Synthesis, characterization and biological activities of homo-binuclear Cu(II) and Zn(II) complexes derived from 2-hydroxy-5-methyl-1,3-benzenedicarboxaldehyde derivatives

Article abstract of DOI:10.2478/s11532-009-0103-2

Few novel binuclear Schiff base metal complexes [M₂LCl₃], where M = Cu(II) and Zn(II); L= 2,6-bis-({2-[(3-hydroxy-4-nitrobenzylidene)amino]ethylimino}methyl)-4-methylphenol (BHEM), 2,6-bis-({2-[(3,4-dimethoxybenzylidene)amino]ethylimino} methyl)-4-methylphenol (BDEM) and 2,6-bis-({2-[(2,3,5-trichlorobenzylidene)amino]ethylimino}methyl)-4-methylphenol (BTEM), have been synthesized and characterized by analytical and spectral data. The data suggest that BHEM/BDEM/BTEM ligands afford square-pyramidal/distorted square-pyramidal geometry on metalation with Zn(II)/Cu(II). The binding behaviour of these complexes with DNA has been investigated using electronic absorption spectroscopy as well as viscosity and voltammetric measurements; the results show that they interact with DNA through intercalating way. From the DNA cleavage study of these complexes, investigated by gel electrophoresis, we found that they efficiently cleave supercoiled pUC19 DNA in the presence of a reducing agent (3-mercaptopropionic acid) and on irradiation with UV light of 360 nm wavelength. The mechanism reveals that singlet oxygen (¹O₂) plays a significant role in the photo cleavage. The superoxide dismutase (SOD) mimetic activity of the synthesized complexes demonstrates that most of the complexes have promising SOD-mimetic activity. The antimicrobial study indicates that the complexes inhibit the growth of bacteria and fungi more than the free ligands. Versita Warsaw and Springer-Verlag Berlin Heidelberg.

Full text of DOI:10.2478/s11532-009-0103-2

Cent. Eur. J. Chem. • 8(1) • 2010 • 96–107
DOI: 10.2478/s11532-009-0103-2
Central European Journal of Chemistry
Synthesis, characterization and biological
activities of homo-binuclear Cu(II) and Zn(II)
complexes derived from 2-hydroxy-5-methyl-1,3-
benzenedicarboxaldehyde derivatives
Research Article
Natarajan Raman*, Arumugam Sakthivel, Ramaraj Jeyamurugan
Research Department of Chemistry, VHNSN College,
Virudhunagar-626001, India
Received 30 April 2009; Accepted 17 July 2009
Abstract: Few novel binuclear Schiff base metal complexes [M2LCl3], where M = Cu(II) and Zn(II); L= 2,6-bis-({2-[(3-hydroxy-4-
nitrobenzylidene)amino]ethylimino}methyl)-4-methylphenol (BHEM), 2,6-bis-({2-[(3,4-dimethoxybenzylidene)amino]ethylimino}
methyl)-4-methylphenol (BDEM) and 2,6-bis-({2-[(2,3,5-trichlorobenzylidene)amino]ethylimino}methyl)-4-methylphenol (BTEM),
have been synthesized and characterized by analytical and spectral data. The data suggest that BHEM/BDEM/BTEM ligands afford
square-pyramidal/distorted square-pyramidal geometry on metalation with Zn(II)/Cu(II). The binding behaviour of these complexes with
DNA has been investigated using electronic absorption spectroscopy as well as viscosity and voltammetric measurements; the results
show that they interact with DNA through intercalating way. From the DNA cleavage study of these complexes, investigated by gel
electrophoresis, we found that they efficiently cleave supercoiled pUC19 DNA in the presence of a reducing agent (3-mercaptopropionic
acid) and on irradiation with UV light of 360 nm wavelength. The mechanism reveals that singlet oxygen (1O2) plays a significant role
in the photo cleavage. The superoxide dismutase (SOD) mimetic activity of the synthesized complexes demonstrates that most of the
complexes have promising SOD-mimetic activity. The antimicrobial study indicates that the complexes inhibit the growth of bacteria
and fungi more than the free ligands.
Keywords: Binuclear complexes • Photo cleavage • DNA binding • SOD activity
© Versita Warsaw and Springer-Verlag Berlin Heidelberg.
1. Introduction
are interested in designing multimetal models [5,6];
complexes with polyaza ligands are of special interest
The design and synthesis of binucleating ligands and
their complexes has been of much interest in the recent
past [1]. The studies done on binuclear metal complexes
in which the two metal centers are held in close
proximity have addressed [2,3] the ligand environment,
redox behaviour, magnetic exchange interactions and
spectroscopic properties. Considerable interest has also
been placed on synthesis and biomimetic reactions of
model systems related to metalloenzymes [4]. In the
area of development of artificial nucleases researchers
due their high DNA cleavage activity [7,8].
DNA cleavage by metal complexes generally occurs
viaoxidativeandhydrolyticpathways[9,10].Theoxidative
DNA cleavage involves abstraction of sugar hydrogen
atom and/or damage of DNA by nucleobase oxidation
whereas hydrolytic cleavage involves hydrolysis of the
phosphodiester bond. The photo-activated oxidative
cleavage of DNA has recently received considerable
attention due to its utility in highly targeted photo-
chemotherapeutic applications. For example porphyrin
* E-mail: drn_raman@yahoo.co.in
96

