Spectral, magnetic, biocidal screening, DNA binding and photocleavage studies of mononuclear Cu(II) and Zn(II) metal complexes of tricoordinate heterocyclic Schiff base ligands of pyrazolone and semicarbazide/thiosemicarbazide based derivatives

Article abstract of DOI:10.1016/j.saa.2010.03.007

We depict the synthesis and characterization of copper(II) and zinc(II) coordination compounds of 4-(3′,4′-dimethoxybenzaldehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5-semicarbazone (1a), 4-(3′,4′-dimethoxybenzaldehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5

Full text of DOI:10.1016/j.saa.2010.03.007

Spectrochimica Acta Part A 76 (2010) 161–173
Contents lists available at ScienceDirect
Spectrochimica Acta Part A: Molecular and
Biomolecular Spectroscopy
journal homepage: www.elsevier.com/locate/saa
Spectral, magnetic, biocidal screening, DNA binding and photocleavage studies
of mononuclear Cu(II) and Zn(II) metal complexes of tricoordinate
heterocyclic Schiff base ligands of pyrazolone and
semicarbazide/thiosemicarbazide based derivatives
N. Ramana,∗, A. Selvana, P. Manisankarb
a Research Department of Chemistry, VHNSN College, Virudhunagar, Tamil Nadu 626001, India
b Department of Industrial Chemistry, Alagappa University, Karaikudi 630003, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We depict the synthesis and characterization of copper(II) and zinc(II) coordination compounds
of 4-(3ꢀ,4ꢀ-dimethoxybenzaldehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5-semicarbazone (1a), 4-
(3ꢀ,4ꢀ-dimethoxybenzaldehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5-thiosemicarbazone (1b), 4-(3ꢀ-
hydroxy-4ꢀ-nitrobenzaldehydene)2-3-dimeth yl-1-phenyl-3-pyrazolin-5-semicarbazone (1c) and 4-(3ꢀ-
hydroxy-4ꢀ-nitrobenzal dehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5-thiosemicarbazone (1d). All
the remote compounds have the general composition [ML2] (M = Cu(II) and Zn(II)); L = Schiff base (1a–1d).
All the complexes were characterized by elemental analysis, molar conductivity, IR, 1H NMR, UV–vis,
ESI-Mass, magnetic susceptibility measurements, cyclic voltammetric measurements, and EPR spectral
studies. It has been originated that the Schiff bases with Cu(II) and Zn(II) ions form mononuclear com-
plexes on 1:2 (metal:ligand) stoichiometry. Distorted octahedral environment is suggested for the metal
complexes. The conductivity data confirm the non-electrolytic nature of the complexes. The interaction of
CuL21a–1d complexes with CT DNA was investigated by spectroscopic, electrochemical and viscosity mea-
surements. Results suggest that the copper complexes bind to DNA via an intercalative mode. Moreover,
the complexes have been found to promote the photocleavage of plasmid DNA pBR322 under irradiation
at 365 nm. The Schiff bases and their metal complexes were screened for their antifungal and antibac-
terial activities against different species of pathogenic fungi and bacteria and their biopotency has been
discussed.
Received 7 January 2010
Received in revised form 24 February 2010
Accepted 11 March 2010
Keywords:
Schiff base
Metal complexes
DNA interaction
Photocleavage
Tricoordinate ligand
Antimicrobial study
© 2010 Elsevier B.V. All rights reserved.
1. Introduction
increased or decreased activities in relation to the non-complexed
semicarbazones/thiosemicarbazones have been reported for sev-
Schiff bases are important class of ligands in coordination chem-
istry and found extensive application in different fields of science.
Schiff bases of semicarbazones/thiosemicarbazones have aroused
with significant interest in the field of chemistry and biology
due to their antibacterial, antifungal, antimalarial, antineoplastic
and antiviral activities [1]. The biological activities of semicar-
bazones/thiosemicarbazones are considered to be related to their
ability to form chelates with metals. The chemistry of transition
metal complexes of semicarbazones/thiosemicarbazones based
Schiff bases has been receiving significant attention because of their
pharmacological properties. The biological activities of metal com-
plexes differ from those of either the free ligands or metal ions and
eral transition metal complexes [2]. Among the metal complexes of
semicarbazones/thiosemicarbazones, the Cu(II) and Zn(II) chelates
have been especially studied regarding their antitumour potentials
[3]. Moreover, Cu(II) and Zn(II) complexes with oxygen, sulphur and
nitrogen containing ligands are the subject of intensive biological
evaluation in the search for less toxic and more selective anticancer
therapies [4,5]. Especially heterocyclic thiosemicarbazones have
been the subject of extensive investigation because of their wide
use in biological field [6–8].
The investigation of metal complexes with Schiff bases derived
from pyrazole, e.g., 4-amino-2-3-dimethyl-1-phenyl-3-pyrazolin-
5-one, is interesting because of the fact that these compounds can
serve as models for studying a wide range of biological reactions
which are catalyzed by enzymes. It has been shown that in the pres-
ence of metal ions, free pyrazole can catalyze many reactions like
carbonylation, hydroformylation, oxidation, reduction and epoxi-
dation [9]
∗
Corresponding author.
E-mail address: drn raman@yahoo.co.in (N. Raman).
1386-1425/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.saa.2010.03.007

162
N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
Considering the significance of semicarbazones/thiosemicar-
bazones and pyrazolone derivatives, we have synthesized the Schiff
bases (1a–1d) and their Cu(II) and Zn(II) complexes. It is well known
that a general study on structure and bonding can help in better
understanding of the complex life processes. Hence, the structures
of the synthesized Schiff bases and their complexes were eluci-
dated by analytical and spectral studies. Their biological studies
like antimicrobial and DNA binding and cleavage were carried out
in order to understand the mode of interaction of the synthesized
Schiff bases and their complexes with Calf thymus DNA (CT DNA).
Scheme 1. Preparation of 4-(3ꢀ,4ꢀ-benzaldehydene)2-3-dimethyl-1-phenyl-3-
pyrazolin-5-one.
2. Experimental
2.1. Chemicals
stolonifer, Candida albicans and Rhizoctonia bataicola. For the detec-
tion of the biocidal activities, the filter paper disc agar diffusion
method was used [12–14]. The minimum inhibitory concentra-
tion (MIC) was considered to be the lowest concentration, which
exhibited the same turbidity as the blank tube. Pure Streptomycin
and Nystatin were used separately as standards for antibacterial
and antifungal activity tests respectively. Nutrient agar (NA) was
used as basal medium for the cultured bacteria. A series of differ-
ent concentrations of the compounds in dimethylformamide (DMF)
solvent were placed on the surface of the culture and incubated
at 37 ◦C for 24 h for the bacterial culture. The antifungal activities
were performed with potato agar medium in DMF as solvent and
incubated at 37 ◦C for 48–72 h.
All reagents and solvents were of AR grade, purchased com-
mercially. Tris–HCl buffer salts were purchased from Qualigens
(India); CT DNA and pBR322 DNA from Bangalore Genei, India.
Tris–HCl buffer solution [5 mM Tris–HCl and 50 mM NaCl (pH 7.2);
Tris = Tris(hydroxymethyl) aminomethane] was prepared using
deionised double distilled water.
All DNA stock solutions (1000 mg/L) were prepared with the
Tris–HCl buffer solution and kept frozen. More dilute solutions
were prepared with 50 mM Tris–HCl (pH 7.2). The concentration
of CT DNA was measured by using its known extinction coefficient
at 260 nm (6600 M−1 cm−1) [10]. The absorbance at 260 nm (A260
)
and at 280 nm (A280) for CT DNA was measured to ensure its purity.
The ratio A260/A280 was found to be 1.84, indicating that CT DNA
was satisfactorily free from protein.
2.2. Physical measurements
2.4. Synthesis
2.4.1. Preparation of 4-(3ꢀ,
UV–vis spectra were recorded on a Shimadzu Model 1601 UV-
Visible Spectrophotometer. IR spectra were recorded (KBr) with
a PerkinElmer FTIR-1605 spectrophotometer. 1H NMR spectra
were taken on a Varian XL-300 MHz spectrometer with tetram-
ethylsilane (TMS) as the internal standard at room temperature.
The complexes were analyzed for their metal contents, follow-
ing standard procedures [11] after decomposition with a mixture
of conc. HNO3 and HCl, followed by conc. H2SO4. Microanalyses
were carried out on a PerkinElmer 240 elemental analyzer. Mass
spectrometry experiments were performed on a JEOL-AccuTOF
JMS-T100LC mass spectrometer equipped with a custom-made
electrospray interface (ESI). The X-band EPR spectra of the com-
plexes were recorded at RT (300 K) and LNT (77 K) using TCNE as
the g-marker. Room temperature magnetic susceptibility measure-
ments were carried out on a modified Gouy-type magnetic balance,
Hertz SG8-5HJ. The molar conductivity of the complexes in DMF
solution (10−3 M) was measured using a conductometer model
601/602.
Voltammetric experiments were performed on a CHI 620C
electrochemical analyzer in freshly distilled DMF solutions. 0.1 M
tetrabutylammonium perchlorate (TBAP) was used as the sup-
porting electrolyte. The three-electrode cell comprised a reference
Ag/AgCl, auxiliary Pt and the working glassy carbon electrodes.
All the solutions examined by electrochemical techniques were
purged with nitrogen for 10 min prior to each set of experiments.
All measurements were carried out at room temperature (25 ◦C).
4ꢀ-benzaldehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5-one
The reaction of 4-amino-2-3-dimethyl-1-phenyl-3-pyrazolin-
5-one with 3,4-dimethoxybenzaldehyde and 3-hydroxy-4-nitro-
benzaldehyde, carried out for the preparation of 4-(3ꢀ,4ꢀ-benzal-
dehydene)2-3-dimethyl-1-phenyl-3-pyrazolin-5-one is shown in
Scheme 1.
The Schiff bases under investigation were prepared
by mixing an ethanol solution (25 mL) of 4-amino-2-3-
dimethyl-1-phenyl-3-pyrazolin-5-one (2.032 g, 0.01 mol) with
3,4-dimethoxybenzaldehyde (1.66 g, 0.01 mol) and 3-hydroxy-
4-nitrobenzaldehyde (1.67 g, 0.01 mol) in the same volume of
ethanol and refluxing for 3 h in water bath, the precipitate formed
was collected and recrystallized from ethanol.
4-(3ꢀ,4ꢀ-Dimethoxybenzaldehydene)2-3-dimethyl-1-phenyl-3-
pyrazolin-5-one is light yellow crystal and its yield is 89%. Anal.
calcd. C20H21N3O3 M.W.: 351.40, C(68.36%), H(6.02%), N(11.95%),
found: C(68.25%), H(5.92%), N(11.48%); 1H NMR (DMSO-d6) ı:
3.28(s, 3H, PZ–N–CH3), 2.26(s, 3H, PZ–C–CH3), 3.92(s, 3H, O–CH3),
6.97–7.40(m, 3H, Ph), 6.90–7.37(m, 5H, Ph), 8.01(s, 1H, N CH); IR
(KBr) ꢀ (cm−1): 1634(strong, s) (HC N), 1308(s) (O–CH3), 1281(s)
(PZ–C–CH3), 1181(s) (PZ–N–CH3), 1714(s) (PZ–C O), 1471(s)
(Ph–C C), 1545(medium, m) (Ph–C–C), 3048(s) (Ph–C–H).
4-(3ꢀ-Hydroxy-4ꢀ-nitrobenzaldehydene)2-3-dimethyl-1-
phenyl-3-pyrazolin-5-one is yellow powder and its yield is
85%. Anal. calcd. C18H16N4O4 M.W.: 352.35, C(61.35%), H(4.57%),
N(15.90%), found: C(68.25%), H(4.43%), N(15.48%); 1H NMR
(DMSO-d6) ı: 3.11(s, 3H, PZ–N–CH3), 2.58(s, 3H, PZ C–CH3),
10.28(s, 1H, OH), 6.77–7.15(m, 3H, Ph), 6.90–7.37(m, 5H, Ph),
8.00(s, 1H, N CH); IR (KBr) ꢀ (cm−1): 1614(s) (HC N), 1608(weak,
w) (C–NO2), 3442(broad, b) (OH), 1293(s) (PZ–C–CH3), 1145(s)
(PZ–N–CH3), 1651(s) (PZ–C O), 1423(s) (Ph–C C), 1530(m)
(Ph–C–C), 3075(s) (Ph–C–H).
2.3. Biocidal screening
Antibacterial and antifungal activities of the antibiotics and the
metal complexes were screened against four pathogenic bacte-
ria, viz. Staphylococcus aureus, Bacillus subtilis, Escherichia coli, and
Pseudomonas putita and four fungi, viz. Aspergillus niger, Rhizopus

