Novel metal based anti-tuberculosis agent: Synthesis, characterization, catalytic and pharmacological activities of copper complexes

Article abstract of DOI:10.1016/j.ejmech.2012.01.006

Copper complexes of molecular formulae, [CuL¹(OAc)], [CuL ²(H₂O)], [CuL³(H₂O)], [CuL ⁴(H₂O)], [CuL⁵(H₂O)] where L ¹-L⁵ represents Schi

Full text of DOI:10.1016/j.ejmech.2012.01.006

European Journal of Medicinal Chemistry 49 (2012) 151e163
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Novel metal based anti-tuberculosis agent: Synthesis, characterization, catalytic
and pharmacological activities of copper complexes
*
J. Joseph , K. Nagashri, G. Boomadevi Janaki
Department of Chemistry, Noorul Islam Centre for Higher Education, Kumaracoil 629 180, Tamilnadu, India
a r t i c l e i n f o
a b s t r a c t
Copper complexes of molecular formulae, [CuL1(OAc)], [CuL2(H2O)], [CuL3(H2O)], [CuL4(H2O)],
[CuL5(H2O)] where L1eL5 represents Schiff base ligands [by the condensation of 3-hydroxyflavone with
4-aminoantipyrine (L1)/o-aminophenol (L2)/o-aminobenzoic acid (L3)/o-aminothiazole (L4)/thio-
semicarbazide (L5)], have been prepared. They were characterized using analytical and spectral tech-
niques. The DNA binding properties of copper complexes were studied using electronic absorption
spectra and viscosity measurements. Superoxide dismutase and antioxidant activities of the copper
complexes have also been studied. Furthermore, the copper complexes have been found to promote
pUC18 DNA cleavage in the presence of oxidant. Anti-tuberculosis activity was also performed.
Ó 2012 Elsevier Masson SAS. All rights reserved.
Article history:
Received 19 August 2011
Received in revised form
16 November 2011
Accepted 5 January 2012
Available online 18 January 2012
Keywords:
Screening
Antioxidant
Inhibition
Superoxide dismutase
Cleavage
Copper
1. Introduction
Metal complexes of Schiff base ligands have been investigated as
models for active sites of enzymes [1,2], including DNA cleavage
Several classes of antimicrobial compounds are presently
available; microorganism’s resistance to these drugs constantly
emerges. In order to prevent this serious medical problem, the
elaboration of new types of antibacterial agents or the expansion of
bioactivity of the naturally known biosensitive compounds is a very
interesting research problem. The synthesis and characterization of
metal complexes with organic bioactive ligands is one of the
promising fields for the search, in particular, of copper complexes
with derivatives of hydroxyflavone.
Deoxyribonucleic acid (DNA) is the primary target molecule for
most anticancer and antiviral therapies according to cell biologist.
Investigations on the interaction of DNA with small molecules are
important in designing new types of pharmaceutical molecules.
The important criteria for the development of metallodrugs as
chemotherapeutic agents are the ability of the metallo drug to
bring about DNA cleavage. A large number of transition metal
complexes have been found to promote DNA cleavage because of
their redox properties. Transition metal complexes have been re-
ported to bring about DNA cleavage either oxidatively or hydro-
lytically or photolytically.
systems [3,4], and as antibacterial [5e7] and anticancer [8] drugs.
Metal complexes of S-, N-, and O-chelating ligands have attracted
the considerable attention because of their interesting
physicoechemical properties, pronounced biological activities, and
as models of metalloenzyme active sites [9,10]. Schiff bases and
their transition metal complexes have been used as anticancer,
antituberculer, antibacterial, antifungal, hypertensive and hypo-
thermic reagents [11e13].
Copper has a wide spectrum of effectiveness against a variety of
microorganisms and thus copper-containing compounds such as
Bordeaux mixture, copper sulphate, etc., have been used for the
control of plant pathogenic fungi and bacteria on most agricultural
crops [14]. Natural chelating agents can reduce copper toxicity by
binding part of the available copper and protect against the toxicity
of copper towards bacteria and fungi [15]. As transition metal ions
play a vital role in the initiation of free radical processes (via the
Fenton reaction), metal chelation is widely considered as another
mechanism of the antioxidant activity of flavonoids. The interaction
of metal ions with flavonoids may change the antioxidant proper-
ties and some biological effects of the flavonoids [16].
The structural modifications of promising lead compounds are
still a major line of approach to develop new therapeutic agents. It
involves an intensive effort to condense the different pharmaco-
phoric groups of bioactive active moieties into one compound, and
* Corresponding author. Tel.: þ91 4651 366295, þ91 9629474150.
E-mail address: chem_joseph@yahoo.co.in (J. Joseph).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2012.01.006

152
J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
it may behave as chemotherapeutic agents. Due to the diversified
nature, hydroxyflavone derivatives is a useful substance in medic-
inal research. In the present study, we report the synthesis and
biochemical activities of copper complexes with hydroxyflavone
derivatives.
ethanol, benzene and chloroform, but their considerable solubility
hasbeennoticed inDMFand DMSO. Thecomplexesarestableatroom
temperature. They are non-hygroscopic and can be stored for a long
length of period without decomposition. The structures of the
synthesized compounds were confirmed by analytical and spectral
data (IR, 1H & 13C NMR, ESR and FAB mass).
2. Chemistry
3. Pharmacology
Ligands were synthesized bythereactionof 3-hydroxyflavone and
4-aminoantipyrine/o-aminophenol/o-aminobenzoicacid/o-amino-
thiazole/thiosemicarbazide (targeted compounds are shown in
Scheme 1). The complexes were obtained by refluxing the equimolar
solutionsofcopperacetate and ligand (s). Thecoppercomplexes were
obtained as solids in different yields. The purity of synthesized
compounds was confirmed by TLC and elemental analysis. C, N and H
analysis were carried out micro analytically. Magnetic moments were
determined at room temperature. They are insoluble in acetone,
The in vitro evaluation of antimicrobial activity was carried out.
The purpose of the screening program is to provide antimicrobial
efficiencies of the investigated compounds. The prepared
compounds were tested against some fungi and bacteria to provide
the minimum inhibitory concentration (MIC) for each compound.
MIC is the lowest concentration of solution to inhibit the growth of
a test organism. The in vitro antimicrobial activities of the investi-
gated compounds were tested against the bacterial species,
O
O
OH
OH
N
N
O
OH
N
N
2
L
1
L
O
O
OH
N
OH
N
COOH
SH
O
3
L
4
L
OH
N
5
L
HN
S
H2N
Scheme 1. Targeted compounds (L1eL5).