N. Raman, A. Sakthivel, R. Jeyamurugan
andphthalocyanin-basedcompoundshavefoundclinical oxalate. In a typical experiment, about 0.3 g of the
applications in photodynamic therapy (PDT) of cancer dried complex was accurately weighed in a previously
[11,12]. Among the non-porphyrinic species, the DNA weighed silica crucible. AnalaR ammonium oxalate,
photo-cleavage activity of 4d and 5d metal complexes roughly 3 parts by weight of the complex was added
has been studied in detail [13,14]. In comparison, the and the mixture was incinerated slowly at first and
chemistry of 3d metal based model photonuclease has then more vigorously using a Bunsen burner for
remained relatively unexplored [15,16]. Hence, in this 3 h. It was then cooled in a desiccator and weighed. The
work we report the synthesis, characterization, and procedure was repeated till the final weight of the oxide
antimicrobial, DNA binding and DNA cleavage activities was constant. The percentage of metal in the complex
of binuclear Cu(II) and Zn(II) complexes having tertraaza was calculated from the weight.
ligands.
2.3. Synthesis of ligands
The
precursor
(2-hydroxy-5-methyl-1,3-
2. Experimental procedure
benzenedicarboxaldehyde) was synthesized by the
method described in [19]. The outline of the synthesis of
the ligands using this precursor is shown in Scheme 1.
2.1. Materials and Methods
All reagents and chemicals were procured from
Merck products. Solvents used in electrochemical and
spectroscopic studies were purified using standard
procedures [17]. DNA was purchased from Bangalore
Genei (India). Agarose (molecular biology grade) and
ethidium bromide were obtained from Sigma (USA).
C, H and N analyses of the complexes were carried
out on a CHN analyzer Calrlo Erba 1108, Heraeus.
The IR (KBr discs) of the samples was recorded on
a Perkin–Elmer 783 series FTIR spectrophotometer.
The UV-Vis, spectra of the samples were recorded on
1
a Shimadzu UV-1601 spectrophotometer. H and 13C
NMR spectra (300 MHz) of the ligands and their zinc
complexes were recorded on a Bruker Avance DRX
300 FT-NMR spectrometer in CDCl3 and DMSO-d6
respectively using trimethylsilane (TMS) as the internal
standard. The fast atom bombardment (FAB) mass
Scheme 1. The outline of the synthesis of the ligands
spectra of the complexes were recorded on a JEOL SX 2.3.1. Preparation of N΄-(3,4-dimethoxybenzylidene)
102/DA-6000 mass spectrometer using Argon/Xenon ethane-1,2-diamine
(6 kV, 10 mA) as the FAB gas. The accelerating voltage An ethanolic solution (20 mL) of ethylenediamine
was 10 kV and the spectra were recorded at room (0.60 g; 10 mM) was slowly added, with stirring, to an
temperature using m-nitrobenzyl alcohol as the matrix. ethanolicsolution(40mL)of3,4-dimethoxybenzaldehyde
Molar conductance of 10-3M solution of the complexes (1.67 g; 10 mM). The mixture was refluxed for 1 h and
in N,N-dimethylformamide (DMF) was measured at then the clear solution was allowed to cool to room
room temperature with an Deepvision Model-601 digital temperature. The solid material formed was removed by
direct reading deluxe conductivity meter. Magnetic filtration and recrystallised from EtOH.
susceptibility measurements were carried out by
employing the Gouy method at room temperature on diamine
N΄-(2,3,5-trichlorobenzylidene)ethane-1,2-
and N΄-(3-hydroxy-4-nitrobenzylidene)
powder sample of the complex using CuSO4•5H2O as ethane-1,2-diamine were synthesized according to the
the calibrant. Electrochemical measurements were procedure described above by the replacement of 3,4-
performed on a CHI 620C electrochemical analyzer. dimethoxybenzaldehyde by 2,3,5-trichlorobenzaldehyde
The purity of ligands and their complexes was evaluated (2.09 g; 10 mM) and 3-hydroxy-4-nitrobenzaldehyde
by column and thin layer chromatography.
(1.67 g; 10 mM), respectively.
2.2. Estimation of metal
2.3.2. Synthesis of BDEM ligand
The metals, in general, were estimated gravimetrically N΄-(3,4-dimethoxybenzylidene)ethane-1,2-diamine
as their oxides [18] by fusion with AnalaR ammonium (2.08 g; 10 mM) and 2-hydroxy-5-methyl-1,3-
97

