JOURNAL OF

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Journal of Inorganic Biochemistry 101 (2007) 881–892

InorganicBiochemistry

Synthesis, X-ray crystal structures and biomimeticand anticancer activities of novel copper(II)benzoate

complexes incorporating 2-(4 0-thiazolyl)benzimidazole(thiabendazole), 2-(2-pyridyl)benzimidazole and 1,10-phenanthroline

as chelating nitrogen donor ligands

Michael Devereux a,*, Denis O Shea a, Andrew Kellett a,c, Malachy McCann b,Maureen Walsh c, Denise Egan c, Carol Deegan c, Kinga Kedziora c, Georgina Rosair d,

Helge Muller-Bunz e

a The Inorganic Pharmaceutical and Biomimetic Research Laboratory, Dublin Institute of Technology, Cathal Brugha Street, Dublin 1, Irelandb Department of Chemistry, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland

c Pharma R&D Team, Department of Applied Science, Institute of Technology Tallaght, Dublin 24, Irelandd School of Engineering and Physical Sciences, Heriot Watt University, Edinburgh, EH14 4AS, UK

e School of Chemistry and Chemical Biology, University College Dublin, Dublin 4, Ireland

Received 25 July 2006; received in revised form 6 February 2007; accepted 7 February 2007Available online 14 February 2007

Abstract

Cu(BZA)2(EtOH)0.5 (1) was generated by the reaction of copper(II) hydroxide with benzoic acid (BZAH). [Cu(TBZH)2(BZA)]-(BZA) Æ 0.5TBZH Æ H2O (2) and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)] Æ 1.66EtOH (3) were obtained when 1 reacted with Thiabendazole(TBZH) and 2-(2-pyridyl)benzimidazole (2-PyBZIMH), respectively. [Cu(BZA)2(phen)(H2O)] (4) was isolated from the reaction of ben-zoic acid and 1,10-phenanthroline (phen) with copper(II)acetate dihydrate. Molecular structures of 2, 3 and 4 were determined crystal-lographically. 2 and 3 are hydrogen bonded dimers and trimers, respectively. The copper centres in complexes 2 and 3 are bis-chelatederivatives that have N4O ligation and their geometry is very similar being approximately square-pyramidal. However whereas in com-plex 2 both TBZH ligands are neutral in 3 one of the 2-PyBZIMH chelators is deprotonated on each copper. The structural results for 4

represent a re-examination of this crystallographically known compound for which no hydrogen atom coordinates have been previouslyreported. It crystallises as a hydrogen bonded dimmer and is a mono-chelate of phen with each copper centre possessing N2O3 ligationand square pyramidal geometry. The catalase and superoxide dismutase (SOD) activities of the four complexes along with those of theknown phenanthroline complexes [Cu(mal)(phen)2] and [Cu(phendione)3](ClO4)2 (malH2 = malonic acid and phendione = 1,10-phenan-throline-5,6-dione) were investigated. Complexes 1–4, the metal free ligands and a simple copper(II) salt were assessed for their cancerchemotherapeutic potential against the hepatocellular carcinoma (Hep-G2) and kidney adenocarcinoma (A-498) cell lines. TBZH, 2-PyB-ZIMH and benzoic acid when uncoordinated to a metal centre offer poor chemotherapeutic potential. copper(II) benzoate is significantlymore active than the free acid. The bis-chelate derivatives [Cu(TBZH)2(BZA)](BZA) Æ 0.5TBZH Æ H2O (2) and [Cu(2-PyBZIMH)(2-PyB-ZIM)(BZA)] Æ 1.66EtOH (3) elicit a significant cytotoxic response to the cancer cell lines tested. Replacing TBZH and 2-PyBZIMH withphen to give [Cu(BZA)2(phen)(H2O)] (4) does not significantly increase the anti-cancer activity.� 2007 Elsevier Inc. All rights reserved.

Keywords: Copper(II)benzoates; Thiabendazole; 2-(2-Pyridyl)benzimidazole; 1,10-Phenanthroline; Catalase; SOD; Anticancer activity

0162-0134/$ - see front matter � 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.jinorgbio.2007.02.002

* Corresponding author. Tel.: +353 1 4024486; fax: +353 4024495.E-mail address: Michael.Devereux@dit.ie (M. Devereux).

