Synthesis, Antioxidant Properties and Antiproliferative

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

Synthesis, Antioxidant Properties and Antiproliferative Activities of Tetrameric Copper and Copper-Zinc Metal Complexes of Catecholamine Schiff Base Ligand Sabari Dutta, Ratnamala Bendre, and Subhash Padhye Department of Chemistry, University of Pune, Pune, India

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

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 dismutase (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 treatment have been shown to cause increased cellular ROS generation. These therapeutic agents include anthracyclines, cisplatin, bleomycin, and the synthetic retinoid viz. N-(4hydroxyphenyl) 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 membranes, offering no immunogenicity, possessing longer lifetime 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 manganese, 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

Modulation of the intrinsic oxidative stress in invasive metastatic cancer cells using two superoxide dismutase mimicking conjugates of catecholamine Schiff base ligand 3-[(20 – (200 pyridylethyl)iminoethyl] benzene-1,2 diol] (1) results in superior killing of malignant cells. The homometallic copper compound as well as its heterometallic copper-zinc counterpart (3) are characterized by spectroscopic, electrochemical and magnetic susceptibility measurements where the latter shows remarkable SOD activity (IC50 5 0.94 mM) as well as a potent antiproliferative activity against estrogen independent breast (BT-20) and androgen independent prostate cancer (PC-3) cell lines. Keywords

Schiff base complexes; copper tetramer; copper-zinc mixed-metal complex; SOD activity; antiproliferative activity

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;

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 measurements and their interpretations. Address correspondence to Subhash Padhye, Department of Biochemistry and Molecular Biology, School of Medicine, Wayne State University, Detroit, MI 48201, USA. E-mail: sbpadhye@chem. unipune.ernet.in

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complexes that mimic the spectroscopic, magnetic, and structural 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 imidazolate-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 heterodinuclear copper(II) – zinc(II) complexes have been rather scarce and the coordination of superoxide ion has been confirmed 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 macrocyclic 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 2,3dihydroxybenzaldehyde 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. Manganese 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 characterization 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 copperzinc moiety. Both compounds are evaluated for their superoxide 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 - (200 pyridylethyl)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 equivalent to one unit of the native SOD enzyme. MTT Assay The number of viable cells remaining after an appropriate 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 overnight. The test agent was then added to each well at final concentrations 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

ANTIOXIDANT SCHIFF BASE COMPLEXES HAVING ANTIPROLIFERATIVE ACTIVITIES

optical density (OD)570 values and expressed as percent survival using the following formula: % cell viability ¼

OD570 of drug treated sample 100 OD570 of untreated control sample

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 carboxylate absorptions. Finally, the difference in the symmetric and asymmetric modes of carboxylate stretches in the present

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compounds (D ¼ 130 2 160 ncm21) indicates a bidentate symmetric 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 antiferromagnetically 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.

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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 2Jsˆ1sˆ2. In the linear chain model (Bu et al., 2000), the full Spin Hamiltonian involves two super-exchange pathways and two exchange integrals that would be required to describe the molar susceptibilities as follows: Hex ¼ 2½J1 ðS1 S2 þ S3 S4 2J2 ½ðS2 S3 Þ xm ¼ fðJ1 ; J2 ; g, TÞ þ Na

½1 ½2

In the former case, any superexchange interaction between Cu(1). . . . . .Cu(3), Cu(2). . . . . .Cu(4) would be considered to

where Na ¼ temperature independent paramagnetism. In Eq. [1], out of the two exchange integrals, J2 is considered 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 observation suggests that substitution of the two Zinc (II) ions in this cluster takes place at the site of inner pair.

FIG. 3.Temperature dependence of the magnetic susceptibility of tetramic copper clusters [Cu21(CH3COO)ZgZ].H2O.

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 CuII3 CuI/CuII2 CuI and CuII4 /CuII3 CuI, 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 environment 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.

short and two longer contacts (Bu et al., 2000), as shown below: Cu(1)------------Cu(2)--------Cu(3)---------Cu(4) J1

J2

J3


FIG. 4. Cyclic voltammograms in DMSO solvent at sweep rates of 100 mv/s for: (a) ligand 1 and (b) compound 2.

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

FIG. 5. X-band EPR spectra of compound 2 as polycrystalline solid from 4 k to 300 K.

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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 anisotropic “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 predicting 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. 6. X-band powder EPR spectrum for compound 3 at 300 K.

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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 .300 mM, indicating the crucial role played by the metal centers in interaction with the radical species.

FIG. 7. Plot of % inhibition of NBT reduction at various concentrations of compound 3.

