
DNA is shown in Fig. 4. On increasing the amounts of
[Co2(Cip)2(bpdmed)2(pip)(H2O)2]ꢀ3H2O, the relative vis-
cosity of DNA increases apparently. The result suggests
that the dinuclear complex [Co2(Cip)2(bpdmed)2(pip)(H2O)2]ꢀ
3H2O (I) can bind to DNA by the intercalation mode,
similarly complexes III, V, and VI can bind via interca-
lation mode, while in case of complexes II and IV the
relative viscosity of DNA decreases, which can be attrib-
uted to binding of DNA via non-classical intercalation
mode. Which may act as a wedge to pry apart one side of a
base-pair stack, as observed for the [Ru(phen)3]2?, but not
fully separate the stack as required by the classical inter-
calation mode. This would cause a static bend or kink in
the helix and decrease the viscosity of DNA. Such non-
classical intercalative mode was not visualized for
Viscosity measurements
Optical photophysical probes provide necessary, but not
sufficient, clues to support a binding model. To further
clarify the interactions between the two complexes and
DNA, viscosity measurements were carried out. Viscosity
measurements that are sensitive to length change are
regarded as the least ambiguous and the most critical tests
of a binding model in solution in the absence of crystal-
lographic structural data (Satyanarayana et al., 1992,
1993). A classical intercalation model demands that the
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Med Chem Res (2011) 20:220–230
225
complexes formed with ligands; dcbd, bmbbd, bepded,
benen, bmpded, and bipym (Patel et al., 2009); and that
was concluded by raise in viscosity data in case of all the
above complexes.
Table 6 Gel electrophoresis data of the compounds
Compounds
% SC
% NC
% OC
DNA Control
100
62
52
34
19
20
26
26
34
20
14
20
21
20
22
–
–
–
38
36
66
48
49
74
48
38
52
49
37
50
51
71
DNA ? Co(NO3)2ꢀ6H2O
DNA ? Cip.
12
–
Cleavage of plasmid pBR322 DNA
DNA ? I
DNA ? II
33
31
–
There has been considerable interest in DNA cleavage
reactions that are activated by transition metal complex
(Liu et al., 2005). The delivery of metal ion to the helix, in
locally generating oxygen or hydroxide radicals, yields an
efficient DNA cleavage reaction. Figure 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
(Santra et al., 2002). When circular plasmid DNA is con-
ducted 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 ciproflox-
acin (Table 6). 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. The complexes III and IV have highest nuclease
activity among all including complexes cited in our earlier
work (Patel et al., 2009). The maximum interconversion of
DNA from SC to OC, is the root cause of explaining above
facts. One of the most interesting electrophoretic results of
the complexes takes place when experiment is done in the
presence of H2O2 in TAE buffer. The DNA ? com-
plex ? H2O2 systems (Fig. 5, Lanes 10–15) cleave more
supercoiled 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.
DNA ? III
DNA ? IV
DNA ? V
26
28
28
37
29
29
29
7
DNA ? IV
DNA ? I ? H2O2
DNA ? II ? H2O2
DNA ? III ? H2O2
DNA ? IV ? H2O2
DNA ? V ? H2O2
DNA ? VI ? H2O2
Conclusion
Antimicrobial activity shows that the complexes have been
found more potent compared with our previous effort
(Patel et al., 2009) against gram(?ve) than gram(-ve) spe-
cies. DNA-binding study indicates that intrinsic binding
constant of all complexes varies in the range of 1.0 9 104–
3.0 9 104 M-1, which is comparable to those of some
DNA intercalative polypyridyl Ru(II) complexes
1.0 9 104–4.8 9 104 M-1 and with our previous work on
cobalt complexes (Patel et al., 2009). All the complexes
exhibit good binding and cleavage activity. The complexes
I, III, V, and VI can bind via intercalation mode, while II
and IV can bind via non-classical intercalation mode. The
oxidation promoting reactivity of cobalt(II) complexes in
presence of H2O2 have been studied at pH 7.5 using
pBR322 DNA. The mixture of complex with H2O2 have
been found to be efficient oxidant by generating more
strand breaks in plasmid DNA compare to complex alone.