J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
153
Table 1
Table 3
Electronic spectral data of ligands and their copper complexes.
Cyclic voltammetric data of copper complexes.
S.No Compound Solvent Absorption (nm) Band assignment Geometry
Complex
Epa
Epc
DEp
Potential assignment
1
2
L1
DMSO 268
DMSO 282
498
DMSO 248
342
DMSO 234
342
DMSO 246
358
INCT
INCT
INCT
INCT
INCT
INCT
INCT
INCT
INCT
INCT
INCT
2B1g
INCT
INCT
2B1g
INCT
INCT
2B1g
e
e
[CuL1(OAc)]
ꢁ0.258
ꢁ0.592
1.16
ꢁ0.166
ꢁ0.748
e
118
158
Cu(II)/Cu(I)
Cu(II)/Cu(I)
Ligand oxidation
Cu(II)/Cu(I)
Cu(II)/Cu(III)
Cu(II)/Cu(I)
Cu(I)/Cu(0)
Cu(II)/Cu(I)
Cu(I)/Cu(0)
L2
[CuL2(H2O)]
3
4
5
6
L3
L4
L5
e
[CuL3(H2O)]
[CuL4(H2O)]
[CuL5(H2O)]
ꢁ0.632
ꢁ0.772
e
140
e
0.465
e
ꢁ0.606
0.436
ꢁ0.778
ꢁ0.597
ꢁ0.768
ꢁ0.568
169
e
e
ꢁ0.655
116
0.424
[CuL1(OAc)] DMSO 251
Square planar
342
578
/
/
/
/
/
2A1g
2A1g
2A1g
2A1g
2A1g
7
[CuL2(H2O)] DMSO 248
Square planar
Square planar
Square planar
Square planar
5. Discussion
352
530
8
[CuL3(H2O)] DMSO 254
The elemental analysis and mass spectrum were good agreement
with the formation of 1:1 (metal:ligand) stoichiometry. All
complexes were decomposed above 280 ꢀC indicating their thermal
stability. The molar conductance data for the copper complexes were
measured in DMSO solution are given in experimental section. The
348
572
9
[CuL4(H2O)] DMSO 241
INCT
INCT
2B1g
INCT
INCT
2B1g
348
544
[CuL5(H2O)] DMSO 260
values of complexes fall in the range of 8e29 U
ꢁ1 cm2 molꢁ1, which
10
351
538
is the expected range of 1e35 Uꢁ1 cm2 molꢁ1 for the complexes to
behave as non-electrolytes [18,19]. Thus, the complexes have non-
electrolytic nature as evidenced by the involvement of acetate
group in coordination.
Staphylococcus aureus, Escherichia coli, Klebsiella pneumoniae,
Proteus vulgaris and Pseudomonas aeruginosa and fungal species,
Aspergillus niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia
bataicola and Candida albicans. The Superoxide dismutase and
antioxidant activities of copper complexes were measured [17]. The
DNA binding and cleavage studies of copper complexes were also
studied. Anti-tuberculosis activity was also performed. In addition,
catalytic activity of copper complexes was also performed by the
conversion of benzene into phenol in the presence of hydrogen
peroxide.
Mass spectra provide a vital clue for elucidating the structure of
compounds. The mass spectra of the ligand (L1) and its copper
complex [CuL1(OAc)] were recorded and their stoichiometric
compositions were compared. The intensity of these peaks reflects
the stability and abundance of the ions [20]. The molecular ion peak
for the ligand (L1) is observed at424 m/z whereas its copper
complex shows the molecular ion peak at 515 m/z, which confirms
the stoichiometry of the metal complexes to be [CuL1(OAc)] type.
Elemental analysis values are in close agreement with the values
calculated from molecular formula assigned to these complexes,
which is further supported by the FAB-mass studies of respective
complexes. Similar mass spectral features were assigned for other
ligands and their copper complexes. The different pathways of the
fragments of the parent molecular ion peaks are given in Scheme 2.
The stoichiometry is confirmed by the mass spectra of complexes.
4. Results
The UVeVis., and ESR spectral data of ligands and their copper
complexes are recorded in DMSO and are presented in Tables 1 and
2. The redox behaviour of copper complexes were measured in
DMSO and summarized in Table 3. In addition, the inhibitory values
obtained for compounds against bacteria and fungi and are tabu-
lated in Tables 4 and 5. The superoxide dismutase (SOD) mimetic
activity (IC50) of the synthesized complexes are summarized in
Table 6. The binding constant for copper complexes with CT DNA
are presented in Table 7. Furthermore, in vitro anti-tubercular
screening data of synthesized compounds are summarized in
Table 8.
The IR spectrum of the ligands show a
n
(C]N) peak in the region
(C]N)
1645-1632 cmꢁ1. The IR spectra of all complexes show
n
bands shifted to lower frequency of at 1629e1590 cmꢁ1 [20] which
indicates that the azomethine nitrogen is one of the coordinating
atoms in the Schiff bases. However, the spectra of complexes show
two characteristic bands at 1630e1602 and 1404e1344 cmꢁ1
attributed to yasy(COOꢁ) and ysy(COOꢁ) respectively, indicating
the participation of the carboxylate oxygen atom in the complexes.
The mode of coordination of carboxylate group has often been
Table 2
ESR spectral data of the copper complexes.
Complex
g
jj
gt
giso
A
jj
At
K
Kt
a2
b2
g2
f(g /A )
jj jj
jj
[CuL1(OAc)] at 300 K
[CuL1(OAc)] at 77 K
[CuL2(H2O)] at 300 K
[CuL2(H2O)] at 77 K
[CuL3(H2O)] at 300 K
[CuL3(H2O)] at 77 K
[CuL4(H2O)] at 300 K
[CuL4(H2O)] at 77 K
[CuL5(H2O)] at 300 K
[CuL5(H2O)] at 77 K
2.08
e
2.24
2.24
2.22
2.20
2.21
2.05
2.06
2.05
2.05
2.05
150
146
154
156
154
42
38
50
52
54
0.91
0.85
0.78
0.76
0.75
0.56
0.42
0.52
0.51
0.51
0.74
0.78
0.83
0.83
0.80
1.3
1.5
1.4
1.2
1.4
0.75
0.72
0.81
0.80
0.82
150
153
144
141
143
2.12
e
2.06
e
2.10
e
2.08
e