882 M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892

1. Introduction

Cancer, of which there are over 100 different forms, is aleading cause of death. The clinical success of cisplatin, andrelated platinum based drugs, as anti-cancer agents consti-tutes the most impressive contribution to the use of metalsin medicine [1]. However major problems associated withthese anti-cancer metallo-drugs include serious toxicityand other side-effects, and major problems with resistance.New potent and selective anti-cancer drugs are urgentlyrequired. Recently novel metal based compounds contain-ing metals such as titanium, copper, ruthenium, tin andrhodium have been reported with promising chemothera-peutic potential, and which have different mechanisms ofaction to the Platinum based drugs [2]. In our laboratories,we have shown that the N,N 0-donor ligands 1,10-phenan-throline (phen) and 1,10-phenanthroline-5,6-dione (phendi-one) (Fig. 1) and a range of their Cu(II), Mn(II) and Ag(I)carboxylate complexes are significantly more active (in-vitro) anti-cancer agents than cisplatin against selected can-cer cell lines [3–6]. The active complexes were found toinhibit DNA synthesis in a concentration-dependent man-ner through a mechanism that did not involve intercalationand it is possible that they may have a different mode ofaction to that of cisplatin [4,5]. Importantly, the phenand phendione complexes are non-mutagenic (unlike cis-platin) and are not readily expelled from cells. One possiblemode of action for the copper and manganese complexescould involve superoxide dismutase (SOD) mimetic activitywhich can lead to oxidative damage to cellular compo-nents. A number of copper and manganese SOD mimeticshave been shown to possess antitumor activity and havebeen proposed as a new class of potential anticancer agents[7]. Conversion of intracellular O��2 to H2O2 by these com-plexes and subsequent Fenton-like reaction with H2O2 gen-erating the cytotoxic �OH radicals is their proposedmechanism of action. Catalase (CAT) mimetic activity isnot desirable in such systems as they would simply convertthe H2O2 to water and molecular oxygen preventing theproduction of the hydroxyl radicals.

In an effort to further develop this novel class of poten-tial anti-cancer agent we have studied the synthesis andchemotherapeutic potential of a range of novel copper(II)carboxylate complexes derived from the chelating benz-imidazole ligands 2-(4 0-thiazolyl)benzimidazole {thiaben-

N Nphendione

N Nphen

OO

Fig. 1. The structure of the phen and the phendione ligands.

dazole} (TBZH) and 2-(2-pyridyl)benzimidazole (2-PyBZIMH) (Fig. 2). Like the phenanthroline based ligandsthese compounds are known to chelate to metal centresalthough their coordination chemistry has not been asextensively explored. Furthermore, because of the presenceof the benzimidazole moiety their potential as ligands isenhanced by their ability to act as both acids and bases[8] suggesting that they can be either neutral or anionic che-lators or they may simply be found in complexes as cationiccounterions [9]. Complexes of TBZH, whether in solutionor in the solid state, tend to have the ligand bound to themetal centres (regardless of the nature of the metal ion)through the imidazolic and thiazole nitrogen atoms [10].Furthermore, the bis-chelate complexes of neutral TBZHare predominantly cis-isomers and indeed the only knownstructurally characterised trans isomer is a sodium complexin which both the neutral TBZH and anionic TBZ� arefound [10]. No examples have been reported in the litera-ture where the heterocyclic sulfur interacts with a metalpresumably due to its weak Lewis base nature. Howeverwe have recently found spectroscopic evidence for suchan interaction in an iron complex formulated as[Fe(TBZH)3] Cl3 [11]. As is the case for 1,10-phenanthro-line and similar chelating ligands tris-chelate compoundsof TBZH are known for metals such as cobalt, nickeland cadmium [10]. The X-ray crystal structure of[Rh(H)2(PPh3)2(TBZH)]ClO4 was recently reported and itis the only known structurally characterised mono-chelatecomplex of TBZH [12].

Like TBZH the synthesis of mono-, bis- and tris-chelatecompounds of 2-PyBZIMH have been reported [13–19].The only structures reported so far for this ligand are formono-chelate derivatives of copper(II) [16] and Palla-dium(II) [17], and tetrakis-chelate Lanthanide complexesin which it is found in both the neutral and anionic states[20].

