1
CHAPTER V
BIOLOGICAL AND PHARMACOLOGICAL STUDIES OF LIGANDS AND
THEIR COPPER COMPLEXES
5.1 General introduction
The Schiff bases with sulphur and nitrogen donor atoms in their structures act as
superior chelating agents for the transition and non-transition metal ions [183]. When
such heterocyclic ligands are complexes with metal ions, the resulting complexes
showed enhanced activity. DNA-metal complex interaction has become a subject of
intense research [184]. This interaction is essentially non-covalent, either by
intercalation, groove binding or external electrostatic binding [185]. The binding of
DNA to metal complex is closely related to the structure of the complex.
The stable non toxic metal complexes which catalase the superoxide anion show
considerable promise as SOD mimics for pharmaceutical application, especially copper
(II) with square planar geometry capable of protecting cells against O2- attack [186].
The success of Cu complexes as potential therapeutics will most likely be due to their
ability to increase SOD activity, leading to relief of oxidative stress in the generation of
free radicals and ROS describe oxidative damage to DNA and lipid peroxidation as the
main effects of oxidative stress [187].
Metal complexes have a higher position in medicinal chemistry. The therapeutic
use of metal complexes in cancer and leukemia are reported from the sixteenth century.
In 1960 an inorganic complex cisplatin was discovered, today more than 50 years, it is
still one of the world’s best selling anticancer drug. Metal complexes formed with other
metals like copper, gold, gallium, germanium, tin, ruthenium, iridium was shown
significant antitumor activity in animals. Formation of DNA adducts with cancer cell
and results in the inhibition of DNA replication. In the treatment of ovarian cancer
ruthenium compounds containing arylazopyridine ligands show cytotoxic activity. Now
2
a day’s metal complex in the form of nanoshells are used in the treatment of various
types of cancer.A number of copper and manganese SOD mimetics have been shown to
possess antitumor activity and have been proposed as a new class of potential anticancer
agents [188].
In this chapter, the antimicrobial, anti-oxidant, SOD, DNA studies (binding &
cleavage), anti-inflammatory studies of 2-aminobenzothiazzole derivatives and their
complexes were performed and summarized. The biological results are discussed
separately in different sections.
5.2. Antimicrobial activity
The in vitro antimicrobial activities of the investigated compounds were tested
against the bacterial species by well diffusion method [189,190]. Their antibacterial
activities against Gram-negative bacteria (Escherichia coli and Klebsiellapneumoniae)
and Gram-positive bacteria (Staphylococcus aureus, Proteus vulgaris and Pseudomonas
aeruginosa) have been investigated. The inhibitions around the antibiotic discs were
measured after incubation and Streptomycin was used as standard drug. It is suggested
that the synthesized copper complexes of 2-aminobenzothiazole derivatives showed
more activity than its free ligand.
The enhanced activity of the complexes can be explained on the basis of
Overtone’s concept [191] and Tweedy’s Chelation theory [192]. According to
Overtone's concept of cell permeability, the lipid membrane that surrounds the cell
favours the passage of only the lipid-soluble materials makes which liposolubility is an
important factor, which controls the antifungal activity. On chelation, the polarity of the
copper 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 copper ion with donor groups. Further,
it increases the delocalization of π-electrons over the whole chelate ring and enhances
the lipophilicity of the complexes. This increased lipophilicity enhances the penetration
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
3
cell and thus block the synthesis of the proteins that restricts further growth of the
organism.
The nature of substitutents on the ligand also plays a significant role in
determining antimicrobial properties. Of all the test compounds attempted, the presence
of electron withdrawing substituent (nitro group) on the aromatic ring in general
increases the antimicrobial activities of the tested metal complexes compared to
complexes having other substituents. In the present study, the order of the antimicrobial
activity of the synthesized compounds (based on the substituent present in the phenyl
ring) as follows:
Cinnamaldehyde > 4-NO2 > 3-NO2 > 4-Cl > 3-Cl > 2-Cl > OCH3 > 3-OH -
4-OCH3 > 3-OH > 4-N-(CH3)2.
[CuL9(OAc)2] possessing superior activity due to its extended conjugation. It is
inferred from the results that electron withdrawing nitro group ([CuL10
(OAc)2] and
[CuL4(OAc)2]), have effective and direct impact on selective antimicrobial activities
against bacteria. The complexes, however, with electron-releasing substituents such as
methoxy and hydroxyl groups, are lesser active compared to unsubstituted phenyl ring.
