Synthesis, characterization, DNA cleavage and in vitro antimicrobial activities of copper(II) complexes of Schiff bases containing a 2,4-disubstituted thiazole

Article abstract of DOI:10.1007/s11243-010-9412-8

Six Cu(II) complexes of Schiff base ligands of arylidene-2-(4-(4-bromo/ methoxy-phenyl)thiazol-2-yl) hydrazines have been synthesized, characterized and screened for DNA cleavage and antimicrobial activities. The chemical structures of the complexes were

Full text of DOI:10.1007/s11243-010-9412-8

Transition Met Chem (2010) 35:917–925
DOI 10.1007/s11243-010-9412-8
Synthesis, characterization, DNA cleavage and in vitro
antimicrobial activities of copper(II) complexes of Schiff bases
containing a 2,4-disubstituted thiazole
•
Sanjay K. Bharti Saurabh K. Patel
•
•
Gopal Nath Ragini Tilak Sushil K. Singh
•
Received: 25 May 2010 / Accepted: 9 August 2010 / Published online: 27 August 2010
ꢀ Springer Science+Business Media B.V. 2010
Abstract Six Cu(II) complexes of Schiff base ligands
of arylidene-2-(4-(4-bromo/methoxy-phenyl)thiazol-2-yl)
hydrazines have been synthesized, characterized and
screened for DNA cleavage and antimicrobial activities.
The chemical structures of the complexes were deduced by
physicochemical and spectroscopic methods. Elemental
analyses indicated that the stoichiometry of the complexes
is CuL2 (L = Schiff base ligand). The DNA cleavage
activities of the complexes were evaluated by agarose gel
electrophoresis in the presence and absence of oxidant
(H2O2) and free radical scavenger (DMSO). All the six
complexes showed significant nuclease activity in the
presence of H2O2, and two of the complexes showed
moderate nuclease activity even in the absence of oxidant.
The complexes did not show nuclease activity in the
presence of free radical scavenger. The compounds were
tested for activity against selected bacteria and fungi.
other antibiotic-resistant human pathogenic microorganisms
[1]. Metal complexes of some antibiotics have shown
improved results against these microorganisms [2, 3].
Complexes of the quinoline group antibiotics, namely, cip-
rofloxacin, norfloxacin and tetracycline have been found to
possess enhanced activity compared to the antibiotic alone.
Thus, a Pd(II) complex of tetracycline has sixteen times
more potency than the parent compound against E.coli
HB101/pBR322, a bacterial strain resistant to tetracycline,
while a Pd(II) complex of doxycycline is two times more
potent than doxycycline against resistant strains [4]. Schiff
bases and their transition metal complexes are well known as
antibacterial, antifungal, anticancer and antiviral agents, etc.
[5, 6]. The significant antimicrobial activity of Schiff bases
containing a 2,4-disubstituted thiazole ring reported by us
earlier [7] prompted us to synthesize their copper complexes
as possible antimicrobial agents.
Transition metal complexes with Schiff base ligands
have shown promising nucleolytic activity. The interaction
of transition metal complexes with DNA has been studied
extensively for their use as probes for DNA structure and
their potential applications in chemotherapy [8]. Antican-
cer drugs can bind to DNA via covalent and/or non-cova-
lent interaction(s), causing DNA damage leading to the
inhibition of uncontrolled growth of cancerous cells.
Bis(1,10-phenanthroline)copper(I) was the first transition
metal complex shown to possess DNA cleavage activity
[9], followed by derivatives of ferrous-EDTA [10, 11],
various metalloporphyrins [12], cis-diamino dichloro plat-
inum complexes [13] and ruthenium complexes of 4,7-
diphenyl-1,10-phenanthroline [14]. Recently, some Cu(II)
complexes have been reported to be active in DNA strand
scission [15]. Keeping the above facts in mind, the DNA
cleavage activity of the present Cu(II) complexes has been
studied by gel electrophoresis.
Introduction
Recent clinical reports have highlighted the increasing
occurrence of methicillin-resistant Staphylococcus aureus
(MRSA), vancomycin-resistant enterococci (VRE) and
S. K. Bharti ꢀ S. K. Singh (&)
Department of Pharmaceutics, Institute of Technology,
Banaras Hindu University (BHU), Varanasi 221005, India
e-mail: sksingh.phe@itbhu.ac.in
S. K. Patel ꢀ G. Nath ꢀ R. Tilak
Department of Microbiology, Institute of Medical Sciences,
Banaras Hindu University (BHU), Varanasi 221005, India
123

918
Transition Met Chem (2010) 35:917–925
Results and discussion
DMSO for 10-3 M solutions at room temperature indicate
their non-electrolytic nature [18].
The synthesis of Schiff base ligands containing a 2,4-
disubstituted thiazole ring, HL1–HL6, is outlined in
Scheme 1. The corresponding copper complexes [Cu(L)2]
were obtained in three steps. In the first step, the Schiff
base thiosemicarbazone was synthesized by condensing
equimolar quantities of the substituted aryl aldehyde/
ketone with thiosemicarbazide in methanol or ethanol in
the presence of a few drops of glacial acetic acid as cata-
lyst. In the second step, equimolar quantities of the thio-
semicarbazone and substituted phenacyl bromide (4-
bromo/-methoxy phenacyl bromide) were refluxed to
obtain Schiff bases HL1–HL6, containing a 2,4-disubsti-
tuted thiazole. This reaction proceeds via the cyclization of
thiosemicarbazone to the corresponding 2,4-disubstituted
thiazole [7]. The thiazoles HL1–HL6 were then refluxed
with copper(II) acetate in 2:1 M ratio, in accordance with
the reported methods for similar compounds [16, 17], to
obtain the desired complexes in 68–85% yields. The
products were characterized by TLC, UV, FTIR, 1H NMR,
13C NMR, Mass spectrometry, electronic, magnetic, ther-
mogravimetric (TG/DTA), ESR and elemental analyses.