154
J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
Table 4
Table 6
Minimum inhibitory of concentration of the synthesized compounds against growth
of bacteria ( M).
Superoxide dismutase activity of some copper(II) complexes.
m
S.No
Complex
IC50 (mM)
S.No Compound
E. coli K. pneumoniae S. typhi P. aeruginosa S. aureus
1
2
3
4
5
6
[CuL1(OAc)]
[CuL2(H2O)]
[CuL3(H2O)]
[CuL4(H2O)]
[CuL5(H2O)]
Native SOD
0.72
0.67
0.56
0.66
0.08
0.04
1
2
3.
4
5
L1
L2
L3
L4
L5
60
24
28
60
24
64
26
36
64
26
38
28
54
38
28
10
14
10
66
20
26
66
20
32
30
58
32
30
08
12
04
66
16
32
66
16
28
26
60
28
26
04
10
06
72
28
30
72
28
42
48
63
42
48
06
08
14
6
7
8
9
10
11
12
13
[CuL1(OAc)] 34
[CuL2(H2O)] 26
[CuL3(H2O)] 52
[CuL4(H2O)] 34
[CuL5(H2O)] 26
Ampicillin
Vancomycin 06
Ofloxacin 08
shifts observed in the wave number region 1142e980 cmꢁ1 are in
agreement with the structural changes observed in the molecular
carbon skeleton after complexation, which cause some changes in
(CeC) bond lengths. Conclusive evidence of the bonding is also
shown by the observation that new bands in the spectra of all
copper complexes appear in the low frequency regions at 550e516,
12
504e498 and 486e448 cmꢁ1 characteristic to
(MeS) stretching vibrations, respectively, that are not observed in
the spectra of both free ligands [22]. The IR bands at 810e854 and
784e799 cmꢁ1
(H2O) of coordinated water, is an indication of the
y(MeO), y(MeN) and
deduced from the magnitude of the observed separation between
the yasy(COOꢁ) and ysy(COOꢁ), the separation value (A) between
yasy(COOꢁ) and ysy(COOꢁ) in metal complexes was more than
200 cmꢁ1 (260e216 cmꢁ1) suggests the coordination of carboxylate
group in copper complexes of the ligands in a monodentate fashion
[21]. The Schiff base ligand (L4) displays band around 844 cmꢁ1
y
,
y
binding of the water molecules to the metal ions. Another band
located at 522 cmꢁ1 in the spectrum of [Cu(L)(OAc)] complex may
be due to y(MeO) of the acetate group. The IR spectrum of the
ligand (L5) and its copper complex are shown in figs
(Supplementary material).
ascribed to y CeSeC [21] which shifts to lower frequencies in their
spectra of copper complexes in the region 838e832 cmꢁ1, respec-
tively, suggesting the coordination of copper ions through the
The electronic absorption spectra of the Schiff base ligands
and their copper complexes in DMSO were recorded at room
temperature and the band positions of the absorption maxima;
band assignments and the proposed geometry are shown in
experimental section. The electronic spectra of the ligands
and their complexes were recorded in DMSO as a solvent.
The absorption spectrum for L1 shows band at 268 nm
(Fig, Supplementary material). These bands can be attributed to
sulphur atom of thioazole moiety. The band at 3466 cmꢁ1 for
y(OH)
in the free ligand disappeared on complexation, indicating coor-
dination through a deprotonated oxygen. In the IR spectra of the
ligand (L5), two sharp bands at ca. 3436 and 3352 cmꢁ1, probably
due to asymmetric and symmetric vibrations of the NH2 group, do
not undergo any change in the spectra of the complexes indicating
the non-involvement of the NH2 group on coordination. In the
spectrum of copper complexes, the coordination of azomethine
nitrogen atom is further substantiated by the observed positive
shift of 34e48 cmꢁ1 in the NeN stretching mode in the complexes.
Monodentate coordination of the NeN moiety is reported to
increase its stretching frequency by this amount. The absence of
n-p* transitions within the Schiff base molecule. The electronic
spectrum of the corresponding [CuL1(OAc)] complex in DMSO
(Fig, Supplementary material) reveals a broad band at 498 nm
assignable to 2B1g/2A1g transition [23] which is characteristic of
square planar environment around the copper(II) ion. Similar
spectral features were assigned for other complexes and are
tabulated (Table 1).
thioamide band
n
(HNeC]S) at ca 782e813 cmꢁ1 and the appear-
ance of new band at ca. 610e631 cmꢁ1 confirmed the conversion of
n
(C]S) into n(CeS) band. The reduction of thioamide band n(N]
CeSH) observed at ca 982 cmꢁ1 suggests that coordination occurs
through S atom. The IR spectra of complexes show new band due to
the stretching vibrations of >C]NeN]C< bonds at
5.1. Stability of the complexes in solution
1568e1560 cmꢁ1
.
The band observed in the region
C]C stretching of the aromatic ring
The stability of these targeted complexes in aqueous solution
has been studied by observing the UVeVis spectra and estimating
the molar conductivities at different time intervals for any possible
change [24]. The tested compounds were prepared in DMSO and for
experiments freshly diluted in phosphate buffer system. Then, the
UVeVis., spectra and molar conductivities were measured at
different time intervals. It can seen form the results show that there
are no change absorption bands and their molar conductance
values for freshly prepared solutions. It indicates these three
complexes are quite stable in phosphate buffer.
1534e1526 cmꢁ1 is due to the
n
system. In all the coppereSchiff base complexes, most of the band
Table 5
Minimum inhibition of concentration of the synthesized compounds against growth
of fungi (mM).
S.No Compound A. niger R. stolonifer A. flavus R. bataicola C. albicans
1
2
3
4
L1
L2
L3
L4
L5
60
72
85
69
88
66
84
76
79
90
30
26
55
30
26
16
10
72
63
69
88
96
34
46
68
34
46
8
80
77
64
82
70
38
36
80
38
36
14
06
50
65
102
86
78
32
38
50
32
38
12
12
Table 7
5
Absorption spectral properties of copper complexes.
6
7
8
9
10
11
12
[CuL1(OAc)] 28
[CuL2(H2O)] 32
[CuL3(H2O)] 52
[CuL4(H2O)] 28
[CuL5(H2O)] 32
Complex
lmax
Free bound
Dl (nm)
Kb (Mꢁ1
)
[CuL1(OAc)]
[CuL2(H2O)]
[CuL3(H2O)]
[CuL4(H2O)]
[CuL5(H2O)]
386
368
388
385
376
380
360
381
378
371
6
8
7
7
5
1.4 ꢂ 106
2.1 ꢂ 106
1.9 ꢂ 106
2.4 ꢂ 105
3.0 ꢂ 105
Nystatin
Bavstatin
10
14
08

J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
155
Table 8
The NMR spectra of the synthesized compounds give informa-
tion about the skeleton of the compounds. The 1H and 13C NMR
spectrum of ligands were recorded in CDCl3 and are given in
experimental section.
In vitro anti-tubercular screening data of synthesized compounds.
S.No
Compound
MIC (
m
M ꢂ 10ꢁ3
)
1
2
3
4
5
6
7
8
L1
44
40
52
60
34
18
24
12
4
The 1H NMR spectrum of the ligand (L1) shows different signals
and are assigned as follows: 6.6e7.5 ppm (14H, m, AreH),
1.6 ppm(3H, s, H3CeC), 1.9 ppm(s, 3H, H3CeN) and the peak at
10.8 ppm(1H, s, OeH, D2O exchangeable) is attributable to the eOH
group present in the 3-hydroxyflavone moiety, respectively. The 13C
NMR ligand (L1) shows different signals and are assigned as
follows: 12.5 ppm (H3CeC), 19.6 ppm (H3CeN), 143.5 ppm (H3CeC),
162.8 ppm (C]O), 154.5 ppm (¼CeN), 124.6 ppm (C-2), 118.8 ppm
(C-3), 152.6 ppm (C-4),119.2 ppm (C-5), 146.4 ppm (C-6), 125.2 ppm
(C-7), 126.4 ppm (C-8), 157.3 ppm (C-9), 121.6 ppm (C-10),
133.8 ppm (C-10), 127.4 ppm (C-20, 60), 141.8 ppm (C-30, 50),
133.5 ppm (C-40), 127.2 ppm (C-100), 119.6 ppm (C-200, 600), 118.9 ppm
(C-300, 500) and 121.6 ppm (C-400), respectively. All the protons
L2
L3
L4
L5
[CuL1(OAc)]
[CuL2(H2O)]
[CuL3(H2O)]
[CuL4(H2O)]
[CuL5(H2O)]
INH
9
10
11
12
10
2
9
CFL
HN
S
N
O
H2N
HO
O
S
H2N
OH
NH2
NH
O
S
NH2
OH
OH
OH
-OH
OH
Scheme 2. Mass fragment pattern of L5.