In the present study, the complexes containing the methoxy group showed increased
activity than that of hydroxyl group due to the comparatively faster diffusion of copper
complex into cell membrane in the presence of methoxy group. The significant activity
of the Schiff base ligand may arise from the two imine groups which import in
elucidating the mechanism of transformation reaction in biological system. All the
copper complexes are found to have higher antibacterial activity than Schiff base
ligands. The antibacterial results evidently showed that the activity of the Schiff base
compounds becomes more pronounced when coordinated to the copper ion. The MIC
values indicate that all the compounds tested exhibit moderate to strong antimicrobial
activity on the tested microorganisms. In the scheme 1, the order of activities as
follows:
4
[CuL9(OAc)2] [CuL
10(OAc)2] [CuL
4(OAc)2] [CuL
11(OAc)2] - [CuL
5(OAc)2]-
[CuL7(OAc)2] [CuL
1(OAc)2] [CuL
3(OAc)2] > [CuL
6(OAc)2] [CuL
2(OAc)2]
[CuL8(OAc)2]
In the copper complexes, the ligands have few uncoordinated hetero atoms
(Nitrogen, oxygen and sulfur-containing heterocycles such as pyrrole, furfural, 5-methyl
thiazole) which enhance the activity of the complexes by bonding with trace elements
present in microorganisms may combine with the uncoordinated site and may inhibit the
growth of antimicrobial pathogens. The mode of action of the compounds may involve
the formation of a hydrogen bond through the azomethine group amd imine grsoup with
the active centers of cell constituents, resulting in interferences with the normal cell
process [193].
It is concluded that the heterocyclic compounds of furfuraldehyde moiety or
pharmacophore exibits higher activity. The inhibitory action gets enhanced with the
introduction of oxygen donor atom present in the heterocyclic ring. From the
antimicrobial screening observation, heteroaromatic ring systems have higher activity as
compared to aromatic ring systems in the order (Scheme 1to Scheme 6).
5
Fig. 5.1 Minimum inhibitory concentration of the synthesized ligands
(L1-L
11) and their corresponding copper complexes against growth
of bacteria (μg/mL)
Fig. 5.2 Minimum inhibitory concentration of the synthesized ligands
(L12
-L14
) and their corresponding copper complexes against growth
of bacteria (μg/mL)
6
Fig. 5.3 Minimum inhibitory concentration of the synthesized ligands
(L15
-L24
) and their corresponding copper complexes against growth
of bacteria (μg/mL)
Fig. 5.4 Minimum inhibitory concentration of the synthesized ligands
(L25
-L34
) and their corresponding copper complexes against growth
of bacteria (μg/mL)
7
Fig. 5.5 Minimum inhibitory concentration of the synthesized ligands
(L35
-L44
) and their corresponding copper complexes against growth
of bacteria (μg/mL)
Fig. 5.6 Minimum inhibitory concentration of the synthesized ligands
(L45
-L54
) and their corresponding against growth of bacteria
(μg/mL)
8
5.3. DNA binding experimentss
5.3.1 Cyclic Voltammetric Studies
The electrochemical investigations of DNA binding are an acceptable method
for the determination of metallointercalation and coordination of the metal ions with the
DNA base pairs. It is also the complement to UV–Vis spectroscopy. The changes in the
peak currents observed for the complexes upon addition of CT DNA may indicate that
the complexes possess a higher DNA binding affinity.
The cyclic voltammogram of copper complexes of fixed concentration of the
complex with increasing concentration of DNA in the solution causes a considerable
decrease in the voltammetric current with very significant potential shift was observed
in almost for all these complexes, which is consistent with the binding of copper
complexes of ligand moiety between the DNA base pairs as also evidenced by the
spectral results. There is a considerable decrease in peak current as well as in the ipa/ipc
values. The formal potential, E1/2 (voltammetric) taken as the average of Epc and Epa
shifts slightly towards the positive side on binding to DNA suggest that both Cu(II) and
Cu(I) forms bind to DNA intercalative mode at different rates. The Nernst equation for
the reversible redox reactions of the free and bound species and the corresponding
equilibrium constants for binding of each oxidation state to DNA for one electron redox
process are given as follows:
The ratio of equilibrium constants, K2+/K+ for the binding of the Cu(II) and
Cu(I) forms of complexes to DNA can be estimated from the net shift in E1/2, assuming
reversible electron transfer. For an electron transfer system in which both the oxidized
and reduced forms associated with a third species such as DNA in solution, the ratio
K2+/K+ could be calculated from the net shift in E1/2 values from the equation,
2+DNAe- b
K
2++ e- +
+-DNA +
-
K2+
+
CuL CuL E
o ,
of,
ECuL CuL
9
The cyclic voltammograms of the complexes in the absence of DNA reveal a
non-Nernstian but quasi-reversible one electron redox process involving the
Cu(II)/Cu(I) couple, as judged from the peak potential separation of 87 mv (93 mVin
the presence of DNA) for [CuL25
Cl2].For all these complexes K2+ is higher than
K+(Table.), suggests that the interaction of Cu(II) complexes with DNA tends to
stabilize the Cu(II) over the Cu(I) state. The low Ipa value over the Ipc is also in
consistent with this observation.The ratio of the binding constants (K2+/K+) was
calculated which indicates that the Cu(I) displaying higher DNA binding affinity than
Cu(II) form (Table ) [194, 195].
Thus, K+/K2+ values for electron-withdrawing group, containing copper complex
were less than unity suggesting the preferential stabilization of Cu(II) form. However,
and interestingly enough, we observed the ratio of binding constant for -OH and -OCH3
group substituted complexes was approximately unity suggesting that each oxidant state
interacts with DNA to the same extent.