N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
163
required molar ratio (2:1). The reaction mixture was refluxed on
a water bath for 2–3 h and then concentrated to a small volume
on a hot plate at ∼50 ◦C. After cooling, the complexes obtained
were filtered, recrystallized from ethanol and dried in vacuum over
CaCl2.
1a
CuL2
is light green powder and its yield is 65%. Anal.
calcd. CuC42H46N12O6 M.W.: 878.45, C(57.42%), H(5.27%),
N(19.13%), Cu(7.23%) found: C(57.03%), H(4.95%), N(18.91%),
Cu(6.85%); IR (KBr) ꢀ (cm−1): 3182–3219(b) (NH2), 1060(m) (C–O),
1564(s) (C N), 1326(m) (O–CH3), 1262(s) (PZ–C–CH3), 1164(s)
(PZ–N–CH3), 1421(m) (Ph–C C), 1521(s) (Ph–C–C), 2930(s)
(Ph–C–H), 456(s) (Cu–N), 557(m) (Cu–O).
1b
Scheme 2. Preparation of 4-(3ꢀ,4ꢀ-benzaldehydene)2-3-dimethyl-1-phenyl-3-
CuL2
is yellow powder and its yield is 75%. Anal. calcd.
pyrazolin-5-semicarbazone/thiosemicarbazone.
CuC42H46N12O4S2 M.W.: 910.57, C(55.40%), H(5.09%), N(18.45%),
S(7.04%), Cu(6.97%) found: C(55.13%), H(4.85%), N(18.15%),
S(6.85%), Cu(6.75%); IR (KBr) ꢀ (cm−1): 3186–3275(b) (NH2), 694(s)
(C–S), 1598(m) (C N), 1332(s) (O–CH3), 1261(m) (PZ–C–CH3),
1129(b) (PZ–N–CH3), 1421(s) (Ph–C C), 1531(m) (Ph–C–C),
2924(b) (Ph–C–H), 458(s) (Cu–N), 364(m) (Cu–S).
2.4.2. Preparation of 4-(3ꢀ,4ꢀ-benzaldehydene)2-3-dimethyl-1-
phenyl-3-pyrazolin-5-semicarbazone/thiosemicarbazone
The
reaction
of
4-(3ꢀ,4ꢀ-benzaldehydene)2-3-dimethyl-
with semicarbazide (l.11 g,
1-phenyl-3-pyrazolin-5-one
1c
CuL2 is light yellow powder and its yield is 60%. Anal. calcd.
0.01 mol)/thiosemicarbazide (0.91 g, 0.01 mol) in ethanol was
refluxed for 4–5 h to produce Schiff bases (1a–1d). This is shown in
Scheme 2. After cooling to room temperature, light yellow product
obtained was filtered and washed with ethanol.
CuC38H36N14O8 M.W.: 880.33, C(51.84%), H(4.12%), N(22.27%),
Cu(7.21%), found: C(51.20%), H(3.95%), N(21.85%), Cu(6.95%); IR
(KBr) ꢀ (cm−1): 3262(s) (NH2), 1075(m) (C–O), 1595(m) (C N),
1602(w) (C–NO2), 3354(s) (OH), 1273(s) (PZ–C–CH3), 1146(s)
(PZ–N–CH3), 1416(m) (Ph–C C), 1509(s) (Ph–C–C), 2963(b)
1a is light yellow powder and its yield is 75%. Anal. calcd.
C
21H24N6O3 M.W.: 408.45, C(61.75%), H(5.92%), N(20.57%), found:
(Ph–C–H), 456(s) (Cu–N), 552(m) (Cu–O).
C(61.20%), H(6.04%), N(19.65%); 1H NMR (DMSO-d6) ı: 2.21(s, 3H,
PZ–C–CH3), 2.87(s, 3H, PZ–N–CH3), 6.85(s, 2H, NH2), 3.71(s, 3H,
O–CH3), 6.91–7.36(m, 5H, Ph), 7.59–7.68(m, 3H, Ph), 8.28(s, 1H,
1d
CuL2
is light greenish yellow powder and its yield is 55%.
Anal. calcd. CuC38H36N14O6S2 M.W.: 912.46, C(50.02%), H(3.97%),
N(21.49%), S(7.02%), Cu(6.96%) found: C(49.75%), H(3.65%),
N(20.95%), S(6.95%), Cu(6.85%); IR (KBr) ꢀ (cm−1): 3279(s) (NH2),
702(m) (C–S), 1591(m) (C N), 1604(w) (C–NO2), 3465(s) (OH),
1263(m) (PZ–C–CH3), 1192(s) (PZ–N–CH3), 1455(m) (Ph–C C),
1543(s) (Ph–C–C), 2947(s) (Ph–C–H), 452(s) (Cu–N), 367(m)
(Cu–S).
N
CH), 9.66(s, 1H, NH-sc); IR (KBr) ꢀ (cm−1): 3180–3224(b) (NH2),
3069(b) (–NH–Sc),1698(s) (Sc–C O), 1589(s) (C N), 1324(m)
(O–CH3), 1266(s) (PZ–C–CH3), 1167(s) (PZ–N–CH3), 1422(m)
(Ph–C C), 1518(s) (Ph–C–C), 2932(s) (Ph–C–H).
1b is also light yellow powder and its yield is 80%. Anal.
calcd. C21H24N6O2S M.W.: 424.52, C(59.41%), H(5.69%), N(19.79%),
S(7.55%), found: C(59.20%), H(6.04%), N(19.65%), S(7.45%); 1H NMR
(DMSO-d6) ı: 2.15(s, 3H, PZ C–CH3), 2.85(s, 3H, PZ–N–CH3), 6.39(s,
2H, NH2), 3.92(s, 3H, O–CH3), 7.08–7.48(m, 5H, Ph), 7.56–7.81(m,
3H, Ph), 8.41(s, 1H, N CH), 9.69(s, 1H, NH-tsc); IR (KBr) ꢀ
(cm−1): 3188–3273(b) (NH2), 3163(b) (–NH-tsc), 834(m) (C S),
1626(m) (C N), 1335(s) (O–CH3), 1262(m) (PZ–C–CH3), 1133(b)
(PZ–N–CH3), 1420(s) (Ph–C C), 1534(m) (Ph–C–C), 2929(b)
(Ph–C–H).
2.4.3.2. Zn(II) complexes of 1a–1d. A general method was used for
the preparation of the complexes. A hot ethanolic solution of the
corresponding zinc(II) salt was mixed with a hot ethanolic solution
of the respective ligand (in 1:2 molar ratio). The reaction mixture
was refluxed on a water bath for 2–3 h. On cooling at room tem-
perature, the desired complexes were precipitated out in each case.
They were filtered, recrystallized, washed with ethanol and finally
dried over CaCl2 under vacuum.
1c is light yellow powder and its yield is 65%. Anal. calcd.
1a
ZnL2 is light yellow powder and its yield is 56%. Anal. calcd.
C
19H19N7O4 M.W.: 409.40, C(55.74%), H(4.67%), N(23.94%), found:
ZnC42H46N12O6 M.W.: 880.28, C(57.30%), H(5.26%), N(19.09%),
Zn(7.42%); found: C(56.65%), H(5.15%), N(18.87%), Zn(7.34%); 1H
NMR (DMSO-d6) ı: 2.28(s, 3H, PZ–C–CH3), 2.90(s, 3H, PZ–N–CH3),
6.08(s, 2H, NH2), 3.93(s, 3H, O–CH3), 6.90–7.26(m, 5H,Ph),
7.60–7.75(m, 3H, Ph), 8.92(s, 1H, N CH); IR (KBr) ꢀ (cm−1):
3184–3221(b) (NH2), 1098(m) (C–O), 1569(s) (C N), 1323(m)
(O–CH3), 1263(s) (PZ–C–CH3), 1164(s) (PZ–N–CH3), 1421(m)
(Ph–C C), 1519(s) (Ph–C–C), 2929(s) (Ph–C–H), 459(s) (Zn–N),
553(m) (Zn-O).
C(54.85%), H(4.27%), N(23.45%); 1H NMR (DMSO-d6) ı: 2.24(s,
3H, PZ C–CH3), 2.93(s, 3H, PZ–N–CH3), 6.43(s, 2H, NH2), 10.56(s,
1H, OH), 6.91–7.26(m, 5H, Ph), 7.44–7.60(m, 3H, Ph), 8.45 (s,1H,
N
CH), 9.88(s, 1H, NH-Sc); IR (KBr) ꢀ (cm−1): 3268(s) (NH2),
3127(b) (–NH–Sc), 1618(m) (C N), 1603(w) (C–NO2), 3357(s)
(OH), 1270(s) (PZ–C–CH3), 1147(s) (PZ–N–CH3), 1651(w) (Sc–C O),
1418(m)(Ph–C C), 1511(s) (Ph–C–C), 2966(b) (Ph–C–H)
1d is also a light yellow powder and its yield is 65%. Anal.
calcd. C19H19N7O3S M.W.: 425.46, C(53.63%), H(4.50%), N(23.04%),
S(7.53%), found: C(54.85%), H(4.27%), N(23.45%), S(7.32%); 1H
NMR (DMSO-d6) ı: 2.51(s, 3H, PZ C–CH3), 3.02–3.41(s, 3H,
PZ–N–CH3), 6.38(s, 2H, NH2),10.75(s, 1H, OH), 7.35–7.58(m, 5H,Ph),
7.85–7.98(m, 3H, Ph), 8.18(s, 1H, N CH), 9.57(S, 1H, NH-tsc); IR
(KBr) ꢀ (cm−1): 3279(s) (NH2), 3126(b) (–NH-tsc), 1615(m) (C N),
1606(w) (C–NO2), 3469(s) (OH), 1265(m) (PZ–C–CH3), 1195(s)
(PZ–N–CH3), 828(m) (C S), 1455(m) (Ph–C C), 1545(s) (Ph–C–C),
2948(s) (Ph–C–H).
ZnL21b is light colourless powder and its yield is 72%. Anal. calcd.
ZnC42H46N12O4S2 M.W.: 912, C(55.28%), H(5.08%), N(18.42%),
S(7.02%), Zn(7.16%); found: C(55.12%), H(4.85%), N(18.28%),
S(6.93%), Zn(7.07%); 1H NMR (DMSO-d6) ı: 2.01(s, 3H, PZ–C–CH3),
3.15(s, 3H, PZ–N–CH3), 5.93(s, 2H, NH2), 3.97(s, 3H, O–CH3),
6.85–7.33(m, 5H, Ph), 7.54–7.68(m, 3H, Ph),8.87(s, 1H, N CH); IR
(KBr) ꢀ (cm−1): 3185–3274(b) (NH2), 649(s) (C–S), 1602(m) (C N),
1331(s) (O–CH3), 1264(m) (PZ–C–CH3), 1129(m) (PZ–N–CH3),
1419(s) (Ph–C C), 1531(m) (Ph–C–C), 2926(b) (Ph–C–H), 465(s)
(Zn–N), 357 (s) (Zn-S).
1c
2.4.3. Synthesis and characterization of the complexes
ZnL2
is yellow powder and its yield is 65%. Anal. calcd.
2.4.3.1. Cu(II) complexes of 1a–1d. All the complexes were pre-
pared by mixing ethanolic solution of ligands and metal salts in
ZnC38H36N14O8 M.W.: 882.17, C(51.73%), H(4.11%), N(22.22%),
Zn(7.41%); found: C(51.65%), H(4.05%), N(21.98%), Zn(7.38%); 1H