Experimental
Materials and methods
All the chemicals used were of analytical grade. Thio-
phene-2-carboxaldehyde, 1,8-diaminonaphthalene were
purchased from Lancaster, England. Ciprofloxacin hydro-
chloride was purchased from Bayer AG (Wyppertal,
Germany). Anthranilic acid, benzoyl chloride, ethylenedi-
amine, 2,3-butanedione, aniline, hydrazine hydrate, pyri-
dine, cyclohexanone, benzil, p-anisidine, glycerol, and
cobalt nitrate were purchased from E. Merck (India) Ltd.
Fig. 5 Agarose gel electrophoresis of pBR322 plasmid DNA: 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
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Med Chem Res (2011) 20:220–230
4-Methoxy-N-(thiophene-2-ylmethylene)aniline
(A2*mtma)
Mumbai. Xylene cyanol FF, ethidium bromide, and Luria
broth was purchased 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. (Furniss et al., 2004)
An ethanolic solution (100 ml) of thiophene-2-carbalde-
hyde (1.12 g, 10 mmol) was added dropwise to an etha-
nolic solution (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
recrystallized in ethanol, washed with n-hexane and dried
in air. Yield: 68%, m.p.: 2758C, Found %: C, 66.38, H,
5.18, N, 6.47. C12H11NOS (217.29) requires %: C, 66.33,
H, 5.10, N, 6.45. IR (cm-1): 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).
Instrumentation
Infrared spectra were recorded on a FT-IR Shimadzu
spectrophotometer as KBr pellets in the range of
4000–400 cm-1. C, H, and N elemental analyses were
performed with a model 240 Perkin Elmer elemental ana-
lyzer. 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 (Vogel,
1978) 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 analyses was obtained
with 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-
160A UV–Vis. spectrophotometer, Shimadzu (Japan). The
magnetic moments were measured by Gouy’s method
using mercury tetrathiocyanatocobaltate(II) as the calibrant
(vg = 16.44 9 10-6 cgs units at 208C), Citizen Balance.
The diamagnetic correction was made using Pascal’s con-
stant (Pascal, P. Compt Rend 57, 218, 1944). 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.
3-Amino-2-phenyl-3H-quinazolin-4-one (A3*apq)
The solution of anthranilic acid (1.37 g, 0.1 mol) was
prepared in pyridine (100 ml) and 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.: 1208C, the obtained 2-phenyl-3,1-benz-
oxazin-4-one (0.557 g, 0.05 mol) in ethanol (50 ml) was
mixed with hydrazine hydrate (0.125 g, 0.05 mol) in eth-
anol (50 ml) and refluxed for 3 h. The obtained product
was crystallized in ethanol. Yield: 85%, m.p.: 1968C,
Found %: C, 70.67, H, 4.62, N, 17.59. C14H11N3O (237.26)
requires %: C, 70.87, H, 4.67, N, 17.71. IR (cm-1): 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).
Synthesis and physical properties of Schiff bases
N,N0-Bis-(1-phenylethylidene)-ethane-1,2-diimine
(A4*bpeed)
N,N0-Bis-(phenyl)-1,2-dimethyl-ethane-1,2-diimine
(A1*bpdmed)
An ethanolic solution of ethylene diamine (0.60 g,
10 mmol) and acetophenone (2.40 g, 20 mmol) were
refluxed on water bath for 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.: 1358C, Found %: C, 81.70, H, 7.68, N, 10.67.
C18H20N2 (264.36) requires %: C, 81.78, H, 7.63, N, 10.60.
IR (cm-1): 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).
An ethanolic solution (100 ml) of aniline (1.86 g,
20 mmol) was added dropwise 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 re-
crystallized in ethanol, washed with n-hexane and dried in
air. Yield: 58%, m.p.: 1148C, Found %: C, 81.49, H, 6.71,
N, 11.69. C16H16N2 (236.31) requires %: C, 81.32, H, 6.82,
1
N, 11.85. IR (cm-1): 1613 (C=N), 1572 (C=C); H NMR
(ppm): 6.81–7.42 (10H, m, Ar–H), 2.19 (6H, s, Al–H); 13
C
NMR (ppm): 118.8–129.0 (Ar–C), 168.3 (C=N), 150.9 (C–
N), 15.4 (Al–C).