Fig. 5.7 Cyclic voltammogram of [CuL13
Cl2] in the presence
and absence of different concentrations of DNA
10
The cyclic voltammograms of the complexes in the absence of DNA reveal a
non-nernstian but quasi-reversible one electron redox process involving the Cu(II)/Cu(I)
couple for [CuL13
Cl2] , in which the first segment, cathodic and anodic peaks were
observed at -0.806 mV and -0.388mV, respectively. This showed reduction from +2 to
+1 form at a cathodic peak potential with the scan rate of -0.416. Also, the second
segments of cathodic peaks and anodic peaks Epc2 and Epa2 at +0.260 mV and
+0.499mV with the scan rate of 0.239 mV which corresponds to ligand oxidation and
reduction behaviour, respectively.
The cyclic voltammogram of [CuL13
Cl2] in the presence of different
concentration of DNA causes a considerable decrease in the voltammetric current. In
addition, the both peak potentials, both Epc1(-0.802 mV) and Epa1(-0.399) as well as E1/2
have a shift to positive potential with scan rate of -0.403 mV which is shown in Fig.
The decrease extents of the peak currents observed for metal complex upon addition of
CT-DNA may indicate that the binding affinity of copper complex and thus copper
complex interacts with CT-DNA through intercalation binding mode [196]. In the
presence of DNA, the significant reduction in peak currents on the addition of DNA is
due to slow diffusion of an equilibrium mixture of the free and DNA-bound complexes
to the electrode surface. Also from the fact of copper ion has strong coordination with
guanine bases. It is deduced that the strong affinity of copper complex and DNA was
also likely caused by the coordination interaction of CuII ion in [CuL
13Cl2]with guanine
bases of DNA [199].The electrochemical parameters of the Cu(II) complexes are shown
in Table . It was concluded that the present ligand systems stabilize the unusual
oxidation states of copper ion during electrolysis. Other copper complexes were also
showed similar electrochemical behaviour.
11
Table 5.1
Electrochemical parameters for the interaction of DNA with copper complexes
Table 5.2 Binding constant of copper complexes on interaction with DNA
S.No Copper complexes Kb K2+/K+
1 [CuL1(OAc)2] 1.6 0.87
2 [CuL7(OAc)2] 2.5 1.29
4 [CuL9(OAc)2] 2.8 1.19
5 [CuL10
(OAc)2] 1.6 0.79
Copper
complexes
Redox couple
E1/2 (V)
mV Ep (V)
mV ipa/ipc
Free Bound Free Bound
[CuL1(OAc)2] Cu(II)Cu(I) -1.017 -1.007 -0.414 -0. 423 1.14
[CuL2(OAc)2] Cu(II)Cu(I) -1.049 -0.949 -0.392 -0.409 1.25
[CuL3(OAc)2] Cu(II)Cu(I) -0.672 -0.684 -0.284 -0.298 1.17
[CuL4(OAc)2] Cu(II)Cu(I) -1.018 -0.764 -0.388 -0.363 1.17
[CuL5(OAc)2] Cu(II)Cu(I) -0.978 -0.923 -0.479 -0.488 0.93
[CuL6(OAc)2] Cu(II)Cu(I) -1.194 -1.104 -0.465 -0.481 1.14
[CuL7(OAc)2] Cu(II)Cu(I) -1.114 -1.083 -0.390 -0.402 1.25
[CuL8(OAc)2] Cu(II)Cu(I) -1.049 -0.974 -0.451 -0.450 1.12
[CuL9(OAc)2] Cu(II)Cu(I) -1.117 -1.088 -0.406 -0.410 0.99
[CuL10
(OAc)2] Cu(II)Cu(I) -1.011 -0.941 -0.401 -0.405 1.11
12
5.3. 2 Absorption spectral titrations
Electronic absorption spectroscopy is one of the most useful techniques for
DNA binding studies of metal complexes. The binding of copper(II) complexes to DNA
helix has been characterized through absorption spectral titrations, by following
changes in absorbance and shift in wavelength. The experiments were performed by
maintaining a constant concentration of the complex while varying the DNA
concentration.
A compound can bind to DNA either via covalent (in which a labile ligand is
replaced with a nitrogen atom of DNA base, such as N7 of guanine) or non-covalent
(such as intercalative, electrostatic and groove binding) interaction. Normally, a
compound bound to DNA through intercalation results in hypochromism (decrease in
absorbance) and bathochromism (red shift). It is due to the fact that intercalative mode
involves a strong stacking interaction between aromatic chromophore and the base pairs
of DNA. It is believed that the extent of hypochromism depends on the strength of
intercalation.
A fixed concentration of the complexes was titrated with increasing
concentration of DNA. It was observed that the Cu(II) complex of L2exhibited
‘hypochromism’ in the intra-ligand region (at 242 nm) with a noticeable red shift. This
indicates strong binding intensity of the complex towards CT DNA leading to the
damaged DNA double helix structure. While significant hypochromism with a red shift
of 10 nm (bathochromism) of absorption band implicates intercalative mode of binding
and is likely that the copper complexes with aromatic chromophore stabilizes the DNA
duplex.