156
J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
were found as to be in their expected region [25]. The number
of protons calculated from the integration curves and those
obtained from the values of the expected CHN analyses agree with
each other.
rate 100 mV sꢁ1)) at 300 K were examined. The electrochemical data
of copper complexes are given in Table 3. The cyclic voltammogram
of the [CuL1(OAc)] complex in DMSO solution at 300 K in the
potential range þ0.4 to ꢁ0.8 V at scan rate 0.1 Vsꢁ1 was recorded
(Fig. 1). It shows a well defined redox process corresponding to the
formation of the quasi-reversible Cu(II)/Cu(I) couple. The anodic
peak at Epa ¼ ꢁ0.258 mV versus Ag/AgCl and the associated
cathodic peak atEpc ¼ ꢁ0.166 mV correspond to the Cu(II)/Cu(I)
The ESR spectrum of the [CuL1(OAc)] complex was recorded in
DMSO at 300 and 77 K. The spectrum at 300 K shows one intense
absorption band at high field, which is isotropic due to the
tumbling motion of the molecules. However, this complex in the
frozen state shows four well resolved peaks with low intensities in
the low field region and one intense peak in the high field region.
The magnetic susceptibility value reveals that the copper complex
has a magnetic moment of 1.94 B.M. corresponding to one unpaired
electron, indicating that the complex is mononuclear in nature. This
fact was also evident from the absence of half field signal, observed
in the spectrum at 1600G due to the ms ¼ ꢃ2 transitions, ruling out
any CueCu interaction [26] and confirms that the synthesized
copper complexes are monomeric in nature [27]. The ESR spectral
data are given in Table 2.
couple. The [CuL1(OAc)] complex exhibits
behaviour.
a quasi-reversible
On comparing the cyclic voltammograms, we observed that the
variation in oxidation and reduction potential may be due to
distortion in the geometry of the complexes which arises due to
different donor atoms coordinated to the copper ion. It is concluded
that the present ligand systems stabilize the unusual oxidation
states of copper ion during electrolysis. It is essential factor for
pharmacological activities and plays a crucial role in curing or
prevention of untreatable diseases. Similar electrochemical
behaviour was observed and assigned for other complexes.
The value of g < 2.3 in the present complex gives a clear indi-
jj
cation of covalent character of the metaleligand bond and deloc-
alisation of the unpaired electron into the ligand. The trend of
g (2.24) > gt(2.05) > ge(2.0036) describes the axial symmetry
jj
5.2. Thermal analysis
with the unpaired electron residing in the dx2ꢁy2 orbital. For the
present Cu(II) complex, the observed g values are gjj(2.24)>
gt(2.05)>ge(2.0036), which suggest that the unpaired electron lies
The thermogravimetric analyses for the copper complexes were
carried out within a temperature range from 200 to 650 ꢀC in N2
atmosphere at a rate of 10 ꢀC per minute in order to establish their
compositional differences as well as to ascertain the nature of
associated water molecules [29]. Water molecules were lost in
between 50 and 300 ꢀC and metal oxides were formed above 550 ꢀC
for all complexes. The copper chelates degrade into two stages. The
first step corresponds to the loss of coordinated water molecule and
acetate ion, respectively. The second step corresponds to the loss of
ligand molecule that leads to the formation of copper oxide as a final
product from which the percentage of copper content was calculated
and compared with those obtained from AAS. The degradation
pathway for all complexes may be represented as follows:
in the dx2ꢁy2 orbital. The A and At values in the order: A
jj
jj
(150) > At(42) also indicate that the complex has square planar
geometry and the system is axially symmetric [28]. In the present
study, the f values for copper complexes were observed in the range
of 141e153 cmꢁ1. It is suggested that the synthesized copper
complexes exhibiting appreciable square planar distortion is ex-
pected to show high SOD like activity.
Molecular orbital coefficients
a
2(in-plane
s-bonding), b
2(in-
plane -bonding) and p
p
g
2(out-plane
-bonding) were calculated
using Eqs. (1)e(3).
ꢀ
ꢁ
ꢀ
ꢁ
a2 ¼ ꢁ A =0:036
þ
g ꢁ 2:0036 þ 3=7ðgt ꢁ 2:0036Þ
k
k
þ 0:04
(1)
(2)
(3)
ꢀ
ꢁ
b2
¼
g ꢁ 2:0036 E= ꢁ 8la2
k
g2 ¼ ðgt ꢁ 2:0036ÞE=ꢁ2la2
The a2 value of 0.5 indicates complete covalent bonding, while
that of 1.0 suggests complete ionic bonding. The observed value of
0.74 for the present complex indicates that the copper complex
has some covalent character. The observed b2 and g2 values of 1.3
and 0.75 indicate that there is interaction in the out-of-plane
bonding, whereas the in-plane -bonding is predominantly ionic.
Significant information about the nature of bonding in the Cu(II)
p-
p
complex can be derived from the relative magnitudes of K
and Kt.
jj
K
¼
a2b2
a2g2
(4)
k
Kt
¼
(5)
For the present complex, the observed order: K (0.91)
Kt(0.56) implies a greater contribution from out-of plane p-bonding
>
jj
than from in in-plane p-bonding in metaleligand p bonding.
The electrochemical behaviours of the Schiff base Cu(II)
complexes in DMSO (0.1 M of TBAP as supporting electrolyte (scan
Fig. 1. Cyclic voltammogram of [CuL1(OAc)] complex.

J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
157
5.3. Powder XRD
[32] by creating lesions in DNA strands by oxidative rupture and by
binding the nitrogen bases or DNA or RNA, hindering or blocking
base replication. The inhibition growth may be due to the effect on
the biosynthesis of phospholipids in cell membrane and proteins.
Among the studied compounds, [CuL1(OAc)] complex presented
good activity against C. albicans. Since this fungi is very harmful to
humans we consider this result of major importance. This confirms
that antibacterial and antifungal activities are dependent on the
molecular structure of the ligands and the type of the complex
formed. It appears from the above results that Cu(II) Schiff base
complex may be able to maintain a good antibacterial and antifungal
activities and be an effective antibacterial broad-spectrum drug that
may be able to solve some problem of antibacterial resistance.
In the present study, the observed cyclic voltammetric behav-
iour of copper complexes showed redox cycle may also contribute
to their inherent toxicity. For example, redox cycling between Cu(II)
and Cu(I) can catalyze the production of highly reactive hydroxyl
radicals, which can subsequently damage lipids, proteins, DNA and
other biomolecules. Further, the potential reduction of the Cu(II)/
Cu(I) process is related to the potential SOD mimetic activity. The
synthesized copper complexes also have reduction potential
greater than ꢁ0.6 V, it is possible for electron uptake to occur in the
biologic milieu, followed by donation to an acceptor.
The crystallite size of the complex was calculated from Scherre’s
formula:
dXRD ¼ 0:9 =
l b q
where is the wavelength,
l
b is the full-width half maximum of the
characteristic peak and is the diffraction angle for the hkl plane.
q
From the observed dxRD patterns, the average crystallite sizes for
the [Cu(L1)(OAc)], [CuL2(H2O)], [CuL3(H2O)], [CuL4(H2O)],
[CuL5(H2O)] complexes are found to be 75, 84, 92, 64 and 52 nm,
respectively. The smaller grain sizes were found from XRD sug-
gesting that these complexes are polycrystalline with nanosized
grains.
5.4. SEM
The surface morphology of the copper complexes exhibited
different morphology. The particle sizes of the Cu(II) complexes
were in the diameter range of few microns. However, particles with
sizes less than 100 nm were also observed which groups to form
agglomerates of larger size. Such facts are in good agreement with
the obtained powder XRD results.
The antibacterial activity of the title compounds distinctly
depends on the nature of pharmacophoric moieties and their
location in the 3-hydroxyflavone.
5.5. Antimicrobial activity
The in vitro antimicrobial activities of the investigated
compounds were tested against the bacterial species S. aureus, E.
coli, K. pneumaniae, P. vulgaris and P. aeruginosa and fungal species
A. niger, R. stolonifer, A. flavus, R. bataicola and C. albicans. The MIC
values of the compounds are summarized in Tables 4 and 5. A
comparative study of the ligands and their complexes indicates that
complexes exhibit higher antimicrobial activity than the free
ligands. In this study, the antimicrobial activity of the ligands may
be due to the heteroaromatic residues. Compounds containing
>C]N group have enhanced antimicrobial activity than >C]C<
group. The growth of certain microorganisms takes place even in
the absence of O2. Hence, compounds containing >C]C< group
though capable of absorbing O2 are not related with the growth of
microorganisms.
The enhanced activity of the complexes can be explained on the
basis of Overtone’s concept [30] and Tweedys Chelation theory
[31]. According to Overtone’s concept of cell permeability, the lipid
membrane that surrounds the cell favours the passage of only the
lipid-soluble materials which makes liposolubility an important
factor which controls the antimicrobial activity. On chelation, the
polarity of the metal ion will be reduced to a greater extent due to
the overlap of the ligand orbital and partial sharing of the positive
charge of the metal ion with donor groups. Further, it increases the
A Incorporation of thiosemicarbazide moiety in complex L5
demonstrated higher antimicrobial activity than other
complexes.
A Introduction of 4-aminoantipyrine moiety in the 3-
hydroxyflavone derivatives, complex L1 showed higher anti-
microbial activity than those complexes L2eL4.
A Introduction of thiazole group in the 3-hydroxyflavone deriv-
atives, the complex L3 showed higher antimicrobial activity
than that of complexes of L1 and L4.
A Introduction of carboxylic acid moiety in the 3-hydroxyflavone
derivatives, the complex L4 showed higher antimicrobial
activity than that of complexes of L2.
The increasing order of antimicrobial activity for the designed
targeted complexes as shown below:
[CuL5(H2O)] > [CuL4(OAc)] > [Cu(L1)(OAc)] > [CuL3(H2O)] >
[CuL2(H2O)]
5.6. DNA binding studies
DNA binding studies are important for the rational design and
construction of new and more efficient drugs targeted to DNA
[33e35]. A variety of small molecules interact reversibly with
double-stranded DNA, primarily through three modes: (i) electro-
static interactions with the negative charged nucleic
sugarephosphate structure, which are along the external DNA
double helix and do not possess selectivity; (ii) binding interactions
with two grooves of DNA double helix; and (iii) intercalation
between the stacked base pairs of native DNA. In order to explore
the mode of the Cu(II) complex binding to DNA, the following
experiments have been carried out.
delocalisation of
p-electrons over the whole chelate ring and
enhances the lipophilicity of the complexes. This increased lip-
ophilicity enhances the permeation of the complexes into lipid
membranes and blocking of the metal binding sites in the enzymes
of microorganisms. These complexes also disturb the respiration
process of the cell and thus block the synthesis of the proteins that
restricts further growth of the organism and as a result microor-
ganisms die.
The increased activity of the complexes can also be explained on
the basis of their high solubility, fitness of the particles, size of the
metal ion and the presence of the bulkier organic moieties. Another
mechanism of toxicity of these complexes to microorganisms may
be due to the inhibition of energy production or ATP production, by
inhibiting respiration or by the uncoupling of oxidative phosphor-
ylation. The biological activity involves inhibition of DNA synthesis
5.7. Electronic absorption study
The absorption spectra of the [CuL1(OAc)] complex in the
absence and presence of DNA is shown in Supplementary figure. In

