Synthesis, Antioxidant Properties and AntiproliferativeActivities of Tetrameric Copper and Copper-Zinc MetalComplexes of Catecholamine Schiff Base Ligand

Sabari Dutta, Ratnamala Bendre, and Subhash PadhyeDepartment of Chemistry, University of Pune, Pune, India

Fakhara Ahmed and Fazlul SarkarWayne State University, School of Medicine, Detroit, MI, USA

Modulation of the intrinsic oxidative stress in invasivemetastatic cancer cells using two superoxide dismutase mimickingconjugates of catecholamine Schiff base ligand 3-[(20 –(200 pyridyl-ethyl)iminoethyl] benzene-1,2 diol] (1) results in superior killing ofmalignant cells. The homometallic copper compound as well asits heterometallic copper-zinc counterpart (3) are characterizedby spectroscopic, electrochemical and magnetic susceptibilitymeasurements where the latter shows remarkable SOD activity(IC50 5 0.94mM) as well as a potent antiproliferative activityagainst estrogen independent breast (BT-20) and androgen inde-pendent prostate cancer (PC-3) cell lines.

Keywords Schiff base complexes; copper tetramer; copper-zincmixed-metal complex; SOD activity; antiproliferativeactivity

INTRODUCTION

The disparities in the generation and metabolism of reactive

oxygen species (ROS) in cancer cells versus normal cells seem

to offer a biochemical basis for developing new anticancer

agents that can kill malignant cells selectively (Hileman

et al., 2004). Although an optimum amount of ROS has been

found to be necessary and important to maintain appropriate

redox balance and to stimulate cellular proliferation

(McCord, 1995; Murrell et al., 1990; Nicotera et al., 1994;

Preeta and Nair, 1999), alterations in the cellular ROS status

have been shown to play a crucial role in apoptotic cell death

(Armstrong, 2002; Green and Reed, 1998; Raha, 2001). An

important aspect of the metabolic process is the continuous

production of superoxide by the mitochondria (Saybasili et al.,

2001; Staniek et al., 2002), which is subsequently converted to

hydrogen peroxide (Boveris and Chance, 1973; Fridovich,

1995) and other ROS that are considered the major sources of

cellular damages. Antioxidant enzymes such as superoxide dis-

mutase (SOD), catalase, and various peroxidases can effectively

remove ROS and are critical in regulating ROS-mediated

cellular damage (Halliwell and Gutteridge, 1999).

Since cancer cells produce high levels of ROS and are under

increased oxidative stress, it is reasonable to expect that the

malignant cells should be more dependent on normal cells.

Consequently, inhibition of antioxidant enzymes or exposure

to further exogenous ROS stress would cause more damage

to cancer cells.

Several anticancer agents currently used for cancer treat-

ment have been shown to cause increased cellular ROS

generation. These therapeutic agents include anthracyclines,

cisplatin, bleomycin, and the synthetic retinoid viz. N-(4-

hydroxyphenyl) retinamide (Hug et al., 1997; Miyajima

et al., 1997; Serrano et al., 1999; Suzuki, 1999). Added to

this list are the synthetic copper conjugates, which mimic the

superoxide dismutase enzyme (SOD).

These are expected to provide advantages over the natural

enzymes in respect of their ability in crossing the cell mem-

branes, offering no immunogenicity, possessing longer life-

time of the active forms, possibility of oral administration,

and comparative lower costs. Among the three classes of

SOD mimics that have been investigated so far include manga-

nese, iron, and copper compounds, and their chemistry has

been reviewed recently (Dillon, 2003).

Most of the work reported in literature on copper SOD

mimics has involved designing of Cu–Cu and Cu–Zn dinuclear

Received 29 September 2004; accepted 27 October 2004.SD and RB would like to thank CSIR for SRF Fellowship. SBP

would like to acknowledge help from Dr. B.L. Ramakrishna,Arizona State University, Tempe in magnetic and EPR measurementsand their interpretations.

Address correspondence to Subhash Padhye, Department of Bio-chemistry and Molecular Biology, School of Medicine, Wayne StateUniversity, Detroit, MI 48201, USA. E-mail: sbpadhye@chem.unipune.ernet.in

Synthesis and Reactivity in Inorganic, Metal-Organic and Nano-Metal Chemistry, 35:3–10, 2005

Copyright # 2005 Taylor & Francis, Inc.