The observed molar conductances of the complexes in
The tentative assignments of the IR bands useful for
determining the ligand’s mode of coordination are listed in
Table 1. The FTIR spectra of the thiosemicarbazones
showed bands in the range of 3,120–3,330 cm-1 for m(NH)
and m(NH2), and 1,590–1,670 cm-1 for the azomethine
group (HC=N). The absence of a band in the range of
1,700–1,750 cm-1 confirms the conversion of the alde-
hyde/ketone group to azomethine in the product. Absorp-
tion bands in the range of 3,124–3,282 cm-1 for NH, and
1,604–1,663 cm-1 for the azomethine group were observed
for the Schiff bases containing 2,4-disubstituted thiazoles.
The stretching frequencies of the azomethine group
m(HC=N) in the free ligands was shifted by 14–63 cm-1
,
being present in the range of 1,562–1,655 cm-1 in the
complexes, which suggests participation of the azomethine
nitrogen in coordination [19]. The broad peaks of the ortho
phenolic OH for HL3, HL4, HL5 and HL6 were present in
the region of 3,403–3,425 cm-1 in the free ligands, but
absent from the spectra of the complexes, indicating
coordination between the phenolic oxygen and copper. The
phenolic m(C–O) stretching vibration that was present in
the region of 1,227–1,335 cm-1 for the free Schiff base
H
N
NH2
H2N
R
Br
R
S
R1
AcOH
Reflux, 4 - 6 h
O
R1
HN
N
C
+
N
N
C
R2
HN
R1
R2
Reflux, 4 - 10 h
H2N
R2
O
C
S
S
Scheme 1 Synthetic route to the Schiff base ligands. HL1, R=Br, R1=Ph,
R2=–CH2CH(OH)Ph; HL2, R=OMe, R1=Ph, R2=–CH2CH(OH)Ph; HL3,
R=Br, R1=H, R2=–C6H3–2,4–(OH)2; HL4, R=OMe, R1=H, R2=–C6H3–
2,4–(OH)2; HL5, R=Br, R1=H, R2=–C6H4OH; HL6, R=OMe, R1=H,
R2=–C6H4OH
Table 1 Infrared spectroscopic assignment (KBr, mmax cm-1) for HL1–HL6 and [Cu(L1)2]–[Cu(L6)2]
Compound
m(O–H)
m(N–H)
m(C=N) azomethine
m(C=N) thiazole
m(C–O)
m(Cu–N)
m(Cu–O)
HL1
HL2
HL3
HL4
HL5
HL6
[Cu(L1)2]
[Cu(L2)2]
[Cu(L3)2]
[Cu(L4)2]
[Cu(L5)2]
[Cu(L6)2]
3,340
3,423
3,423
3,425
3,403
3,406
–
3,171
3,124
3,282
3,170
3,153
3,167
3,169
3,122
3,280
3,169
3,150
3,168
1,663
1,622
1,604
1,635
1,604
1,635
1,633
1,608
1,655
1,572
1,562
1,575
1,521
1,508
1,545
1,446
1,511
1,521
1,520
1,508
1,546
1,452
1,508
1,521
1,241
1,354
1,227
1,243
1,240
1,335
1,126
1,255
1,245
1,259
1,257
1,241
–
–
–
–
–
–
–
–
–
–
–
–
680
700
671
623
684
639
490
596
470
521
528
493
–
–
–
–
–
123

Transition Met Chem (2010) 35:917–925
919
1
Table 2 The H NMR spectral dataa (d, ppm)b of selected free ligands and their copper(II) complexes
Compound [empirical formula]
OHc
NH
HC=N (azomethine)
Aromatic protons
HL1 [C23H18BrN3OS]
HL3 [C16H12BrN3O2S]
HL5 [C16H12BrN3OS]
[Cu(L1)2][CuC46H34Br2N6O2S2]
[Cu(L3)2][CuC32H22Br2N6O4S2]
[Cu(L5)2][CuC32H22Br2N6O2S2]
12.1
7.7 (s, 1H)
8.2 (s, 1H)
8.0 (s, 1H)
7.7 (d, 2H)
8.3 (s, 2H)
8.0 (s, 2H)
–
6.9–7.7 (m, 14H)
7.2–8.0 (m, 7H)
7.3–7.6 (m, 8H)
6.7–7.2 (m, 28H)
6.9–7.7 (m, 14H)
6.4–7.3 (m, 16H)
12.1 (s, 1H)
9.9 (s, 1H)
7.2 (s, 1H)
–
9.7 (s, 1H)
–
–
–
11.5 (s, 2H)
7.7 (s, 2H)
a
Solvent DMSO-d6/CDCl3
b
Relative to TMS
c
At ortho position
Table 3 Magnetic moments and electronic spectra of HL1–HL6 and [Cu(L1)2]–[Cu(L6)2]
Compound [empirical formula]
Magnetic moment, leff (B.M.)a at 300 K
kmax (nm)
Assignments
HL1 [C23H18BrN3OS]
HL2 [C24H21N3O2S]
–
240, 335
260, 340
p–p*, n–p*
–
p–p*, n–p*
HL3 [C16H12BrN3O2S]
–
296, 320
p–p*, n–p*
HL4 [C17H15N3O3S]
HL5 [C16H12BrN3OS]
HL6 [C17H15N3O2S]
[Cu(L1)2][CuC46H34Br2N6O2S2]
[Cu(L2)2] [CuC48H40N6O4S2]
[Cu(L3)2][CuC32H22Br2N6O4S2]
[Cu(L4)2] [CuC34H28N6O6S2]
[Cu(L5)2][CuC32H22Br2N6O2S2]
[Cu(L6)2] [CuC34H28N6O4S2]
–
257, 340, 392
235, 262, 310
230, 265, 340
260, 275, 350, 500
244, 252, 295, 457
252, 392,457, 510
260, 340, 490
283, 320, 410, 500
295, 457, 490
p–p*, n–p*
–
p–p*, n–p*
–
p–p*, n–p*
1.54
1.79
1.74
1.88
1.70
1.84
p–p*, n–p*, L–M, d–d
p–p*, n–p*, L–M, d–d
p–p*, n–p*, L–M, d–d
p–p*, n–p*, L–M, d–d
p–p*, n–p*, L–M, d–d
p–p*, n–p*, L–M, d–d
a
B.M. Bohr magneton
1
ligands HL3–HL6 was shifted by 16–94 cm-1 in the spectra
of the complexes. The band attributed to m(NH) group
remained almost unchanged in the complexes, suggesting
non-coordination of the N atom of this group to the metal.