158
J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
the UV region, the Cu(II) complex exhibits two absorption bands:
one at ca. 354 nm and another at ca. 297 nm. With increasing DNA
concentration, the absorption bands of the complexes were
affected, resulting in a hypochromism tendency and slight shifts to
longer wavelengths, which indicates that the Cu(II) complex can
interact with DNA. The observed hypochromism and bath-
ochromism for the Cu(II) complex are large compared to those
observed for potential intercalators. The intrinsic binding constant
(Kb) was obtained by monitoring the change in absorbance with
increasing concentrations of DNA for the Cu(II) complexes
(Table 6).As a result, binding constants of the copper complexes to
DNA follow the order from high to low:
Fig. 3. Agarose gel electrophoresis of copper complexes lane 1: DNA alone; lane 2:
DNA þ [CuL1(OAc)H2O] complex þ H2O2 lane 3: DNA þ [CuL2(H2O)] complex þ H2O2;
lane 4: DNA
þ
[CuL3(H2O)] complex
þ
H2O2; lane 5: DNA
þ
[CuL4(H2O)]
[CuL5(H2O)] > [CuL4(OAc)] > [Cu(L1)(OAc)] > [CuL3(H2O)] >
complex þ H2O2; lane 6: DNA þ [CuL5(H2O)] complex þ H2O2.
[CuL2(H2O)]
The cleavage of pUC19DNA (Fig. 3) in the presence of H2O2
(5 mM) has been studied by gel electrophoresis using supercoiled
(SC) pUC19DNA (0.2
m
g, 33.3
m
M) in 50mMTriseHCl/50 mM NaCl
M). In the present
5.8. Viscosity measurements
buffer (14 L, pH 7.2) and the complexes (50
m
m
study, the pUC19DNA gel electrophoresis experiment was con-
ducted at 35 ꢀC using our synthesized complexes in the presence
of H2O2 as an oxidant. As can be seen from the results in Fig. 3,
copper complexes exhibit nuclease activity in the presence of
H2O2. Control experiment using DNA alone (lane 1) does not
show any significant cleavage of pUC19DNA even on longer
exposure time. From the observed results, we conclude that the
copper complexes (lane 2 and 6) were completely degraded as
compared to control DNA. This shows that a slight increase in
concentration over the optimal value (i.e., the value at which
100% cleavage efficiency was observed) led to extensive degra-
dations, resulting in disappearance of bands on agarose gel (Lane
6). As a result, the DNA cleavage abilities of copper complexes in
the following order:
To further confirm the interaction mode of the Cu(II) complex
with DNA, a viscosity study was carried out. The viscosity
measurement is based on the flow rate of a DNA solution through
a capillary viscometer. The specific viscosity contribution (g) due to
the DNA in the presence of a binding agent was obtained. 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. As seen in Fig. 2 the
viscosity of CT DNA increases as increase in the ratio of complexes
to CT DNA. The result further suggested an intercalative binding
mode of the complexes with CT DNA and also parallelled to the
above spectroscopic results, such as hypochromism, blue-shift of
complexes in the presence of DNA. The viscosity studies provide
a strong evidence for intercalation. The viscosity increase of DNA is
ascribed to the intercalative binding mode of the drug because this
could cause the effective length of the DNA to increase [36].
[CuL5(H2O)] > [CuL4(OAc)] > [Cu(L1)(OAc)] > [CuL3(H2O)] >
[CuL2(H2O)]
It is evident that the complexes cleave DNA more effectively in
the presence of an oxidant (except lane 2), which may be due to the
reaction of hydroxyl radical with DNA. These hydroxyl free radicals
participate in the oxidation of the deoxyribose moiety, followed by
hydrolytic cleavage of the sugar phosphate backbone [37].
5.9. Chemical nuclease activity
Chemical nuclease activity of copper complexes were studied
using gel electrophoresis It is known that chemical nuclease
activity is controlled by relaxation of supercoiled circular confor-
mation of pUC19DNA to nicked circular and/or linear conformation.
The DNA cleavage of ligand alone is inactive in the presence and
absence of any external agents (data not shown). The results indi-
cate the importance of the copper in the complex for observing the
chemical nuclease activity.
In order to confirm the result obtained from 2-deoxy-D-ribose
degradation & Rhodamine B dye degradation methods and to
compare the ability of the copper complexes to generate hydroxyl
radicals under physiological conditions. Quantification by 2-deoxy-
D-ribose under the conditions of 2-mercaptoethanol and H2O2
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
R
Fig. 2. Plot of relative viscosity versus [complex]/[DNA] effect of copper complex on the viscosity of CT DNA at 25 ꢃ 0.1 ꢀC. Copper complex ¼ 0 ꢁ 100
mM [DNA] ¼ 50
mM.