Synthesis, characterization and biological activities
of homo-binuclear Cu(II) and Zn(II) complexes derived from
2-hydroxy-5-methyl-1,3-benzenedicarboxaldehyde derivatives
benzenedicarboxaldehyde (0.82 g; 5 mM) were refluxed (– NO2), 1598 (–CH=N), 2838 (methyl –CH3), 497
for 3 h in ethanolic solution (40 mL). The pale yellow (M–O), 428 (M–N) and 282 (M–Cl) m/z: 780, Anal.
precipitate that crystallized out from the clear solution Calcd for [Cu2C27H25N6O7Cl3] : Cu, 16.3, C, 41.6, H, 3.2,
upon cooling was filtered off, washed with EtOH and N, 10.8: Found: Cu, 15.6, C, 41.1, H, 3.3, N 10.1(%). λM
dried in vacuo. Yield: 1.85 g; (64%). IR (KBr, cm-1): 10-3 (ohm-1 cm2 mol-1), 10.4; µeff (BM), 1.48. UV-visible
2855 (methyl –CH3), 1628 (–CH=N), 3250-3500 (–OH). [λmax, nm, (ε, dm3 mol–1 cm–1)] in DMF, 262 (28459), 355.5
1H-NMR (CDCl3): (phenyl multiplet) 6.6-7.5 δ, (–OCH3) (12983) and 628 (179); IC50, 31µmol/ dm–1. [Cu2(BTEM)
3.8 δ, (–CH3) 2.2 δ, (–CH2–) 3.4 δ (t, J = 7.25 Hz, 4H), Cl3]: Yield: 2.87 g; (59 %). IR (KBr, cm-1): 1605 (–CH=N),
(–OH) 12.3 δ, (–CH=N–) 8.3 δ; m/z: 546; Anal. Calcd for 2862 (methyl–CH3), 485 (M–O), 441 (M–N), 271
[C31H37N4O5]: C, 68.3, H, 6.8, N, 10.2; Found: C, 67.7, (M–Cl); m/z: 864, Anal. Calcd for [Cu2C27H21N4OCl9] :
H 6.7, N. 9.7 (%); UV-visible [λmax, nm, (ε, dm3 mol–1 cm–1)] Cu, 14.7, C, 37.5, H, 2.4, N, 6.4; Found: Cu, 14.2, C,
in EtOH, 272 (32740) and 384 (11789).
37.1, H 2.4, N, 5.9(%). λM 10-3 (ohm-1 cm2 mol-1), 7.8; µeff
Ligands BHEM and BTEM were synthesized (BM), 0.98. UV-visible [λmax, nm, (ε, dm3 mol–1 cm–1)] in
according to the procedure described above by the DMF, 260 (27451), 363 (10789) and 647 (174), IC50,
replacement of N΄-(3,4-dimethoxybenzylidene)ethane- 27 µmol/ dm–1
.
1,2-diamine
ethane-1,2-diamine (2.89 g; 10 mM) and N΄-(2,3,5- 2.3.4. Synthesis of zinc complexes
trichlorobenzylidene)ethane-1,2-diamine (2.51 g; Zinc chloride (1.36 g; 10mM) in EtOH (40 mL) was
by
N΄-(3-hydroxy-4-nitrobenzylidene)
10 mM), respectively. BHEM: Yield: 1.92 g; (52%). IR added, with stirring, to an ethanolic solution (40 mL)
(KBr, cm-1): 3250-3500 (–OH), 1365 (– NO2), 1635 of the ligand (BDEM) (2.73 g; 5 mM) and refluxed for
1
(–CH=N), 2834 (methyl –CH3), H-NMR (CDCl3): 6.4- 2 h. The dirty white compound that precipitated was
7.6 δ (phenyl multiplet), (–CH3) 2.2 δ, (–CH2–) 3.4 δ (t, filtered off, washed with EtOH and dried in vacuo. Yield:
J = 2.5 Hz, 4H), (–OH) 11.1 δ, (–OH) 12.4, (–CH=N–) 7.9 1.68 g; (41%). IR (KBr, cm-1): 2855 (methyl–CH3), 1602
δ; m/z: 548: Anal. Calcd for [C27H26N6O7]: C, 59.3, H, 4.8, (–CH=N), 471 (M–O), 445 (M–N), 285 (M–Cl). 1H-NMR
N, 15.4; Found: C, 58.4, H, 4.8, N, 14.9 (%); UV-visible (DMSO-d6): (phenyl multiplet), 6.6-7.5 δ, (–OCH3) 3.8 δ,
[λmax, nm, (ε, dm3 mol–1 cm–1)] in EtOH, 284 (29758) and (–CH3) 2.2 δ, (–CH2–) 3.4 δ (t, J = 8.1 Hz, 4H), (–CH=N–),
379 (14589). BTEM: Yield: 2.47 g; (75%). IR (KBr, cm-1): 8.1 δ; m/z: 782. Anal. Calcd for [Zn2C31H36N4O5Cl3] : Zn,
3250-3500 (–OH), 1631 (–CH=N), 2858 (methyl–CH3); 16.7, C, 47.6, H, 4.6, N 7.1: Found: Zn 16.1, C 47.4, H
1H-NMR (CDCl3): (phenyl multiplet) 6.4-7.6 δ, (–CH3) 4.6, N, 6.8(%). λM 10-3 (ohm-1 cm2 mol-1), 6.2 UV-visible
2.2 δ, (–CH2–) 3.3 δ (t, J = 4.8 Hz, 4H), (–CH=N–) 8.2 [λmax, nm, (ε, dm3 mol–1 cm–1)] in DMF, 254 (28758) and
δ; m/z: 631; Anal. Calcd for [C27H22N4OCl6]: C, 51.4, H, 340 (14358), IC50, 74µmol/ dm–1.
3.5, N, 8.9; Found: C, 50.5, H, 3.5, N, 8.4 (%); UV-visible
Similarly,complexes[Zn2(BHEM)Cl3]and[Zn2(BTEM)
[λmax, nm, (ε, dm3 mol–1 cm–1)] in EtOH, 275 (31458) and Cl3] were synthesized according to the procedure
387 (15438).
described above. [Zn(BHEM)Cl3]: Yield: 1.43 g; (35%).
IR (KBr, cm-1): 3250-3500 (–OH), 1371 (– NO2), 1609
(–CH=N), 2824 (methyl–CH3), 494 (M–O), 430 (M–N),
2.3.3. Synthesis of [Cu2(BDEM)Cl3] complex
Copper(II) chloride (1.72 g; 10 mM) in EtOH (30 mL) 288 (M–Cl). 1H-NMR (DMSO-d6): (phenyl multiplet)
was added, with stirring, to an ethanolic solution 6.4-7.6 δ, (–CH3) 2.2 δ, (–CH2–) 3.4 δ (t, J = 2.8 Hz,
(40 mL) of the BDEM (2.73 g; 5 mM) and refluxed for 4H), (–OH) 11.1 δ, (–CH=N–) 7.9 δ; m/z: 784 Anal.
3 h. The dark green compound that precipitated was Calcd for [Zn2C27H25N6O7Cl3] : Zn, 16.7, C, 41.4, H, 3.2,
filtered off, washed with EtOH and dried in vacuo. Yield: N, 10.7: Found: Zn, 15.9, C, 40.7, H, 3.3, N, 10.4(%).
2.31 g; (52 %). IR (KBr): 2860 (methyl –CH3), 1601 λM 10-3 (ohm-1 cm2 mol-1), 11.9; UV-visible [λmax, nm, (ε,
(–CH=N), 489 (M–O), 420 (M–N) and 249 (M–Cl) cm-1. dm3 mol–1 cm–1)] in DMF, 242 (17248), 352 (6331), IC50,
m/z: 778, Anal. Calcd for [Cu2C31H36N4O5Cl3] : Cu, 16.3, 72 µmol/ dm–1: [Zn2(BTEM)Cl3]: Yield: 1.94 g; (43 %).
C, 47.8, H, 4.6, N, 7.2: Found: Cu, 15.8, C, 47.2, H, IR (KBr, cm-1): 1602 (–CH=N), 2842 (methyl–CH3), 492
1
4.6, N, 7.1 (%). λM 10-3 (ohm-1 cm2 mol-1) 7.7; µeff (BM), (M–O), 447 (M–N) and 275 (M–Cl); H-NMR (DMSO-
1.08. UV-visible [λmax, nm, (ε, dm3 mol–1 cm–1)] in DMF, d6): (phenyl multiplet) 6.4-7.6 δ, (–CH3) 2.2 δ, (–CH2–)
274 (31452), 343.5 (11235), 452 (19785) and 642 (110); 3.3 δ (t, J = 5.1 Hz, 4H), (–CH=N–) 8.0 δ; m/z: 867;
IC50, 45µmol/ dm–1.
Similarly, complexes
Anal. Calcd for [Zn2C27H21N4OCl9] : Zn, 15.0, C, 37.3, H,
and 2.4, N, 6.4: Found: Zn, 14.6, C, 36.9, H, 2.4, N, 6.1 (%).
[Cu2(BHEM)Cl3]
[Cu2(BTEM)Cl3] were synthesized according to the λM 10-3 (ohm-1 cm2 mol-1), 12.4; UV-visible [λmax, nm, (ε,
procedure described above. [Cu2(BHEM)Cl3]: Yield: dm3 mol–1 cm–1)] in DMF, 262 (21498), 367 (9507), IC50,
3.07 g; (69%). IR (KBr, cm-1) : 3250-3500 (–OH), 1368 58µmol/ dm–1.
98