SOD Activity The SOD-like activities of the ligand and its copper compounds were determined by the NBT method in the concentration 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 34.56 mM (for compound 2) and 0.94 mM (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

Anticancer Activity The two cell lines selected for the evaluation of antiproliferative activities of the present compounds include estrogen independent BT-20 breast cancer cell line and androgen independent 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 2 3 [Cu(im)CuL]ClO4 . 0.5 H2Oa [Cu(im)ZnL-2H) (CuimZnL-H)](ClO4)a3 [Cu(im)ZnL0 ]3þa [Cu2(bdpi)(CH3CN)2]3þb [Cu2(Me4bdpi)(H2O)2]3þc [CuZn(Me4bdpi)(H2O)2]3þc [Cu2(bpzbiap)(Cl)3]d Native Cu2Zn2 SOD

þ IC50

34.60 0.94 0.62 0.26 0.50 0.32 1.1 0.24 0.52 0.04

Ref. This work This work (Sato et al., 1986) (Sato et al., 1986) (Bodini et al., 1983) (Bodini et al., 1983) (Bodini et al., 1983) (Bodini et al., 1983) (Strothkamp and Lippard 1982) (Bodini et al., 1983)

The legends for ligands are: (a) L ¼ 3,6,9,16,19,22-hexaaza6,19-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.


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 concentrations 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 5.8 mM, respectively, suggesting that it needs to be investigated further for mechanical details. REFERENCES Armstrong, J. S.; Steinauer, K. K.; Hornung, B.; Irish, J. M.; Lecane, P.; Birrell, G. W.; Peehl, D. M.; Knox, S. J. Role of Glutathione Depletion and Reactive Oxygen Species Generation in Apoptotic Signaling in a Human B Lymphoma Cell Line. Cell Death Differ. 2002, 9, 252–63. Bhirud, R. G.; Shrivastava, T. S. Superoxide Dismutase Activity of Cu(II)2(aspirinate)4 and its Adducts with Nitrogen and Oxygen Donors. Inorg. Chim. Acta 1990, 173, 121– 125. Bodini, M. E.; Copia, G.; Robinson, R.; Sawyer, D. T. Redox Chemistry of Metal-Catechol Complexes in Aprotic Media. 5. 3,5-Ditert-butylcatecholato and 3,5-di-tert-butyl-o-benzosemiquinonato Complexes of zinc(II). Inorg. Chem. 1983, 22, 126– 129. Bodini, M. E.; Arancibia, V. Polyhedron. Manganese complexes with 2-hydroxy-3(3-methyl-2-butenyl)-1,4-naphthoquinone (Lapachol). Redox Chemistry and Spectroscopy in Dimethylsulphoxide 1989, 8, 1407–1412. Boveris, A.; Chance, B. The Mitochondrial Generation of Hydrogen Peroxide. Biochem J 1973, 134, 707– 716. Bu, X. H.; Du, M.; Shang, Z. L.; Zhang, R. H.; Liao, D. Z.; Shionoya, M.; Clifford, T. Varying Coordination Modes and Magnetic Properties of Copper(II) Complexes with Diazamesocyclic Ligands by Altering Additional Donor Pendants on 1,5-diazacyclooctane. Inorg. Chem. 2000, 39, 4190– 4199. Buchanan, R. M.; Wilson, C.; Blumenberg, C.; Larsen, S. K.; Greene, D. L.; Pierpont, C. G. Counter Ligand Dependence of Charge Distribution in Copper-Quinone Complexes. Structural and Magnetic Properties of (3,5-di-tert-butylcatecholato)(bipyridine)copper(II). Inorg. Chem. 1986, 25, 3070– 3076. Chen, J. S. K.; Konopleva, M.; Andreeff, M.; Multani, A.; Pathak, S.; Mehta, K. Drug-resistant breast carcinoma (MCF-7) cells are paradoxically. J. Cell Physiology 2004, 200, 223. Christou, G.; Perlepes, S. P.; Libby, E.; Folting, K.; Huffman, J. C.; Webbs, R. J.; Hendrickson, D. N. Preparation and Properties of the Triply Bridged, Ferromagnetically Coupled Dinuclear Copper(II) Complexes [Cu2(OAc)3(bpy)2](ClO4) and [Cu2(OH)(H2O)(OAc)(bpy)2](ClO4)2. Inorg Chem. 1990, 29, 3657– 3666. Correa, P. The Role of Antioxidants in Gastric Carcinogenesis. Crit. Rev. Food. Sci. Nutr. 1995, 35, 59 – 64.

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