In the far infrared region, bands in the region of
623–700 cm-1 were attributed to m(M–N) and in the region
of 470–596 cm-1 to m(M–O) [20].
range of 3.6–3.9 ppm. The H NMR spectra of the copper
complexes exhibited peaks which were either shifted
downfield or absent compared to the free ligands. Thus, the
phenolic –OH peak was absent in the spectra of the com-
plexes. The peak at 7.7–8.2 ppm due to NH remained at
almost the same position, indicating the non-coordination
of NH to the metal.
The 1H NMR spectra were recorded in DMSO-d6/
CDCl3, and spectral data of the free ligands and their
copper(II) complexes are presented in Table 2. The aro-
matic protons were present as multiplets in the range of
6.9–8.0 ppm for the free Schiff base ligands, which were
slightly shifted downfield for the complexes. This was
assigned to a decrease in the local electron density in the
complexes [21]. The azomethine proton of all the six Schiff
base ligands was observed in the range of 7.2–9.9 ppm,
while peaks at 7.7–8.2 ppm were observed for the NH
group. A singlet in the range of 9.7–12.1 ppm was assigned
to the ortho phenolic group proton. The Ph-OCH3 protons
of Schiff bases HL2, HL4 and HL6 were present in the
The 13C NMR spectra were consistent with the 1H NMR
spectra. Changes in the chemical shifts for the azomethine
carbon and carbon attached to the phenolic –OH were
observed in the complexes. The aryl carbon and azomethine
carbon of all the Schiff base ligands exhibited peaks in the
region of 111–135 and 132–143 ppm, respectively. Peaks in
the region of 100–108, 147–157 and 160–171 ppm were
assigned to C5, C4 and C2 of the thiazole ring, respectively.
A peak at 55–56 ppm was observed for the methoxy carbon
of the ligands HL2, HL4 and HL6. In the complexes, the
usual azomethine resonances were shifted ca. 5–14 ppm
downfield, suggesting involvement of the nitrogen lone pair
in coordination. The aryl carbons showed peaks in the
123

920
Transition Met Chem (2010) 35:917–925
111–135 ppm region. The phenolic aryl carbon was present
at 158–160 ppm, hence shifted ca. 3–7 ppm downfield,
consistent with coordination of the phenolic oxygen.
The electronic spectra of the compounds were recorded
in DMF and are presented in Table 3. The spectra of the
free Schiff base ligands showed two main absorption bands
in the region of 230–392 nm, assigned to p–p* transitions
of the aromatic ring and phenolic chromophore. This band
was shifted in the spectra of the complexes. A band in the
region of 310–392 nm was assigned to the n–p* transition
of the azomethine chromophore. The equivalent transitions
of the complexes were shifted in comparison with the free
ligands, due to coordination of the imine nitrogen atom to
the metal [22]. A transition in the range of 410–457 nm for
the complexes was assigned to ligand to metal charge
transfer (LMCT), while a band at 490–510 nm was
attributed to metal d–d transitions, which is compatible
with the complexes having a planar or distorted tetrahedral
structure [23].
interaction between two Cu(II) centres is explained by the
Hathaway expression G = (g|| - 2.0023)/(g\ - 2.0023).
The deviation of ‘g’ values from the free electron value
(2.0023) may be due to angular momentum contribution in
the complexes.
According to Hathaway, if G \ 4.0, a considerable
exchange coupling is present in the solid complex [27]. In
the present case, the G values of the complexes [Cu(L3)2],
[Cu(L5)2] and [Cu(L6)2] are in the range of 1.982–3.593,
indicating considerable exchange interaction. If the G[ 4.0,
the exchange interaction is negligible which is the case for
complexes [Cu(L1)2], [Cu(L2)2] and [Cu(L4)2]. Kivelson
and Neiman showed that for an ionic environment g|| is
normally 2.3 or larger, but for a covalent environment g|| is
less than 2.3. In the present study, the g|| values for the
complexes are in the range of 2.111–2.213; consequently,
the environment is essentially covalent.
The mass spectra of the free ligands showed the
expected peaks. For example, HL1 showed the molecular
ion peak at m/z 464 (M?, 80), which matches with its
molecular formula C23H18BrN3OS. A peak at m/z 416
(M ? 1, 100%) was observed for HL2 which is in con-
formity with the molecular formula C24H21N3O2S. A peak
at m/z 326 (M ? 1, 100%) for HL6 fits with the molecular
formula C17H15N3O2S. Similarly, the complex [Cu(L1)2]
showed a molecular ion peak M? at m/z 990 which is
equivalent to its molecular weight. Molecular ion peaks
M? at m/z 892 for [Cu(L2)2] and at m/z 841 for [Cu(L3)2]
were observed and are equivalent to their molecular
weights. Complexes [Cu(L4)2], [Cu(L5)2] and [Cu(L6)2]
showed molecular ion peaks M? at m/z 744, 810, and 712,
respectively. In addition, the spectra also showed fragment
ion peaks including demetallated products of the copper
complexes. For example, peaks at m/z 648 and 340 were
assigned to removal of two methoxy and two hydroxyl
groups [M-C2H8O4] and demetallation, respectively, in the
case of [Cu(L4)2].
The magnetic moments of the complexes were deter-
mined at room temperature and are presented in Table 3.
All of these copper(II) complexes were paramagnetic, with
magnetic moments in the range of 1.54–1.88 B.M., con-
siderably lower than the value expected for the spin-only
contribution in a d9 system. This suggests a probable
antiferromagnetic interaction between Cu(II) atoms and
consequently a planar or distorted tetrahedral arrangement
of the four donor atoms around the metal [24]. Thus, the
electronic spectra and magnetic moments suggest a planar
or distorted tetrahedral geometry for the complexes [25].