164
N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
Table 1
NMR (DMSO-d6) ı: 2.57(s, 3H, PZ–C–CH3), 3.20–3.60(s, 3H,
PZ–N–CH3), 6.34(s, 2H, NH2), 10.59(s, 1H, OH), 7.31–7.42(m, 5H,
Ph), 7.80–7.98(m, 3H, Ph), 8.58(s, 1H, N CH); IR (KBr) ꢀ (cm−1):
3264(s) (NH2), 1058(m) (C–O), 1591(m) (C N), 1598(w) (C–NO2),
3351(s) (OH), 1271(s) (PZ–C–CH3), 1149(s) (PZ–N–CH3), 1420(m)
(Ph–C C), 1507(s) (Ph–C–C), 2961(b) (Ph–C–H), 472(s) (Zn–N),
Electronic absorption spectral data of the ligands and their compounds.
Complex
Solvent
Absorption
(cm−1
Band
assignment
Geometry
)
1a
EtOH
31,645
36,231
41,322
INCTa
INCT
INCT
–
–
561(m) (Zn-O).
1d
ZnL2
is light yellow powder and its yield is 53%. Anal.
38H36N14O6S2 M.W.: 914.29, C(49.92%), H(3.96%),
1b
1c
EtOH
EtOH
EtOH
DMF
DMF
DMF
DMF
25,974
34,965
38,910
INCT
INCT
INCT
calcd. Zn
C
–
–
N(21.44%), S(7.01%), Zn(7.15%); found: C(49.85%), H(3.82%),
N(21.25%), S(6.87%), Zn(7.03%); 1H NMR (DMSO-d6) ı: 2.60(s, 3H,
PZ–C–CH3), 3.09–3.36(s, 3H, PZ–N–CH3), 6.43(s, 2H, NH2),10.82(s,
1H, OH), 7.35–7.68(m, 5H, Ph), 7.82–7.96(m, 3H, Ph), 8.61(s, 1H,
24,271
33,003
42,016
INCT
INCT
INCT
N
CH); IR (KBr) ꢀ (cm−1): 3273(s) (NH2), 706(m) (C–S), 1597(m)
1d
22,573
31,545
39,215
INCT
INCT
INCT
(C N), 1602(w) (C–NO2), 3464(s) (OH), 1262(m) (PZ–C–CH3),
1197(m) (PZ–N–CH3), 1453(m) (Ph–C C), 1539(s) (Ph–C–C),
2951(s) (Ph–C–H), 462(s) (Zn–N), 345(m) (Zn-S).
1a
[CuL2
[CuL2
[CuL2
]
]
]
]
11,148
26,178
40,161
2Eg
INCT
INCT
←
2T2g
2T2g
2T2g
2T2g
Distorted octahedral
Distorted octahedral
Distorted octahedral
Distorted octahedral
3. Results and discussion
1b
1c
1d
11,521
25,840
42,735
2Eg
INCT
INCT
←
3.1. Physicochemical properties of the synthesized compounds
15,385
27,397
39,683
2Eg
INCT
INCT
←
The ligands (1a–1d) were prepared by refluxing an appropri-
ate amount of respective 4-(3ꢀ,4ꢀ-benzaldehydene)2-3-dimethyl-
1-phenyl-3-pyrazolin-5-one with the corresponding semicar-
bazide/thiosemicarbazide in ethanol. The structures of the
synthesized ligands were established with the help of their IR,
NMR, and microanalytical data. All metal(II) complexes of Cu(II)
and Zn(II) of these ligands were prepared by using the respective
metal salts as chloride with the corresponding ligands in molar ratio
of metal:ligand as 1:2 as obtainable in the following reaction.
[CuL2
9709
28,249
38,168
2Eg
INCT
INCT
←
a
INCT – intraligand charge transfer band.
of the C S group appears at 828–834 cm−1 in the ligand [17]. The
absence of this band in the IR spectra of the metal complexes can
be explained by the tautomerism of the C S group with one of
the imino groups to form the C–SH and the coordination of sul-
phur after deprotonation. The bands that appear in the range of
649–706 cm−1 and 1058–1098 cm−1 are thus assigned to the ꢀ(C–S)
and ꢀ(C–O) respectively in the IR spectra of the metal complexes.
These overall data suggest that the azomethine-N and enol/thiol-O
or S groups are involved in coordination with the Cu(II) and Zn(II)
ion complexes. In the low-frequency region, spectra of the Cu(II)
and Zn(II) complexes exhibit [18] new bands which are not present
in the spectra of the ligands. These bands are recognized to ꢀ(M–O),
ꢀ(M–N) and ꢀ(M–S).
M·Cl2·nH2O + 2L → ML2 + nH2O + nHCl
where M = Cu(II) and Zn(II).
All these complexes are intensively coloured, air and mois-
ture firm amorphous solids. They are insoluble in common
organic solvents and only soluble in DMF and DMSO. Molar
conductance values of the soluble complexes in DMF (10−3 M
solution at 25 ◦C) indicate that the complexes have molar ratio
of metal:ligand as 1:2. The lesser molar conductance values
(0.93–4.32 ohm−1 cm−2 mol−1) indicate that they are all non-
electrolytic in nature. The elemental analyses data concur well with
the planned formulae for the ligands and also recognized the [ML2]
composition of the metal(II) chelates.
3.3. 1H NMR spectral studies
The 1H NMR spectral data are reported along with the possible
assignments in “experimental.” All the protons were found as to
be in their expected region [19]. The conclusions drawn from these
studies lend further support to the mode of bonding discussed in
their IR spectra. In the spectra of diamagnetic Zn(II) complexes, the
resonance arising from the NH proton disappears, whereas that
arising from the azomethine (HC N) proton shifts to downfield
values indicating the chelation of the ligands through the depro-
tonated enol/thiol-O/S groups and azomethine-N is established by
downfield shifting of these signals in the Zn(II) complexes due to
the increased conjugation and coordination [20].
3.2. Infrared spectral studies
In the current studies, semicarbazone/thiosemicarbazone
related compounds are capable of exhibiting keto-enol tau-
tomerism and react with metal cations to form metal complexes.
The assignments of IR spectra of the ligands and their Cu(II)
and Zn(II) complexes beside with their assignments are reported
in Section 2. The broad band that appears in the range of
3069–3163 cm−1 is assigned to the stretching vibration of (N–H).
A
new prominent band at 1589–1626 cm−1
, due to azome-
thine ꢀ(C N) association appeared in all the ligands, indicates
[15] that condensation between ketone moiety of 4-amino-2-3-
dimethyl-1-phenyl-3-pyrazolin-5-one and that of amino group of
semicarbazone/thiosemicarbazone has taken place ensuing into
the creation of the desired ligands (1a)–(1d).
Moreover, on comparison of the IR spectra of the ligands with
their Cu(II) and Zn(II) complexes showed [16] a major shift to lower
wave numbers by 15–25 cm−1 in azomethine ꢀ(C N) suggesting
involvement of the azomethine-N with Cu(II) and Zn(II) ion in the
complexation. The band corresponding to the stretching vibration
3.4. Electronic spectral studies
The electronic spectra of the Cu(II) complexes (Table 1) showed
one low-energy weak band around 9709–15,385 cm−1 and two
strong high energy bands around 25,840–42,735 cm−1. The low-
energy band in this position typically expected for an octahedral
configuration may be assigned to 10 Dq corresponding to the
2
transition 2Eg ← T2g [21]. The strong high-energy band, in turn,