J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
159
1.4
1.2
1
0.8
0.6
0.4
0.2
0
Complex L1
Complex L2
Complex L3
Complex L4
Complex L5
Fig. 4. Assay of hydroxyl radical production of 2-deoxy-D-ribose (400
m
M) by formation 2-thiobarbiturate with copper complexes (100
m
M) in 1 mm phosphate buffer. Reaction
conditions: 1. activation by 50 mM 2-mercaptoethanol and 50 mM H2O2, simultaneous addition with each copper complex; 2. activation by 50 mM 2-mercaptoethanol and 50 mM
H2O2, simultaneous addition with each copper complex; 3. activation by 50 mM 2-mercaptoethanol and 50 mM H2O2, after each copper complex has been reacted with DNA for
30 min and 4. quenching of activation reaction of 50 mM 2-mercaptoethanol and 50 mM H2O2 by 250 mM mannitol.
activations is shown in Fig. 4. It demonstrated that the production
power of the hydroxyl radical decrease in the following order:
In the present study, the higher SOD mimetic activity of copper
complexes than that of native enzyme is due to the presence of
easily labile acetate ion and also azomethine containing stabilize
the the Cu(I) complex formed during superoxide dismutation
reaction which further reacts with superoxide ion to give hydrogen
peroxide. The distorted geometry of these complexes may favour
the geometrical change, which is essential for the catalysis as the
geometry of copper in the SOD enzyme also changes from distorted
square planar geometry. The difference in reactivities of the
synthesized complexes may be attributed to the coordination
environment and the redox potential of the couple Cuþ/Cuþþ in
copper(II) complexes during the catalytic cycle. The above results
also supported from the “f” factor obtained from ESR spectra.
Antioxidants are defined as substances that even at low
concentration significantly delay or prevent oxidation of easy
oxidizable substrates. There is an increased interest of using anti-
oxidants for medical purposes in the recent years. Thus, drugs
possessing antioxidant and free radical scavenging properties are
considered for preventing and/or treatment of diseases, which are
directly related to the lack of the antioxidant capacity of the
organisms. Many non-steroidal anti-inflammatory drugs have been
reported to act either as inhibitors of free radical production or as
radical scavengers [38]. It is well known that free radicals play an
important role in inflammatory action [39]. Consequently,
compounds with antioxidant properties could be expected to offer
protection in inflammation and lead to potentially effective drugs.
In fact, many non-steroidal anti-inflammatory drugs have been
[CuL5(H2O)] > [CuL4(OAc)] > [Cu(L1)(OAc)] > [CuL3(H2O)] >
[CuL2(H2O)]
The degradation of the Rhodamine B dye (552 nm) provides
a direct measure of the concentrations of hydroxyl radicals in the
reaction mixture (Fig. 5). From the observed results, it suggested
that the reactive oxygen species (hydroxyl radical) can be produced
by the copper complex in the presence of oxidant.
The SOD mimetic activity values (IC50) of synthesized complexes
are given in Table 6. In a variety of complexes which act as Super-
oxide dismutase enzymes [37], there is either one-electron oxida-
tion followed by reduction of a metal ion or formation of
a Superoxide complex which then is reduced to peroxide by
another Superoxide ion. In order to explore the mechanism,
absorption spectrum of complexes were recorded in the presence
and absence of alkaline DMSO (alkaline DMSO acts as a source of
O2e). The spectrum peaks became suppressed in alkaline DMSO
containing buffer (pH 8.6). However upon addition of NBT, which
acts as O2e scavenger, these peaks were reverted to their original
position. Thus, this experiment indicates that O2e is initially
attached to the metal complexes which later are reduced by
another O2e ion. The results report the scavenging ability of each
complex, giving its final concentration that produced efficient
quenching of the superoxide anion radical.
1
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0
0.2
0.4
0.6
0.8
1
1.2
Time (min)
Fig. 5. Rhodamine B degradation followed by decrease of absorbance at 552 nm, pH 8.1 in10 mM phosphate buffer: (a) In presence of 0.1 mM copper complex and (b) In presence of
0.1 mM copper complex, 1 mM H2O2 and 10 mM ascorbic acid.

160
J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
80
70
60
50
40
30
20
10
0
L1
L2
L3
L4
L5
Complex Complex Complex Complex Complex Vitamin RUTIN
L1 L2 L3 L4 L5
Tested compounds
C
Fig. 6. Inhibition of DPPH by ligands and complexes, with rutin and C vitamin as control. All compounds were at 10 mM.
reported to act either as radical scavengers or as inhibitors of free
radical production.
The DPPH method is described as a simple, rapid and convenient
method independent of sample polarity for screening many
samples for radical scavenging activity [39]. The antioxidant
hydroxyflavone derivatives. It implied that copper complex of L4
might be considered as new promising lead candidates for further
design and synthesis of antioxidants.
5.10. Anti-tuberculosis activity
activity of complexes is presented in Fig. 6. At 10 mM concentration,
complex L4 showed a much stronger DPPH scavenging activity
(58.2%) than other ligands and their complexes. The positive
controls, rutin and vitamin C showed 58% and 46% DPPH scav-
enging, respectively. The marked antioxidant activity of complex L4,
in comparison to free ligands and other complexes, could be due to
the coordination of copper in the 4 and 5 positions of the
condensed ring system (A and C ring), increasing its capacity to
stabilize unpaired electrons and, thereby, to scavenge free radicals.
In addition, incorporation of thiazole moiety in the hydroxyflavone
derivatives further enhanced the antioxidant activity. Complex of L1
showed higher antioxidant activity than vitamin C is due to the
introduction of p-substituted (eOCH3) phenyl moiety in the
It is seen from the results of antimycobacterial activity (Table 8),
complexes were found to be highly active with MIC values, which is
well below the MIC values of standard drugs. Complex of L4 was
found to be a more active antimycobacterial agent than ethambutol
(due to the presence of SH group at ortho position).
5.11. Catalytic activity
The catalytic conversion of benzene to phenol was performed
in acetonitrile under argon atmosphere in the presence of
hydrogen peroxide as the oxidant, copper complexes act as cata-
lysts and o-dichlorobenzene as internal standard for quantitative
O
N
O
N
O
H O
2
2
Cu
Cu
First step
O
OH
O
OAc
1
[CuL (OAc)] complex
Homolytic cleavage
Second step
O
N
O
Cu
.
O
.
OH
Scheme 3. Proposed reaction mechanism for catalytic conversion.