Herein we report the synthesis, X-ray structures andanti-cancer activities of the two new bis-chelate copper(II)benzoate derivatives [Cu(TBZH)2(BZA)](BZA) Æ 0.5TBZ-H Æ H2O and [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)] Æ 1.66Et-OH. Furthermore, in an effort to compare theirchemotherapeutic potential to that of a similar copper(II)phen complex we have also generated, structurally re-examined and biologically tested the known compound[Cu(BZA)2(phen)(H2O)]. In an effort to assess the potentialof the complexes to perturb cellular redox balance theirin vitro catalase and superoxide dismutase activities were

N

HNS

N

TBZH

N

HN

N

2-PyBZIMH

Fig. 2. The structure of the TBZH and 2-PyBZIMH ligands.

M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892 883

also examined and compared to those of the copper(II) bis-phen and copper(II) tris-phendione complexes.

2. Experimental

2.1. Chemistry

Chemicals were purchased from commercial sources andused without further purification. IR spectra were recordedin the region 4000–400 cm�1 on a Nicolet-400 Impact spec-trometer. UV spectra were recorded in the region of 400–1000 nm on a Specord 200 spectrophotometer. Magneticsusceptibility measurements were made using a JohnsonMatthey Magnetic Susceptibility balance. [HgCo(SCN)4]was used as a reference. Conductivity readings wereobtained using an Orion model 150 plus conductivitymeter. Satisfactory microanalytical data for the complexeswere reported by the Microanalytical Laboratory, Univer-sity College Cork, Ireland. [Cu(mal)(phen)2], [Cu(phendi-one)3](ClO4)2 and [Cu2(indo)4(H2O)2] were synthesisedaccording to published methods [4,5,21].

2.2. Cu(BZA)2(EtOH)0.5 (1)

To a solution of freshly prepared Cu(OH)2 (1.0 g,10.25 mmol) in ethanol (50 mL) was added an ethanolicsolution (25 mL) of benzoic acid (2.5 g, 20.5 mmol) andthe resulting suspension was refluxed for 3 h. The blue-green powder that deposited was filtered off, washed witha small volume of ethanol, and then air-dried. Yield:0.61 g 16%). % Calc: C, 54.7; H, 3.9. % Found: C, 54.4; H3.5. IR (KBr): 3061, 2970, 1597, 1561, 1520, 1509, 1445,1402, 1309, 1179, 1146, 1071, 1026, 929, 845, 718, 685,510, 481 cm�1. leff: 1.56 BM. Solubility: insoluble in water,ethanol, methanol, acetone, and trichloromethane. Solublein DMSO. kd–d(Nujol Mull) = 718 nm. kd–d(DMSO) =720 nm, e = 125 M�1 cm�1. KM (DMSO) = 1.2 S cm2 mol�1.All attempts to obtain crystals of 1 from the mother liquorwere unsuccessful.

2.3. [Cu(TBZH)2(BZA)](BZA) Æ 0.5 TBZH Æ H2O (2)

To a hot solution of 1 (0.5 g, 0.8 mmol) in ethanol/water(100 mL) was added Thiabendazole (TBZH) (0.67 g,3.3 mmol) and the resulting green solution was refluxedfor 3 h. The green powder that deposited was filtered off,washed with a small volume of ethanol, and then air-dried.Yield: 0.55 g (41.5%). % Calc: C, 56.6; H, 3.59; N, 11.9. %Found: C, 56.4; H 3.7; N, 11.6. IR (KBr): 3099, 3058,1634, 1592, 1524, 1481, 1425, 1428, 1395, 1398, 1331,1302, 1279, 1227, 1176, 1011, 992, 940, 832, 745, 719, 674,626, 570, 566, 526, 485, 431 cm�1. leff: 1.73 BM. Solubility:insoluble in water, ethanol, methanol, acetone, trichlorom-ethane. Soluble in DMSO. kd–d(Nujol Mull) = 690 nm.kd–d(DMSO) = 698 nm, e = 40 M�1 cm�1. KM(DMSO) =4.6 S cm2 mol�1. A small quantity of green crystals of 2,

suitable for X-ray structural analysis, were obtained fromthe filtrate upon standing for one week.