The ESR spectra of the complexes were recorded as
polycrystalline samples at room temperature (RT), and the
data are presented in Table 4. The spectra exhibited aniso-
tropic signals with gjj ¼ 2:111 ꢁ 2:213; g? ¼ 2:017 ꢁ 2:059
and giso=av ¼ 2:065 ꢁ 2:110 calculated from the formula
1=3½gjj þ 2g?ꢂ, characteristic of axial symmetry [26]. Since
the g|| and g? values are close to 2 and gjj [ g?, a planar
geometry is suggested around Cu(II), corresponding to
elongation along the four fold symmetry Z-axis [27]. The
trend g|| [ g\ [ ge (2.0023) shows that the unpaired elec-
tron is localized in the dx2-y2 orbital of the Cu(II) atom in
these complexes [28]. In addition, exchange coupling
Thermal decomposition of the complexes was studied by
TG/DTA. The complexes did not show any weight loss up to
200 ꢁC, which reveals that both crystalline and coordinated
water were absent from the complexes. The TG curves in the
range of 210–330 ꢁC suggested that the loss in weight
Table 4 ESR spectral data
Compound [empirical formula]
of the Cu(II) complexes as
Temperature
g||
g\
gaiso/av
Gb
[Cu(L1)2][CuC46H34Br2N6O2S2]
polycrystalline sample
RT
RT
RT
RT
RT
RT
2.193
2.162
2.212
2.213
2.204
2.111
2.033
2.017
2.059
2.046
2.056
2.056
2.086
2.065
2.110
2.101
2.105
2.074
5.848
9.529
3.593
4.630
3.642
1.982
[Cu(L2)2 [CuC48H40N6O4S2]
[Cu(L3)2][CuC32H22Br2N6O4S2]
[Cu(L4)2] [CuC34H28N6O6S2]
[Cu(L5)2][CuC32H22Br2N6O2S2]
[Cu(L6)2] [CuC34H28N6O4S2]
a
giso=av ¼ 1=3½gjj þ 2g?ꢂ
G ¼ ðgjj ꢁ 2:0023Þ=
b
ðg? ꢁ 2:0023Þ
123

Transition Met Chem (2010) 35:917–925
921
corresponds to decomposition of the ligands. In all cases,
the remaining residues were metal oxide (CuO).
complexes showed enhanced antibacterial activity com-
pared to their free ligands. Ciprofloxacin (100 lg/ml) was
used as standard.
The results of antifungal screening of all the Schiff base
ligands and their complexes are presented in Table 5. The
free ligands showed moderate or no antifungal activity. The
complex [Cu(L1)2] showed significant antifungal activity
against all the four fungal strains tested. In contrast, no
significant change in the antifungal activity of [Cu(L2)2]
was observed compared with its free ligand. Except for
[Cu(L2)2], the results showed enhanced antifungal activity
of the copper(II) complexes compared to their free ligands.
Fluconazole (100 lg/ml) was used as standard.
Antibacterial and antifungal activities
Antibacterial activities of the complexes were evaluated
against various Gram-negative and Gram-positive patho-
genic bacterial strains, namely, Escherichia coli, Staphylo-
coccus aureus, Salmonella typhi, Pseudomonas aeruginosa,
Klebsiella pneumoniae and Vibrio cholerae. Antifungal
activities were evaluated against Candida albicans, Cryp-
tococcus neoformans, Aspergillus flavus and Chrysospori-
um tropicum. The antibacterial and antifungal activities
were evaluated by the agar disc diffusion method, and
minimum inhibitory concentrations (MIC) were determined
by serial dilution as per the guidelines of the National
Committee for Clinical Laboratory Standards (NCCLS)
[29]. The solvent, DMSO, did not show any inhibition
against the tested organisms.
Nuclease activity
The DNA cleavage activities of the copper(II) complexes
were evaluated by agarose gel electrophoresis. To inves-
tigate the probable mechanism of nuclease activity,
experiments were also performed in the presence and
absence of oxidant (H2O2) and free radical scavenger
(DMSO). Cleavage in plasmid DNA was detected by UV
illumination in the presence of ethidium bromide (EB),
which is the most widely used intercalative agent and
fluorescence probe for DNA structure [30]. When the
supercoiled (SC) form of DNA is nicked, it gives rise to an
open circular (OC) relaxed form and further cleavage gives
The results of antibacterial screening of the free Schiff
bases and their copper(II) complexes are presented in
Table 5. The free ligands showed moderate or no anti-
bacterial activity. The complex [Cu(L1)2] showed signifi-
cant antibacterial activity against both S. aureus and
V. cholerae. [Cu(L2)2] showed good antibacterial activity
against V. cholerae, while [Cu(L5)2] showed good
antibacterial activity against K. pneumoniae. Thus, the
Table 5 Antibacterial and antifungal activities of the ligands HL1–HL6 and the complexes [Cu(L1)2]–[Cu(L6)2]
Compound
Microbial species
Bacteria
Fungi
E.coli
S.aureus S.typhi
P. aeruginosa K. pneumoniae V. cholerae C. albicans C. neoformans A. flavus C. tropicum
HL1
HL2
HL3
HL4
HL5
HL6
[Cu(L1)2]
[Cu(L2)2]
[Cu(L3)2]
[Cu(L4)2]
[Cu(L5)2]
[Cu(L6)2]
–
–
–
–
–
–
–
–
–
–
–
–
20 (12.5) –
–
–
20 (12.5)
22 (6.25)
21 (6.25)
20 (6.25) 19 (6.25)
–
–
–
–
–
–
–
–
–
–
–
–
19 (25)
–
–
16 (25)
–
–
–
–
–
–
–
–
–
–
–
\10 (50)
–
–
–
–
–
–
\10 (50)
–
–
–
–
–
–
–
14 (25)
15 (25)
16 (25)
22 (6.25) –
–
–
21 (6.25)
24 (6.25)
23 (6.25)
22 (6.25) 20 (6.25)
–
–
–
–
–
–
–
–
–
–
–
–
21 (12.5)
–
–
17 (25)
–
–
–
–
–
–
–
–
–
–
–
–
\10 (50)
–
–
–
–
–
–
14 (25)
–
–
–
–
–
–
–
16 (12.5)
16 (25)
18 (12.5)
–
Ciprofloxacin 29 (6.25) 28 (6.25) 22 (6.25) 27 (3.12)
26 (6.25)
22 (6.25)
–
–
Fluconazole
DMSO
–
–
–
–
–
–
–
–
–
–
–
–
25 (6.25)
–
25 (6.25)
–
23 (6.25) 24 (6.25)
–
–
Diameter of inhibition zone was measured in mm. MIC values are given in brackets. MIC (lg/ml) = Minimum Inhibitory Concentration, i.e. the
lowest concentration of drug which completely inhibits bacterial or fungal growth. – indicates no activity. Ciprofloxacin and fluconazole were used
as standard for antibacterial and antifungal activity, respectively
123

922
Transition Met Chem (2010) 35:917–925
a linear form. In gel electrophoresis, SC shows the fastest
migration, followed by linear form and lastly the OC form.