ISSN: 1553-3174 print/1553-3182 online

DOI: 10.1081/SIM-200047494

3

complexes that mimic the spectroscopic, magnetic, and struc-

tural properties of the active site of the native Cu2-Zn2-SOD

enzyme. These include imidazolate-bridged di-copper(II)

complexes involving ligands like tetramethyldithylenetriamine,

4,5 bis[(2-pyridyl)ethyl]imino]methylimidazole (Strothkamp

and Lippard, 1982) or the macrocyclic moieties like cyclam

(Coughlin, 1984) or the cryptand ligand like (1, 4, 12, 15, 18.

26, 31, 39)-octaazapetacyclo[13, 13, 1, 3, 13], tetratetracontance

(8, 10, 20, 22, 24, 33, 35, 37) nonaene, respectively (Sato,

1986). Reedjik et al. have recently reported one of the best imi-

dazolate-bridged dicopper(II) complex containing ligand, viz.

1,5-bis(1-pyrazoyl)’-3-bis(2-imidazolyl)methyl-3-azapentene,

which shows high catalytic rate constants for dismutation

of superoxide anion to hydrogen peroxide (Tabbi et al.,

1997). Structurally characterized imidazolate-bridged hetero-

dinuclear copper(II)–zinc(II) complexes have been rather

scarce and the coordination of superoxide ion has been con-

firmed at least in the case of ligand 4,5-bis(di(2-pyridylmethyl)

aminomethyl)-imidazole with the help of electronic and ESR

spectra (Ohtsu and Fukuzumi, 2000). More recently Li et al.

have described a Cu(II)-Zn(II) complex containing a macro-

cyclic ligand with two hydroxyethyl pendant arms that can

catalyze the dismutation of superoxide with high efficiency

(Li et al., 2003).

In our laboratory, we have been investigating catecholimine

Schiff base ligand viz. 3-[(20–(200 pyridylethyl)iminoethyl]

benzene-1,2 diol] (1), prepared by condensation of 2,3-

dihydroxybenzaldehyde with 2-(20-aminoethylpyridine), which

is capable of stabilizing multinuclear metal complexes

through catecholate and caroxylate bridges (Kulkarni et al.,

2001). The ligand was first described by Labtour et al.

(1987), who had also prepared its copper conjugates. Manga-

nese and iron complexes of 1 have also been recently

reported by Padhye and co-workers (Theil et al., 1997)

although the biological activities of these compounds have

remained unexplored.

In the present work, we describe preparation and charac-

terization of two tetrameric compounds of 1, wherein one of

the conjugates is a homometallic compound containing all

copper atoms, while the other is a heterometallic copper-

zinc moiety. Both compounds are evaluated for their super-

oxide radical scavenging activities and are subsequently

examined for their antiproliferative activities against highly

metastasizing hormone independent BT-20 and PC-3 cancer

cell lines.

EXPERIMENTAL

All reagents were obtained from commercial suppliers

and were used without purification unless otherwise noted.

Solvents were purified prior to their use according to literature

methods (Perrin, Armargo, and Perrin, 1966). The details of

physical measurements have been described previously

(Murugkar, 1999).

Preparation of Ligand

The ligand 3-[(20- (200pyridylethyl)iminoethyl]benzene-1,

2-diol (1) was prepared as described in the literature

(Strothkamp and Lippard, 1982). Yield: 82%. Anal. Calc. for

C14H14N2O2: C, 69.42; H,5.74; N,11.57% Found: C,69.16;

H, 5.92; N,11.92 %.

Preparation of the Complexes

The tetrameric copper complex [fCu21(CH3COO)2g2] . H2O

(2) was prepared by reacting methanolic solution of 1 (4.84 g,

0.02 mol) with copper (II) acetate (2.17 g, 0.01 mol) in 2 : 1

stoichiometric ratio with a constant stirring over a period of

1 hr. The precipitated complex was filtered and washed by

cold methanol to eliminate the unreacted ligand. It was

stored in vacuum desicator over a desiccant. Yield: 13.49 g,

68%. Anal. Calc. for C36H42N4O13Cu4: C, 35.99; H, 3.49;

N, 4.66; Cu, 21.17 % Found: C, 35.73; H, 3.70; N, 4.84;

Cu, 21.70%.