13
Fig. 5.8 UV Absorption spectrum for [CuL2(OAc)2] in the presence and
absence of DNA
In the UV region, the Cu(II) complex of L2
also exhibits a band at ca. 413, 433
nm. With increasing DNA concentration, the absorption bands of the complexes were
affected, resulting in a hypochromism tendency and slight shifts to longer wavelengths,
which indicate that the Cu(II) complex can interact with DNA (Fig.5.8) (guanine N7) of
base pair. The observed hypochromism and bathochromism for the Cu(II) complex are
also observed for the complexes. These hypochromism and bathochromism are lower
when compared to those for potential intercalators [197, 198]. The intrinsic binding
constant (Kb) was obtained by monitoring the change in absorbance with increasing
concentrations of DNA for the Cu(II) complexes.
The observed Kb values obtained for the copper complexesthose compared
lower when observed for typical classical intercalators (EthBr, Kb, 1×4 106
M–1
in 25
mM Tris-HCl/40 mM. NaCl buffer, pH 7.9) [199] and suggesting that the diimine
complexes is involved in DNA binding engaged in complete insertion in between the
base pairs of DNA. The strongest binding affinity exhibited by the complex is expected
on the basis of the additional aromatic ring of aldol condensation which enhances the
extent of stacking of the diimine with the DNA base pairs. We can conclude that the
14
free ligand and the Cu(II) complex can interact with CT-DNA through the intercalation
mode of binding and The binding strength of the synthesized complexes with DNA is
shown in the following order: -NO2> -OH > -OCH3> 4-N-(CH3)2.
The intrinsic binding constant (Kb) values of copper complexes of L1 – L
11 are
1.6 × 106M
–1, 1.7 × 10
6M
–1, 3.2 × 10
6M
–1, 1.6 × 10
6M
–1, 2.4 × 10
6M
–1, 1.9 × 10
6M
–1,
2.5 × 106M
–1, 2.9 × 10
6M
–1, 2.8 × 10
6M
–1, 1.6 × 10
6M
–1, 2.3 × 10
6M
–1, respectively and
compared with classical intercalator (EthBr-DNA). The prepared copper complexes are
less binding strength than classical intercalator. These data implies that the compounds
interact with DNA by appreciable intercalation binding mode. A similar spectral
behaviour was obtained for all other complexes shown in the Fig. and their
corresponding binding constants are listed in the Table.
The intrinsic binding constant (Kb) value of DNA–curcumin binding constant
value is observed in the order of 1.1 × 106 M
–1. These binding constants indicate finite
interaction, but it was lower compared to the typical intercalators like ethidium
bromide. From these binding constants it is obvious that the synthesized copper
complexes intercalate more preferentially to DNA than that of curcumin. The binding
free energy values of the above complexes were calculated using the equation and are
given in Table .
∆G = -RT ln Kb
Where,
∆G - Binding free energy
R - Gas constsnt
T - Temperature
Kb-Intrinsic binding constant ---------- (16)
15
Table. 5.3 The binding constant of the copper complexes with DNA
S.No Copper complexes Kb
(106M
–1)
1 [CuL1(OAc)2] 1.6
2 [CuL2(OAc)2] 1.7
3 [CuL3(OAc)2] 3.2
4 [CuL4(OAc)2] 1.6
5 [CuL5(OAc)2] 2.4
6 [CuL6(OAc)2] 1.9
7 [CuL7(OAc)2] 2.5
8 [CuL8(OAc)2] 2.9
9 [CuL9(OAc)2] 2.8
10 [CuL10
(OAc)2] 1.6
11 [CuL11
(OAc)2] 2.3
5.4 Viscosity Measurements
Viscosity is considered as least ambiguous and most critical testin predicting the
nature of binding of the complexes to CT-DNA [200]. A classical intercalator causes
significant increase in the viscosity of DNA solution due to the increase in the
separation in overall DNA contour length. A partial/or non-classical intercalation of
metal complexes causes a bend or kink in the DNA helix reducing its effective length
and, as a result, DNA solution viscosity is decreased or remains unchanged [201].
Hydrodynamic measurements (viscosity) were carried out to further clarify the
interaction of copper complexes and DNA [202, 203]. The viscosity measurement is
determined from the flow rate of a DNA solution through a capillary viscometer. The
specific viscosity contribution (η) due to the DNA in the presence of a binding agent
16
was obtained. The values of (η/η0)1/3
were plotted against [compound]/[DNA]. The
effects of the ligand and the Cu(II) complex on the viscosity of CT-DNA are shown in
Fig. 5.9, indicate that the absence and the presence of the metal complex have a marked
effect on the viscosity of the DNA.The significant increase in viscosity of the complex,
which, however, is less than that for the potential intercalator viz., EthBr [204], leading
to small change in relative viscosity of DNA. However, interestingly, an increase in
viscosity of DNA as much as for the complex ([CuL21
Cl2]) is observed, this increase in
separation of base pairs at intercalation sites and hence an increase in overall DNA
contour length.
As expected, the known DNA-intercalator EthBr increased the relative viscosity
of DNA due to its strong intercalation. Compared with EthBr, complexes exhibit minor
increase in the relative viscosity of DNA, suggesting an intercalation mode between the
complex and DNA. Thus the viscosity studies suggest that the central rings of copper
and imine group are involved in intercalative modeof DNA binding. The results from
the viscosity experiments confirm the mode of these compounds intercalating into DNA
base pairs and already established through absorption spectroscopic studies such as
hypochromism and red shift of the complexes in the presence of DNA.