The present complexes at micromolar concentrations, after
2 h incubation with DNA, showed the nucleolytic activities
presented in Fig. 1a, b and c. To investigate the mechanism
of nucleolytic activity, experiments were also performed in
the presence of H2O2 as oxidant and DMSO as a free
radical scavenger. The complexes exhibited significant
nucleolytic activity in the presence of H2O2.
a
Many copper(II) complexes are known to cleave DNA
more efficiently in the presence of oxidants such as
hydrogen peroxide and ascorbic acid. Possible reaction
mechanisms of DNA cleavage by the copper(II) complexes
in the presence of H2O2 have been proposed by several
research groups [31, 32]. The more pronounced nuclease
activity of these complexes in the presence of oxidant
(Fig. 1b), compared to that without oxidant (Fig. 1a), may
be due to the increased production of hydroxyl radicals.
The complexes Cu(L3)2 and Cu(L6)2 showed moderate
nuclease activity (Fig. 1a, lanes 3 and 6) while Cu(L1)2,
Cu(L2)2, Cu(L4)2 and Cu(L5)2 did not show any nuclease
activity in the absence of oxidant (Fig. 1a, lanes 1, 2, 4 and
5). The nuclease activity of the complexes did not change
in the presence of free radical scavenger (Fig. 1c, lanes
1-6). The purity of the isolated plasmid DNA was checked
by UV–Vis spectroscopy. The ratio of UV absorbance at
260 and 280 nm was 1.68, indicating that the DNA was
sufficiently free from RNA and/or protein.
C
M
1
2
3
4
5
6
b
C1
1
2
3
4
5
6
C
c
C
1
2
3
4
5
6
C
1
Conclusion
Six copper(II) complexes of Schiff base ligands of arylid-
ene-2-(4-(4-bromo/methoxy-phenyl) thiazol-2-yl) hydra-
zines have been synthesized, and their chemical structures
were deduced by physicochemical and spectroscopic
methods. The complexes were formulated as [CuL2], where
the ligand L acts as bidentate chelate with O and N as
donating atoms. Gel electrophoresis experiments showed
pronounced DNA cleavage activity of the complexes in the
presence of an oxidant. Antimicrobial screening showed
enhanced antibacterial and antifungal activities of the
complexes compared to the free ligands. Hence, the present
work contributes to the development of novel copper
complexes as nucleolytic and antimicrobial agents.
Fig. 1 a Gel electrophoresis photograph showing the effects of
Cu(II) complexes on plasmid DNA on 1% agarose gel. Lane C:
Control (Untreated DNA), Lane M: Copper salt, Lane 1: DNA ?
Complex I, Lane 2: DNA ? Complex II, Lane 3: DNA ? Complex
III, Lane 4: DNA ? Complex IV, Lane 5: DNA ? Complex V, Lane
6: DNA ? Complex VI. Complexes I–VI represent [Cu(L1)2]–
[Cu(L6)2], respectively. b Gel electrophoresis photograph showing
the effects of Cu(II) complexes on plasmid DNA in the presence of
H2O2 on 1% agarose gel. Lane C1: Untreated DNA ? H2O2, Lane 1:
DNA ? Complex I ? H2O2, Lane 2: DNA ? Complex II ? H2O2,
Lane 3: DNA ? Complex III ? H2O2, Lane 4: DNA ? Complex
IV ? H2O2, Lane 5: DNA ? Complex V ? H2O2, Lane 6: DNA ?
Complex VI ? H2O2, Lane C: Control (untreated DNA). Complexes
I–VI represent [Cu(L1)2]–[Cu(L6)2], respectively. c Gel electropho-
resis photograph showing the effects of Cu(II) complexes on plasmid
DNA in the presence of DMSO on 1% agarose gel. Lane C1:
Untreated DNA ? DMSO, Lane 1: DNA ? Complex I ? DMSO,
Lane 2: DNA ? Complex II ? DMSO, Lane 3: DNA ? Complex
III ? DMSO, Lane 4: DNA ? Complex IV ? DMSO, Lane 5:
DNA ? Complex V ? DMSO, Lane 6: DNA ? Complex VI ?
DMSO, Lane C: Control (Untreated DNA). Complexes I–VI represent
[Cu(L1)2]–[Cu(L6)2], respectively
Experimental
Chemicals and solvents were obtained from Merck, Genei
(Bangalore), S.D. Fine, Himedia (Mumbai) and Aldrich
and were of analytical reagent grade or purified by standard
methods prior to use. Melting points were determined in
open-glass capillaries on a Stuart-SMP10 melting point
apparatus and are uncorrected. IR spectra were recorded on
a Shimadzu FTIR-8400 s spectrophotometer using KBr
123

Transition Met Chem (2010) 35:917–925
923
pellets in the range of 4,000–400 cm-1. Electronic
absorption spectra were recorded on a Shimadzu UV-1700
spectrophotometer using DMSO as solvent in the range of
200–800 nm. Magnetic moment measurements of powder
samples were carried out on a Vibrating Sample Magne-
tometer (Lakeshore 7410) at room temperature. Molar
conductances of the complexes were measured in DMSO
1-(2,4-dihydroxybenzylidene)thiosemicarbazide: M.P.