The mixed-metal complex viz. [fCuZn1(CH3COO)2g2] .

H2O (3) was prepared by interacting 2.17 g (0.01 mol) of

Cu(CH3CO2)2. 2H2O and 2.19 g (0.01 mol) of Zn(CH3CO2)2

.

2H2O with 1 (2.42g, 0.01mol) in methanol in a similar

manner. Yield: 73% (7.26 g). Anal. Calc. for

C36H42N4O13Cu2Zn2: C, 35.88; H, 3.49; N, 4.65; Cu, 10.55%

Found: C, 35.37; H, 3.72; N, 4.88; Cu, 10.04%.

SOD Activity

The SOD activity was evaluated in DMSO solvent using

the nitro blue tetrazolium method (NBT) assay (Bhirud and

Shrivastava, 1990). Potassium superoxide stabilized in

18-crown-6 ether was used as the superoxide source (Lu

et al., 1990). The IC50 values calculated for the compounds

represent concentrations which exhibit SOD activity equiva-

lent to one unit of the native SOD enzyme.

MTT Assay

The number of viable cells remaining after an appro-

priate treatment with test compounds was determined by

the MTT assay involving 3-(4,5-dimethylthiazol-2-yl)-2,

5-diphenyltetrazolium bromide (Sigma Chemical Co.) (Chen,

2004).

Briefly cells were plated (4,000 cells/well per 0.2 ml RPMI

1640 medium) in 96-well microtiter plates and incubated over-

night. The test agent was then added to each well at final con-

centrations to quadruplicate wells. After 48 h, MTT was added

to each well at a final volume of 0.5 mg/ml, and microplates

were incubated at 378C for 3 hr. After the supernatant was

removed, the formazan salt resulting from the reduction of

MTT was solubilized in dimethyl sulfoxide (DMSO; Sigma

Chemical Co.) and the absorbance was read at 570 nm using

an automatic plate reader (Molecular Devices Corporation,

Sunnyvale, CA). The cell viability was extrapolated from

S. DUTTA ET AL.4

optical density (OD)570 values and expressed as percent

survival using the following formula:

% cell viability ¼OD570 of drug treated sample

OD570 of untreated control sample� 100

RESULTS AND DISCUSSION

The Schiff base ligand 1 is a bright yellow compound that

undergoes complexation reactions with many metal ions

although the products of such reactions vary depending upon

the nature of the metal salts used. For example, when copper

nitrate was employed in the reaction, a tetrameric compound

with a distorted cubane structure was obtained by Gojon

et al. (1987). In the present work, we have employed metal

acetates as the starting material that leads to tetrameric

species having two-fold symmetry promoted by the acetate,

as well as catecholate bridges similar to the one observed in

case of manganese compound reported by us earlier (Theil

et al., 1997). Satisfactory elemental analyses were obtained

for all synthesized compounds. Both the metal complexes are

insoluble in common organic solvents, but soluble in highly

polar solvents like DMF and DMSO (Figure 1).

IR Spectra

The IR spectrum of the ligand shows a broad band around

2700 cm21 attributable to the hydroxyl stretches of the

catechol moiety (Buchanan et al., 1986) that are absent in the

present metal conjugates indicating their involvement in

metal coordination. This is accompanied by shifting of the

nC – O stretches of the catechol moiety from 1480 cm21 to

1460 cm21. The imino nC¼N stretching frequency appearing

at 1635 cm21 for 1 is also found to be displaced upon metal

complexation (Stalling et al., 1981). Both metal conjugates 2

and 3 show broadening of the bands between 1600 and

1450 cm21, probably due to overlapping of carbonyl and car-

boxylate absorptions. Finally, the difference in the symmetric

and asymmetric modes of carboxylate stretches in the present

compounds (D ¼ 130 2 160 ncm21) indicates a bidentate sym-

metric bridging mode for the acetate groups (Christou et al.,

1990).