N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
165
Table 2
The spin Hamiltonian parameters of Cu(II) complexes in DMSO at 300 and 77 K.
Complex
g-tensor
Hyperfine constant
(×10−4 cm−1
)
g
||
g
⊥
giso
A
||
A
⊥
Aiso
1b
[CuL2
[CuL2
]
]
2.6141 2.1289
2.5969 2.1405
2.2906
2.2926
80
90
250
260
193
203
1c
loss of weights is observed up to 675 and 690 ◦C respectively. The
final solid product of thermal decomposition is identified as respec-
1d
tive metal oxide (found: 83.25%, calcd.: 83.39% for CuL2
and
found: 84.52%, calcd.: 84.75% for ZnL21d). The CuL2 and ZnL2
complexes show no mass loss up to 182 ◦C, indicating the absence
of water molecules and any other adsorptive solvents molecules in
coordination sphere. The continuous loss of weight is observed up
to 750 and 800 ◦C respectively; after that the weight of the product
remains constant. The final weight losses in these cases agree with
the formation of the respective metal oxides (found: 83.30%, calcd.:
1a
1a
1a
1d
83.52% for CuL2 and found: 84.57%, calcd.: 84.72% for ZnL21a).
Fig. 1. The TGA curve of CuL2 complex.
From the above thermogravimetric analyses, the overall weight
losses for the CuL21d, ZnL21d, CuL2 and ZnL2 complexes agree
well with the proposed formula obtained by elemental analyses, IR,
1H NMR, Mass and magnetic susceptibility measurements.
1a
1a
is assigned to metal → ligand charge transfer. Also, the mag-
1a–1d
netic moment values (1.65–1.83 BM) for the CuL2
complexes
are indicative of antiferromagnetic spin–spin interaction through
1a–1d
molecular association. Hence, the CuL2
complexes appear to
3.7. EPR studies of Cu(II) complexes
be in the distorted octahedral geometry with dx2 –d ground state
2
y
[22].
To obtain further information about the stereochemistry and
the site of the metal–ligand bonding and to determine the mag-
netic environment in the metal complexes, EPR studies of copper(II)
complexes were carried out. Powder samples were used to record
X-band EPR spectra of the Cu(II) complexes and the spectra were
recorded in DMSO at liquid nitrogen temperature (LNT) and at room
temperature (RT). The spin Hamiltonian parameters and bonding
parameters of the Cu(II) complexes were calculated and are sum-
marized in Tables 2 and 3.
3.5. ESI-mass spectral studies
The electron impact mass spectrum of 1b Schiff base shows
a molecular ion peak m/z at 424 (M+) with a relative intensity
15% which is equivalent to its molecular weight. The different
pathways of the fragments of the parent molecular ion peaks are
given in Scheme 3. The other molecular ion peaks appeared in the
mass spectrum (abundance range from 1% to 100%) is attributed
to the fragmentation of 1b molecule obtained from the rupture of
different bonds inside the molecule as shown in Scheme 3. The
In the present case, Cu(II) complexes, measured in polycrys-
talline sample at room temperature, give the following values:
1b
g = 2.6141, g = 2.1289 for the CuL
and g = 2.5969, g = 2.1405
||
⊥
|| ⊥
2
CuL2 complex gives a molecular ion peak at m/z 910(M+) and
1b
for CuL21c. The trend exhibits an auxiliary symmetric g-tensor
parameters with g > g > 2.0027 indicating that the copper site has
m/z 911(M+1) with a relative intensity 10% and 7% respectively.
The intensities of these peaks give the idea of the stability and
abundance of the fragments. This type of stoichiometry (ML2) is
confirmed by the mass spectra of other complexes. This is in good
agreement with the microanalytical data.
||
⊥
a dx –y ground state characteristic of octahedral geometry [26].
2
2
The parameter G, determined as G = (g − 2)/(g⊥ − 2), which
||
measures the exchange interaction between the metal centers in a
polycrystalline solid, has been calculated. According to Hathaway et
al. [27] if G > 4, the exchange interaction is negligible, but G < 4 indi-
cates considerable exchange interaction in the solid complexes. The
Cu(II) complexes reported in this paper give the “G” values which
are greater than 4 indicating the exchange interaction is absent in
solid complexes.
3.6. Thermal studies
Thermal stability and thermal behaviors of all complexes were
studied by thermogravimetric analysis (TGA) at the atmosphere
of nitrogen in the temperature range 25–900 ◦C. The correlations
between the different decomposition steps of the complexes with
the corresponding weight losses are discussed in terms of the pro-
posed formula of the complexes [23–25]. The thermal behavior
studies of all the complexes are almost same. Hence, the repre-
The observed values of ˛2 and ˇ2 parameters indicate that the
complexes have some covalent character and there is interaction
in the out-of-plane ␲-bonding. The lower values of ˛2 compared
to ˇ2 indicate that the in-plane ␴-bonding is more covalent than
the in-plane ␲-bonding. For the present copper(II) complexes, the
sentative CuL21d, ZnL21d, CuL2 and ZnL2 complexes have been
discussed. The representative thermogram of CuL21d is depicted in
Fig. 1.
1a
1a
observed order K is greater than K implying a greater contribu-
||
⊥
tion from out-of-plane ␲-bonding than from in-plane ␲-bonding
in metal–ligand ␲-bonding. Based on these observations, a dis-
torted octahedral geometry is proposed for Cu(II) complexes. The
The TGA profiles over the temperature range 30–250 ◦C are usu-
ally due to loss of water of moisture, hydration and coordination.
1d
1d
The complexes CuL2 and ZnL2 have first decomposition stage
in the range 35–250 ◦C. This dehydration process probably is due to
the loss of hydration water, which may be bound to hydroxyl group
Table 3
The bonding parameters of Cu(II) complexes in DMSO solution.
2
Complex
˛
ˇ2
ꢁ2
K
||
K
⊥
G
of the ligand by hydrogen bonds. Above 250 ◦C, CuL2 and ZnL2
1d
1d
1b
[CuL2
[CuL2
]
]
0.9276
0.9432
1.1463
1.4632
0.9465
1.3573
1.0632
1.3800
0.8779
1.2802
4.8446
4.3120
decompose in a gradual manner, which may be due to fragmenta-
tion and thermal degradation of the organic moiety. The continuous
1c