208 ꢁC; Yield: 80%; IR (KBr, mmax cm-1): 3,541 (-OH, br.),
3,220 (-NH2), 3,140 (-NH), 1,620 (HC=N azomethine),
1
1,087 (C=S); H NMR (DMSO-d6, 300 MHz) d (ppm):
7.1–7.4 (m, 3H, aromatic H), 8.2 (s, 1H, –N=CH), 9.0 (s, 1H,
NH), 11.4 (s, 1H, –OH); 13C NMR (DMSO-d6) d (ppm):
124.8–128.2 (Aryl-CH), 132.0–134.8 (Aryl-C), 140.0
1
(10-3M) using a digital conductivity meter. H NMR and
(HC=N); % Elemental analysis found (Calc.) for
13C NMR spectra were recorded on a JEOL AL300
FTNMR spectrometer operating at 300/75 MHz and TMS
(tetramethylsilane) was used as internal standard. 1H NMR
and 13C NMR chemical shifts are reported in ppm down-
field from TMS. Mass spectra were recorded on a VG-
AUTOSPEC spectrometer. ESR spectra were recorded on a
JEOL JES-FA200 ESR spectrometer at room temperature.
TG and DTA measurements were recorded on a Labsys
TG-DTA16 instrument at a heating rate of 10 ꢁC min-1. In
all cases, the 50–800 ꢁC temperature range was studied
under air. Elemental analyses were done on a Heraeus
CHN rapid analyser. Copper contents were determined by
standard methods.
C8H9N3O2S: C, 45.3 (45.4); H, 4.3 (4.2); N, 19.7 (19.8).
1-(2-hydroxybenzylidene)thiosemicarbazide: M.P. 227 ꢁC;
Yield: 82%; IR (KBr, mmax cm-1): 3,403 (-OH, br.), 3,230
(-NH2), 3,174 (-NH), 1,570 (HC=N azomethine), 1,052
(C=S); 1H NMR (DMSO-d6, 300 MHz) d (ppm): 7.2–7.5 (m,
4H, aromatic H), 7.6 (s, 1H, –N=CH), 8.6 (s, 1H, NH), 11.2
(s, 1H, –OH); 13C NMR (DMSO-d6) d (ppm): 121.5–126.9
(Aryl-CH), 132.0–133.6 (Aryl-C), 136.5 (HC=N); % Ele-
mental analysis found (Calc.) for C8H9N3OS: C, 49.1 (49.2);
H, 4.6 (4.6); N, 21.4 (21.5).
Synthesis of HL1–HL6
The compounds HL1–HL6 were prepared by the reported
method [7] with slight modification. To a solution of
the appropriate thiosemicarbazone in ethanol or methanol,
an equimolar quantity of substituted phenacyl bromide
(4-bromo/methoxy phenacyl bromide) in methanol was
added and the mixture was stirred for 10 min. The mixture
was refluxed on a water bath for 4–10 h with monitoring of
progress of the reaction by TLC. The product was recrys-
tallized from ethanol or chloroform. Characterization data
of the compounds are given in Tables 1, 2 and 3.
General procedure for the synthesis
of the thiosemicarbazone Schiff bases
The thiosemicarbazones were prepared by our earlier
reported method [7]. To a solution of thiosemicarbazide
(1.82 g, 20 mmol) in aqueous ethanol (40 ml), a solution
of aryl aldehyde/ketone (20 mmol) in ethanol or methanol
(40 ml) was added slowly at 60–70 ꢁC with continuous
stirring. The mixture was refluxed, and the progress of
reaction was monitored by TLC. After the completion of
the reaction (4–6 h), the mixture was cooled, the precipi-
tate filtered off and washed with ice-cold water then dried
in air. Finally, the product was recrystallized from aqueous
ethanol (1:3). The compounds were soluble in MeOH,
EtOH, DMSO and DMF.
Synthesis of [Cu(L1)2]–[Cu(L6)2]
To a hot (60–70 ꢁC) solution of HL (10 mmol) in ethanol
or methanol (30 ml), a solution of Cu(AcO)2 (5 mmol) in
absolute ethanol (30 ml) was added dropwise with stirring.
The reaction mixture started to change colour immediately
upon addition of Cu(AcO)2. Stirring was then continued for
1–2 h at 80 ꢁC. The resulting precipitate was filtered off,
washed with ethanol, dried in vacuo over P4O10 and stored
in a desiccator over CaCl2. Analytical and physicochemical
data for the complexes are given in Table 6, and their
proposed molecular structures are presented in Fig. 2.
Characterization data of the compounds are given
below
1-(2-hydroxy-1,2-diphenylethylidene)thiosemicarbazide:
M.P. 169 ꢁC; Yield: 72%; IR (KBr, mmax cm-1): 3,320
(–OH), 3,240 (-NH2), 3,132 (–NH), 1,658 (C=N azo-
1
methine), 1,034 (C=S); H NMR (DMSO-d6, 300 MHz)
d (ppm): 7.0–7.5 (m, 10H, aromatic H), 8.4 (s, 1H, NH),
9.5 (s, 1H, –OH); 13C NMR (DMSO-d6) d (ppm):
114.4–127.8 (Aryl-CH), 131.4–133.9 (Aryl-C); % Ele-
mental analysis found (Calc.) for C15H14N3OS: C, 63.0
(63.1); H, 5.4 (5.3); N, 14.7 (14.7).
