Metal-based molecular design tuning biochemical behavior: Synthesis, characterization, and biochemical studies of mixed ligand complexes derived from 4-aminoantipyrine derivatives

Article abstract of DOI:10.1080/00387010.2013.776087

Biosensitive mixed ligand complexes of metals (Fe(III), Co(II), Ni(II), Cu(II), and Zn(II)) with the Schiff bases of L¹ and HL² (L¹: obtained through the condensation of 4-aminoantipyrine with furfuraldehyde; HL²

Full text of DOI:10.1080/00387010.2013.776087

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Metal-Based Molecular Design Tuning Biochemical
Behavior: Synthesis, Characterization, and Biochemical
Studies of Mixed Ligand Complexes Derived From 4-
Aminoantipyrine Derivatives
J. Joseph a & G. Ayisha Bibin Rani a
a Department of Chemistry , Noorul Islam Centre for Higher Education , Kumaracoil ,
Tamilnadu , India
Accepted author version posted online: 05 Mar 2013.Published online: 03 Dec 2013.
To cite this article: J. Joseph & G. Ayisha Bibin Rani (2014) Metal-Based Molecular Design Tuning Biochemical Behavior:
Synthesis, Characterization, and Biochemical Studies of Mixed Ligand Complexes Derived From 4-Aminoantipyrine Derivatives,
Spectroscopy Letters: An International Journal for Rapid Communication, 47:2, 86-100, DOI: 10.1080/00387010.2013.776087
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Spectroscopy Letters, 47:86–100, 2014
Copyright # Taylor & Francis Group, LLC
ISSN: 0038-7010 print=1532-2289 online
DOI: 10.1080/00387010.2013.776087
Metal-Based Molecular Design Tuning
Biochemical Behavior: Synthesis,
Characterization, and Biochemical
Studies of Mixed Ligand Complexes
Derived From 4-Aminoantipyrine
Derivatives
J. Joseph
and G. Ayisha Bibin Rani
ABSTRACT Biosensitive mixed ligand complexes of metals (Fe(III),
Co(II), Ni(II), Cu(II), and Zn(II)) with the Schiff bases of L1 and HL2
(L1: obtained through the condensation of 4-aminoantipyrine with furfur-
aldehyde; HL2: derived from 2-aminophenol and 3-nitrobenzaldehyde)
were synthesized. They were characterized using elemental analysis, mag-
netic susceptibility, molar conductance, proton nuclear magnetic reson-
ance spectroscopy, ultraviolet–visible spectroscopy, infrared, and
electron spin resonance techniques. Cyclic voltammogram of the com-
plexes in dimethylsulfoxide solution at 300 K was recorded and their sali-
ent features were summarized. The X-band electron spin resonance
spectrum of the copper complex in dimethylsulfoxide solution at 300
and 77 K was recorded. The in vitro biological screening effects of the
investigated compounds were tested against the bacterial species Sta-
phylococcus aureus, Escherichia coli, Klebsiella pneumoniae, Proteus vul-
garis, and Pseudomonas aeruginosa and the fungal species Aspergillus
niger, Rhizopus stolonifer, Aspergillus flavus, Rhizoctonia bataicola, and
Candida albicans by disc diffusion method. Comparative study of inhi-
bition values of the Schiff bases and their complexes indicates that the
complexes exhibit higher antimicrobial activity than the free ligands.
DNA binding studies of metal complexes using ultra-violet-visible spec-
troscopy and cyclic voltammetry paved the way to probe their DNA bind-
ing abilities. The solvatochromism and superoxide dismutase activity of
these complexes have also been examined.
Department of Chemistry,
Noorul Islam Centre for Higher
Education, Kumaracoil,
Tamilnadu, India
Received 14 January 2013;
accepted 11 February 2013.
Address correspondence to J. Joseph,
Department of Chemistry, Noorul
Islam Centre for Higher Education,
Kumaracoil-629180, Tamilnadu, India.
E-mail: chem_joseph@yahoo.co.in
KEYWORDS 4-aminoantipyrine, disc diffusion, DNA binding, mixed ligand
metal complexes, SOD
86