2.4. [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)] Æ 1.66 EtOH

(3)

To a solution of 1 (1.0 g, 1.6 mmol) in ethanol (75 mL)was added 2-(2-Pyridyl)-benzimidazole (2-PyBZIMH)(1.95 g, 6.6 mmol) and the resulting green solution wasrefluxed for 3 h. The green powder that deposited was fil-tered off, washed with a small volume of ethanol, and thenair-dried. Yield: 2.10 g (55%). % Calc: C, 62.9; H, 4; N,14.2. % Found: C, 63.1; H, 3.7; N, 14.1. IR (KBr): 3054,1602, 1544, 1514, 1493, 1478, 1456, 1445, 1427, 1384,1332, 1278, 1232, 1151, 1097, 1006, 986, 819, 792, 744,726, 678, 647, 627, 574, 433 cm�1. leff: 1.44 BM. Solubility:insoluble in water, ethanol, methanol, acetone, trichloro-methane. Soluble in DMSO. kd–d(Nujol Mull) = 660(890 sh) nm. kd–d(DMSO) = 720 nm, e = 460 M�1 cm�1.KM (DMSO) = 63.75 S cm2mol�1. A small quantity ofgreen crystals of 3, suitable for X-ray structural analysis,were obtained from the filtrate upon standing for 24 h.

2.5. [Cu(BZA)2(phen)(H2O)] (4)

To a solution of [Cu2(CH3CO2)4(H2O)2] (0.5 g,1.25 mmol) in EtOH (75 mL) was added 1,10 phenanthro-line (0.45 g, 2.5 mmol) and benzoic acid (0.31 g, 2.5 mmol)and the resulting solution was heated and stirred for 2 hand then left to stand overnight. The blue/green powderthat deposited was filtered off, washed with a small volumeof ethanol, and then air-dried. Yield: 0.26 g (21.6%).% Calc: C, 61.96; H, 3.99; N, 5.55. % Found: C, 61.75;H, 3.86; N, 5.21. IR (KBr): 3087, 3059, 1598, 1558, 1519,1426, 1385, 1115, 1067, 1019, 848, 827, 721, 676,434 cm�1. leff: 2.31 BM. Solubility: insoluble in water, eth-anol, methanol, acetone, and trichloromethane. Soluble inDMSO. kd–d(Nujol) = 690 nm. kd–d(DMSO) = 688 nm,e = 192 M�1 cm�1. KM (DMSO) = 9.4 S cm2 mol�1. Asmall quantity of blue crystals of 4, suitable for X-raystructural analysis, were obtained from the filtrate uponstanding for 24 h.

2.6. X-ray crystallography

For complexes 2 and 3 crystal data were collected usinga Bruker SMART APEX CCD area detector diffractome-ter. A full sphere of the reciprocal space was scanned byphi–omega scans. Pseudo-empirical absorption correctionbased on redundant reflections was performed by the pro-gram SADABS [22]. All programmes used in the structuresolution and refinement are contained in the SHELXTLprogrammes [23,24]. Hydrogen atoms were added at calcu-lated positions and refined using a riding model. Their iso-tropic temperature factors were fixed to 1.2 times (1.5 timesfor methyl groups and hydrogen atoms attached to oxygen)the equivalent isotropic displacement parameters of the

Table 1Crystal data and structure refinement for 2 and 3

Compound (2) (3)

Empirical formula C78H59N15O10S5Cu2 C103H96N18O11Cu3

Formula weight 1653.78 1952.60Temperature (K) 100(2) 100(2)Wavelength (A) 0.71073 0.71073Crystal system Triclinic MonoclinicSpace group P�1(#2) Cc(#9)a (A) 11.9838(8) 32.670(2)b (A) 12.2282(8) 16.5187(11)c (A) 14.4085(10) 18.3721(12)a (�) 104.443(10) 90b (�) 100.3190(10) 113.395(1)c (�) 110.9800(10) 90Volume (A3) 1823.1(2) 9099.7(10)Z 1 4Density (calc) (Mg/m3) 1.506 1.425Absorption coefficient

(mm�1)0.799 0.769

F(000) 850 4060Crystal size (mm3) 0.40 · 0.30 · 0.20 0.35 · 0.20 · 0.15Crystal description Green block Green blocksh Range (�) 1.90–28.55 1.67–28.52Index ranges �16 6 h 6 15 �41 6 h 6 42

�16 6 k 6 16 �21 6 k 6 21�19 6 l 6 19 �24 6 l 6 23

Reflections collected 31364 38550Independent reflections 8533 [0.0206] 19867 [ 0.0221]

884 M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892

atom the H-atom is attached to. In 2, the hydrogen atomsH3N and H6N were located in the difference fourier mapand allowed to refine freely with isotropic displacementparameters. The hydrogen atoms of the water molecule(H1O5 and H2O5) in this structure were located in the dif-ference fourier map, too, and there positions were allowedto refine. Their isotropic displacement parameters werefixed to 1.5 times the Ueq of the water oxygen, O5. Aniso-tropic displacement parameters were used for all non-hydrogen atoms. In the structure of 2 all but the sulfuratom in the free ligand could only be refined with isotropicthermal displacement parameters. As it is disordered overan inversion centre it could only be refined as a rigid group,using one of the complexong ligands as a template. Neithera free refinement nor the application of a SAME restraintled to a sensible result. Details of the data collection andrefinement for 2 and 3 are given in Table 1.