17
Fig. 5.9 Effect on relative viscosity of CT-DNA under the influence of
increasing amount of the complexes at 25 ± 0.1 °C.
The viscosity studies provide a strong evidence for intercalation. The increase in
viscosity of DNA is ascribed to the intercalative binding mode of the copper complexes
because this could cause the effective length of the DNA to increase [205].
5.5 Thermal denaturation
The thermal behaviour of CT-DNA in the presence of complexes gave insight
into their conformational changes when temperature is raised when temperature is
raised and information about the interaction strength of the complexes with DNA. The
double-stranded DNA tends to gradually dissociate to single strands on increase in the
solution temperature and generates a hyperchromic effect on the absorption spectra of
DNA bases (at 316 nm).
18
Fig. 5.10 Melting curves of CT-DNA in the absence and presence of
copper complexes
In the present study, melting temperature (Tm) of DNA in the absence of copper
complexes was found to be 54 ± 1°C. Under the same set of experimental conditions,
addition of complexes increased the melting temperature Tm (± 1°C) from 10°C to
10.3°C, for all copper complexes respectively [206]. This experimental data indicates
that the all Cu(II) complexes of peptides has interaction with double helix CT-DNA.
The intercalation of small molecules into the double helix has as a result an increase of
melting temperature at which the double helix denatures into single helix DNA, The
significant increase of Tm (∆Tm = 10.3ºC) suggests that the interaction of the all copper
complexes with DNA is performed through intercalation shown in the Fig. 5.10. The
DNA melting curves obtained in the presenceof DNA reveal a monophasic and
irreversible melting of the DNA strands. The insertion of the planar aromatic ligand in
19
between the DNA base pairs via interclation cause stabilization of base stack and hence
raises the melting temperature of the double-stranded DNA [207].
5.6 Lipophilicity test
Lipophilicity is one of the most important parameters for quantitative structure
activity and relationship. The design of drugs significantly depends on the accuracy of
lipophilicity determinations [208]. It is the most informative and successful
physiochemical property in medicinal chemistry. The partition coefficient (log P) was
indicated the lipophilic nature of copper complexes. The absorption maximum of the
copper complexes was determined using UV-Visible double beam spectrophotometer.
The λmax for n-octanol is 263 nm. All the observation showed that the complexes have
enchanced bioavailability than its corresponding ligands. This liphophilicity tends to
increases the efficiency across the lipoidal bacterial membrane due to its highly
conjugated system of synthesized copper complexes and have a relatively thin cell wall
consisting of a few layers of peptidoglycan surrounded by a second lipid membrane
containing lipopolysaccharides and lipoproteins. These differences in cell wall structure
can produce differences in antibacterial susceptibility.
The log P value is an important criterion to evaluate the drug likeness of
substances, especially for the anti-alzheimer’s agents which must possess the ability to
penetrate the blood–brain-barrier (BBB). In order to evaluate whether the synthesized
compounds possess such ability, the log P value of each compound was calculated. The
calculated log P values of the compounds are around 4.00, ranging from 3.70 to 4.4,
suggesting a good lipophilicity and a potential ability to penetrate the BBB. According
to the Lipinski’s Rule of Five which suggests the optimal log P value of drug candidate
should be not higher than 5, it can be expected that the synthesized compounds, possess
a good potential to behave as drug candidates.
20
Table 5.4 Partition coefficients (log P) values of copper complexes
Compound Partition coefficients
(log P)
[CuL12
Cl2] 4.32
[CuL13
Cl2] 4.36
[CuL14
Cl2] 4.4
[CuL15
Cl2] 4.21
[CuL16
Cl2] 4.18
[CuL17
Cl2] 4.10
[CuL18
Cl2] 4.25
[CuL19
Cl2] 3.9
[CuL20
Cl2] 4.26
[CuL21
Cl2] 4.38
[CuL22
Cl2] 4.4
[CuL23
Cl2] 4.2
[CuL24
Cl2] 4.22
[CuL25
Cl2] 3.84
[CuL26
Cl2] 3.81
[CuL27
Cl2] 3.92
[CuL28
Cl2] 3.75
[CuL29
Cl2] 3.95
[CuL30
Cl2] 3.70
[CuL31
Cl2] 3.8
[CuL32
Cl2] 3.7
21
5.7 Antioxidant assay
5.7.1 Superoxide dismutase activity
The superoxide dismutase activity (SOD) of the complexes was investigated by
the NBT assay method [209]. The chromophore concentration value required to yield
50% inhibition of the reduction of NBT (IC50). The IC50 of present copper complexes
was found at the range of 25-69 mol dm-3
which are higher than the value exhibited
by the native enzyme (IC50 = 0.04 mol dm-3
). All the tested compounds show SOD
activity. Similar values obtained for all compounds. The SOD values of Cu(II)
complexes were listed in the Table and graphically presented in Fig. 5.11.