toxic agents as well as the origins of some diseases
like gene mutation. Many compounds expose their
antitumor activity through binding to DNA and can
cause DNA damage in cancer cells by blocking the
division of cancer cells and resulting in cell death.
Superoxide dismutase activity (SOD) in conjunc-
tion with catalase appears to be the most effective
enzymatic defense against the toxicity of oxygen
metabolism. Among the known SOD enzymes,
CuZn(SOD) is the most efficient catalytic species. It
catalyzed the disproportionation of the cytotoxic
superoxide radical Oꢀ2ꢁ, to oxygen and hydrogen per-
oxide, through one electron redox cycle involving its
copper center. It has been known since three decades
ago that cancer cells have less than normal SOD
activity and treatment with bovine-native CuZn-SOD
decreased the growth of several solid tumors.[8]
The superoxide radical anion Oꢀ2ꢁ is formed as a
byproduct of normal cellular respiration. Its decom-
position produces undesired harmful species like
hydroxyl radical and hydrogen peroxide. To control
this, nature has created a family of enzymes that remove
them from the cellular environment. One of these,
SOD, catalyzes the disproportionation of superoxide
to dioxygen and hydrogen peroxide, which is decom-
posed via being catalyzed to water and dioxygen.[9]
Inspired from the literature facts, the present study
focused on the mixed ligand transition metal com-
plexes of Schiff bases of L1 and HL2 toward tuning
of biochemical potentials. They were characterized
using analytical and spectral techniques. The bio-
chemical studies of complexes were also performed.
INTRODUCTION
Transition metal complexes with oxygen and
nitrogen donor ligands are of great interest for their
ability to possess unusual configurations, their struc-
tural lability, and their sensitivity to molecular envir-
onments.[1] Ternary complexes formed between
metal ions and two different types of bioligands,
namely, heteroaromatic nitrogen bases and Schiff
bases, may be considered as models for substrate
metal ion–enzyme interactions and other metal
ion–mediated biochemical interactions.[2] Antipyrine
(AP) was first synthesized by Knorr in 1883, and
there has been a continued interest in the studies
of antipyrine derivatives (APDs). APDs have been
accepted as important biomodel compounds in the
biological systems, and research works have been
carrying on with new compound findings. In the
coordination chemistry, APDs were extensively used
as a group of important ligands due to the coordi-
nated function of the keto or aza-groups.[3] APDs
have exhibited attractive multifunctional properties
as coordinate, antioxidant, antiputrefactive, and
optical characteristics in chemical and material
fields.[4] Schiff bases of 4-aminoantipyrine and its
complexes are known for their variety of applica-
tions in the area of catalysis, clinical applications,
and pharmacology. New kinds of chemotherapeutic
agents containing Schiff bases have gained signifi-
cant attention among biochemists, and of those ami-
nopyrines are commonly administered intravenously
to detect liver disease in clinical treatment.[5] Schiff
bases are potential anticancer drugs and, when admi-
nistered as their metal complexes, the anticancer
activity of these complexes is enhanced in compari-
son to the free ligand. Schiff bases of 4-aminoantipyr-
ine and its complexes present a great variety of
biological activities, including antitumor, fungicide,
bactericide, antiinflammatory, and antiviral activi-
ties.[6] Aminophenols are also important to the phar-
maceutical industry, since they have antibacterial and
antitubercular action. Schiff bases obtained from the
condensation of 2-aminophenol with some alde-
hydes and ketones have also been used widely as
antituberculosis compounds, and their importance
is mainly due to their ability to form metal chelates.[7]
To discover new therapeutic agents that specifi-
cally target DNA, it is essential to understand the
action mechanism of some DNA-targeted drugs and
MATERIALS AND METHODS
Materials
All chemicals used in the present work, viz.,
4-aminoantipyrine, furfuraldehyde, 2-aminophenol,
3-nitrobenzaldehyde, Fe(III), Co(II), Ni(II), Cu(II),
and Zn(II) chlorides, were of analytical reagent grade
(Merck, Darmstadt, Germany). The solvents used
were distilled before use. Calf thymus DNA was
purchased from Genie Biolab (Bangalore, India).
Instrumentation
The elemental analysis was performed using a
PerkinElmer 2400 CHN elemental analyzer (Perkin-
Elmer, Waltham, Massachusetts, USA). IR spectra of
Metal-Based Molecular Design Tuning Biochemical Behavior
87