Electronic Spectra

The electronic spectrum of 1 (1.0 mM in DMSO) exhibits

intra-ligand transitions while its tetrameric copper compound

(2) (1.0 mM in DMSO) exhibits a broad d-d band in the

visible region around 700 nm (14,285 cm21), with a shoulder

absorption at 500 nm (20,000 cm21), as shown in Figure 2a, b.

The former can be assigned to the dxz,yz ! dx22y2 transition

of Cu(II) ions in a square pyramidal geometry (Lintvedt

et al., 1988) while the latter absorption is thought to arise

from the catechol ! copper (II) charge transfer transition

observed for several monomeric, as well as copper catecholate

complexes (Bodini et al., 1983). The d-d band for the compound

3 is observed at 720 nm (Figure 2c). Additional absorptions at

390 nm in compound 2 is due to acetate to Cu(II) charge

transfer transition, while the band at 360 nm in compound 3

can be assigned to the transitions involving delocalization

within the azomethine chromophore (Hathway, 1984).

Magnetic Susceptibility Studies

The magnetic susceptibility measurements were carried out

on both complexes in the temperature range 4–300 K. For the

tetrameric copper compound 2, the xT values were found to

decrease with temperature (Figure 3), typical of antiferromag-

netically coupled metal centers (Goodson et al., 1990). Two

distinct magnetic domains are observed for this compound

with a plateau around 0.8 cm3/mol for temperatures lower

than 60 K, indicative of the presence of two independent

spins S ¼ 1/2. These compounds show an increase of xT in

the higher temperature range revealing antiferromagnetically

coupled system that begins contributing at temperatures

higher than 60 K. The tetranuclear arrangement of copper

ions in 2 can be regarded either as magnetically isolated

pairs of binuclear centers (Halvarson et al., 1987; Tandon

et al., 1992) or as a linear chain arrangement with one relatively

FIG. 1. Schematic structures of ligand 1 and its copper (2) and copper-zinc (3) complexes.

ANTIOXIDANT SCHIFF BASE COMPLEXES HAVING ANTIPROLIFERATIVE ACTIVITIES 5

short and two longer contacts (Bu et al., 2000), as shown

below:

Cu(1)------------Cu(2)--------Cu(3)---------Cu(4)

J1 J2 J3

In the former case, any superexchange interaction between

Cu(1). . . . . .Cu(3), Cu(2). . . . . .Cu(4) would be considered to

be negligible. The significant exchanges would be considered

only within each of the binuclear units, i.e., Cu(1)-----Cu(2),

Cu(2)-----Cu(3) and Cu(3)-----Cu(4), respectively, via the

superexchange mechanism involving acetate and catecholate

bridges. In this case, magnetic properties are dictated by the

Bleaney-Bowers expression (Bu et al., 2000) for an interacting

S ¼ 1/2 pair defined by the spin Hamiltonian H ¼ 2 2Js1s2. In

the linear chain model (Bu et al., 2000), the full Spin Hamil-

tonian involves two super-exchange pathways and two

exchange integrals that would be required to describe the

molar susceptibilities as follows:

Hex ¼ �2½J1ðS1 � S2 þ S3 � S4� � 2J2½ðS2S3Þ� ½1�

xm ¼ fðJ1; J2; g, TÞ þ Na ½2�

where Na ¼ temperature independent paramagnetism.

In Eq. [1], out of the two exchange integrals, J2 is con-

sidered to be the dominant exchange integral because of the

shorter copper-copper separation. The major spin exchange

interaction (J2) takes place between the inner copper centers

of the tetranuclear cluster where super-exchanges are

promoted by the catecholate single atom bridges, while a

weak coupling is observed between the outermost copper

pair of each dimer where the exchanges are dictated by the

multi-atom carboxylate bridges.

The mixed-metal compound 3 exhibits negligible magnetic

exchanges interactions as revealed from its very low J value

(ca. 21 cm21) when fitted to the Bleany-Bowers equation for

the dimeric species (Bu et al., 2000). The magnetic data for

this compound can be fitted to a simple Curie-Weiss law

which yields u ¼ 2 0.78 (juj , 1 k) indicative of the absence

of any exchange interactions in the total molecule. This obser-

vation suggests that substitution of the two Zinc (II) ions in this

cluster takes place at the site of inner pair.