Intensity data were collected on a single crystal of com-plex 41 mounted on a glass fibre on a Bruker Nonius X8Apex2 diffractometer. All programmes used in the struc-ture solution and refinement are contained in the SHEL-XTL programmes [23,24]. Hydrogen atoms for the boundwaters were included in the model and coordinates freelyrefined {O–H restrained to 0.9(2) A} and displacementparameters treated as riding (1.5 · Ueq O5).

[Rint]Completeness to h (=) (=28.55�) 91.9% (=28.56�) 92.5%Absorption correction Semi-empirical from

equivalentsSemi-empirical fromequivalents

Refinement method Full-matrix least-squares on F2

Full-matrix least-squares on F2

Data/restraints/parameters 8533/0/469 19867/2/1227Goodness-of-fit on F2 1.026 1.049Final R indices [I > 2r(I)] R1 = 0.0684 R1 = 0.0359

wR2 = 0.1822 wR2 = 0.0852R indices (all data) R1 = 0.0720 R1 = 0.0384

wR2 = 0.1854 wR2 = 0.0870Largest difference in peak

and hole (e A�3)2.048 and �1.973 0.647 and �0.311

2.7. Catalase (CAT) activity

The ability of the complexes to disproportionate hydro-gen peroxide was assayed using a method previously pub-lished [25] as follows: To a solid sample (ca 10 mg) of thecopper complex was added aqueous H2O2 (35% w/w,10 mL, 114 mmol). The mixture was stirred and thermo-stated at 25 �C and the evolved O2 was measured volumet-rically (volume ±0.10 mL). In Cases where imidazole(50 mg) was added this was introduced into the reactionvessel before the addition of H2O2.

2.8. Superoxide dismutase (SOD) activity

The O��2 dismutase activities of the metal complexes wereassessed using a modified nitro-blue-tetrazolium (NBT)

1 Crystal data and refinement for 4: Empirical formula: C26H20CuN2O5.Formula weight: 1007.99. Temperature: 100(2) K. Wavelength (A):0.71073. Crystal system: Monoclinic. Space group: P21/c(#14).a (A): 10.4668(6); b (A): 21.1414(13); c (A): 11.1302(6); a (A): 90; b (A):117.645(2); c (A): 90. Volume (A3): 2181.8(2). Z: 4. Density (calc) (Mg/m3): 1.534. Absorption coefficient (mm�1): 1.044. F(000): 1036. Crystalsize (mm3): 0.32 · 0.28 · 0.24. Crystal description: blue blocks. h range (�):2.28–32.95. Index ranges: �15 6 h 6 15; �32 6 k 6 32; �17 6 l 6 16.Reflections collected: 35,743. Independent reflections [Rint]: 7994 [0.0369].Completeness to h (=): (=25.00�) 99.9%. Absorption correction: Semi-empirical from equivalents. Refinement method: Full-matrix least-squareson F2. Data/restraints/parameters: 7994/0/313. Goodness-of-fit on F2:1.039. Final R indices [I > 2r(I)]: R1 = 0.0365; wR2 = 0.0951. R indices (alldata): R1 = 0.0549; wR2 = 0.1016. Largest difference peak and hole(e A�3): 0.886 and �0.396.

assay with xanthine–xanthine oxidase system as the sourceof O��2 [26]. The quantitative reduction of NBT to blue for-mazan by the O��2 was followed spectrophotometrically at550 nm. All reagents were obtained from Sigma–AldrichChemical Co. Ltd and assays were run in 3 mL of solution.Results are graphed as the % inhibition of NBT reductionfor three concentrations. Tabulated results were derivedfrom linear regression analyses and are given as the concen-tration (lM) equivalent to 1unit of bovine erythrocyteSOD activity. A unit SOD activity is the concentration ofthe complex or enzyme which causes 50% inhibition inthe reduction of NBT (referred to as the IC50 value).