the Schiff base ligands and their metal complexes
were recorded on a PerkinElmer FT-IR 783 spectro-
photometer (PerkinElmer, Waltham, Massachusetts,
USA) in the 4000–300cmꢁ1 range using KBr disc.
1H-NMR spectra were recorded on a Bruker Avance
Dry 300 MHz FT-NMR spectrometer (Bruker,
Rheinstetten, Germany) in dimethylsulfoxide (DMSO)
with tetramethylsilane (TMS) as the internal reference.
The FAB mass spectrum of the Schiff base ligands and
their complexes were recorded on a JEOL S ꢂ 102=
DA-6000 mass spectrometer=data system (JEOL,
Vernon Hills, Illinois, USA) using argon=xenon
(6 kV, 10mA) as the FAB gas. The electron paramag-
netic resonance spectrum of the mixed ligand copper
complex was recorded on a Varian E 112 EPR spec-
trometer (JEOL, Japan) in DMSO solution both at
room temperature (300K) and at liquid nitrogen tem-
perature (77 K) using TCNE (tetracyanoethylene) as
the g marker. Electronic absorption spectra were
The Schiff base FAAP was prepared by a dropwise
addition, with stirring, of ethanolic solution (20 cm3)
of furan-2-carboxaldehyde (1.0 g, 0.01 M) to an etha-
nolic solution (25 cm3) of 4-aminoantipyrine (2.1 g,
0.01 M), respectively. The reaction mixture was
refluxed on a water bath for 1–2 hr. On cooling,
the solid products were separated and filtered. Both
Schiff physical measurement bases were recrystal-
lized from ethanol and dried in vacuo over P4O10,
to yield yellow crystals.
HL2: The ligand (HL2) was prepared as reported
previously.[11]
The Schiff base was synthesized by stirring a mix-
ture of 3-nitro benzaldehyde (1.51 g, 0.01 M) with
2-aminophenol (1.09, 0.01 M) in 50 mL of ethanolic
medium. The solid product formed was removed
by filtration and recrystallized from ethanol.
Preparation of Metal Complexes
recorded in DMSO using
a
Systronics 2201
=
An ethanolic solution of metal (II) (M FeCl3.6H2O,
double-beam UV-Vis spectrophotometer (Systronics,
Ahmedabad, India). Molar conductance of the copper
complexes was measured in DMSO solution using a
Coronation digital conductivity meter (Coronation,
Ahmedabad, India). The magnetic susceptibility
CoCl2.6H2O, NiCl2.6H2O, CuCl2.2H2O & ZnCl2)
(1 mM) was stirred with an ethanolic solution of
ligands (L1 and HL2) (1 mM) and the resultant mix-
ture was refluxed for ca. 6–8 hr. Then the volume
of solution was reduced to one-third on a water bath.
The solid complex precipitated was filtered, washed
thoroughly with ethanol, and dried in vacuum.
The schematic route for synthesis of Schiff base
ligands and their metal complexes is given in
Scheme 1.
values were calculated using the relation meff
¼
2.83(vm .T)1=2 BM. The diamagnetic corrections were
made by Pascal’s constant and Hg[Co(SCN)4] was
used as a calibrant. The amount of metal present in
the metal complexes was estimated using the
ammonium oxalate method. Electrochemical experi-
ments were performed on a CHI 604D electrochemical
analysis system (CH Instruments, Inc., Austin, Texas,
USA) with a three-electrode system consisting of a
glassy carbon working electrode, Pt wire auxiliary
electrode, and Ag=AgCl reference electrode. Tetrabu-
tylammoniumperchlorate (TBAP) was used as the sup-
porting electrolyte. All solutions were purged with N2
for 30min prior to each set of experiments. Calf thy-
mus DNA was purchased from Genie Biolab
(Bangalore, India). Tris–HCl buffer solution used for
binding studies was prepared using deionized
double-distilled water.
DNA-Binding Assay
Interaction of the complex with calf thymus DNA
has been studied by recording electronic absorption
spectra. A solution of CT-DNA in 5 mM Tris–HCl=
50 mM NaCl (pH 7.0) gave a ratio of UV absorbance
at 260 and 280 nm (A260=A280) of 1.8–1.9, indicating
that the DNA was sufficiently free of proteins. A con-
centrated stock solution of DNA was prepared in
5 mM Tris–HCl=50 mM NaCl in water at pH 7.0 and
the concentration of CT-DNA was determined per
nucleotide by taking the absorption coefficient
(6600 dm3 molꢁ1 cmꢁ1) at 260 nm. Doubly distilled
water was used to prepare buffer solutions. Solu-
tions were prepared by mixing the complex and
CT-DNA in DMF medium. After equilibrium was
reached (ca. 5 min) the spectra were recorded
against an analogous blank solution containing the
Preparation
Preparation of Ligands
L1: The ligand (L1) was prepared as reported
previously.[10]
88
J. Joseph & G. Ayisha Bibin Rani

respectively. A plot of [DNA]=(Eaꢁ Ef) versus [DNA]
gave a slope of 1=(Ebꢁ Ef) and Y-intercept equal to
1=Kb(Ebꢁ Ef); Kb is the ratio of the intercept.
Antimicrobial Activity
The in vitro evaluation of antimicrobial activity
was carried out. The synthesized compounds were
tested against some fungi and bacteria to provide
the minimum inhibitory concentration (MIC) for
each compound. The MIC is the lowest concen-
tration of solution to inhibit the growth of a test
organism. The in vitro biological screening effects
of the investigated compounds were tested against
the bacterial species and fungal species by the disc
diffusion method. One day prior to the experiment,
the bacterial and fungal cultures were inoculated in
nutrient broth (inoculation medium) and incubated
overnight at 37ꢄC. Inoculation medium containing
24-hr-grown culture was added aseptically to the
nutrient medium and mixed thoroughly to get uni-
form distribution. This solution was poured (25 mL
in each dish) into Petri dishes and then allowed to
attain room temperature. Wells (6 mm in diameter)
were cut in the agar plates using proper sterile tubes.
Then, the wells were filled up to the surface of agar
with 0.1 mL of the test compounds dissolved in
DMSO (200 mg=mL). The plates were allowed to
stand for an hour to facilitate the diffusion of the
drug solution. Then the plates were incubated at
37ꢄC for 24 hr for bacteria and 48 hr for fungi and
the diameter of the inhibition zones was measured.
MICs were detected by the serial dilution method.
The lowest concentration (mg=mL) of compound,
which inhibits the growth of bacteria after 24-hr incu-
bation at 37ꢄC and of fungi after 72-hr incubation at
37ꢄC, was taken as the MIC.
SCHEME 1 Schematic route for synthesis of schiff base
ligands and their metal complexes.
same concentration of DNA. UV spectral data were
fitted into Eq. (1) to obtain the intrinsic binding
constant (Kb).
Superoxide Dismutase (SOD) Activity
In vitro SOD activity was measured using alkaline
DMSO as a source of superoxide radical ion (Oꢀ2ꢁ)
and nitrobluetetrazolium (NBT) as Oꢀ2ꢁ scavenger.[12]
In general, 400 mL of the sample to be assayed was
added to a solution containing 2.1 mL of 0.2 M
potassium phosphate buffer (pH 7.8) and 1 mL of
56 mM NBT. The tubes were kept in ice for 20 min
and then 1.5 mL of alkaline DMSO solution was
added while stirring. The absorbance was then
monitored at 540nm against a sample prepared under
½DNAꢃ=ðea ꢁ efÞ ¼ ½DNAꢃ=ðeb ꢁ efÞ þ Kbðeb ꢁ efÞ
ð1Þ
where [DNA] is the concentration of DNA in base
pairs; Ea, Eb, and Ef are apparent extinction coeffi-
cients (Aobs=[M]), the extinction coefficient for the
metal (M) complex in the fully bound form and
the extinction coefficient for free metal (M),
Metal-Based Molecular Design Tuning Biochemical Behavior
89