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Med Chem Res (2011) 20:220–230
227
N,N0-Dicyclohexylidene-naphthalene-1,8-diamine
(A5*dcnd)
Found %: C, 56.12; H, 5.08; N, 8.44; Co, 8.92.
C62H66Cl2Co2F2N8O11 (1326.00) requires %: C, 56.16; H,
5.02; N, 8.45; Co, 8.89.
An ethanolic solution (100 ml) of cyclohexanone (1.96 g,
20 mmol) was added to ethanolic solution (100 ml) of 1,8-
diaminonaphthalene (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.: 1358C, Found %: C, 83.00, H,
8.09, N, 8.74. C22H26N2 (318.45) requires %: C, 82.97, H,
[Co2(Cip)2(mtma)2(pip)(H2O)2]ꢀ3H2O (II)
It was prepared by using mtma(A2) (2.17 g, 10 mmol).
Yield: 71.2%, m.p.: 3008C (dec.), Found %: C, 50.29; H,
4.57; N, 6.54; Co, 9.10. C54H58Cl2Co2F2N6O13S2
(1289.97) requires %: C, 50.28; H 4.53; N, 6.51; Co, 9.14.
1
8.23, N, 8.80. IR (cm-1): 1600 (C=N), 1570 (C=C); H
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).
[Co2(Cip)2(apq)2(pip)(H2O)2]ꢀ3H2O (III)
It was prepared by using apq(A3) (2.37 g, 10 mmol).
Yield: 69.5%, m.p.: 2808C, Found %: C, 52.32; H, 4.52; N,
10.57; Co, 8.87. C58H60Cl2Co2F2N10O13 (1331.93) requires
%: C, 52.30; H 4.54; N, 10.52; Co, 8.85.
N,N0-(1,2-Diphenyl-ethane-1,2-diylidene)dianiline
(A6*dpeda)
[Co2(Cip)2(bpeed)2(pip)(H2O)2]ꢀ3H2O (IV)
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.:
1658C, Found %: C, 86.60, H, 5.57, N, 7.70. C26H20N2
(360.45) requires %: C, 86.64, H, 5.59, N, 7.77. IR (cm-1):
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).
It was prepared by using bpeed(A4) (2.64 g, 10 mmol).
Yield: 67.8%, m.p.: 3708C, Found %: C, 57.39; H, 5.41; N,
8.14; Co, 8.50. C66H74Cl2Co2F2N8O11 (1382.11) requires
%: C, 57.35; H 5.40; N, 8.11; Co, 8.53.
Synthesis and physical properties of the mixed-ligand
complexes
[Co2(Cip)2(bpdmed)2(pip)(H2O)2]ꢀ3H2O (I)
A
methanolic solution of Co(NO3)2ꢀ6H2O (2.91 g,
10 mmol) was added to methanolic solution of bpdmed(A1)
(2.36 g, 10 mmol), followed by addition of a previously
prepared solution of CipꢀHCl (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 (Wu
et al., 2003). The resulting red solution was refluxed for
8–10 h on a steam bath, and then was kept overnight at
room temperature. A fine yellowish crystalline product was
obtained which was washed with ether and dried in a
vacuum desiccator. The physical parameters of the com-
plexes are shown in Table 3 and the reaction scheme is
shown in Scheme 1. Yield: 69.5%, m.p.: 3008C (dec.),
Scheme 1 Reaction scheme of the complex (I)
123

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Med Chem Res (2011) 20:220–230
[Co2(Cip)2(dcnd)2(pip)(H2O)2]ꢀ3H2O (V)
Inoculation procedure
It was prepared by using dcnd(A5) (3.18 g, 10 mmol).
Yield: 68.2%, m.p.: [3708C, Found %: C, 59.67; H, 5.86;
N, 7.53; Co, 7.94. C74H86Cl2Co2F2N8O11 (1490.29)
requires %: C, 59.64; H 5.82; N, 7.52; Co, 7.91.