Fig. 5.11 Superoxide dismutase activity of Cu(II) complexes in (mol dm-3
)
Compounds with antioxidant properties could be expected to offer protection in
inflammation and lead to potentially effective drugs. Lower IC50 value, greater the
hydrogen donating ability. Copper complex of L9 showed greater antioxidant activity.
Copper complex of L2
and L1
also showed a good antioxidant activity is due to the
presence of OH group (efficient hydrogen donors to stabilize the unpaired electrons and
SOD ACTIVITY
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100 120
CONCENTRATION mg/ml
PE
RC
EN
TA
GE
OF
IN
HIB
ITIO
N
[CuL1(OAc)2] [CuL2(OAc)2] [CuL3(OAc)2] [CuL4(OAc)2] [CuL5(OAc)2] [CuL6(OAc)2]
22
there by scavenging free radicals). The introduction of –NO2 group (L10
and L4 )
in the
ligand system markedly increases the antioxidant efficiency of the complexes with
careful selection of the substituents on the ligands, the antioxidant behavior of the
complexes can be improved. The activity was found in the order of
[CuL9(OAc)2]<[CuL
2(OAc)2][CuL
1(OAc)2][CuL
10(OAc)2][CuL
4(OAc)2]
<[CuL7(OAc)2]<[CuL
5(OAc)2]<[CuL
11(OAc)2]<[CuL
3(OAc)2]<[CuL
6(OAc)2]
< [CuL8(OAc)2].
Most of the synthesized complexes show the negative reduction potential,
typically seen in many other simple square planar complexes. Complex shows an
irreversible peak for the couple Cu II/Cu I. The Cu I/Cu II, indicating the planar
geometry with a consequent negative reduction Cu(II) state over Cu(I) serve as the
model for the copper proteins [210].
Cu complexes with their redox potentials are in the suitable range for superoxide
scavenging. Here, bioactive ligands of N, S donor set of Cu-complexes, which have
wide spectrum of metal–ligand combinations. Accordingly, reactivity of these copper
complexes toward O2•− and H2O2 was systematically studied and redox behaviour of
the complexes responsible for its antioxidant activity. Electrochemical properties have
best correlations with antioxidant properties due to their redox potentials [211, 212]. It
is found that compounds with strong scavenging capabilities are oxidized at relatively
low potentials [50]. The redox potential of almost all the complexes falls between 0 V
to -1.6 V, results reveal that the redox potential values of these complexes fall into the
redox potential range that resembles the SOD enzyme [213]. Besides that the
synthesized copper complexes have higher antioxidant activity due to the presence of
highly conjugated curcumin analog system containing two imine groups and redox
properties of metal can serve as the structural models as well as good functional models
of the enzyme that can decompose superoxide, similarly to the native enzyme [214].
23
It is found that copper complexes possessing Cinnamaldehyde including
[eg. CuL34
Cl2] can behave like potent antioxidants oxidants due to their strong lipid
peroxidation. In these series of copper complexes of Scheme-4, [CuL26
Cl2] exhibit
excellent SOD mimic activity due to the presence of hydroxyl group enhanced lipid
peroxidation and oxidative damage to proteins [215], Although superior to copper
complexes of methoxy substituted ([CuL27
Cl2], [CuL32
Cl2]), [CuL4Cl2] complexes.
Certainly complexes with nitro groups ([CuL29
Cl2]&[CuL29
Cl2]) showed loweranti-
oxidantactivity when compared to [CuL26
Cl2].
Native Enzyme
CuIIZn
II SOD + O2•− O2 + Cu
IZn
II SOD
CuIZn
II SOD+O2•− +2H
+ H2O2 + Cu
IIZn
II SOD
Synthesized copper complex
[CuL26
Cl2] + O2•− O2 + [CuL26
Cl2]
[CuL26
Cl2] + O2•− +2H+
H2O2 + [CuL26
Cl2]
All the tested compounds show SOD activity. Similar values obtained for all
compounds. The SOD Values of Cu(II) complexes were listed in the (Table ). The
antioxidant activities of synthesized complexes possessing hydroxy derivatives are in
the order of
[CuL26
Cl2] [CuL16
Cl2] [CuL35
Cl2]
[CuL26
Cl2]>[CuL16
Cl2]>[CuL35
Cl2]
24
Copper complexes having g||/A|| values found in the range of 154-174 cm-1
are
in agreement with significant deviation from planarity which is further confirmed by the
bonding parameter α2 whose value is less than unity. The covalency parameters α
2
(covalent in-plane r-bonding) and β2 (covalent in-plane p-bonding) have been calculated
from Hathaway equation [197]. The g||/A|| values found in the range of 170- 250 cm are
indicative of distortion in square planar geometry.
Inspection of the spectral data reveals that the dichloro complexes show
increased distortion. Fig. shows most of the available data for CuN2S2 and
representative species with various CuN4, CuN2O2 and CuO4 centres [55-57]. A
distortion of a square planar geometry of the complex into a distorted tetrahedron with
any of the biomimetic (N,O,S) donors reduces A║ and increases g║. The A║values of the
complexes are in the border line between the naturally occurring blue copper proteins
and typical square planar complexes.