N. Raman, A. Sakthivel, R. Jeyamurugan
2.4. DNA binding study
The binding of complexes to DNA using electronic
absorption,electrochemical,andviscositymeasurements
was done according to the methods described in [20].
microorganisms was affected. The inhibition zone was
developed, at which the concentration was noted.
2.7. Superoxide dismutase activity
The superoxide dismutase activity of the complexes
was assayed using nitrobluetetrazolium chloride (NBT)
as reported previously [22]. The concentration of the
complex required to yield 50% inhibition of the reduction
of NBT (named IC50) was determined.
2.5. DNA cleavage study
The extent of cleavage of super coiled (SC) pUC19
DNA (33.3 µM, 0.2 µg) to its nicked circular (NC) form
was determined by agarose gel electrophoresis in
50 mM Tris–HCl buffer (pH 7.2) containing 50 mM NaCl.
For photo-induced DNA cleavage studies, the reactions
were carried out under illuminated conditions using
UV sources at 360 nm. After exposure to light, each
sample was incubated for 1 h at 37oC and analyzed for
the photo-cleaved products using gel electrophoresis
as discussed below. The inhibition reactions for the
“chemical nuclease” reactions were carried out under
dark conditions by adding the reagents (distamycin
50 µM and DMSO 4 µL) prior to the addition of each
complex and the reducing agent, 3-mercaptopropionic
acid (MPA). The inhibition reactions for the photo-
induced DNA were carried out at 360 nm adding the
reagents (NaN3 100 µM and DMSO 4 µL) prior to the
addition of each complex. For the D2O experiment, the
solvent (D2O) was used for the dilution of the sample
to 18 µL. The samples, after incubation for 1 h at 37oC
in a dark chamber, were added to the loading buffer
containing 25% bromophenol blue, 0.25% xylene
cyanol and 30% glycerol (3 µL) and the solution was
finally loaded on 0.8% agarose gel containing 1 µg mL-1
ethidium bromide. Electrophoresis was carried out in a
dark chamber for 3 h at 50V in Tris-acetate-EDTA buffer.
Bands were visualized by UV light and photographed.
3. Results and Discussion
The ligands (L) and their complexes are found to be
stable in air. The ligands are soluble in common organic
solvents but the complexes are soluble only in DMF and
DMSO. The elemental analytical and mass spectral data
of the ligands and their complexes are in agreement
with the formulae [M2LCl3]. The magnetic moment of
Cu(II) complexes lie in the range 0.98-1.48 BM at room
temperature. The observed magnetic moment values
are less than the spin only value (1.73 to 2.20 BM) for
Cu(II) (3d9) mononuclear complexes having no major
interactions between the moieties. The low magnetic
moments in Cu(II) complexes are usually attributed
to spin-coupling within a dimer brought about by the
bridging of monomeric units of paramagnetic centers
through the phenolic oxygen or chlorine atoms. This
oxygen or chlorine bridging often leads to change in
electronic structure of the attached atoms resulting in
reduction of paramagnetism, partially or completely, of
the metal atoms bridge. Hence the subnormal magnetic
moments observed for the studied Cu(II) complexes may
be accounted for by assuming a dimeric structure in the
solid state, in which a considerable antiferromagnetic
exchange occurs through a super exchange mechanism
as there is little possibility of a direct interaction.
The IR spectra of the ligands exhibit a strong band
at 1624-1635 cm−1 assigned to ע(C=N) stretching
frequency. This band shifts to lower wave numbers
(∼25 cm-1) in all the complexes, suggesting the
coordination of the azomethine nitrogen to the metal
centre [23]. The phenolic ע(OH), found as a weak broad
band in the 3250-3500 cm–1 region, disappears in all
complexes indicating deprotonation and coordination
to the metal. This is further supported by the shift of
phenolic ע(C–O) from 1280 cm-1 recordedfor the ligands
to higher frequency, by about 40-50 cm-1, in complexes.
The low frequency bands in the 500-470 and 420-
450 cm–1 regions are assigned to ע(M-O) and ע(M-N)
respectively [24]. The presence of a chloro bridge is
evident from the IR bands observed in the 290-240 cm–1
2.6. Antimicrobial activity
Theinvitrobiologicalscreeningeffectsoftheinvestigated
compounds were tested against the bacteria Salmonella
typhi, Staphylococcus aureus, Escherichia coli, and
Bacillus subtilis by the disc diffusion method, using
agar nutrient as the medium and streptomycin as the
standard. The antifungal activities of the compounds
were evaluated by the disc diffusion method against the
fungi viz., Aspergillus niger, Aspergillus flavus, Candida
Albicans and Rhizoctonia bataicola cultured on potato
dextrose agar as medium and nystatin as the standard.
The stock solution (10-2 M) was prepared by dissolving
the compounds in DMSO and the solutions were
serially diluted in order to find the minimum inhibitory
concentration (MIC) values. In a typical procedure
[21], the disc was filled with the test solution using a
micropipette and the plate was incubated 24 h for
bacteria and 72 h for fungi at 35oC. During this periodthe
test solution diffused and the growth of the inoculated
region. The ligand (BHEM)
gives a broad band for the
99