166
N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
Scheme 3. Mass fragment pattern of 1b.

N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
167
EPR study of the Cu(II) complexes has provided supportive evidence
to the conclusion obtained on the basis of electronic spectrum and
magnetic moment value.
3.8. Biological activity
The antimicrobial activity results presented in Table 4 show
that the newly synthesized compounds (1a–1d) and their Cu(II)
and Zn(II) complexes possess biological activity. These new deriva-
tives obtained by condensation of 4-(3ꢀ,4ꢀ-benzaldehydene)2-
3-dimethyl-1-phenyl-3-pyrazolin-5-one with the corresponding
semicarbazone/thiosemicarbazone (Scheme 2) were screened for
their antibacterial activity against microorganisms. These results
exhibited markedly an enhancement in activity on coordination
with the metal ions against four testing bacterial strains. This
enhancement in the activity is rationalized on the basis of the struc-
tures of (1a–1d) by possessing an additional azomethine (C N)
linkage which imports in elucidating the mechanism of transami-
nation and resamination reactions in biological system [28]. It has
also been suggested [29,30] that the ligands with nitrogen and oxy-
gen donor systems might restrain enzyme production, since the
enzymes which require these groups for their activity appear to
be especially more susceptible to deactivation by the metal ions
upon chelation. Chelation reduces the polarity [29,30] of the metal
ion mainly because of the partial sharing of its positive charge
with the donor groups and possibly the ␲-electron delocalization
within the whole chelate ring system, thus formed during coordi-
nation. This process of chelation thus increases the lipophilic nature
of the central metal atom, which in turn favors its permeation
through the lipoid layer of the membrane. This in turn is respon-
sible for increasing the hydrophobic character and liposolubility of
the molecule in crossing cell membrane of the microorganism, and
hence enhances the biological utilization ratio and activity of the
testing drug/compound.
Fig. 2. Gel electrophoresis diagram showing the oxidative cleavage of SC pBR322
DNA (30 ␮M) by the Cu(II) and Zn(II) complexes of 1a–1d in the presence of H2O2 in
50 mM Tris–HCl/NaCl buffer (pH 7.2) containing DMF. Lane 1, DNA control; lane 2,
DNA + CuL21a + H2O2; lane 3, DNA + ZnL21a + H2O2; lane 4, DNA + CuL21b + H2O2; lane
5, DNA + ZnL21b + H2O2; lane 6, DNA + CuL21c + H2O2; lane 7, DNA + ZnL21c + H2O2;
lane 8, DNA + CuL21d + H2O2; lane 9, DNA + ZnL21d + H2O2.
Fig. 3. Agarose gel electrophoresis pattern of supercoiled pBR322 DNA in the
presence of concentration of CuL21d, [H2O2] = 10 mM and light after irradiation at
365 nm for 30 min in 50 mM Tris–HCl/NaCl buffer (pH 7.2). Lane 1, DNA control;
1d
1d
1d
lane 2, DNA + CuL2 (0.5 ␮M); lane 3, DNA + CuL2 (1.0 ␮M); lane 4, DNA + CuL2
1d
1d
(1.5 ␮M); lane 5, DNA + CuL2 (2.0 ␮M); lane 6, DNA + CuL2 (2.5 ␮M); lane 7,
DNA + CuL2 (3.0 ␮M); lane 8, DNA + CuL2 (3.5 ␮M).
1d
1d
The order of inhibition with respect to metal ions of 1a–1d is
Cu > Zn. It is found that these complexes show strong antimicrobial
activity at lower concentration when compared to earlier reported
1d
Fig. 4. Cleavage of supercoiled pBR322 DNA in the presence of CuL2 (15 ␮M)
complex and light after irradiation at 365 nm for 30 min in 50 mM Tris–HCl/NaCl
buffer (pH 7.2). Lane 1, DNA control; lane 2, DNA + H2O2 (5 mM); lane 3, DNA + H2O2
(10 mM); lane 4, DNA + H2O2 (15 mM); lane 5, DNA + H2O2 (20 mM); lane 6,
DNA + H2O2 (25 mM); lane 7, DNA + H2O2 (30 mM); lane 8, DNA + H2O2 (35 mM).
1b
1c
literature [31]. But some ZnL2 and ZnL2 have higher antibacte-
1a
rial activity for B. subtilis and P. putita respectively and ZnL2 has
higher antifungal activity for R. stolonifer, compared to the corre-
1a
sponding CuL2 complex.
1a
1a
1c
CuL2 and ZnL2 exhibit lesser activity than CuL21d, CuL2 and
CuL21b. These five complexes can only switch supercoiled form to
nicked form. The difference in cleavage characteristics of these five
complexes appears may be due to the attendance of methoxy moi-
ety and nature of metal ions. The control experiments were carried
out in the attendance of individual complexes together with DNA.
3.9. Oxidative cleavage of DNA
3.9.1. DNA cleavage activity comparison of the synthesized Cu(II)
and Zn(II) complexes
In order to assess the competence of the Cu(II) and Zn(II) com-
plexes for DNA strand scission, pBR322 DNA was incubated with
individual Cu(II) and Zn(II) complexes under identical reaction
conditions. The cleavage reaction was monitored by gel elec-
trophoresis. The delivery of high concentrations of metal ion to the
helix, in locally generating oxygen or hydroxide radicals, leads to an
efficient DNA cleavage reaction. When circular pBR322 DNA is sub-
jected to electrophoresis, DNA cleavage is monitored by relation of
supercoiled circular pBR322 (form I) into nicked circular (form II)
and linear (form III).
As shown in Fig. 2 all of the examined complexes are capable
of promoting oxidative damage of DNA in the presence of H2O2
under physiological conditions (pH 7.2, 37 ◦C), but display differ-
ent cleavage activities [32]. CuL2 and CuL2 reveal the highest
DNA cleavage activity. Under the conditions we used, it can com-
pletely convert supercoiled form to nicked and linear form of DNA.
CuL21b also can switch supercoiled DNA to nicked and linear forms
of DNA, but the supercoiled form is still seen. The difference in
3.9.2. Effects of complex concentration on DNA cleavage
In order to investigate the influence of the concentration of
CuL21d or H2O2 on DNA cleavage reactions, two independent exper-
iments were explored. In the first experiment, the concentration of
H2O2 was kept constant and the concentration of the complexes
was varied (Fig. 3); in the second experiment, the concentration of
the complexes was kept constant and the concentration of H2O2
was varied (Fig. 4). All of the results indicate that the DNA cleav-
age activities of the complexes are obviously both complex and
H2O2 concentration dependent. With the increase in complex or
H2O2 concentration, the supercoiled DNA (SC DNA) decreases and
is finally completely converted to nicked and linear forms. As also
verified, the appearance of forms II and III depends on concentration
of copper complex and H2O2.
1d
1c
3.9.3. Effect of irradiation time on DNA cleavage
cleavage performance of the two complexes appears to be due to
Fig. 5 shows the gel electrophoresis separation of SC pBR322
DNA after incubation with the complexes CuL2 and CuL2 and
1c
1d
1c
the attendance of nitro moiety in 1d and 1c. ZnL21d, ZnL21b, ZnL2
,