The distorted geometry of these complexes may favour the geometrical change,
which is essential for the catalysis as the geometry of copper in the SOD enzyme also
changes from distorted square planar geometry. The difference in reactivity of the
synthesized complexes may be attributed to the coordination environment and the redox
potential of the couple CuI/Cu
II in copper(II) complexes during the catalytic cycle.
The marked antioxidant activity of copper complexes, in comparison to free
ligands and other complexes, could be due to the coordination of copper in the
condensed ring system, increasing its capacity to stabilizeunpaired electrons and,
thereby, to scavenge free radicals. Inaddition, incorporation of aromatic moiety in the β-
diketone derivatives further enhanced the antioxidant activity. Cu(II) complex of L
showed higher antioxidant activity than vitamin C. It implied that copper complexes
possessing the β-diketimine might be considered as new promising lead candidate for
design and synthesis of antioxidants.
25
5.7.2 H2O2 scavenging assay
Hydroxyl radical is a highly oxygen-centered radical formed from the reactions
of various hydroperoxides with transition metal atoms. Among all the free radicals,
hydroxyl radical is by far the most potent and therefore the most dangerous oxygen
metabolic and hence the elimination of this radical is one of the major aims of
antioxidant administration [216]. It attacks proteins, DNA, polyunsaturated fatty acid in
membranes and most biological molecules [217]. Hydroxyl radical is known to be
capable of abstracting hydrogen atoms from membrane lipids and brings about peroxide
reaction of lipids. Scavenging activity of the free ligand and copper(I) complex on
hydroxyl radical has been investigated and compared with the standard ascorbic acid.
The synthesized compounds scavenged the radical in a concentration dependent
manner by causing oxidative damage to biological targets mediated through Fenton type
reaction or Haber-Weiss reaction and produce OH at the site. With increase production
of OH, vigorously damage DNA (with multiple hit effect) and convert them into highly
reactive radicals. However it causes damage to the cell even at a very low concentration
(20l) because they liberally soluble in aqueous solution and easily penetrate through
biological membrane. Results of percentage of free radical scavenging activity are
shown in Fig. 5.12
26
Fig. 5.12 Anti oxidant activity of Cu(II) complexes in (mol dm-3
)
5.8 Catalase activity
5.8.1 Absorption titration experiment
In the present study, a rapid and sensitive modification of the standard kinetic
spectrophotometric assay for catalase activity were performed. Using this method, up
to 6 reactions can be performed simultaneously within 5 min. To evaluate the sensitivity
and accuracy of this method using bovine catalase of known concentrations as a
proficiency control. The rate of decomposition of H2O2 was linear throughout the
reaction, with catalase levels ranging from 0.01 to 0.18 units in a total reaction volume
of 450 μL (Fig. 5.13).
ANTIOXIDANT ACTIVITY
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80 90
CONCENTRATION
PE
RC
EN
TA
GE
OF
IN
HIB
ITIO
N
[CuL1(OAc)2] [CuL2(OAc)2] [CuL3(OAc)2] [CuL4(OAc)2] [CuL5(OAc)2] [CuL6(OAc)2]
27
Fig. 5.13 Catalase activity of Catalase enzyme
It is found that catalase at quantities above 0.54 units, resulted in excessive
oxygen formation and consequent bubbling, hence limiting the assay. The lower limit of
the assay was found to be 0.01 units, making the assay comparable in sensitivity to the
standard Beers and Sizer assay [2]. The catalase activity of replicate samples was
calculated based on the rate of decrease in absorbance at λ = 240 nm using the molar
extinction coefficient of hydrogen peroxide, and corrected for pathlength. To assess the
accuracy of the assay method, the calculated values were compared with the actual
values of the Catalase as standard (Fig. 5.13). To evaluate the applicability of the
microtiter assay to other types of samples, we measured the catalase activities activities
of various copper complexes, among them we found compounds having moderate to
superior activity like catalase enzyme. The catalase activities of the complexes were
calculated from the rate of decrease in absorbance at λ = 240 using and the curve
established previously (Fig. 5.14). As expected, the complexes also exhibited the
highest levels of catalase activity, as it possesses both functional catalase/peroxidase.
28
Fig. 5.14 Catalase activity of copper complexes
For the complete decomposition, the rate of decomposition of H2O2was linear
throughout the reaction, with catalase levelsranging from 0.01 to 3.2units in a total
reaction volume of 1100 μL (Fig. 5.15).
Fig. 5.15 Complete Decomposition of H2O2 by Catalase enzyme and
copper complexes
29
5.8.2 Electrochemical Behaviour
Electrochemical properties of the catalase(CAT) enzyme and synthesised copper
complexes were investigated in DMSO solution using cyclic voltammetry technique.
The copper complexes were also studied under the same conditions for a direct
comparison of the results. The enzyme show one-electron redox wave in the plotted
potential range, like Cu(II)/Cu(I) redox couple with Epc= -0.0085V and Epa = 0.0328 V
and its peak to peak separation (ΔEp=0.0413 V) and proportion of the anodic peak
current and the cathodic peak current mostly indicates a quasi reversible process.
Fig. 5.16 Electrochemical response of catalase enzyme
(A) is potassium phosphate buffer (0.1M) at pH 7
(B) is Catalase enzyme (10 µg/ml) in potassium phosphate buffer (0.1M)
at pH 7
(C) is Catalase enzyme (10 µg/ml) in potassium phosphate buffer (0.1M)
at pH 7 in H2O2 concentrations.