similar conditions except that NaOH was absent
in DMSO. The percentage inhibition (g) of NBT
reduction was calculated using the following equation:
shows that the following signals are assigned as phe-
=
nyl multiplet at 7.1–7.9 d (8H), -CH N at 8.5 d (due to
phenyl moiety), and -OH at 10.4 ppm (s, 1H). The azo-
=
methine proton (-CH N) signal in the spectrum of zinc
ð% Inhibition of NBT reductionÞ ¼ ð1 ꢁ k0=kÞ ꢂ 100
(II) complex is shifted downfield compared to the free
ligands, suggesting deshielding of the azomethine
group due to the coordination with metal ions. The
phenolic OH proton in the ligand (HL2) disappeared
in the zinc (II) complex, suggesting that the -OH
where, k0 and k represent the slopes of the straight line
of absorbance values as a function of time in the pres-
ence and in the absence of SOD mimic or a model
compound, respectively. The IC50 value of the
complex was determined by plotting the graph of per-
centage inhibition of NBT reduction against an
increase in the concentration of complex. The concen-
tration of the complex that causes 50% inhibition of
NBT reduction is reported as IC50.
1
proton is involved in coordination.[11] The H-NMR
spectrum of the zinc (II) complex is shown in Fig 1.
IR Spectra
To study the binding mode of the Schiff base to
the metal complexes, the IR spectra of the free
ligands were compared with the spectra of the com-
RESULTS AND DISCUSSION
plexes. The absorption band at 1647 cmꢁ1 is assigned
The Schiff base ligands and their mixed ligand
complexes have been synthesized and characterized
by spectral and analytical techniques. The color, ana-
lytical data, molar conductance, and magnetic
moment values are given in Table 1. The elemental
analyses of the Schiff base ligands support the
chemical composition of the ligands and their com-
plexes. In the present study, the prepared complexes
are 1:1 electrolytic in nature, except the iron complex
(nonelectrolytic in nature). All the complexes are
stable in air. The antipyrine derivatives are found
to be soluble in chloroform, dimethylformide, tetra-
hydrofuran, and dimethylsulphoxide.
1
=
n(C O) in L free ligand. The shift of bands to lower
wavenumber 20–32 cmꢁ1 in the spectra of all com-
plexes suggests the involvement of the pyrazolone
oxygen in chelation. The strong absorption bands
located at 1616 and 1603 cmꢁ1 in the spectrum of
the free ligands L1 and HL2 are attributed to
=
n(-CH N) vibrations. These bands are shifted
(by ꢅ 10–40 cmꢁ1) toward lower frequencies in the
spectra of all the complexes, which clearly indicated
that the complexation has taken place through the
nitrogen atom of the azomethine group.
The IR spectra of the ligands show a strong band
in the 3050–3400 cmꢁ1 region, assigned to the phe-
nolic -OH group. The disappearance of this band
in the spectra of the complexes indicates the depro-
tonation of the hydroxyl group upon coordination.
The IR spectra of the metal complexes also show
some new bands in the 480–450-cmꢁ1 and
447–410-cmꢁ1 regions, which may probably be due
1H-NMR Spectra
The ligand (L1) shows the following signals and
their assignments are given below: phenyl multiplet
=
at 7.3–7.5 d (5H), -CH N at 7.9 (due to furfuryl moi-
ety), -C-CH3 at 2.4 d, N-CH3 at 3.2 d. The ligand (HL2)
TABLE 1 Physical and Analytical Data of the Synthesized Complexes
Elemental analysis
Molar
conductance
(Xꢁ1 cm2 molꢁ1
Compound
Color
Yellow
Yellow
Yield %
C
H
N
M
)
meff (BM)
L1
HL2
85%
81%
80%
82%
78%
82%
84%
68.30 (68.2)
5.38 (5.37) 14.94 (14.92)
—
—
—
—
6
—
—
64.44 (64.41) 4.16 (4.12) 11.57 (11.60)
[FeL1L2 Cl2] Brown
50.77 (50.75) 3.68 (3.65) 10.22 (10.25) 8.15 (8.12)
53.29 (53.28) 3.86 (3.81) 10.72 (10.75) 9.02 (9.05)
53.31 (53.34) 3.86 (3.85) 10.73 (10.70) 8.99 (8.96)
52.92 (52.90) 3.83 (3.80) 10.65 (10.63) 9.66 (9.65)
52.77 (52.72) 3.82 (3.85) 10.62 (10.60) 9.92 (9.95)
5.6
[CoL1L2] Cl
[NiL1L2] Cl
[CuL1 L2]Cl
[ZnL1L2] Cl
Brown
38
54
45
38
3.12
Dia
1.81
Dia
Brown
Brown
Dark Yellow
90
J. Joseph & G. Ayisha Bibin Rani