Synthesis, characterization and biological activities
of homo-binuclear Cu(II) and Zn(II) complexes derived from
2-hydroxy-5-methyl-1,3-benzenedicarboxaldehyde derivatives
–OH group at ca. 3250-3500 cm-1, which is attributable
to the phenolic –OH group present in the 3-hydroxy-4-
nitrobenzaldehyde moiety. The appearance of this peak
in their copper and zinc complexes indicates that the
chelation does not take place via the –OH group.
In general typical Cu(II) complexes of square-
pyramidal or distorted square-pyramidal geometries
exhibit a band in the 550-650 nm range [25-26]. The
electronic absorption spectra of the Cu(II) complexes,
recorded in DMF solution, show one broad band in the
visible region around 628-647 nm, which is assigned to
distorted-square pyramidal geometry around the Cu(II)
ion.
The decoupled 13C NMR spectra of BDEM (in
CDCl3) and its zinc complex (in DMSO-d6) confirm the
presence of azomethine (-C=N-) and methoxy (–OCH3)
groups. In the zinc complex, the imine carbon signal
was deshielded compared to the free ligand suggesting
the coordination of azomethine nitrogen to the metal
ion. Comparison of all other macrocyclic carbon peaks
of the ligand with those of the zinc complex [Zn2LCl3]
shows some up-field and down-field shifts which are not
very significant. The data for the free ligand and its zinc
complex given in Table 1 are in good agreement with the
assigned structure of the ligand and its complex.
Based on the above spectral and analytical data,
the structure of the Schiff base complexes is given in
Fig. 1.
3.1. DNA binding studies
3.1.1. Absorption titration
The electronic absorption spectrum of copper complex
[Cu2(BDEM)Cl3] in the presence of increasing amounts
of CT DNA in 5 mM Tris-HCl and 50 mM NaCl buffer
(pH 7.2) is shown in Fig. 2. In the UV region, the intense
absorption bands observed in the region 340-367 nm
were attributed to intra ligand π-π* transition. Increase in
concentration of CT-DNA resulted in the hypochromism
and blue-shift in UV-Vis spectrum of the copper complex.
These spectral characteristics suggest that the copper
complex might bind to DNAby an intercalative mode due
to a strong stacking interaction between the aromatic
chromophore of the complex and the base pairs of the
DNA. After intercalating the base pairs of DNA, the
π*orbital of the intercalated ligand could couple with
the π orbital of the base pairs, thus decreasing the
π-π*transition energy and further resulting in the blue-
shift. On the other hand, the coupling of the π orbital
was partially filled by electrons, thus decreasing the
transition probabilities and concomitantly, resulting in
the hypochromism. A similar type of binding mode was
observed for other complexes with CT DNA. In order to
compare the binding strength of the complexes with CT
DNA, the intrinsic binding constants, Kb (Table 2), are
obtained by monitoring the changes in the absorbance
for the complexes with increasing concentration of
DNA.
Table 1. 13C- NMR spectral data (ppm) of ligand (BDEM) and its Zn(II) complex
Zn
Assignment
Ligand (BDEM)
complex
C1
C2
21.4
131.1
133.7
128.5
164.4
49.2
21.4
131.1
133.7
128.5
163.7
49.1
C3
C4
C5
C6
C7
45.9
45.5
C8
161.4
129.6
113.4
154.2
148.8
110.2
121.6
54.7
161.1
129.4
113.2
145.1
148.6
110.2
122.4
54.7
C9
C10
C11
C12
C13
C14
C15
C16
54.7
54.7
C17
158.7
158.1
100

N. Raman, A. Sakthivel, R. Jeyamurugan
Table 2. Absorption spectral properties of synthesized complexes with DNA
5
x
λmax
∆λ
Hypochromicity H
(%)
Kb 10
SI.No
Complexes
(nm)
(M-1)
Free
Bound
1
2
3
4
5
6
[Cu2(BDEM)Cl3]
[Cu2(BHEM)Cl3]
[Cu2(BTEM)Cl3]
[Zn2(BDEM)Cl3]
[Zn2(BHEM)Cl3]
Zn2(BTEM)Cl3]
343.5
355.5
363.0
340.0
352.0
367.0
340.5
350.0
357.0
338.0
348.5
360.5
3.0
5.5
6.0
2.0
3.5
6.5
3.8
6.3
7.8
5.6
6.0
8.3
2.2
2.7
3.6
1.6
2.9
3.9
3.1.2. Viscosity measurement
Furthermore, the interactions between the complexes
and DNA were investigated by viscosity measurements.
Optical photophysical and voltammetry probe provided
necessary, but not sufficient clues to support a binding
model. Hydrodynamic measurements that were
sensitive to the length change (i.e., viscosity and
sedimentation) were regarded as the least ambiguous
and the most critical tests of binding mode in solution
in the absence of crystallographic structural data
[27]. 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. 5, the
viscosity of DNA increases with the increase in the ratio
of complexes to DNA. This result further suggested an
intercalative binding mode of the complex with DNA
and in agreement with spectroscopic results, such as
hypochromism and bathochromism of complexes in the
presence of DNA.
Figure 1. The proposed structure of the Schiff base complexes
3.1.3. Electrochemical studies
The application of cyclic voltammetry (CV) to the study
of binding of metal complexes to DNA provides a useful
complement to the above methods of investigations.
Typical cyclic voltammograms of the [Cu2(BDEM)Cl3] in
the absence and presence of DNA are shown in Fig. 3.
In the absence of DNA, the first redox couple
cathodic peak appears at 0.234 V for Cu(III) → Cu(II)
(Epa = 0.573 V, Epc = 0.234 V, ∆Ep = 0.339 V and E1/2
= 0.403 V) and the second redox couple cathodic peak
appears at -0.784 V for Cu(II)→ Cu(I) (Epa = 0.31 V, Epc
= -0.784 V, ∆Ep = 0.474 V and E1/2 = -0.237 V). In these
two redox couples, the ratio of ipc/ipa is approximately
unity. This indicates that the reaction of the complex
on the gold electrode surface is a quasi-reversible
redox process. The incremental addition of DNA to the
complex the second redox couple causes a negative
shift in E1/2 of 37 mV and a decrease in ∆Ep of 73 mV. In
Figure 2. Electronic absorption spectrum of [Cu2(BDEM)Cl3] in the
absence (dash line) and presence (dark line) of increasing
amounts of DNA
101

Synthesis, characterization and biological activities
of homo-binuclear Cu(II) and Zn(II) complexes derived from
2-hydroxy-5-methyl-1,3-benzenedicarboxaldehyde derivatives
Figure 3. Cyclic voltammogram of [Cu2(BDEM)Cl3]in the absence (dash line) and presence (dark line) of different concentration of DNA
Figure 4. Differential pulse voltammogram of [Cu2(BDEM)Cl3] in the absence (dash line) and presence (dash line) of different concentration of
DNA
the presence of DNA the ipc/ipa values decrease in the the voltammetric currents in the presence of CT DNAcan
first couple (Cu(III) → Cu(II)), but there is an increase be attributed to diffusion of the metal complex bound to
in the ipc/ipa values in the second couple (Cu(II) → the large, slowly diffusing DNA molecule. The changes
Cu(I)). In addition, the peak potentials, Epc and Epa, of the peak currents observed for the complexes upon
as well as E1/2 shift to more positive values. The shift addition of CT DNA may indicate that BHEM and BTEM
of the redox potential of the complexes in the presence complexes possess a higher DNA-binding affinity than
of DNA to more positive values indicates a binding the other complex. The results are similar to the above
interaction between the complex and DNA that makes spectroscopic and viscosity data of the complexes in the
the complexes less readily reducible. The changes of presence of DNA.
102