J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
161
analysis by gas chromatography. Solvent, catalyst (1 mM),
substrate (1 M), a small amount of oxidant and o-dichlorobenzene
(2 M) were added successively. The total volume of reaction was
2 ml and it was allowed to stir at 25 ꢀC for 20 h the reaction was
quenched by the addition of excess of triphenylphosphine and
hexane. The reaction mixture was filtered with a silica bed. The
products were analyzed by gas chromatography. The proposed
reaction mechanism was shown in Scheme 3. In the case of copper
complex L1, first step, copperehydroperoxo intermediate species is
formed by the reaction of copper complex L1 with hydrogen
peroxide. Second step, copperehydroperoxo species undergoes
haemolytic cleavage of the OeO bond to generate hydroxyl radical
which reacts with benzene to form phenol. The turnover number
for this reaction is 6.2.
recorded with a Systronics 2201 Double beam UVeVis., spectro-
photometer in the 200e1100 nm region. The magnetic susceptibility
values were calculated using the relation meff ¼ 2.83 (cmT). The
diamagnetic corrections were made by Pascal’s constant and Hg
[Co(SCN)4]was used as a calibrant. The ESR spectra of the copper
complexes were recorded at 300 and 77 K on a Varian E112 X-band
spectrometer. Cyclic voltammetric measurements were performed
using a glassy carbon working electrode, Pt wire auxiliary electrode
and an Ag/AgCl reference electrode. Tetrabutylammoniumper-
chlorate (TBAP) was used as the supporting electrolyte. All solutions
were purged with N2 for 30 min prior to each set of experiments.
The X-ray diffractometer system JEOL JDX 8030 was used to record
powder data for the copper complexes, at Central Electrochemical
Research Institute, Karaikudi. Solutions of CT DNA in 50 mM NaCl/
5 mM TriseHCl (pH ¼ 7.0) gave a ratio of UV absorbance at 260 and
280 nm, A260/A280 of ca. 1.8e1.9, indicating that the DNA was suffi-
ciently free of protein contamination. The DNA concentration was
determined by the UV absorbance at 260 nm after 1:100 dilutions.
The molar absorption coefficient was taken as 6600 Mꢁ1 cmꢁ1. Stock
solutions were kept at 4 ꢀC and used after not more than 4 days.
6. Conclusion
The new Schiff base ligands and their copper complexes have
been synthesized and characterized. The DNA binding properties
of copper complexes were studied by using absorption spectra,
viscosity and thermal denaturation experiments. The results show
that the complexes were interacting with CT DNA. We also carried
out the DNA cleavage by using gel electrophoresis techniques.
From the antimicrobial study, the presence of lipophilic and polar
substituent’s such as C]N, SeH and NH2 are expected to enhance
the fungal and bacterial toxicity and therefore copper(II)
complexes have a greater chance of interaction with the nucleo-
tide bases. It also has been observed that some moieties such as
azomethine linkage or heteroaromatic nucleus introduced into
such compounds exhibit extensive biological activities that may be
responsible for the increase in hydrophobic character and lip-
osolubility of the molecules in crossing the cell membrane of the
microorganism and enhance biological utilization ratio and
activity of complexes. The present work has thus shown that
copper complexes of Schiff base derivatives of 4-aminoantipyrine
yield highly potent SOD mimics. The observed correlation
between the SOD activity and the redox potential of the Cuþ/Cu2þ
emphasizes the roles played by the electronic as well as stereo
chemical factors in the biological activities of these complexes.
Further work to investigate this role of copper ions by determining
the lethal dose of the complex in biological systems and their
pharmacological screening is in progress and will be reported in
due course.
7.1. Preparation of ligand (L1eL5)
Equimolar amount of 3-hydroxyflavone and 4-aminoantipyrine
(L1)/o-aminophenol (L2)/o-aminobenzoic acid (L3)/o-aminothiazole
(L4)/thiosemicarbazide (L5) were dissolved in ethanol (40 mL).
Acetic acid (1.0 mL) was added to this solution. The solution was
stirred for 3 h and precipitates formed. The precipitate was filtered
and washed with water and ethanol.
L1: Yield: 78%. Anal. Calcd for C26H21N3O3: C, 73.74; H, 4.99; N,
9.90. Found: C, 73.67; H, 4.91; N, 9.84. FAB mass spectrometry (FAB-
MS): m/z 424 [Mþ1]. 1H NMR (300 MHz, CDCl3,
d, ppm) : 6.6e7.5
(14H, m, AreH), 1.6 (3H, s, H3CeC), 1.9 (s, 3H, H3CeN) and 10.8
(1H, s, OeH, D2O exchangeable). 13C NMR (300 MHz, CDCl3, ppm):
12.5 (H3CeC), 19.6 (H3CeN), 143.5 (H3CeC), 162.8 (C]O), 154.5 (]
CeN), 124.6 (C-2), 118.8 (C-3), 152.6 (C-4), 119.2 (C-5), 146.4 (C-6),
125.2 (C-7), 126.4 (C-8), 157.3 (C-9), 121.6 (C-10), 133.8 (C-10), 127.4
(C-20, 60), 141.8 (C-30, 50), 133.5 (C-40), 127.2 (C-100), 119.6 (C-200, 600),
118.9 (C-300, 500) and 121.6 (C-400).
L2: Yield: 62%. Anal. Calcd for C21H15NO3: C, 76.58; H, 4.59; N,
4.25. Found: C, 76.52; H, 4.56; N, 4.20. FAB mass spectrometry
(FAB-MS), m/z 330 [Mþ1]. 1H NMR (300 MHz, CDCl3)
d: 6.4e7.6
(13H, m, AreH) and 11.5 and 10.6 (2H, s, OeH, D2O exchange-
able, 3-hydroxyflavone and o-aminophenol moities). 13C NMR
(300 MHz, CDCl3, ppm): 151.6 (C-2), 103.8 (C-3), 155.2 (C-4), 143.5
(C-5), 145.8 (C-6), 122.6 (C-7), 125.3 (C-8), 153.5 (C-9), 119.4 (C-
10), 131.5 (C-10), 123.8 (C-20, 60), 125.6 (C-30, 50), 127.2 (C-40), 132.6
(C-100), 116.4 (C-200), 121.8 (C-300), 120.6 (C-400), 127.4 (C-500) and
141.6 (C-600).
7. Experimental
The chemicals used were of AnalaR grade. Copper(II) acetate
was obtained from Merck. Micro analytical data and FAB Mass
spectra of the compounds were recorded at the Regional Sophis-
ticated Instrumentation Center, Central Drug Research Institute
(RSIC, CDRI), Lucknow. The amount of copper present in the copper
complexes was estimated using ammonium oxalate method. The
NMR spectra of the ligands were recorded using TMS as internal
standard (Model: Mercury Plus 300 MHz NMR SPECTROMETER).
L3: Yield: 66%. Anal. Calcd for C22H15NO4: C, 73.94; H, 4.23; N,
3.91. Found: C, 73.89; H, 4.18; N, 3.90. FAB mass spectrometry (FAB-
MS), m/z 358 [Mþ1]. 1H NMR (300 MHz, CDCl3)
d : 6.8e7.6 (13H, m,
AreH), 11.4 and 10.8 (2H, s, OeH, D2O exchangeable, 5-hydroxy-
flavone and o-aminobenzoic acid moities). 13C NMR (300 MHz,
CDCl3, ppm): 148.2 (C-2), 111.5 (C-3), 155.4 (C]N), 116.2 (C-5), 147.1
(C-6), 123.5 (C-7), 127.2 (C-8),153.4 (C-9), 120.6 (C-10), 133.6 (C-10),
126.3 (C-20, 60), 128.6 (C-30, 50), 127.2 (C-40), 149.1 (C-100), 114.0 (C-
200), 132.6 (C-300), 117.5 (C-400), 129.4 (C-500), 141.8 (C-600) and 167.5
(COOH).
Chemical shifts (d) are expressed in units of parts per million
relative to TMS. The FAB mass spectrum of the ligands and their
complexes were recorded on a JEOL SX 102/DA-6000 mass spec-
trometer/data system using argon/xenon (6 kV, 10 mA) as the FAB
gas. The accelerating voltage was 10 kV and the spectra were
recorded at room temperature using m-nitrobenzylalcohol (NBA)
as the matrix. Molar conductance of the copper complexes was
measured in DMSO solution using a coronation digital conductivity
meter. The IR spectra of the ligands and their copper complexes
were recorded on a PerkineElmer 783 spectrophotometer in
4000e200 cmꢁ1 range using KBr disc. Electronic spectra were
L4: Yield: 68%. Anal. Calcd for C21H15NO2S: C, 67.49; H, 3.78; N,
8.75. Found: C, 67.42; H, 3.72; N, 8.69. Fast atom bombardment
mass spectrometry (FAB-MS), m/z 322 [Mþ1]. 1H NMR (300 MHz,
CDCl3)
d
: 5.1 & 5.4 (2H, dd, J, 10.5 Hz,eCH ¼ CH-), 5.9e7.9 (9H, m,
AreH), 12.9 (1H, s, OeH, D2O exchangeable). 13C NMR (300 MHz,
CDCl3) : 148.5 (C-2), 111.4 (C-3), 155.6 (C]N), 116.8 (C-5), 148.2 (C-