FIGURE 1 1H-NMR spectrum of zinc (II) complex. (Color figure available online.)
to the formation of n(M-O) and n(M-N) bands,
respectively. In the case of the Fe(III) complex,
the weak band that appeared at 350 cmꢁ1 is due to
the formation of Fe–Cl bond. IR spectral data of the
ligands (L1 [Fig. 2a] and HL2 [Fig. 2b]) and copper
complex (Fig. 2c) are presented in Table 2.
value of 5.6 BM suggested high-spin octahedral
geometry for the Fe(III) complex.[13]
The spectrum for the Co(II) complex shows a d–d
band at 18,651 (537 nm) and 31,532 cmꢁ1 (317 nm)
1
1
and may be assigned to A1g! Bg, respectively, in
square planar stereochemistry. Together with the
magnetic moment value of 3.12 BM, a square planar
geometry for the Co(II) complex was proposed.
The electronic spectrum of the nickel (II) complex
shows two d–d bands at 20,126 (497 nm) and
Electronic Absorption Spectra
The electronic absorption spectral data of the
ligands and their complexes were recorded in DMSO
and are presented in Table 3. The electronic spec-
trum of free Schiff base ligand show bands at
31,300 (320 nm) and 30,581 cmꢁ1(327 nm), which is
assigned to the p-pꢆ transitions of the azomethine
1
18,670 cmꢁ1(536 nm), which are assigned as 1A1g! B1g
1
1
and A1g! B2g transitions, indicating the square planar
geometry.[14] This complex is diamagnetic in nature.
Therefore, a square planar geometry has been
suggested.
=
(>C N–) chromophore. On complexation this band
In the present study, the copper (II) complex exhi-
bits bands at 18,302 cmꢁ1(546 nm) and 20,823 cmꢁ1
was shifted to lower wavelengths, suggesting the
coordination of azomethine nitrogen to the central
metal ion.
2
2
(480 nm) assigned to B1g! A1g transitions charac-
2
2
teristic of a square planar geometry with dx ꢁy
The electronic spectrum of the Fe(III) complex
exhibits bands at 19,064 (525 nm) and 20,265 cmꢁ1
ground state.[15–17] These data and the magnetic
moment value of 1.81 BM suggest square planar
geometry around Cu(II).
6
4
(494 nm) and are attributable to A1g! T1g(G) and
4
6A1g! T1g(G) transitions, respectively. In these
In general, Zn(II) is a d10 metal ion; no band is
expected in the visible region and it is also found as a
diamagnetic complex. However, a strong band
observed at 22,172cmꢁ1 (451nm) is assignable to the
L!M charge transfer transition,[18] which is compatible
with this complex having a square planar geometry.
transitions two intense bands may be assigned
spin-forbidden d–d transitions and the other
charge-transfer transitions. These transitions are
assigned for octahedral Fe(III) complexes. The elec-
tronic transitions together with the magnetic moment
Metal-Based Molecular Design Tuning Biochemical Behavior
91

FIGURE 2 IR spectrum of ligands (L1(2) and HL2 (b)) and copper complex (c).
92
J. Joseph & G. Ayisha Bibin Rani

FIGURE 2 CONTINUED.
of g < 2.2 in the present copper complex gives
k
a clear indication of the covalent character of
ESR Spectra
The ESR spectrum of the copper complex was
recorded in DMSO at 300 and 77 K (Fig. 3). The
the metal–ligand bond and delocalization of the
unpaired electron into the ligand.
The EPR parameters g , g , and A and the energies
observed trend of gk(2.21) > g (2.02) > ge(2.0023)
?
describes the axial symmetry with the unpaired
k
?
k
of d–d transition were used to evaluate the bonding
2
electron residing in the dx2-y2 orbital.[19] The value
parameters a , b2, and c2, which may be regarded
as measures of the covalency of the in-plane r bonds,
in-plane p bonds, and out-of-plane p bonds.
Molecular orbital coefficients a2 (covalent in-plane
r bonding), b2 (covalent in-plane p bonding), and c2
(out-plane p bonding) were calculated using the
following equations:
TABLE 2 IR Spectral Data (cmꢁ1) for the Free Ligands and Their
Metal Complexes
Compound
n
n
n
n
n
M-Cl
C¼O
C¼N
M-O
M-N
L1
1647
—
1616
1603
1579
1584
1575
1576
1596
1593
1583
1578
1591
1589
—
—
—
—
—
—
HL2
[FeL1L2 Cl2]
1637
471
453
480
450
478
434
429
434
410
447
350
ꢀ
ꢁ
À
Á
a2 ¼ A =0:036 þ g ꢁ 2:0027
jj
jj
[CoL1L2] Cl
[NiL1L2] Cl
[CuL1L2]Cl
[ZnL1L2] Cl
1641
1632
1643
1639
—
ð2Þ
þ 3=7ðg? ꢁ 2:0023Þ þ 0:04
—
2
If the a
value is 0.5, it indicates complete
covalent bonding, while the value of a2 ¼ 1.0
suggests complete ionic bonding. The observed
value of a2 (0.72) indicates that the complex has
some covalent character.
—
—
Metal-Based Molecular Design Tuning Biochemical Behavior
93