168
N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
Table 4
The in vitro antimicrobial activity of ligand and their metal complexes (MIC in ␮g/mL).
Complex
Antibacterial activitya
Antifungal activitya
S. aureus
B. subtilis
E. coli
P. putita
A. niger
R. stolonifer
C. albicans
R. bataicola
1a
1b
1c
21.43
21.34
31.14
28.65
6.25
32.10
34.51
27.35
36.12
7.18
13.25
6.74
5.98
20.43
28.36
36.73
23.76
12.5
18.33
5.42
13.0
9.21
12.4
7.67
11.54
43.28
22.41
35.41
29.54
7.98
10.2
8.67
11.8
10.98
9.21
27.64
36.19
29.61
31.02
4.28
7.29
4.28
8.52
6.98
9.48
5.67
9.58
25.13
37.24
29.48
41.21
4.88
3.54
12.4
9.48
4.29
41.23
27.38
36.40
27.54
9.76
13.2
5.37
8.12
9.54
8.48
4.89
9.94
24.51
45.23
24.85
26.81
3.26
1d
1a
[CuL2
[ZnL2
[CuL2
[ZnL2
[CuL2
[ZnL2
[CuL2
[ZnL2
]
]
]
]
]
]
]
]
1a
1b
1b
1c
1c
11.5
7.28
8.51
11.40
10.51
7.68
9.20
7.84
17.23
9.63
20.11
10.94
17.0
11.02
14.5
9.76
11.87
7.63
16.52
1d
11.5
12.14
1d
10.15
12.01
a
The MIC of standard drugs for antibacterial activity (Streptomycin) and antifungal activity (Nystatin) was found to be <2.45 ␮g/mL.
Fig. 5. Gel electrophoresis diagram showing the cleavage of SC pBR322 (0.5 ␮g)
1d
1c
in the presence of CuL2 and CuL2 complexes (20 ␮M) in 50 mM Tris–HCl/NaCl
buffer (pH 7.2) on photo-irradiation irradiation at 365 nm for 0, 20, 40, 60 and 80 min
(lanes 2–6) for CuL2 complex and (lanes 7–11) for CuL2 complex, lane 1, DNA
alone.
Fig. 7. Gel diagram showing the photocleavage of SC pBR322 DNA (0.5 ␮g) by
1d
1c
using different reagents on monochromatic irradiations of wavelength at 365 nm
1d
for 30 min exposure with CuL2 (25 ␮M) complex, [H2O2] = 10 mM in the presence
of 50 mM Tris–HCl/NaCl buffer (pH 7.2). The reactions were carried out under aerobic
conditions: lane 1, DNA control; lane 2, DNA + CuL21d; lane 3, DNA + CuL21d + H2O2;
1d
lane 4, DNA + NaN3 (100 ␮M) + CuL21d; lane 5, DNA + l-histidine (100 ␮M) + CuL2
;
1d
lane 6, DNA + D2O (14 ␮l) + CuL21d; lane 7, DNA + DMSO (2 ␮l) + CuL2
.
on the DNA cleavage activity indicating non-involvement of singlet
oxygen species in the DNA cleavage reaction. The overall above
observations may suggest that formation of hydroxyl radicals in
the DNA cleavage reaction in the presence of MPA.
Fig. 6. Changes in the agarose gel electrophoretic pattern of pBR322 DNA (0.5 ␮g) in
the presence of reducing agent 3-mercaptopropionic acid (MPA, 5 mM) in the dark
in 50 mM Tris–HCl/NaCl buffer (pH 7.2) containing DMF. Lane 1, DNA control; lane 2,
DNA + MPA; lane 3, DNA + CuL21d; lane 4, DNA + CuL21c; lane 5, DNA + CuL21d + MPA;
lane 6, DNA + CuL21c + MPA; lane 7, DNA + CuL21d + DMSO (2 ␮L) + MPA; lane 8,
DNA + KI (5 ␮L) + CuL21c + MPA; lane 9, DNA + NaN3 (5 ␮L) + CuL21d + MPA.
1d
3.9.5. Photoactivated cleavage of pBR322 DNA by CuL2 complex
There has been considerable interest in DNA endonucleolytic
cleavage reactions that are activated by metal ions [38]. The photo-
induced DNA cleavage activity of the complexes in the absence of
MPA has been studied using UV light. Electrophoresis is carried out
in a dark chamber for 15 min at 50 V in 50 mM Tris–HCl/NaCl buffer
(pH 7.2). When circular plasmid DNA is subjected to electrophore-
sis, relatively fast migration will be observed for the supercoiled
form (form I). If scission occurs on one strand (nicking), the super-
coils will relax to generate a slower-moving open circular form
(form II) [39]. If both strands are cleaved, a linear form (form III)
will be generated that migrates between forms I and II [40]. Fig. 7
shows the gel electrophoretic separations of plasmid pBR322 DNA
irradiation at 365 nm. At the same concentration of complex, with
increasing irradiation time Form II and Form III also increase. Com-
plete cleavage of DNA is observed with a 20 ␮M of complexes on
80 min exposure. It is known that ligands containing thio or thione
moieties show efficient intersystem crossing to the triplet state
on photo-irradiation and such an effect is found to be pronounced
only when the sulphur is bonded to the metal center [33,34]. Sim-
ilar results have been reported in other cases [35]. The different
DNA cleavage efficiency of these two complexes may be due to the
different binding affinity of the complexes to DNA [36].
1d
after incubation with CuL2 complex and irradiation at 365 nm in
1d
the presence of varying radical scavengers (lanes 4–7). The CuL2
complex is incubated with DNA, in the absence or in the presence
of hydrogen peroxide as an oxidant agent. H2O2 itself can induce
some single-strand scission in DNA molecules, as indicated by the
small decrease in the concentration of supercoiled plasmid DNA
(form I), and corresponding small increase in the concentration of
relaxed circle (form II) as shown in Fig. 7 (lanes 2 and 3). To test
the possibility that photo-induced cleavage involves the formation
of singlet oxygen, which is known to react with guanine residues
at neutral pH [41], the cleavage is tested in the presence of D2O.
Singlet oxygen would be expected to induce more strand scissions
in D2O than in H2O due to its longer lifetime in the former sol-
vent. It can also be seen in Fig. 7 that neither irradiation of DNA at
3.9.4. Effect of supercoiled pBR322 DNA by 3-mercaptopropionic
acid (MPA)
1d
1c
The DNA cleavage activity of the CuL2 and CuL2 complexes
has been studied in the presence of MPA as reducing agent (Fig. 6).
1d
1c
The CuL2 and CuL2 complexes efficiently cleave SC DNA in the
presence of MPA, whereas, in the absence of MPA, do not show
any significant cleavage of SC pBR322 DNA. Control experiments
indicate the formation of the hydroxyl radical and/or copper-oxo
species as the cleavage active species [37]. Control experiments
1d
1c
using CuL2 and CuL2 have shown inhibition of cleavage in the
presence of hydroxyl radical scavenger like DMSO or KI, while the
singlet oxygen quencher, NaN3, does not show any apparent effect
1d
1d
365 nm without CuL2 nor incubation with CuL2 without light

N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
169
Table 5
Electrochemical behavior of copper(II) complexes in the presence of CT DNA.
Complex
Redox couple
Ipc (A) (× 10−5
)
Epc (V)
E1/2 (V)
ꢂEp (V)
K+/K2+
Free
Bound
Free
Bound
Free
Bound
Free
Bound
1a
[CuL2
]
Cu(III–II)
Cu(II–I)
0.37
0.33
0.31
0.19
0.061
−0.454
0.096
−0.479
0.162
−0.276
0.211
−0.307
0.202
0.344
0.231
0.307
3.9
0.38
1b
1c
[CuL2
[CuL2
]
]
Cu(III–II)
Cu(II–I)
2.28
2.16
1.73
2.01
0.197
−0.965
0.265
−0.942
0.531
−0.791
0.519
−0.770
0.668
0.348
0.509
0.344
14.1
2.44
1d
[CuL2
]
Cu(III–II)
Cu(II–I)
0.23
0.37
–
0.32
0.166
−0.152
–
0.241
–
–
–
0.150
–
–
–
–
0.41
−0.175
yields significant strand scission. The DNA photocleavage studies
of Cu(II) and Zn(II) complexes are consistent with earlier reports
[42,43,10]
indicates that they have distorted octahedral [44] configurations
around metal ions. This is further supported by their electronic
spectral data and CV studies.
3.10. Magnetic measurement studies
3.11. DNA binding studies
The magnetic moments of the complexes were recorded at
room temperature and the observed magnetic moment values for
the Cu(II) complexes of 1a–1d are 1.72, 1.83, 1.65 and 1.78 BM
respectively. These values are agreeable to spin only value. Hence
observed magnetic moment for the Cu(II) complexes under study
DNA binding experiments were carried out in 0.5 mL
Tris–HCl/NaCl buffer [50 mM Tris–HCl and 5 mM NaCl (pH
7.2)] using DMF solution (15 ␮L) of the complexes. Absorption
titration measurements were done by varying the concentration of
CT DNA but keeping the metal complex concentration as constant.
Fig. 8. Electronic spectra of Cu2+ complexes of (A) 1a, (B) 1b, (C) 1c and (D) 1d (10−3 M) in dimethylformamide in the absence (- - -) and presence (– – –)of CT DNA. Arrow
shows that the absorbance changes upon increasing DNA concentrations. Inset: plots of [DNA]/(εa – εf) versus [DNA] for the titration of CT DNA with the complexes.

170
N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
Fig. 9. Cyclic voltammogram at scan rate varying 0.05–0.250 V s−1 at 25 ◦C for Cu2+ complexes of (A) 1a, (B) 1b, (C) 1c and (D) 1d (10−3 M) in DMF containing 0.05 M n-Bu4NClO4
as supporting electrolyte at a Pt disc working electrode.
The different modes of interaction of a metal complex with DNA
can be studied not only by this technique but also by cyclic
voltammetry (CV) and differential pulse voltammetry (DPV). These
were employed to probe the binding of metal complexes to DNA
in solution. DPV data were used to obtain quantitative information
about the interaction of these metal complexes with CT DNA.
Viscosity experiments were carried out using an Ubbelodhe vis-
cometer maintained at a constant temperature at 30.0 0.1 ◦C in
a thermostatic water bath. Flow time was measured with a digi-
tal stopwatch, and each sample was measured three times, and an
average flow time was calculated. Data were presented as (Á/Á0)1/3
versus binding ratio [45], where Á is the viscosity of CT DNA in the
presence of complex, and Á0 is the viscosity of CT DNA alone.
3.11.1. Absorption spectra studies
The application of electronic absorption spectroscopy in DNA
binding studies is one of the most useful techniques [46] is carried
out at 25 2 ◦C. The electronic spectral trace of Cu(II) complexes
titrated with DNA are given in Fig. 8. Complex binding with DNA
through intercalation usually results in hypochromism and blue
shift, due to the intercalative mode involving a strong stacking
interaction between an aromatic chromophore and the base pairs
of DNA. The extent of the hypochromism commonly parallels the
intercalative binding strength. In order to compare quantitatively
the binding strength of the copper complexes, the intrinsic binding
constants Kb of the copper complexes with DNA were obtained by
monitoring the changes in absorbance with increasing concentra-
Scheme 4. A Nernstian electron transfer to a system in which both the oxidized and reduced forms are associated with a third species in solution (DNA).

