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