Nuclease activity
DNA cleavage experiments were performed according to
the method described in the literature [33]. Luria–Bertani
(LB) broth was used for the culture of S. typhi. A 50 ml
123

924
Transition Met Chem (2010) 35:917–925
Table 6 Analytical and physicochemical data of the Cu(II) complexes
Cu(II) complex Empirical formula
Colour
M.W.a M.P. (ꢁC)b Yield (%) Elemental analysis, found (Calc.)c [%]
C
H
N
Cu
[Cu(L1)2]
[Cu(L2)2]
[Cu(L3)2]
[Cu(L4)2]
[Cu(L5)2]
[Cu(L6)2]
a
[CuC46H34Br2N6O2S2] Green
[CuC48H40N6O4S2] Green
990.26 [280
892.52 [280
80
68
85
83
70
72
55.7 (55.8) 3.4 (3.4)
64.6 (64.5) 4.5 (4.5)
45.6 (45.6) 2.6 (2.6)
8.4 (8.4)
6.4 (6.4)
7.1 (7.1)
7.5 (7.5)
9.4 (9.4)
9.9 (9.9)
[CuC32H22Br2N6O4S2] Dark brown 842.04 [280
[CuC34H28N6O6S2] Dark brown 744.30 [280
[CuC32H22Br2N6O2S2] Brown
[CuC34H28N6O4S2] Brown
54.9 (54.9) 3.8 (3.8) 11.2 (11.3) 8.5 (8.5)
47.3 (47.4) 2.7 (2.7) 10.3 (10.3) 7.8 (7.8)
57.3 (57.4) 3.9 (3.9) 11.7 (11.8) 8.8 (8.9)
810.00 [280
712.30 [280
Molecular weight of the compound
b
c
Melting point of the compound at their decomposition
Elemental analyses for C, H and N were within 0.4% of the theoretical value
Fig. 2 Proposed molecular
structures of the Cu(II)
complexes. For HL1/HL3/HL5;
R=Br and R1=OH/H;
For HL2/HL4/HL6: R=OMe and
R1=OH/H
R
R1
N
S
NH
O
R
N
HC
N
H
N
Cu
N
O
O
O
N
Cu
S
HN
N
S
HN
S
N
HC
N
R
R
R1
[Cu(L1)2] and [Cu(L2)2]
batch of broth was autoclaved for 15 min at 121 ꢁC under
15 lb pressure. The medium was then cooled to 45 ꢁC,
inoculated with the seed culture and incubated at 37 ꢁC
for 24 h. Isolation of plasmid DNA from pure cultures of
S. typhi was carried out by conventional methods [34].
Briefly, the fresh bacterial culture (12 ml) was centrifuged
(3,000 rpm, 10 min) to obtain a pellet, which was dis-
solved in 4 ml of lysis buffer (100 mM tris pH 8.0, 50 mM
EDTA, 10% SDS), and 0.5 ml of saturated phenol was
added followed by incubation at 55 ꢁC for 10 min. The
resultant mixture was centrifuged at 10,000 rpm for 10 min
and to the supernatant, an equal volume of chloroform and
isoamyl alcohol (24:1) mixture and 1/10th volume of 3 M
sodium acetate (pH 4.8) were added and the mixture was
further centrifuged under the same conditions. After that, 3
volumes of chilled absolute alcohol were added and the so
obtained DNA was separated by centrifugation. Finally, the
pellet was dried and dissolved in TE buffer (10 mM Tris,
pH 8.0, 1 mM EDTA) and stored cool.
mixtures were mixed thoroughly and incubated at 37 ꢁC for
2 h. The DNA cleavage activities of the complexes were
determined by agarose gel electrophoresis [33]. Solid aga-
rose gel was prepared by dissolving 500 mg of agarose in
50 ml of 1 9 TBE buffer (45 mM Tris, 45 mM H3BO3,
1 mM EDTA) by boiling and poured into a gel cassette
fitted with a comb. When the gel attained ca. 45 ꢁC, 3 ll of
Ethidium bromide solution (10 lg/ml) was added. The gel
was allowed to solidify, and then the comb was removed
carefully to leave the wells intact. The gel was placed in an
electrophoresis chamber flooded with 1 9 TBE buffer.
Subsequently, 10 ll of DNA sample after incubation for 2 h
at 37 ꢁC was added to the loading buffer (5 ll) containing
0.25% bromophenol blue, 0.25% xylene cyanol and 30%
glycerol. The solution was loaded carefully into the wells,
and the electrophoresis was carried out for 90 min at a
constant 60 V in the buffer. Finally, the gel was removed
carefully and the bands were observed under a UV transil-
luminator. The illuminated gel was photographed using an
Alpha Innotech Corporation Instrument. The efficiency of
the DNA cleavage was measured by determining the ability
of the complex to form open circular and nicked circular
Samples of the different Cu(II) complexes were prepared
in DMF (lM), and 4 ll was added separately to the isolated
and purified plasmid DNA of S. typhi. The DNA-sample
123

Transition Met Chem (2010) 35:917–925
925
References
DNA from its supercoiled form by estimating the intensities
of the bands after gel electrophoresis.