162
J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
6), 123.6 (C-7), 127.8 (C-8), 155.5 (C-9), 122.4 (C-10), 133.6 (C-10),
126.2 (C-20, 60), 128.4 (C-30, 50), 127.6 (C-40), 159.4 (C-11), 104.6 (C-
12) and 149.5 (C-13).
DMSO in the medium did not affect the growth of any of the
microorganisms tested.
L5: Yield: 70%. Anal. Calcd for C16H13N3O2S: C, 61.72; H, 4.20; N,
13.49. Found: C, 61.65; H, 4.15; N, 13.40. Fast atom bombardment
mass spectrometry (FAB-MS), m/z 311 [Mþ1]. 1H NMR (300 MHz,
7.3.1. SOD activity
In vitro SOD activity was measured using alkaline DMSO as
a source of superoxide radical ðOꢁ2 Þ and nitrobluetetrazolium
CDCl3)
d: 6.8e7.6 (8H, m, Ar-H), 11.2 (1H, s, eNH), 8.3 (1H, s, br, HN
(NBT) as Oꢁ2 scavenger [40,41]. In general, 400
mL sample to be
of eNH2), 7.6 (1H, s, br, HN of eNH2), 11.9 (1H, s, OeH, D2O
exchangeable). 13C NMR (300 MHz, CDCl3): 167.7 (C]O), 150.6 (C-
2), 112.5 (C-3), 155.8 (C]N), 116.8 (C-5), 147.4 (C-6), 124.6 (C-7),
125.6 (C-8), 153.4 (C-9), 119.5 (C-10), 133.5 (C-10), 126.0 (C-20, 60),
126.8 (C-30, 50), 127.9 (C-40) and 176.2 (C]S).
assayed was added to a solution containing 2.1 mL of 0.2 M
potassium phosphate buffer (pH 8.6) and 1 mL of 56 M NBT. The
m
tubes were kept in ice for 15 min and then 1.5 mL of alkaline
DMSO solution was added while stirring. The absorbance was then
monitored at 540 nm against a sample prepared under similar
condition except NaOH was absent in DMSO. A unit of superoxide
dismutase [SOD] activity is the concentration of complex or
enzyme, which causes 50% inhibition of alkaline dimethylsulph-
oxide (DMSO) mediated reduction of nitrobluetetrazolium chlo-
ride (NBT).
7.2. Preparation of copper complexes of ligands (L1eL5)
The ligand (s) (0.05 mM) and copper acetate (0.05 mM) were
dissolved in acetone (20 mL). Under stirring, triethylamine
(0.075 mM) was then dropped to the mixture with caution. After
stirring for 4 h at room temperature, the precipitate was separated
by suction filtration, purified by washing several times with
acetone and dried in vaccum.
7.4. Absorption titration experiment
Absorption titration experiment was performed by maintaining
a constant concentration of the complex while varying the nucleic
acid concentration. This was achieved by dissolving an appropriate
amount of the copper complex stock solution and by mixing
various amounts of DNA stock solutions while maintaining the total
volume constant. This resulted in a series of solutions with varying
concentrations of DNA but with a constant concentration of the
complex. The absorbance (A) of the most red-shifted band of
complex was recorded after each successive additions of CT DNA.
The intrinsic binding constant, Kb, was determined from the plot of
Complex of L1: Yield: 68%. Anal. Calcd for CuC28H24N3O5: C,
61.57; H, 4.43; N, 7.70, Cu, 11.64. Found: C, 61.50; H, 4.39; N, 7.68;
Cu, 11.65. FAB mass spectrometry (FAB-MS), m/z 545 [Mþ1]. meff
(BM) ¼ 1.96; Lm (mhocm2 molꢁ1) ¼ 20.
Complex of L2: Yield: 76%. Anal. Calcd for CuC21H17NO4: C,
61.36; H, 4.17; N, 3.41, Cu, 15.47. Found: C, 61.31; H, 4.15; N, 3.36; Cu,
15.43. FAB mass spectrometry (FAB-MS), m/z 411 [Mþ1]. meff
(BM) ¼ 1.94; Lm (mhocm2 molꢁ1) ¼ 22.
Complex of L3: Yield: 69%. Anal. Calcd for CuC22H17NO5: C,
60.18; H, 3.91; N, 3.19, Cu,14.49. Found: C, 60.14; H, 3.86; N, 3.15; Cu,
14.43. FAB mass spectrometry (FAB-MS), m/z [Mþ1]. meff
(BM) ¼ 2.04; Lm (mhocm2 molꢁ1) ¼ 16.
[DNA]/(
base pairs,
by calculating Aobs/[complex] and
3
aꢁ
3
f) vs [DNA], where [DNA] is the concentration of DNA in
a, the apparent extinction coefficient which is obtained
corresponds to the extinction
3
3
f
Complex of L4: Yield: 76%. Anal. Calcd for CuC21H17NO3S: C,
54.22; H, 3.42; N, 6.33; Cu, 14.36. Found: C, 74.62; H, 3.35; N, 6.29;
Cu, 14.31. FAB mass spectrometry (FAB-MS), m/z 443 [Mþ1]. meff
(BM) ¼ 1.98; Lm (mhocm2 molꢁ1) ¼ 18.
coefficient of the complex in its free form. The data were fitted to
the following equation where b refers to the extinction coefficient
of the complex in the fully bound form.
3
Complex of L5: Yield: 60%. Anal. Calcd for CuC16H14N3O3S: C,
48.90; H, 3.85; N, 10.70; Cu, 16.18. Found: C, 74.62; H, 3.81; N, 10.63;
Cu, 16.14. FAB mass spectrometry (FAB-MS), m/z 393[Mþ1]. meff
(BM) ¼ 2.06; Lm (mhocm2 molꢁ1) ¼ 20.
[DNA]/(
a3 ꢁ
f3 ) ¼ [DNA]/(
b3 ꢁ
f3 ) þ 1/Kb(
b3 ꢁ
3
)
(6)
f
Each set of data, when fitted to the above equation, gave
a straight line with a slope of 1/( bꢁ f) and a y-intercept of 1/
Kb( f). Kb was determined from the ratio of the slope to
bꢁ
intercept.
3
3
3
3
7.3. Antimicrobial activity
Fixed amounts of complexes were dissolved in DMSO because
the high solubility of the compounds in this solvent allows us to
prepare concentrated solutions and to utilize reduced volumes
The in vitro antimicrobial activities of the investigated
compounds were tested against the bacterial species and fungal
species. One day prior to the experiment, the bacterial and fungal
cultures were inoculated in broth (inoculation medium) and
incubated overnight at 37 ꢀC. Inoculation medium containing 24 h
grown culture was added aseptically to the nutrient medium and
mixed thoroughly to get the uniform distribution. This solution
was poured (25 mL in each dish) into petri dishes and then
allowed to attain room temperature. Wells (6 mm in diameter)
were cut in the agar plates using proper sterile tubes. Then, wells
were filled up to the surface of agar with 0.1 mL of the test
(5 mL) for titrations. It was also verified that the DMSO percentage
(0.7%) added to the DNA solution did not interfere with the nucleic
acid; in fact, the 260 nm absorption band is not subjected to
modifications in intensity and position.
7.5. Viscosity measurements
Viscosity experiments were carried on an Ostwald viscometer,
immersed in a thermostated water-bath maintained at a constant
temperature at 30 ꢃ 0.1 ꢀC. Viscosity measurements were carried
out at room temperature. DNA samples of approximately 0.5 mM
were prepared by sonicating in order to minimize complexities
arising from DNA flexibility. Flow time was measured with a digital
stopwatch three times for each sample and an average flow time
was calculated. Each experiment was performed three times and an
compounds dissolved in DMSO (200
mM/mL). The plates were
allowed to stand for an hour in order to facilitate the diffusion of
the drug solution. Then the plates were incubated at 37 ꢀC for 24 h
for bacteria and 48 h for fungi and the diameter of the inhibition
zones were read. Minimum inhibitory concentrations (MICs) were
determined by using serial dilution method. The lowest concen-
tration (
m
g/mL) of compound, which inhibits the growth of
average flow time was calculated. Data were represented as (
versus binding ratio, where is the viscosity of DNA in the presence
of complex and is the viscosity of DNA alone. Viscosity values
h/h0)
bacteria after 24 h incubation at 37 ꢀC, and of fungi after 48 h
incubation at 37 ꢀC was taken as the MIC. The concentration of
h
h

J. Joseph et al. / European Journal of Medicinal Chemistry 49 (2012) 151e163
163
were calculated after correcting the flow time of buffer alone (t0),
¼ (t ꢁ t0)/t0.
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7.8. Anti-tubercular activity
In vitro evaluation of the anti-tubercular activity was carried out
against Mycobacterium tuberculosis H37Rv by using broth dilution
[42] assay method. The result of anti-tubercular activity is pre-
sented in Table 8.
Acknowledgement
We express our sincere thanks to the Chancellor, Noorul Islam
Centre for Higher Education, Kumaracoil for providing research
facilities. This study was funded by DST, New Delhi under INSPIRE
fellowship (IF 10544) and CSIR, New Delhi (Grant No. 01(2497)/11/
EMR-II).
Appendix. Supplementary material
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Supplementary data related to this article can be found online at
doi:10.1016/j.ejmech.2012.01.006.