N. Raman, A. Sakthivel, R. Jeyamurugan
Figure 5.The effect of [Cu2(BDEM)Cl3] (▲), [Zn2(BDEM)Cl3] (ж), [Cu2(BHEM)Cl3] (♦), [Zn2(BHEM)Cl3] (●), [Cu2(BTEM)Cl3] (×) and [Zn2(BTEM)Cl3]
(■) on the viscosity of DNA; Relative specific viscosity Vs. R = [Complex]/ [DNA]
All the zinc complexes showed a pronounced Cu(I) complex couple in the bound and free form
decrease in peak currents and shift in E1/2 to less respectively. The ratio of the binding constants (K+/K2+)
negative potential upon addition of DNA. [Zn2(BTEM)Cl3] for DNA binding of Cu(II)/Cu(I) complexes was found
produced a more significant decrease in peak currents to be less than unity (Table 3). This indicates that the
and shows a larger shift in potential upon addition of binding of Cu(I) complex to DNA is weak compared to
DNA. The electrochemical parameters of the Cu(II) and that of the Cu(II) complex. The above electrochemical
Zn(II) complexes are shown inTable 3 and 4 respectively. experimentalresultsindicatethepreferentialstabilization
From these data it is understood that all the synthesized of Cu(II) form over Cu(I) form on binding to DNA.
Cu(II) and Zn(II) complexes interact with DNA through
intercalating way.
Differential pulse voltammogram of the studied Zn(II)
complexes shows a negative potential shift along with a
Differential pulse voltammogram of the [Cu2(BDEM) significant decrease in current intensity during addition
Cl3] in the absence and presence of varying amounts of increasing amounts of DNA. It indicates that zinc ions
of [DNA] is given in Fig. 4. Increase in concentration stabilize the duplex (GC pairs) by intercalating way.
of DNA causes a negative potential shift along with a Hence, for the complexes of the electroactive species
significant increase in current intensity. The shift in (Zn(II)) with DNA the electrochemical reduction reaction
potential is related to the ratio of binding constant
can be divided into two steps:
2+
Zn
2 +
Zn
Eob’- Eof’ = 0.0591 log (K+/K2+)
(1)
-
+ DNA
DNA
2 +
0
-
where Eob’ and Eof’ are formal potentials of the Cu(II)/
+
Zn
2e
Zn
Table 3. Electrochemical parameters for the interaction of DNA with Cu(II) complexes
SI.
k[red]/
k[oxd]
E1/2 (V)
∆Ep (V)
Bound
Complexes
Redox couple
Ipc/Ipa
Free
Bound
Free
No
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
Cu(II)/Cu(I)
Cu(III)/Cu(II)
0.403
0.503
0.339
0.345
0.547
0.384
0.425
0.074
---
0.80
1.3
1
[Cu2(BDEM)Cl3]
-0.237
-0.439
-0.600
-0.569
-0.274
-0.438
-0.495
-0.544
0.474
0.383
0.278
0.097
0.64
---
0.75
1.5
2
3
[Cu2(BHEM)Cl3]
[Cu2(BTEM)Cl3]
0.84
0.72
1.4
103

Synthesis, characterization and biological activities
of homo-binuclear Cu(II) and Zn(II) complexes derived from
2-hydroxy-5-methyl-1,3-benzenedicarboxaldehyde derivatives
9
8
7
6
5
4
3
2
1
Figure 6. Gel electrophoresis diagram showing the cleavage of SC pUC 19 DNA (0.2 µg) by the synthesized complexes (50 µM) in the presence
of MPA (5 mM): lane 1, DNA control; lane 2, DNA + [Cu2(BDEM)Cl3] + MPA; lane 3, DNA + [Cu2(BHEM)Cl3] + MPA; lane 4, DNA +
[Cu2(BTEM)Cl3] + MPA; lane 5, DNA + [Zn2(BDEM)Cl3] + MPA; lane 6, DNA + [Zn2(BHEM)Cl3] + MPA; lane 7, DNA + [Zn2(BTEM)Cl3]
+ MPA; lane 8, DNA + [Cu2(BDEM)Cl3] + DMSO (4 µl) + MPA, lane 9, DNA + [Cu2(BDEM)Cl3] + distamycin (50 µM) + MPA
The dissociation constant (Kd) of the Zn(II)-DNA
complex was obtained using the following equation:
The DNA cleavage of a ligand alone is inactive in the
presenceandabsenceofanyexternalagents.Theresults
indicate the importance of the metal in the complex for
observing the chemical nuclease activity. The oxidative
cleavage of pUC19 DNA (Fig. 6) in the presence of
an external reducing agent like 3-mercaptopropionic
i2
i2p )
2
K
ip2
__
__
___d___
[DNA]
(2)
o
+
o
ip
=
(
p
[DNA]
where Kd is a dissociation constant of the complex acid (MPA, 5 mM) (type-I) has been studied by gel
Zn(II)-DNA, i2po and i2p are reduction currents of Zn(II) electrophoresis using supercoiled (SC) pUC19 DNA
in the absence and presence of DNA respectively. The (0.2 µg, 33.3 µM) in 5 mM Tris–HCl/ 50 mM NaCl buffer
dissociation constant was determined using the above (14 µL, pH 7.2) and the synthesized complexes (50 µM).
Eq. 2. The low dissociation constant values (Table 4) Control experiments, using MPA or the synthesized
of Zn(II) ions were indispensable for catalytic function complexes alone, show apparent cleavage of SC DNA
and structural stability of zinc enzymes which participate in the absence of light. This result suggests that the
in the replication, degradation and translation of genetic synthesized complexes are able to bind to DNA. In order
material of all species.
to determine the groove selectivity of the complexes
control experiments were performed using minor groove
3.2. DNA cleavage studies using Gel binder distamycin. The addition of distamycin does not
electrophoresis
inhibit the cleavage for all the complexes. This indicates
It is known that DNA cleavage is controlled by relaxation the major groove binding for the synthesized complexes
of supercoiled circular conformation of pUC19 DNA with DNA. Control experiments show that the hydroxyl
to nicked circular and/or linear conformations. When radical scavenger DMSO inhibits the DNA cleavage
electrophoresis is applied to circular plasmid DNA, suggesting the possibility of hydroxyl radical and/
fastest migration will be observed for DNA of closed or ‘‘copper-oxo’’ intermediate as the reactive species
circular conformations (Form I). If one strand is [29]. SOD addition does not have any apparent effect
cleaved, the supercoiled will relax to produce a slower- on the cleavage activity indicating the non-involvement
moving nicked conformation (Form II). If both strands of the superoxide radical in the cleavage reaction. The
are cleaved, a linear conformation (Form III) will be mechanism involved in the DNA cleavage reactions is
generated that migrates in between [28].
believed to be similar to that proposed by Sigman and
Table 4. Electrochemical parameters for the interaction of DNA with Zn(II) complexes
Kd×10-10
(mol L-1)
Ep (V)
Ip (A)
SI.No
Complexes
Free
Bound
Free
Bound
1
2
3
[Zn2(BDEM)Cl3]
[Zn2(BHEM)Cl3]
Zn2(BTEM)Cl3]
-0.869
-0.858
-0.847
-0.801
-0.795
-0.782
0.205
0.215
0.254
0.151
0.162
0.143
7.2
7.6
8.2
104