TABLE 3 Electronic Absorption Spectral Data of the Complexes in DMSO Solution
Compound
Solvent
Absorption (cmꢁ1
)
Band assignment
INCT
Geometry
L1
HL2
[FeL1L2 Cl2]
[CoL1L2] Cl
[NiL1L2] Cl
[CuL1L2]Cl
[ZnL1L2] Cl
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
DMSO
31,300
—
30,581
INCT
—
4
6
4
19,064, 20,265
18,651, 31,532
20,126, 18,670
18,302, 20,823
22,172
6A1g! T1g(G) and A1g! T1g(G)
Octahedral
Square planar
Square planar
Square planar
Square planar
1
1A1g! Bg INCT
1A1g! B1g and A1g! B2g
1
1
1
2
2
INCT B1g! A1g
L!M
The observed b2 and c2 values of 1.23 and 0.71
indicate that there is interaction in the out-of-plane
g bonding, whereas the in-plane g bonding is pre-
dominantly ionic. Significant information about the
nature of bonding in the Cu(II) complex can be
derived from the relative magnitudes of K and K .
k
?
K ¼ a2b2
ð5Þ
jj
K ¼ a2c2
For the present complex, the observed order
ð6Þ
?
Kk(0.89) > K (0.51) implies a greater contribution
?
from out-of-plane g bonding than from in-plane g
bonding in metal–ligand g bonding.
The empirical factor f ¼ g===A== cmꢁ1 is an index
of tetragonal distortion. Values of this factor may
vary from 105 to 135 for small to extreme distortions
in square planar complexes, and it depends on the
nature of the coordinated atoms.[20] The f values of
copper complexes were found to be in the range
of 144 (Table 4), indicating significant distortion from
planarity.
Mass Spectra
FIGURE 3 ESR spectrum of copper complex at room (a) and
liquid temperature (b).
The FAB mass spectra of the Schiff bases and their
corresponding metal complexes were recorded and
their stoichiometry compositions compared. The
Schiff base ligands L1 and HL2 show a molecular
ion peak at m=z ¼ 281 and 242, respectively. The
mass spectra of Fe(III), Co(II), Ni(II), Cu(II), and
Zn(II) complexes show a molecular ion peak (Mþ)
at m=z 648, 616, 617, 620, and 622 respectively, the
ꢀ
ꢁ
ꢁ
b2 ¼ gjj ꢁ 2:0027 E= ꢁ 8ka2
ð3Þ
ð4Þ
ꢀ
c2 ¼ gjj ꢁ 2:0027 E= ꢁ 2ka2
TABLE 4 ESR Spectral Data of the Copper Complex
2
Complex
g
g
giso
A
A
K
K
a
b2
c2
f ¼ (g =A )
k
?
k
?
k
?
k
k
[CuL1L2] Cl at 300 K
[CuL1L2]Cl at 77 K
—
—
2.09
—
—
—
—
—
—
—
—
—
2.21
2.02
153
31
0.89
0.51
0.72
1.23
0.71
144
94
J. Joseph & G. Ayisha Bibin Rani

TABLE 5 Electronic Absorption Spectral Data of Copper Com-
plex in Various Solvents
stoichiometry of the complexes as supported by the
FAB mass spectra of other complexes. Elemental
analysis values are in good agreement with the
values calculated from molecular formula of these
complexes, which is further supported by the
FAB-mass studies of representative complexes.
Wavelength, nm (kmax
)
S. No
Solvent
Before
After
1
2
3
4
5
6
7
8
9
DMF
682
480
657
450
452
459
578
621
463
766
618
881
578
474
647
815
806
718
DMSO
C6H6
EtOAc
Hexane
CH3OH
CH3C ꢇ N
DCM
Solvatochromism
The complexes are soluble in various organic sol-
vents and presented distinctive solvatochromism.
The observed colors in different solvents after
irradiation of the copper complex in UV light under
a UV transilluminator as illustrated in Fig. 4 and kmax
values are presented in Table 5. The origin of the
color changes are attributed to the shift in the d–d
transition of the copper (II) ions as a result of sol-
vent–solute interactions. The broad structureless
absorption band is related to the transition of the
electron from the lower-energy orbitals to the hole
THF
in the dx2-y2 orbital of the copper (II) ion (d9). The
large shifts observed for the copper complex indicate
that it has larger polarizability due to the electron-
withdrawing group. The obtained results suggest that
the DN (donor nitrogen) parameter of solvent has
the dominate contribution to the shift of the d–d
absorption band of the complexes. The d–d visible
absorption band exhibits a red shift with the increase
of the donor number of the solvents. Similar solvato-
chromic behavior was observed for all other metal
complexes.
DNA-Binding Studies
The ability of complexes to bind DNA was inves-
tigated by electronic absorption spectra and cyclic
voltammetry.
Absorption Spectral Features of DNA
Binding
The application of electronic absorption spec-
troscopy in DNA-binding studies is one of the
most useful techniques. Complex binding with DNA
through intercalation usually results in hypochro-
mism and bathochromism, 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.[21] The absorption
spectra of complexes in the absence and presence
of CT-DNA of complexes are given in Fig. 5a–e.
The binding results show that the bathochromic
shift of 2–4 nm along with significant hypochromi-
city was observed with the addition of DNA to
FIGURE 4 Colors in different solvents after irradiation of cop-
per complex under UV transilluminator. (Color figure available
online.)
Metal-Based Molecular Design Tuning Biochemical Behavior
95