The target microorganism cultures were prepared sepa-
rately in 15 ml of liquid LB medium. Inoculation of 10 ll
of culture was done to the previously prepared corning
tubes with the help of a micropipette using sterilized tip
under sterile condition. Which were then incubated at 37°C
for 24 h.
[Co2(Cip)2(dpeda)2(pip)(H2O)2]ꢀ3H2O (VI)
It was prepared by using dpeda(A6) (3.60 g, 10 mmol).
Yield: 68.2%, m.p.: 2708C, Found %: C, 62.52; H, 4.73; N,
7.15; Co, 7.46. C82H74Cl2Co2F2N8O11 (1574.28) requires
%: C, 62.56; H 4.74; N, 7.12; Co, 7.49.
DNA binding
UV–visible spectroscopy
The concentration of DNA was measured by using its stan-
dard extinction coefficient at 260 nm (6,600 M-1 cm-1
Antibacterial activity
)
(Reichmann et al., 1954; Sathish et al., 2009). Absorption
titration was carried out by varying the DNA concentration
(2–20 lM) and maintaining constant concentration of the
complex (4 lM). Absorption spectra were recorded after
each successive addition of DNA followed by allowing it to
attain equilibrium (approximately 10 min). The observed
data were then utilized to obtain the intrinsic binding con-
stant, Kb using Eq. 1 (Reichmann et al., 1954).
Dilution method
All the bacteria were incubated and activated at 30°C for
24 h before inoculation into Luria broth for 24 h. The
compounds were dissolved in DMSO and then diluted
using Luria broth. It was reported that DMSO does not
exhibit antimicrobial activity against any of the tested
organisms (Altundas et al., 2009). Twofold serial concen-
trations of the compounds were employed to determine the
Minimum Inhibitory Concentration (MIC) ranging from
3,000 to 0.1 lM. Test cultures were incubated at 37°C
(24 h). The lowest concentrations of antimicrobial agents
that resulted in complete inhibition of growth were repre-
sented as MIC (lM). In each case triplicate tests were
performed and the average was taken as the final value
(Jones et al., 1984).
½DNAꢁ
½DNAꢁ
1
¼
þ
ð1Þ
ðea ꢂ efÞ ðeb ꢂ efÞ Kbðeb ꢂ efÞ
where, [DNA] is the concentration of DNA in terms of
nucleotide phosphate [NP], the apparent absorption coef-
ficient ef, ea, and eb correspond to the extinction coefficient
of the free complex, the extinction coefficient for each
addition of DNA to the complex and the extinction coef-
ficient for the complex in the fully bound form, respec-
tively, and Kb is the ratio of the slope to the y intercept.
Preparation of broth media
Viscosity study
The media was made by dissolving Luria broth (20 g) in
1 l of sterilized distilled water. The mixture was autoclave
for 15 min at 120°C and then dispensed into sterilized
corning tube, allowed to cool down to room temperature
under sterile condition.
Viscosity measurements were carried out using an Ub-
belodhe 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 order to minimize complexities
arising from DNA flexibility. 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 (g/g0)1/3 versus binding ratio [Co]/[DNA]
(Cohen and Eisenberg, 1969), where g is the viscosity of
DNA in the presence of complex and g0 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),
g = t - t0.
Application of corning tube
Sterilized stock solutions of complexes with different
volume were added to corning tubes which results in the
desired concentration of the complex in it, i.e., lM. All the
implicated corning tubes are further autoclaved and
brought to room temperature using laminar air flow. The
control experiments were performed with only equivalent
volume of solvents without added test compounds. All
experiments were performed in triplicate and ciprofloxacin
was used as a standard drug.
123

Med Chem Res (2011) 20:220–230
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4948
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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 (supernatant).
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100 V for 2 h in TAE buffer using 1.0% agarose gel
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Acknowledgments We wish to express our gratitude to Head,
Department of Chemistry Vallabh Vidyanagar, Gujarat, India, for
providing the necessary laboratory facilities. The authors wish to
acknowledge the help of Dr. V. Thakkar and Mr. H. S. Bariya,
Department of Biochemistry, Sardar Patel University. M. R. Chha-
satia is highly thankful to the UGC for JRF and financial assistance of
UGC Grant 32-226/2006(SR).
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