N. Raman, A. Sakthivel, R. Jeyamurugan
coworkers [30] for the chemical nuclease activity of the 3.3. SOD activity
metal complexes.
The SOD mimetic activity values (IC50) of synthesized
complexes are given in the experimental part. In a
variety of complexes which act as superoxide dismutase
3.2.1. Photo cleavage
Irradiation of pUC19 DNA containing Cu(II) and Zn(II) enzymes [31], there is either one-electron oxidation
complexes was carried out in the presence and in followed by reduction of a metal ion or formation of a
absence of various ‘inhibitors’ using gel electrophoresis superoxide complex which then is reduced to peroxide
method. All the complexes cleave DNA from its SC to by another superoxide ion. In order to explore the
NC form, even in the absence of inhibitors, on irradiation mechanism, absorption spectrum of complexes were
with UV light at 360 nm. To assess the possibility that recorded in the presence and absence of alkaline DMSO
the photo-activated change involves formation of singlet (alkaline DMSO acts as a source of O2–). The spectrum
oxygen and hydroxyl radical, that are responsible for peaks became suppressed in alkaline DMSO containing
the cleavage of DNA, the experiments were run in the buffer (pH 8.6). However upon addition of NBT, which
presence of inhibitors, D2O and DMSO. Singlet oxygen acts as O2– scavenger, these peaks were reverted to
would be expected to induce more strand scission in their original position. Thus, this experiment indicates
D2O than in H2O, due to its longer life time in the former that O2– is initially attached to the metal complexes
solvent. The same results were observed in the presence which later are reduced by another O2– ion.
of the hydroxyl radical scavenger DMSO. Studies with
The results report the scavenging ability of each
singlet oxygen quencher NaN3 were also carried out. complex, giving its final concentration that produced
The cleavage is slightly inhibited (Fig. 7, Lane 8) in the efficient quenching of the superoxide anion radical. The
presence of NaN3, which further confirmed that singlet lower SOD-like activity (IC50)of the copper complexes is
oxygen may be the reactive species. These results due to the weak resistance of these chelates toward the
indicate that, besides the presence of inhibitors, the aggressive substrate O2– or the product O2. The metal
complexes show cleavage activity upon irradiation with complexes are the most active favorably comparing
UV light at 360 nm.
with a number of synthetic SOD mimics developed for
therapeutic purposes [32].
-1
Table 5. Minimum inhibitory concentration of the synthesized compounds against the growth of four fungi (mg mL )
S.No
Compound
A. niger
A. flavus
C. albicans
R. bataicola
1
2
BDEM
84
75
82
41
28
39
27
22
39
10
73
69
63
55
32
31
33
27
49
8
68
62
73
51
35
39
42
29
54
12
64
79
71
38
33
41
45
31
57
14
BHEM
3
BTEM
4
[Cu2(BDEM)Cl3]
[Cu2(BHEM)Cl3]
[Cu2(BTEM)Cl3]
[Zn2(BDEM)Cl3]
[Zn2(BHEM)Cl3]
[Zn2(BTEM)Cl3]
Nystatin
5
6
7
8
9
10
-1
Table 6. Minimum inhibitory concentration of the synthesized compounds against the growth of four bacteria (mg mL )
S.No
Compound
E. coli
S. typhi
S. aureus
B. subtilis
1
2
BDEM
60
63
68
41
22
33
39
19
33
14
71
67
74
39
27
39
34
22
34
18
67
65
77
28
17
41
19
27
37
12
75
72
80
33
21
33
37
18
28
10
BHEM
3
BTEM
4
[Cu2(BDEM)Cl3]
[Cu2(BHEM)Cl3]
[Cu2(BTEM)Cl3]
[Zn2(BDEM)Cl3]
[Zn2(BHEM)Cl3]
[Zn2(BTEM)Cl3]
Streptomycin
5
6
7
8
9
10
105