N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
171
Fig. 10. Differential pulse voltammograms of (10−3 M) mol L−1 of Cu2+ complexes of (A) 1a (scan rate 0.05 V s−1) and (B) 1b (scan rate 0.250 V s−1) with incremental addition
of DNA in DMF–Tris–HCl/NaCl buffer solution (pH 7.2).
tion of DNA using the following equation:
of 1a, marked decreases in the peak current heights and shifts of
peak potentials to more negative values are observed [55]. The
shift in reduction peak potential observed between the reduc-
tion of Cu(III)/Cu(II) and reduction of Cu(II)/Cu(I) in complex of 1a
(Scheme 4) is indicative for strong copper binding.
[DNA]
εa − εf
[DNA]
εb − εf
1
=
+
Kb(εb − εf)
where [DNA] is the concentration of DNA in base pairs, the appar-
ent absorption coefficients εa, εf and εb correspond to Aobs/[Cu],
the extinction coefficient for the free copper complexes and the
extinction coefficient for the free copper complexes in the fully
bound form respectively. In plots of [DNA]/(εa − εf) versus [DNA],
Kb is given by the ratio of slope to the intercept [47–49]. Intrinsic
binding constants Kb of copper complexes of 1a–1d are obtained as
1.99 × 104, 3.57 × 104, 3.79 × 104 and 3.93 × 104 M−1 respectively.
Our results are consistent with earlier reports on preferential bind-
ing to CT DNA in the copper complexes [50–53].
The cyclic voltammogram recorded for the CuL21b, reveals
one electron quasi-reversible Cu(III)/Cu(II) wave with E1/2 val-
ues as 0.531 and 0.519 V for free and bound forms respectively.
In the absence of DNA, the complex involves single redox step
corresponding to Cu(III)/Cu(II) at Epc = 0.197 V and the associated
anodic peak at Epa = 0.865 V. On addition of CT DNA, the complex
experiences a shift in E1/2 as well as in Ep values. The ratio of
Ipa/Ipc for the bound complex of 1b is less than unity, suggest-
ing that CT DNA is bound strongly to the complex. In addition
to the changes in the formal potential, the voltammetric peak
decreases upon addition of CT DNA to the complex. The decrease
in the current is due to the diffusion of the equilibrium mixture
of free and DNA-bound metal complex to the electrode surface
[56]. In the absence of DNA one extra anodic peak is appeared
at −0.077 V and no reduction peak, suggesting that unstabiliza-
tion of Cu(I) species may be due to no d–␲ interactions between
the copper-d orbitals and the aromatic ␲-system rather than Cu(II)
species.
3.11.2. Electrochemical studies
Table 5 summarizes the electrode potentials for all complexes
of Cu(II) in DMF. The cyclic voltammetric studies provide an insight
to the CT DNA binding. Typical CV behavior of series of Cu(II) com-
plexes of 1a–1d in the absence and presence of CT DNA is shown in
Fig. 9. The difference between forward and backward peak poten-
tials can provide a rough evaluation of the degree of the reversibility
of one electron transfer reaction. The analysis of cyclic voltam-
metric responses with the scan rate varying 50–250 mV/s gives
the evidence for a quasi-reversible one electron oxidation. The
decreased extents of the peak currents observed for all Cu(II) com-
plexes upon addition of CT DNA indicate that complexes interact
with DNA through intercalating way. The Nernst equation can be
used to estimate the ratio of equilibrium constants for the bind-
ing of the oxidative and reductive ions to DNA according to the
literature [54]
1c
In the absence of DNA, the electrochemical behavior of CuL2
shows a redox process corresponding to the Cu(II)/Cu(I) couple at
Epa = −0.617 V and the associated cathodic peak at Epc = −0.965 V.
The addition of CT DNA causes diminution of the peak currents of
the reduction of Cu(II)/Cu(I). It is due to variation of the binding
state and the slower mass transfer of complex bound to DNA frag-
ments. It has been shown that binding of the metal complex to
DNA can bring about a shift in the redox potential due to its inter-
calative binding with DNA which indicates that the reaction of the
complex with DNA on the GC electrode is a diffusion controlled
quasi-reversible redox process.
ꢀ
ꢁ
Kred
Kox
E◦b − E◦ = 0.0591 log
f
1d
The CuL2 exhibits one quasi-reversible peak in the absence
The drop of the voltammetry currents in the presence of DNA
may be attributed to slow diffusion of the metal complex bound to
CT DNA. This in turn indicates the extent of binding affinity of the
complex to DNA.
of DNA. The representative cyclic voltammogram of Cu(II) com-
plex displays two reduction peaks, one at Epc = 0.166 V with an
associated oxidation peak at Epa = 0.316 V, second reduction peak
at Epc = −0.152 V with no associated oxidation peak corresponding
to the Cu(III)/Cu(II) and Cu(II)/Cu(I) respectively. The incremental
addition of CT DNA to the complex the second cathodic peak causes
a shift in the potential and decrease in the current intensity, but in
the first cathodic peak there is no significant change in the poten-
tial as well as current intensity, suggesting that the changes of peak
In the cyclic voltammogram of CuL21a, in the absence of DNA
two well-defined quasi-reversible one electron cyclic responses
have been observed at Epc = 0.061 V with a corresponding oxi-
dation peak at Epa = 0.263 V [Cu(III)/Cu(II)] and at Epc = −0.454 V
with a corresponding oxidation peak at Epa = −0.098 V [Cu(II)/Cu(I)]
respectively. The ratio of cathodic to anodic peak height is less
than one. When CT DNA is added to a solution of Cu(II) complex
1d
currents of the reduction of Cu(II)/Cu(I) observed for CuL2 may

172
N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
Fig. 11. Effect of increasing amounts of EB ( ) and in the presence of increasing
1a
concentrations of copper complexes of CuL21d (ꢀ), CuL21c (ꢁ), CuL21b (ꢂ) and CuL2
(×) on the relative viscosity of CT DNA at 30 ◦C. [DNA] = 1.5 mM, R = [complex]/[DNA]
or [EB]/[DNA].
Fig. 13. Molecular structure of the complex, where M = Cu2+ and Zn2+
.
indicate that complex possesses higher DNA binding affinity with
complex.
binding model of Cu(II) and Zn(II) complexes with DNA. A clas-
sical intercalation mode causes a significant increase in viscosity
of DNA due to an increase in separation of base pairs at inter-
calation sites and hence an increase in overall DNA length [57].
The well-known DNA intercalator ethidium bromide (EB) increases
the viscosity of DNA with increments of the concentration. On
the other hand, the viscosity of DNA increases dramatically upon
addition of the title complexes. The changes in relative viscos-
ity of CT DNA in the presence of complexes Cu(II), Zn(II) and EB
are shown in Figs. 11 and 12. The increased degree of viscosity,
which may depend on its affinity to DNA follows the order of
EB > 1a > 1b > 1c > 1d, which is consistent with the above results
of Cu(II) and Zn(II) proposes to be bound to DNA by intercalation
[42,10].
3.11.3. Differential pulse voltammetry (DPV)
1a
Differential pulse voltammogram (DPV) of the CuL2
and
1b
CuL2 both in the presence and in the absence of DNA is given
in Fig. 10. The peak potential and current of the both complexes are
changed in the presence of DNA. K+/K2+ value for the copper com-
plexes suggesting that the preferential stabilization of Cu(II) form
over Cu(III) and Cu(I) form on binding to DNA. DPV of the com-
plexes as a function of added DNA also indicates a large decrease in
current intensity with a small shift in formal potential due to the
intercalative interaction of complexes.
3.11.4. Viscosity measurements
The bonding between the complexes and DNA is further elu-
cidated by viscosity measurements. Optical photophysical probes
generally provide necessary, but insufficient clues to support a
4. Conclusions
Based on the above observations of the elemental analysis,
molar conductivity, UV–vis, magnetic, IR and 1H NMR spectral
data it is possible to determine the type of coordination of the
ligands in their metal complexes. The spectral data show that all
the Schiff bases exist as tridentate ligands by bonding to the metal
ion through the deprotonated enol/thiol-O/S groups and azome-
thine nitrogen. The analytical data show the presence of one metal
ion per two ligand molecules and suggest a mononuclear structure
for the complexes. The correlation of the experimental data allows
assigning an octahedral stereochemistry to all the above synthe-
sized complexes as shown in Fig. 13. Cu(II) and Zn(II) complexes
have been synthesized and their abilities to induce oxidatively
generated DNA damage are compared. The observed efficient pho-
1d
tonuclease activity of CuL2
under UV light on short exposure
time and with a lesser concentration of the metal complex is
a significant result in the chemistry of copper-based nucleolytic
agents. Electronic spectra, cyclic voltammetry, differential pulse
voltammogram and viscosity measurements indicate that the com-
plexes bind to DNA by intercalative modes. It is found that the
metal complexes have more biological activity than the free lig-
ands.
Fig. 12. Effect of increasing amounts of EB (*) and in the presence of increasing
1d
1c
1b
1a
concentrations of zinc complexes of ZnL2 (×), ZnL2 (ꢂ), ZnL2 (ꢁ) and ZnL2
(ꢀ) on the relative viscosity of CT DNA at 30 ◦C. [DNA] = 1.5 mM, R = [complex]/[DNA]
or [EB]/[DNA].