FIGURE 5 The absorption spectra of complexes in the absence and presence of CT-DNA of complexes (a: Cu(II), b: Ni(II), c: Co(II),
d: Fe(III), e: Zn(II) complexes). (Color figure available online.)
complex solution. When the amount of CT-DNA is
increased, a decrease of intensity in the charge
transfer band at about 65% was observed. Intense
absorption bands observed near 300 nm for com-
plex are attributed to a n!pꢆ or p!pꢆ transition.
To compare the binding strength of the complexes,
their intrinsic binding constants (Kb) with CT-DNA
have been determined from the decay of the
absorbance.
The observed Kb values of metal complexes are lower
binding constants than Distamycin A.
DNA-Binding Study
In the cyclic voltammetric (CV) study, copper
complexes in the presence and absence of CT-DNA
are shown in Fig. 6. From CVs, it could be found that
copper complexes exhibited a pair of redox peaks
for one electron transfer couple of Cu(II)=Cu(I) at
the scan rate of þ 2 to ꢁ 2 V. The cathodic peak
potential (Epc) and the anodic peak potential (Epa)
in the absence of DNA are 0.78 and 0.26 V, respect-
ively. The separation of the anodic and cathodic to
anodic peak potentials (DEp) is 206 mV, and the ratio
The metal complexes can bind to the double-
stranded DNA in different binding modes on the
basis of their structure, charge, and type of ligands.[22]
The observed intrinsic binding constants Kb of
complexes are 4.6 ꢂ 104 Mꢁ1, 4.3 ꢂ 104 Mꢁ1, 3.9 ꢂ
104 Mꢁ1, 3.5 ꢂ 104 Mꢁ1, and 4.1ꢂ 104 Mꢁ1, respectively.
96
J. Joseph & G. Ayisha Bibin Rani

Superoxide Dismutase (SOD)
Mimic Activities
The SOD activities of complexes were investigated
by the NBT assay method. In the present study, we
prepared different mixed ligand complexes. Among
the metal complexes, the copper complex showed
better SOD activity than other complexes. The mech-
anism of SOD activity is given in Eqs. (7) and (8):
ꢀ
Cu2þ þ Oꢁ2 ! Cuþ þ O2
ð7Þ
ð8Þ
Cuþ þ O2ꢁ þ 2Hþ ! Cu2þ þ H2O2
ꢀ
Figure 7 represents the plot of percentage of inhi-
biting NBT reduction with an increase in the concen-
tration of complexes. The copper (II) complexes
showed SOD-like activity, which was evaluated by
the scavenger concentration causes 50% inhibition
in the detector formation, IC50. The observed find-
ings indicate that the superoxide scavenging proper-
ties and oxidative behavior of mixed ligand
complexes were identical to those of complexes sup-
porting the above mechanism, which are reinforced
by ESR spectral data.
FIGURE 6 The cyclic voltammograms of copper complexes in
the absence and in the presence of various DNA concentrations.
of cathodic to anodic peak currents ipc=ipa is 1.03,
indicating a quasi-reversible redox process (DEp of
59 mV for a one-electron diffusion and ipc=ipa of
about one controlled reversible process). The for-
mal potential (E1=2), taken as the average of Epc
and Epa, is 0.483 V in the absence of DNA. The pres-
ence of DNA in the solution at the same concen-
tration of the copper complex causes a negative
shift in E of 0.483 V and a decrease in DE of
0.251 V. The value of ipc=ipa also decreases with
the increase of DNA concentration. The decrease
in peak currents can be explained in terms of an
equilibrium mixture of free and DNA-bound copper
(II) complex to the electrode surface. These results
clearly suggest that copper complex binds to
CT-DNA through an intercalating way. Electro-
chemical parameters for the mixed ligand com-
plexes on interaction with CT-DNA are shown in
Table 6.
Antimicrobial Activity
The in vitro biological screening effects of the inves-
tigated compounds were tested against the bacterial
species and fungal species by the disc diffusion
method. The MIC values of the synthesized compounds
are summarized in Tables 7 and 8. A comparative study
of the mixed ligands and their complexes (MIC values)
indicates that complexes exhibit higher antimicrobial
activity than the free ligands. The enhanced activity of
the complexes can be explained on the basis of Over-
tone’s concept[23] and Tweedy’s chelation theory.[24]
According to Overtone’s concept of cell permeability,
the lipid membrane that surrounds the cell favors
TABLE 6 Electrochemical Parameters for the Mixed Ligand Complexes on Interaction with CT DNA
E1=2(V)
4Ep(V)
Compound
Redox couple
Free
Bound
Free
Bound
Ipa=Ipc
[FeL1L2 Cl2]
[CoL1L2] Cl
[NiL1L2] Cl
[CuL1L2]Cl
[ZnL1L2] Cl
Fe (III)!Fe(II)
Co(III)!Co(II)
Ni(II)!Ni(I)
ꢁ0.218
ꢁ0.245
ꢁ0.231
0.301
ꢁ0.310
ꢁ0.321
ꢁ0.248
0.270
0.439
0.431
0.486
0.441
1.29
1.21
1.02
1.18
0.81
ꢁ0.431
0.483
ꢁ0.416
0.251
Cu(II)!Cu(I)
Zn(II)!Zn(0)
ꢁ0.347
ꢁ0.353
0.338
ꢁ0.310
Metal-Based Molecular Design Tuning Biochemical Behavior
97