2.9. Cytotoxicity testing

Dimethyl sulphoxide (DMSO) and all cell culturereagents and media were purchased from Sigma–AldrichIreland, Ltd, unless otherwise stated.

M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892 885

2.10. Cell lines and cell culture

Cytotoxicity assays were performed using two humanmalignant model cell lines in order to assess the cancer che-motherapeutic potential of the compounds. Hepatatocellu-lar carcinoma (Hep-G2) and kidney adenocarcinoma (A-498) cell lines were purchased from the ATCC. All cell lineswere grown as monolayers in Eagle’s minimum essentialmedium, supplemented with 2 mM L-glutamine and Earle’sbalanced salt solution, containing 1.5 g/L sodium bicar-bonate, 0.1 mM non essential amino acids, 1.0 mM sodiumpyruvate, 100 U/mL penicillin and 100 lg/mL streptomy-cin supplemented to contain 10% (v/v) foetal bovine serum.All cells were grown at 37 �C in a humidified atmosphere,in the presence of 5% CO2 and were in the exponentialphase of growth at the time of assay.

2.11. Assessment of cytotoxicity, using MTT assay

Each of the two cell lines (100 ll) were seeded at a den-sity of 5 · 104 cells/mL into sterile 96 well flat-bottomedplates (Sarstedt) and grown in 5% CO2 at 37 �C. Test com-pounds were dissolved in DMSO and diluted with culturemedia. The maximum percentage of DMSO present in allwells was 0.2% (v/v). Each drug solution (100 lL) wasadded to replicate wells in the concentration range of0.1–1000 lM and incubated for 96 h. A miniaturised viabil-ity assay using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was carried out according tothe method described by Mosman [27]. The IC50 value,defined as the drug concentration causing a 50% reductionin cellular viability, was calculated for each drug. Eachassay was carried out using five replicates and repeatedon at least three separate occasions. Viability was calcu-lated as a percentage of solvent-treated control cells, andexpressed as a % of the control. The significance of anyreduction in cellular viability was determined using one-way ANOVA (analysis of variance). A probability of0.05 or less was deemed statistically significant.

O

HO

Benzoic acid (BZAH)

EtOH

Cu(OH)2

Reflux

[Cu(TBZH)2(BZA)](BZA).0.5TBZH.H2O (2)

Cu(BZA)2.0.5EtOH (1)

EtOH/H2OReflux

Reflux

[Cu(2 2-PyBZIMH

EtOH

3 TBZH

RefEtO

Scheme

3. Results and discussion

3.1. Synthesis and characterisation of the compounds

Synthetic routes to the complexes 1–4 are shown inScheme 1. Cu(BZA)2(EtOH)0.5 (1) was generated in lowyield by the reaction of copper(II) hydroxide with benzoicacid. Powdered samples of [Cu(TBZH)2(BZA)](BZA) Æ0.5TBZH Æ H2O (2) and [Cu(2-PyBZIMH)(2-PyB-ZIM)(BZA)] Æ 1.66EtOH (3) were easily obtained, in turn,when 1 was treated with TBZH and 2-PyBZIMH, respec-tively. [Cu(BZA)2(phen)(H2O)] (4) was isolated, as a pow-der, from the one pot reaction of benzoic acid and phenwith copper(II) acetate dihydrate. Crystals of 2, 3 and 4,suitable for X-ray structural analysis, were obtained fromthe respective mother liquors by slow evaporation.

The formulation of the compounds was assigned on thebasis of their elemental analysis, IR spectra, and X-rayanalysis. In the respective IR spectra the majority of theligand absorption bands, some of them with changed inten-sity, appear again in the compounds. The IR spectrum, themicroanalysis and the magnetic moment of 1 are similar tothose of a copper benzoate complex previously reportedand formulated as Cu(BZA)2(EtOH) [28]. Indeed the pres-ence of a new band at 2970 cm�1(aliphatic C–H stretching)in the spectrum of 1 supports the inclusion of the half eth-anol molecule in the formulation. The magnetic moment(1.56 BM) for 1 is slightly lower than that expected for sim-ple copper(II) species (i.e. those lacking Cu–Cu interac-tions) and some form of metal–metal interaction, possiblyinvolving the benzoate ligands, may be taking place inthe complex [29].