N. Raman et al. / Spectrochimica Acta Part A 76 (2010) 161–173
173
Acknowledgments
[24] M.M. Omar, G.G. Mohamed, Spectrochim. Acta Part A 61 (2005) 929–936.
[25] M. Thankamony, B. Sindhu Kumari, G. Rijulal, K. Mohanan, J. Therm. Anal.
Calorim. 95 (2009) 259–266.
[26] G. Speir, J. Csihony, A.M. Whalen, C.G. Pierpont, Inorg. Chem. 35 (1996)
3519–3524.
[27] B.J. Hathaway, J.N. Bardley, R.D. Gillard (Eds.), Essays in Chemistry, Academic
Press, New York, NY, USA, 1971.
The authors express their sincere thanks to the College Man-
aging Board, Principal and Head of the Department of Chemistry,
VHNSN College, for providing necessary research facilities and
financial support.
[28] P.P. Dholakiya, M.N. Patel, Synth. React. Inorg. Met.-Org. Chem. 34 (2004)
553–563.
[29] Z.H. Chohan, Appl. Organometal. Chem. 20 (2006) 112–116.
[30] Z.H. Chohan, M. Arif, Z. Shafiq, M. Yaqub, C.T. Supuran, J. Enzyme Inhib. Med.
Chem. 21 (2006) 95–103.
[31] P.V. Rao, A.V. Narasaiah, Indian J. Chem. A 42 (2003) 1896–1899.
[32] V.G. Vaidyanathan, B. Unni Nair, J. Inorg. Biochem. 93 (2003) 271–276.
[33] X. Qian, T.B. Huang, D.-Z. Wei, D.-H. Zhu, M.-C. Fan, W. Yao, J. Chem. Soc., Perkin
Trans. 2 (2000) 715–718.
[34] A. Jacobs, J. Piette, J. Photochem. Photobiol. B 22 (1994) 9–15.
[35] K. Li, L.H. Zhou, J. Zhang, S.Y. Chen, Z.W. Zhang, J.J. Zhang, H.H. Lin, X.Q. Yu, Eur.
J. Med. Chem. 44 (2009) 1768–1772.
[36] X.L. Wang, H. Chao, H. Li, X.L. Hong, Y.-J. Liu, L.N. Ji, Trans. Met. Chem. 30 (2005)
305–311.
[37] T.B. Thederahn, M.D. Kuwabara, T.A. Larsen, D.S. Sigman, J. Am. Chem. Soc. 111
(1989) 4941–4946.
[38] R.P. Hertzberg, P.B. Dervan, J. Am. Chem. Soc. 104 (1982) 313–315.
[39] J.K. Barton, A.L. Raphael, J. Am. Chem. Soc. 106 (1984) 2466–2468.
[40] X. QianHe, Q. YueLin, R. DingHu, X. HongLu, Spectrochim. Acta Part A 68 (2007)
184–190.
[41] P.A.N. Reddy, B.K. Santra, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 98
(2004) 377–386.
[42] P. Xi, Z. Xu, F. Chen, Z. Zeng, X. Zhang, J. Inorg. Biochem. 103 (2009) 210–218.
[43] A. Hangan, A. Bodoki, L. Oprean, G. Alzuet, M. Liu-González, J. Borrás, Polyhedron
29 (2010) 1305–1313.
[44] N.S. Biradar, B.R. Havinale, Inorg. Chim. Acta 17 (1979) 157–160.
[45] G. Cohen, H. Eisenberg, Biopolymers 8 (1969) 45–49.
[46] J.K. Barton, J.J. Dannenberg, A.L. Raphael, J. Am. Chem. Soc. 106 (1984)
2172–2176.
[47] L.F. Tan, H. Chao, Y.F. Zhou, L.N. Ji, Polyhedron 26 (2007) 3029–3036.
[48] L.F. Tan, X.H. Liu, H. Chao, L.N. Ji, J. Inorg. Biochem. 101 (2007) 56–63.
[49] Y.J. Liu, H. Chao, Y.X. Yuan, H.J. Yu, L.N. Ji, Inorg. Chim. Acta 359 (2006)
3807–3814.
[50] Y.H. Li, B.D. Wang, Z.Y. Yang, Spectrochim. Acta Part A 67 (2007) 395–401.
[51] A.K. Patra, M. Nethaji, A.R. Chakravarty, J. Inorg. Biochem. 101 (2007) 233–244.
[52] F. Arjmand, M. Aziz, Eur. J. Med. Chem. 44 (2009) 834–844.
[53] N. Raman, R. Jeyamurugan, A. Sakthivel, L. Mitu, Spectrochim. Acta Part A 75
(2010) 88–97.
[54] M.T. Carter, M. Rodriguez, A.J. Bard, J. Am. Chem. Soc. 111 (1989) 8901–8911.
[55] R. Jain, S. Gupta, Bull. Electrochem. 14 (1998) 57–61.
[56] T.W. Weltch, H.H. Tholp, J. Phys. Chem. 100 (1996) 13829–13836.
[57] B. Peng, X. Chen, K.J. Du, B. LeYu, H. Chao, L. NianJi, Spectrochim. Acta Part A 74
(2009) 896–901.
References
[1] M.M.B. Pessoa, G.F.S. Andrade, V.R.P. Monteiro, M.L.A. Temperini, Polyhedron
20 (2001) 3133–3141.
[2] A.E. Liberta, D.X. West, Biometals 5 (1992) 121–126.
[3] N. Raman, R. Jeyamurugan, B. Raj kapoor, V. Mehesh, Appl. Organometal. Chem.
23 (2009) 283–290.
[4] M.A. Jakupec, M. Galanski, B.K. Keppler, Rev. Physiol. Biochem. Pharmacol. 146
(2003) 1–53.
[5] L. Giovagnini, L. Ronconi, D. Aldinucci, D. Lorenzon, S. Sitran, D. Fregona, J. Med.
Chem. 48 (2005) 1588–1595.
[6] J.R. Dimmock, S.C. Vashishtha, J.P. Stables, J. Med. Chem. 35 (2000) 241–248.
[7] T. Klimova, E.I. Klimova, M. Mertinez Garcia, J.M. Mendez stivalet, L.R. Ramirez,
J. Organomet. Chem. 633 (2001) 137–142.
[8] N. Bharti, K. Husain, M.T. Gonzalez Garza, D.E. Cyuz-Vega, J. Castro-Garza,
Bioorg. Med. Chem. Lett. 12 (2002) 3475–3478.
[9] B. Peter, B. Roman, M. Breza, H. Elias, H. Fuess, V. Jorik, R. Klement, I. Svoboda,
Polyhedron 21 (2002) 1561–1571.
[10] A.K. Patra, S. Roy, A.R. Chakravarty, Inorg. Chim. Acta 362 (2009) 1591–1599.
[11] Vogel A Text Book of Quantitative Inorganic Analysis, third ed., ELBS, Longman,
London, 1969.
[12] L.G. Van Waasbergen, I. Fajdetic, M. Fianchini, H.V. Rasika dias, J. Inorg. Biochem.
101 (2007) 1180–1183.
[13] D. Liu, K. Kwasniewska, Bull. Environ. Contam. Toxicol. 27 (1981) 289–294.
[14] R.N. Patel, N. Singh, K.K. Shukla, U.K. Chauhan, S. Chakraborty, J. Niclos-
Gutierrez, A. Castineiras, J. Inorg. Biochem. 98 (2004) 231–237.
[15] L.J. Bellamy, The Infrared Spectra of Complex Molecules, John Wiley & Sons,
New York, NY, 1971.
[16] J.R. Ferrero, Low-Frequency Vibrations of Inorganic and Coordination Com-
pound, John Wiley & Sons, New York, NY, 1971.
[17] P. Bindu, M.R. Kurup, T.R. Satyakeerty, Polyhedron 18 (1998) 321–331.
[18] K. Nakamoto, Infrared Spectra of Inorganic and Coordination Compounds, sec-
ond ed., Wiley Interscience, New York, NY, 1970.
[19] W.W. Simmons, The Sadtler Handbook of Proton NMR Spectra, Sadtler Research
Laboratories, Philadelphia, PA, 1978.
[20] D.J. Pasto, Organic Structure Determination, Prentice Hall, London, UK, 1969.
[21] V. Reddy, N. Patil, T. Reddy, S.D. Angadi, Eur. J. Chem.
538.
[22] C.J. Ballhausen, An Introduction to Ligand Field Theory, McGraw Hill, New York,
NY, 1962.
5 (2008) 529–
[23] F. Karipcin, E. Kabalcılar, Acta Chim. Slov. 54 (2007) 242–247.