FIGURE 7 Plot of percentage of inhibiting NBT reduction with an increase in the concentration of complexes. (Color figure available
online.)
TABLE 7 Minimum Inhibitory Concentration of the Synthesized Compounds Against Growth of Bacteria (lg/mL)
Compound
E .coli
K. pneumonia
S. typhi
P. aeruginosa
S. aureus
L1
60
58
79
81
66
82
75
43
51
49
52
50
10
12
6
64
62
82
68
74
85
81
58
55
56
54
42
15
10
14
10
66
58
86
72
81
87
81
62
61
52
58
55
6
66
59
82
78
85
91
92
59
63
65
60
63
12
4
72
69
91
89
83
88
83
52
51
53
63
51
4
HL2
FeCl3.6H2O
CoCl2.6H2O
NiCl2.6H2O
CuCl2.2H2O
ZnCl2
[FeL1L2 Cl2]
[CoL1L2] Cl
[NiL1L2] Cl
[CuL1L2]Cl
[ZnL1L2] Cl
Penicillin
Ampicillin
Vancomycin
Ofloxacin
8
6
12
4
10
6
8
8
14
TABLE 8 Minimum Inhibitory Concentration of the Synthesized Compounds Against Growth of Fungi (lg/mL)
Compound
A. niger
R. stolonifer
A. flavus
R. bataicola
C. albicans
L1
60
58
82
94
82
91
78
18
15
23
21
22
12
14
10
66
65
82
84
71
85
75
24
28
25
37
26
15
9
72
58
86
82
73
84
81
36
31
32
32
29
5
80
69
78
87
75
82
85
27
24
21
45
33
15
8
50
58
74
83
81
83
86
21
16
25
38
20
16
15
8
HL2
FeCl3.6H2O
CoCl2.6H2O
NiCl2.6H2O
CuCl2.2H2O
ZnCl2
[FeL1L2 Cl2]
[CoL1L2] Cl
[NiL1L2] Cl
[CuL1L2]Cl
[ZnL1L2] Cl
Nystatin
Ketoconazole
Clotrimazole
15
17
6
14
98
J. Joseph & G. Ayisha Bibin Rani

the passage of only the lipid-soluble materials, which
makes liposolubility an important factor, controlling
the antimicrobial activity. On chelation, the polarity of
the metal ion will be reduced to a greater extent due
to the overlap of the ligand orbital and partial sharing
of the positive charge of the metal ion with donor
groups. Further, it increases the delocalization of
g-electrons over the whole chelate ring and enhances
the lipophilicity of the complexes.
activity due to the presence of electron-donating
methyl groups in the phenolic moiety. In conclusion,
the present study has shown that copper conjugation
may be advantageous in designing highly effective
drugs in anti-inflammatory therapy.
ACKNOWLEDGMENTS
We express our sincere thanks to the President,
Noorul Islam Centre for Higher Education, Kumara-
coil, for providing research facilities and financial
support. We also express our sincere thanks to the
Head, Department of Chemistry, Noorul Islam Centre
for Higher Education, Kumaracoil, for providing
valuable research support.
This increased lipophilicity enhances the perme-
ation of the complexes into lipid membranes and
blocking of the metal binding sites in the enzymes
of microorganisms. These complexes also disturb
the respiration process of the cell and thus block
the synthesis of the proteins, which restricts
further growth of the organism, and as a result,
microorganisms will die. The increased activity of
the complexes may also be explained on the basis
of their high solubility, size of the metal ion, and
presence of the bulkier organic moieties. The differ-
ent lipophilic behaviors of aromatic residues such
as antipyrine and furfuraldehyde are involved in
the biological activity mechanisms. The rise in the
antimicrobial activity of mixed ligand complexes
may be owing to the effect of the metal ion on the
normal cell processes.[25] The comparative study of
mixed ligand complexes showed higher activity than
other ligand complexes.
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