Synthesis, characterization and biological activities
of homo-binuclear Cu(II) and Zn(II) complexes derived from
2-hydroxy-5-methyl-1,3-benzenedicarboxaldehyde derivatives
9
8
7
6
5
4
3
2
1
Figure 7.Gel electrophoresis diagram showing the photocleavage of pUC 19 DNA by the synthesized complexes in DMF-Tris buffer medium and
in the presence of various reagents on irradiation with UV light of 360 nm: lane 1, DNA control (60 min); lane 2, DNA + [Cu2(BDEM)Cl3]
(60 min); lane 3, DNA + [Cu2(BHEM)Cl3] (60 min); lane 4, DNA + [Cu2(BTEM)Cl3] (60 min); lane 5, DNA + [Zn2(BDEM)Cl3] (60 min);
lane 6, DNA + [Zn2(BHEM)Cl3] (60 min); lane 7, DNA + [Zn2(BTEM)Cl3] (60 min); lane 8, DNA + sodium azide (100 µM) + [Cu2(BDEM)
Cl3] (30 min); lane 9, DNA + DMSO (4 µL) + [Cu2(BDEM)Cl3] (30 min).
pyramidal/square-pyramidal geometry around the central
3.4. Antimicrobial study
metal ion. The binding mechanism of the complexes with
DNA was not electrostatic binding but intercalation mode
of binding. Noticeably, the complexes have been found
to promote the cleavage of plasmid pUC19 DNA form
I to form II upon irradiation. The cleavage mechanism
between complexes and pUC19 DNA is likely to involve
singlet oxygen and hydroxyl radicals as the reactive
species. The synthesized complexes exhibit significant
SOD mimetic activities. They have higher antimicrobial
activity than the ligands.
For in vitro antimicrobial activity, the investigated
compounds were tested against the above said bacteria
and fungi. The minimum inhibitory concentration (MIC)
values of the investigated compounds are summarized in
Tables5and6.Acomparativestudyoftheligandsandtheir
complexes (MIC values) indicates that complexes exhibit
higher antimicrobial activity than the free ligands. From
the MIC values, it was found that the BHEM complexes
are the most potent among the investigated complexes.
This may be due to the electron withdrawing nature of
the nitro group which enhances the positive charge on
the metal ions and thereby increases the delocalization
of π-electrons over the whole chelate ring and enhances
the lipophilicity of the complexes. Such properties of the
complexes can be explained on the basis of Overtone’s
[19] and Tweedy’s Chelation theory [33].
Acknowledgements
The authors express their heartfelt thanks to the College
Managing Board, VHNSN College, Virudhunagar for
providing research facilities. NR and AS express their
gratitude to the UGC, New Delhi for financial assistance.
4. Conclusion
Three novel Schiff base ligands and their Cu(II) and Zn(II)
complexesweredesigned,synthesizedandcharacterized.
They have adopted bimetallic and distorted square-
References
[1] M. Shakir, N. Begum, Y. Azim, S. Parveen, Synth. [5] A.M. Thomas, G. Neelakanta, S. Mahadevan,
React. Inorg. Met-Org. Chem. 33, 1367 (2003)
[2] B. Adhikari, A.K. Biswas, K. Nag, P. Zanello,
A. Cinquantini, Polyhedron 6, 897 (1987)
M. Nethaji, A.R. Chakravarty, Eur. J. Inorg. Chem.
2720 (2002)
[6] S.C. Zhang, Y. Shao, C. Tu, C.H. Dai, Z.J. Guo,
Chin. J. Inorg. Chem. 20, 1159 (2004)
[3] K.D. Karlin, Z. Tyeklar, In: G.L. Eichhorn, L. Marzilli
(Eds.),AdvancesinInorganicBiochemistry(Prentice [7] Q.L. Li, et al., Bioorg. Med. Chem. 14, 4151 (2006)
Hall, Englewood Cliffs, NJ, 1994) Vol. 9
[4] K.D. Karluvi, Z. Tyeklar (Eds.), Bioinorganic
[8] S.G. Kang, G.R. Jeong, Inorg. Chem. Commun.
12, 62 (2009)
Chemistry of Copper (Chapman and Hall, New [9] K.E. Erkkila, D.T. Odom, J.K. Barton, Chem. Rev.
York, 1993)
99, 2777 (1999)
106

N. Raman, A. Sakthivel, R. Jeyamurugan
[10] L.J.K. Boerner, J.M. Zaleski, Curr. Opin. Chem. Biol. [23] M.C.R. Arguelles et al., J. Inorg. Biochem. 103, 35
9, 135 (2005)
[11] R. Bannet, Chemical Aspects of Photodynamic [24] S. Niu, M. Zhao, R. Ren, S. Zhang, J. Inorg.
Therapy (Gorden and Breach, London, 2001)
Biochem. 103, 43 (2009)
[12] M.R. Detly, S.L. Gibson, S.J. Wagner, J. Med. [25] B.J. Hathaway, G. Wilkinson, R.D. Gillard,
(2009)
Chem. 47, 3897 (2004)
[13] F.S. Mackay et al., Proc. Natl. Acad. Sci. USA 104,
207423 (2007)
J.A. McCleverty, Comprehensive Coordination
Chemistry (Pergamon Press, Oxford, UK, 1987)
vol. 5
[14] L.C. Xu, S. Shi, J. Li, S.Y. Liao, K.C. Zheng, [26] U. Mukhopadhyay, I. Bernal, S.S. Massoud,
L.N. Ji, Dalton Trans. 291 (2008)
F.A. Mautner, Inorg. Chim. Acta 357, 3673 (2004)
[15] A.K. Patra, T. Bhowmick, S. Ramakumar, [27] S. Sathyanarayana, J.C. Daborusak, J.B. Chaires,
A.R. Chakravarty, Inorg. Chem. 46, 9030 (2007)
Biochemistry 32, 2573 (1993)
[16] M.J. Fernandez, B. Wilson, M. Palacios, [28] A. Sitlani, E.C. Long, A.M. Pyle, J.K. Barton, J. Am.
M.M. Rodrigo, K.B. Grant, A. Lorente, Bioconjugate
Chem. 18, 121 (2007)
[17] D.D. Perrin, W.L.F. Armarego, D.R. Perrin,
Chem. Soc. 114, 2303 (1992)
[29] T.B. Thederahn, M.D. Kuwabara, T.A.Larsen,
D.S. Sigman, J. Am. Chem. Soc. 111, 4941 (1989)
Purification of Laboratory Chemicals (Pergamon [30] D.S. Sigman, A. Mazumder, D.M. Perrin, Chem.
Press, Oxford, 1980)
Rev. 93, 2295 (1993)
[18] N. Raman, S.J. Raja, J. Joseph, J.D. Raja, J. Chil. [31] D.T. Sawyer, J.S. Valentine, Acc. Chem. Res. 14,
Chem. Soc. 52, 1138 (2007)
393 (1981)
[19] A.D. Naik, V. K. Revankar, Indian J. Chem. 43A, [32] M. Baudry, S. Etienne, A. Bruce, M. Palucki,
1447 (2004)
E. Jacobsen, B. Malfroy, Biochem. Biophy. Res.
Commun. 192, 964 (1993)
[33] Z.H. Chohan, H. Pervez, A. Rauf, K.M. Khan,
C.T. Supuran, J. Enzy, Inh. Med. Chem. 19, 417
(2004)
[20] N. Raman, A. Sakthivel, K. Rajasekaran, J. Coord.
Chem. 62, 1661 (2009)
[21] N.M. Aghatabay, Y. Mahmiani, H. Cevik, B. Dulger,
Eur. J. Med. Chem. 44, 365 (2009)
[22] R.G. Bhirud, T.S. Srivastava, Inorg. Chim. Acta 173,
121 (1990)
107