In the spectrum of 2 the N–H stretching band at3093 cm�1 has shifted to 3099 cm�1 and has become moreintense. One of the m(C@N)imidazolic and the m(C@N)thiazolic

bands (1577 cm�1 and 1480 cm�1, respectively) are shiftedto 1592 cm�1 and 1524 cm�1 for 2 indicating that theligand is chelating through the imidazolic and the thiazolicnitrogens. The C–S stretching band (at 1228 cm�1 for the

2-PyBZIMH)(2-PyBZIM)(BZA)].1.66EtOH (3)

[Cu2(OAc)4(H2O)2]

luxH

[Cu(BZA)2(phen)(H2O)] (4)

2 Phen

1.

886 M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892

free ligand) remains essentially unchanged suggesting thatthe sulphur atom in the thiazole ring is uncoordinated. Inthe IR spectrum of 3 the N–H stretching band at3061 cm�1 has shifted slightly to 3054 cm�1 and has lostsome intensity (due to the deprotonation of just one ofthe 2-PyBZIMH ligands). The spectrum also has a strongm(C@N)imidazolic band at 1513 cm�1 which is shifted signif-icantly from that of the free ligand (1568 cm�1) indicatingthat the ligand is also coordinated through one of the imi-dazolic nitrogens. The second m(C@N)imidazolic band at1601 cm�1 is essentially unchanged in the complex. Astrong band at 1314 cm�1 in the spectrum of 2-PyBZIMHis shifted towards higher energy (1331 cm�1) indicatingcoordination through the pyridine nitrogen also. The spec-trum of 4 when compared to that of 1 is far more complexand contains new absorption bands at 850 cm�1 and730 cm�1 which are characteristics of the presence of che-lating phen ligands [25].

As well as the bands that have been assigned to thechelating TBZH, 2-PyBZIMH and Phen ligands thespectra of 2–4 all contain bands that are characteristic ofbenzoate anions with typical m(OCO)assym {1524 cm�1 (2),1544 cm�1 (3) and 1557 cm�1 (4)} and m(OCO)sym

{1398 cm�1 (2), 1384 cm�1 (3) and 1384 cm�1 (4)} values.The calculated D(OCO) values {m(OCO)assym � m(OCO)sym}of 126 cm�1 (2), 160 cm�1 (3) and 131 cm�1 (4) are alllower than the values expected for monodentate carboxyl-ate ligands (>200 cm�1) but the relative reduction in theD(OCO) values are typical where such carboxylate groupsare involved in hydrogen bonding [30]. Whereas the roomtemperature magnetic susceptibility values for 2 and 4 areclose to those expected for simple copper(II) species (i.e.those lacking Cu–Cu interactions) the value for 3 is slightlylower and some form of Cu–Cu interaction may be takingplace in this complex [29]. All of the complexes are effec-tively insoluble in common solvents but they were found

Fig. 3. The structure of [Cu(TBZH)2(

to be soluble in DMSO. The molar conductivities of com-plexes 1, 2 and 4 are in the range 1.2–9.4 S cm2 mol�1 indi-cating that they are essentially non-electrolytes and thus donot dissociate in DMSO. Furthermore, the UV spectra ofthese three complexes recorded in DMSO solution as wellas in the solid state as Nujol Mulls show approximatelysimilar shapes and positions of the absorption bands,indicating no appreciable change in the geometry of thecomplexes in solution. Complex 3 on the other hand disso-ciates extensively in DMSO (KM = 63.75 S cm2 mol�1)indicating that it may be a 1:1 electrolyte giving the follow-ing ions in solution:

½Cuð2� PyBZIMHÞð2� PyBZIMÞðBZAÞ�! ½Cuð2� PyBZIMHÞð2� PyBZIMÞ�þ þ BZA�

Furthermore, there are significant differences in theshape and positions of peaks between the UV spectra of3 recorded as a solid and in DMSO solution.

The X-ray crystal structures of 2 and 3 are shown inFigs. 3–6 and selected bond lengths and angles around theircopper centres are listed in Table 2. The copper centres incomplexes 2 and 3 have N4O ligation and their geometry isvery similar being approximately square-pyramidal (seeFigs. 4 and 6 and Table 2). However whereas in complex2 the [Cu(TBZH)2(BZA)]+ moieties are cationic (becausethe TBZH ligands are neutral) the [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)] moieties in 3 are essentially neutral dueto deprotonation of one of the 2-PyBZIMH chelatorsattached to each copper, a feature which has beenpreviously reported for a series of tetrakis-chelated Lantha-nide complexes of this ligand [18]. To the best of ourknowledge, 3 is the first structurally characterised bis-chelate complex incorporating 2-PyBZDH. In 2 the two[Cu(TBZH)2(BZA)]+ cations, two BZA� counter ionsand two water molecules form a hydrogen-bonded dimer

C31C30

O3

C32

C29C28

C33C34

O4 O5

BZA)](BZA) Æ 0.5TBZH Æ H2O (2).