Electrochemistry

The electrochemical properties of the two complexes were

studied by cyclic voltammetry (CV) in de-gassed DMSO

solution. Figure 4 shows the cyclic voltammograms of the

ligand and its homometallic metal complex. Compound 2

shows two redox waves at E1/2a ¼ 20.20 V and E1/2b ¼

þ0.33 V (Vs SCE) corresponding to the Cu3IICuI/Cu2

IICuI and

Cu4II/Cu3

IICuI, respectively (Lange et al., 2000). While the

heterometallic complex 3 exhibits only one reversible CuII/CuI redox couple at E1/2 ¼ þ0.80 V (Vs SCE) (CV profile

not shown), which indicates that the two terminal cupric ions

in this compound are essentially in identical environment.

The difference in the CuII/CuI redox potentials for 2 and 3

demonstrates the subtle differences in coordination environ-

ment around copper(II) ions in these two compounds, which

also provides an explanation for the differences in the SOD

activities of these two complexes. It should be noted that

the redox potentials of both complexes fall within the

FIG. 2. Electronic spectra of (a) ligand 1, (b) compound 2, and (c)

compound 3.

FIG. 3.Temperature dependence of the magnetic susceptibility of tetramic

copper clusters [Cu21(CH3COO)ZgZ].H2O.

S. DUTTA ET AL.6

permissible range normally observed for many SOD mimicks

(Li et al., 2003).

EPR Spectra

The EPR spectrum of the tetranuclear copper compound 2

was recorded as a polycrystalline sample in the temperature

range 11 to 300 k (Figure 5). It is observed that at 300 K, the

spectrum consists of a single derivative peak at g ¼ 2.15,

while at 11 K, it is resolved into a broad resonance around

gk ¼ 2.28 and a sharp signal at g? ¼ 2.10, respectively

(Bodini and Arancibia, 1989). In addition, the compound also

exhibits the spin forbidden D Ms ¼ +2 transitions in their

X-band spectra (data not shown). It has been established by

Reed and colleagues (Mckee et al. 1984) that the intensity of

the D Ms ¼ +2 transition is dependant on the magnitude of

the zero field splitting (ZFS) in the triplet state and that such

a splitting can be both dipolar as well as pseudo-polar in

origin. In the former case, it leads to the dependence of ZFS

on Cu–Cu distances and g tensor while in the latter case it

depends on the exchange interactions in the excited state.

The fact that D Ms ¼ +2 transitions in 2 are of different

relative intensities probably indicates that the variation in the

ZFS in this tetrameric cluster is largely influenced by the g

tensors. The exchange interactions in 2 can be understood in

terms of two “dissimilar” ion pairs in different geometries

undergoing two types of exchange interactions. A rather slow

electron exchange can then give rise to a well-resolved aniso-

tropic “g” tensor with such sharp signals.

A remarkable feature of compound 2 is the presence of a

sharp band at g ¼ 0.57 (H ¼ 11,770 G) whose intensity is

maximum at 4 K (Figure 5) and its origin probably lies in the

cluster aggregation phenomenon. The sharpness and intensity

of this cluster signal is found to diminish with the increase in

temperature and the total signal is lost at and above 20 K.

This signal is found to be coupled with the band due to

D Ms ¼ +1 transition, which undergoes concomitant changes

in intensity and sharpness with temperatures at the expense of

the cluster signal. It is, therefore, reasonable to suggest that the

intensity of this signal may be diagnostically used for predict-

ing the extent of magnetic exchanges in such cluster

compounds.

Interestingly, the heterometallic compound 3 exhibits an

EPR spectrum typical of an axially distorted monomeric

copper (II) species (Figure 6) suggesting that the inner

copper pair is replaced by two diamagnetic zinc ions making

the compound magnetically dilute, which is in agreement

with the magnetic susceptibility data for this compound

discussed earlier.

FIG. 4. Cyclic voltammograms in DMSO solvent at sweep rates of 100 mv/s

for: (a) ligand 1 and (b) compound 2.

FIG. 5. X-band EPR spectra of compound 2 as polycrystalline solid from 4 k

to 300 K. FIG. 6. X-band powder EPR spectrum for compound 3 at 300 K.