Upon the addition of H2O2, the reduction peak current of the CAT increased
(Fig. 5.16), indicating a typical electro-catalytic behavior of the reduction of H2O2. This
is evident that there is a linear dependence of cathodic peak current on the
30
decomposition of H2O2 concentration. It nearly requires (ΔEp = -0.086 V) for the
complete decomposition of H2O2.
In the present study, electrochemical and antioxidant properties of five copper
complexes were evaluated. The majority of Cu(II) complexes, under the experimental
conditions used in this study, were found to be enzyme mimics possessing CAT like
catalytic activities.
Fig. 5.17 Electrochemical response of Copper complexes
The cyclic voltammogram of [CuL16
Cl2] showed Cu(II)/Cu(I) redox couple with
Epc= -1.293 V and Epa = 0.983 V and its peak to peak separation (ΔEp=0.310 V) and
proportion of the anodic peak current and the cathodic peak current mostly indicates a
quasi reversible process. In addition of H2O2, the oxidation peak potential shifts
positively to 0.958 V, and the reduction peak potential shifts positively to 1.151 V for
the [CuL16
Cl2].The enhancement of the peak currents observed for copper complex
upon addition of H2O2concluded that the present ligand systems stabilize the unusual
oxidation states of copper ion during electrolysis. Other copper complexes were also
showed similar electrochemical behaviour.
31
Compared with that at CAT, the remarkable enhancement in the peak currents
and the lowering of overpotential provide clear evidence of the catalytic effects copper
complexes towards H2O2 detoxification. In order to make a copper complex
thermodynamically apt in the H2O2 detoxification, the redox potential of the metal-
centred redox couples should fall within the 0.04 V (O2/H2O2) to 1.61 V (H2O/H2O2)
versus SCE potential range [218]. All the complexes ([CuL2(OAc)2], [CuL
16Cl2],
[CuL26
Cl2], [CuL35
Cl2]>[CuL45
(OAc)2]) have suitable E1/2 and ΔEp potential showed
activity for the catalytic decomposition of H2O2. Among them, [CuL16
Cl2] and
[CuL26
Cl2] complexes are comparably effective as CAT mimics. The [CuL45
(OAc)2]
complex showed negligible CAT-like activity but moderate ability to reduction H2O2
and other complexes have good activity. The electro-catalytic process could be
expressed as follows,
5.9 Anti-inflammatory study
The antiinflammatory effects of the copper-aspirin complex (Cu-Asp) were
more potent than that of Asp in rats or mice with fewer classic adverse effects. The
present study was undertaken to compare the anti-inflammatory activity of metal
complexes in rats. The anti-inflammatory activity of the ligands and their metal
complexes was studied using carrageenan-induced paw edema in rats and measuring the
32
zone of inflammation. This method is most widely used by various research groups as
this method is reliable and cost effective.
The anti-inflammatory activity of copper complexes was assayed in Wistar male
albino rats using Carrageenan-induced rat paw edema method (Table 5.9). Edema,
which develops after carrageenin inflammation, is a biphasic event. The initial phase is
attributed to the release of histamine and serotonin. The edema maintained between the
first and the second phase is due to kinin-like substances. The second phase is said to be
promoted by prostaglandin-like substances. It has been reported that the second phase
of edema is sensitive to drugs like hydrocortisone, phenylbutazone, and indomethacin.
The comparative study of inhibition of paw edema by the metal complexes and the
parent compounds shows that there was a significant increase in anti-inflammatory
activity when the parent compounds was given orally as metal complexes as compared
to the ligand itself. The results show that the copper complex has higher activity than
other complexes is due to the high diffusion of the copper complex. These results are
consistent with the reports that copper complexes of NSAIDs, are more active anti-
inflammatory agents than their parent drugs [29, 30]. Further study would also be
needed to look into the possible mechanism of action of the copper complexes.
Table 5.9 Preliminary pharmacological screening of copper complexes on carrageena-
induced rat hind paw edema
Sl.
No. Drug
Dose
(mg/kg bw)
Increase in paw
volume after three
hours.
% Inhibition in
paw volume
1.
2.
3.
4.
5.
6.
7.
Control
Standard Indomethacin
L1
L2
L3
L4
[CuL1(OAC)2]
0.5 ( Ml/kg)
10
100
100
100
100
100
0.38 0.01
0.16 0.01
0.38 0.01
0.42 0.01
0.34 0.01
0.25 0.01
-
45.2 1.91
16.2 3.04
19.0 3.02
25.6 1.99
28.5*±1.92
33
8.
9.
10.
[CuL2(OAC)2]
[CuL3(OAC)2]
[CuL4(OAC)2]
100
100
100
0.16±0.01
0.22±0.01
0.18±0.01
0.14±0.01
38.0*±1.95
34.6 *±1.75
34.4*±1.80
28.6*±2.34
*P<0.001 as compared to Control (ANOVA followed by Dunnett’s t test)
Each value is the mean ± SEM of six rats weighing 150–170 g
34