1. Viksveen P (2003) Homeopathy 92:99–107
2. Shaikh AR, Giridhar R, Yadav MR (2007) Int
332(1–2):24–30
3. Uivarosi V, Neagoe S, Aldea V, Ni¸tulescu A (2001) Roum Arch
Microbiol Immunol 60(3):267–277
J
Pharm
Antimicrobial activities
4. Guerra W, de Andrade AE, de Souza Monteiro AR, Bucciarelli-
Rodriguez M, Chartone-Souza E, Nascimento AM, Fontes AP,
Moyec L, Pereira-Maia EC (2005) J Inorg Biochem 99:2348–2354
5. Wang M, Wang LF, Li YZ, Li QX, Xu ZD, Qu DM (2001) Trans
Met Chem 26:307–310
6. Radhakrishanan PK, Indrasen P, Nair CG (1994) Polyhedron
3:67–70
7. Bharti SK, Nath G, Tilak R, Singh SK (2010) Eur J Med Chem
45:651–660
8. Liu Y, Wang N, Mei W, Chen F, Li-Xin H, Jian L, Wang R
(2007) Trans Met Chem 32:332–337
9. Travers AA (1993) In: DNA-Protein Interactions. Chapman and
Hall, London
10. Sigman DS (1986) Acc Chem Res 19:180–186
11. Schultz PG, Taylor JS, Dervan PB (1982) J Am Chem Soc
104:6861–6863
12. Ward B, Skorobogaty A, Dabrowiak JC (1986) Biochemistry
25:6875–6883
13. Groves JT, Farrell TP (1989) J Am Chem Soc 111:4998–5000
14. Barnard CF, Clear MJ, Hydes PC (1987) Chem. Britain
22:1001–1004
15. Reddy PR, Rao KS, Satyanarayana B (2006) Tetrahydron Lett
47:7311–7315
16. Cukurovali A, Yilmaz I (2006) Trans Met Chem 31:207–213
17. Yilmaz I, Cukurovali A (2003) Trans Met Chem 28:399–404
18. Geary WJ (1971) Coord Chem Rev 7:81–122
19. Sengupta SK, Pandey OP, Srivastava BK (1998) Trans Met Chem
23:349–353
20. Bellamy LJ (1958) The infrared spectra of complex molecules,
2nd edn. Chapman & Hall, Methuen
21. Rai A, Sengupta SK, Pandey OP (2005) Spectrochim Acta A
61:2761–2765
22. Koksal M, Tumer M, Serin S (1997) Synth React Inorg Met Org
Chem 27(9):775–786
23. Lever ABP (1968) In: Inorganic electronic spectroscopy. Else-
vier, Amsterdam, p 337
The microorganisms were obtained from the culture col-
lection of the Department of Microbiology, Institute of
Medical Sciences, Banaras Hindu University, Varanasi,
India. Antimicrobial activities were evaluated according to
the guidelines of the National Committee for Clinical Lab-
oratory Standards (NCCLS) using the agar disc diffusion
method [29]. Briefly, a 24/48-h-old culture of selected bac-
terium or fungus was mixed with sterile physiological saline
(0.85%), and the turbidity was adjusted to the standard
MacFarland scale inoculum of 0.5 [*106 colony forming
units (CFU) per millilitre]. Petri plates containing 20 mL of
Mueller–Hinton Agar (MHA, Hi-Media) were used for all
the bacteria tested. Fungi were cultured in Sabouraud’s
dextrose agar (SDA)/potato dextrose agar (PDA) (Hi-Media)
and purified by single spore isolation technique. The inoc-
ulum was spread on the surface of the solidified media, and
Whatman no. 1 filter paper discs (6 mm in diameter)
impregnated with the test compound (20 lg/disc) were
placed on the plates. Ciprofloxacin (5 lg/disc, Hi-Media)
was used as positive control for bacteria and fluconazole
(10 lg/disc, Hi-Media), was used as positive control for
fungi. A paper disc impregnated with DMSO was used as
negative control. Plates inoculated with the bacteria were
incubated for 24 h at 37 ꢁC, and the fungal cultures
were incubated for 72 h at 25 ꢁC. Inhibition zone diameters
were measured in millimetres. All the tests were performed
in triplicate, and the average was taken as the final value.
Minimum inhibitory concentrations (MIC), the lowest
concentration which completely inhibits visible growth
(turbidity in liquid media), were determined according to the
guidelines of NCCLS document M27-A [29]. Stock solu-
tions (100 lg/ml) of the test compounds, ciprofloxacin and
fluconazole were prepared in DMSO. From this, serial
dilutions (50, 25… 3.12 lg/ml) were prepared to determine
the MIC. All the determinations were done in triplicate, and
the average was taken as the final value. The standard anti-
biotic ciprofloxacin (100 lg/ml) for bacteria and fluconazole
(100 lg/ml) for fungi were used as positive controls, and
100 ll of DMSO was used as a negative control.
24. Lopez-Garzon R, Moreno-Carrettero MN, Salas-Peregrin MA,
Salas-Peregrin JM (1993) Polyhedron 12:507–512
25. Greenwood NN, Earnshaw A (2001) Chemistry of the elements,
2nd edit, vol 28. Butterworth Heinemann, p 1192
26. Fouda MF, Abd-Elzaher MM, Shadofa MM, El Saied FA, Ayad
MI, El Tabl AS (2008) Trans Met Chem 33:219–228
27. Chandra S, Kumar U (2005) Spectrochim Acta A 61:219–224
28. Gudasi KB, Patil SA, Vadavi RS, Shenoy RV, Nethaji M (2006)
Trans Met Chem 31:586–592
29. NCCLS (National Committee for Clinical Laboratory Standards)
(1997) Performance standards for antimicrobial disc susceptibil-
ity test, 6th edn. Approved Standard, M2-A6. NCCLS, Wayne
30. Wilson WD, Ratmeyer L, Zhao M, Strekowski L, Boykin D
(1993) Biochemistry 32:4098–4104
31. Sigman DS, Mazumder A, Perrin DM (1993) Chem Rev 93:2295
32. Kobayashi T, Kunita M, Nishino S, Matsushima H, Tokii T, Ma-
suda H, Einaga H, Nishida Y (2000) Polyhedron 19:2639–2648
33. Brown TA (1990) Mol Biol 1:51–52
34. Sambrook J, Russell DW (2006) Preparation of plasmid DNA by
alkaline lysis with SDS: minipreparation, molecular cloning, a
laboratory manual, 3 edn, vol 1. Cold Spring Harbor Laboratory
Press, Cold Spring Harbor, pp 1.32–1.34
Acknowledgments The authors are grateful to The Head, Depart-
ment of Chemistry, Faculty of Science, Banaras Hindu University
(BHU), Varanasi, India for 1H NMR and 13C NMR spectrometry,
Indian Institute of Chemical Technology (IICT), Hyderabad for Mass
spectrometry and Indian Institute of Technology (IIT), Guwahati
for providing facility for ESR and magnetic moment measurements.
Mr. S. K. Bharti is grateful to University Grants Commission (UGC),
New Delhi for the award of senior research fellowship.
123