ANTIOXIDANT SCHIFF BASE COMPLEXES HAVING ANTIPROLIFERATIVE ACTIVITIES 7

SOD Activity

The SOD-like activities of the ligand and its copper com-

pounds were determined by the NBT method in the concen-

tration range of 0.01 to 2.0 mM in triplicate and are

presented as the mean % inhibition of NBT reduction

(Figure 7). The IC50 values for the SOD activity determined

for the two tetrameric compounds were found to be

34.56mM (for compound 2) and 0.94mM (for compound 3),

respectively, and are listed in Table 1 along with the values

observed for some other SOD mimics reported in literature

(Pierre et al., 1995; Ohtsu et al., 2000). It is obvious in the

present case that the heterometallic compound (3) is a much

superior antioxidant than 2, due perhaps to a structural motif

resembling that of the native SOD enzyme especially with

respect to its Cu–Zn distances (Ohse et al., 2001) and redox

potentials. Ligand 1 showed no significant SOD mimetic

activity even up to concentrations .300mM, indicating the

crucial role played by the metal centers in interaction with

the radical species.

Anticancer Activity

The two cell lines selected for the evaluation of antiproli-

ferative activities of the present compounds include estrogen

independent BT-20 breast cancer cell line and androgen inde-

pendent PC-3 prostate cancer cell line, which are both

invasive cell types. It has been concluded from studies on

gastric carcinogenesis that such invasive cancers occur as a

consequence of an insufficient control of the oxidative stress

for a prolonged time (Correa, 1995). It has been further

shown that although antioxidant enzyme status of such cells

does not differ too much from that in the primary cell

TABLE 1

SOD activities of model complexes

Complexes� IC50þ Ref.

2 34.60 This work

3 0.94 This work

[Cu(im)CuL]ClO4. 0.5 H2Oa 0.62 (Sato et al., 1986)

[Cu(im)ZnL-2H)

(CuimZnL-H)](ClO4)3a

0.26 (Sato et al., 1986)

[Cu(im)ZnL0]3þa 0.50 (Bodini et al., 1983)

[Cu2(bdpi)(CH3CN)2]3þb 0.32 (Bodini et al., 1983)

[Cu2(Me4bdpi)(H2O)2]3þc 1.1 (Bodini et al., 1983)

[CuZn(Me4bdpi)(H2O)2]3þc 0.24 (Bodini et al., 1983)

[Cu2(bpzbiap)(Cl)3]d 0.52 (Strothkamp and

Lippard 1982)

Native Cu2Zn2 SOD 0.04 (Bodini et al., 1983)

�The legends for ligands are: (a) L ¼ 3,6,9,16,19,22-hexaaza-

6,19-bis (2 hydroxyethyl)tricyclic [22,2,2,2]triaconta-1,11,13,24,27,

29-hexane; (b) bdpi, 4,5-bis(di(2-pyridyl-methyl)aminomethyl)-

imidazolate; (c) Me4bdpi ¼ 4,5-bis(di(6-methyl-2-pyridylmthyl)

aminomethyl)imidazolate; (d) Hbpzbiap ¼ 1,5-bis(1-pyrazolyl)-3-

[bis(2-imidazolyl)methyl]azapentane; þIC50 ¼ concentration of the

compound which exerts the SOD activity equivalent to one unit of

native SOD.FIG. 8. Antiproliferative activities of compound 3 against BT-20 and PC-3

cell lines.

FIG. 7. Plot of % inhibition of NBT reduction at various concentrations of

compound 3.

S. DUTTA ET AL.8

cultures, the variations in tissue and tumor-stage dependence

offer excellent possibilities for modulation of the antioxidant

status either by systematic change of the enzymatic antioxidant

system (Domenicotti et al., 2000) or release of radicals through

SOD mimetic compounds.

In the present study, ligand 1 and its copper compound 2

showed no activity against BT-20 and PC-3 cell lines.

However, compound 3 was found to be highly potent and

hence it was subsequently examined at three different concen-

trations and their cellular effects were noted at three different

time intervals. The results of such experiments (Figure 8)

confirm that heterometallic Cu–Zn compound is effective

against both estrogen independent and androgen independent

cell lines at IC50 values of 4.4 and 5.8mM, respectively,

suggesting that it needs to be investigated further for

mechanical details.

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