C18

C19

C8C9

C17

C7

C20C10

C6

C16C15

C73

C23C24

O2N3

N6

N2

N5

C21C4 C22

C25

C14

CuO1

C27 C26C3

C13

N1

N4

C2

C12

C1

C11

S1

S2

Fig. 4. The arrangement of the neutral TBZH and BZA� ligands aroundone of the cationic copper centres in 2.

C11

C10

C12

C9

C16

C28

C27

O2

C7

C15C17

C8

C29

C25

C26

C14N4

N2

N3

C13

O1Cu1

C30

C31

C6

C18N5

N6

C1

N1

C19C20

C2

C24

C5

C21

C3

C23C22

C4

Fig. 6. The arrangement of the neutral 2-PyBZIMH, the anionic 2-PyBZIM� and the BZA� ligands around one of the neutral copper centresin 3.

M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892 887

(Fig. 3 and Table 3), the two halves are related by an inver-sion centre. These dimeric units pack quite loosely, leavingvoids in which the disordered free ligands are found. Sur-prisingly there does not appear to be any significant p–pstacking involving the TBZH ligands. The structure of 3is essentially trimeric with three independent copper cen-tres, together with the five solvent ethanol molecules form-ing a hydrogen-bond-linked chain-like subunit (Fig. 5 andTable 4). These trimeric subunits are connected to oneanother {via one of the solvent ethanols (represented byO4S) to form an infinite chain running along the ac-facediagonal (direction ½�101�)}. The chains have a hexagonal-close rod packing and again the 2-PyBZIMH ligands donot appear to be involved in any p–p interactions.

O2S

O4S

Cu1

Cu2

O3S

1O

Fig. 5. The structure of [Cu(2-PyBZIMH

The structure of 4 has been determined previously atroom temperature [31] but the hydrogen atom coordinateswere not available and thus the current structural resultsrepresent a re-examination of this crystallographicallyknown compound. In the solid state 4 exists as a looselyassociated dimer (Fig. 7) whose intermolecular link is madeby two O–H� � �O hydrogen bonds formed between thewater ligand on each copper and the uncoordinated car-boxylate oxygen atom of the benzoate ligand of their

O4S

Cu3

O5S

)(2-PyBZIM)(BZA)] Æ 1.66EtOH (3).

Table 2Selected bond lengths [A] and angles [�] around the copper centres for 2

and 3

Complex (2) Complex (3)

Cu–O(1) 2.031(2) Cu(1)–O(1) 1.9799(17)Cu–N(1) 2.002(3) Cu(1)–N(5) 1.940(2)Cu–N(2) 2.062(3) Cu(1)–N(4) 2.095(2)Cu–N(4) 2.223(3) Cu(1)–N(1) 2.237(2)Cu–N(5) 1.960(3) Cu(1)–N(2) 1.984(2)

N(1)–Cu–N(2) 80.86(12) N(5)–Cu(1)–N(4) 80.69(9)N(4)–Cu–N(5) 79.62(10) N(1)–Cu(1)–N(2) 77.84(8)

O(1)–Cu–N(1) 91.78(11) O(1)–Cu(1)–N(5) 95.01(8)O(1)–Cu–N(2) 144.73(10) O(1)–Cu(1)–N(4) 145.25(8)O(1)–Cu–N(4) 104.86(9) O(1)–Cu(1)–N(1) 115.59(7)O(1)–Cu–N(5) 91.95(10) O(1)–Cu(1)–N(2) 77.84(8)

Table 3Hydrogen bonds for [Cu(TBZH)2(BZA)](BZA) Æ 0.5TBZH Æ H2O (2) [Aand �]

D–H� � �A d(D–H) d(H� � �A) d(D� � �A) \(DHA)

O(5)–H(2O5)� � �O(4)#1 0.82(11) 2.09(11) 2.804(6) 146(10)N(6)–H(6N)� � �O(3)#2 0.86(5) 1.76(5) 2.622(4) 172(5)N(3)–H(3N)� � �O(2)#3 0.76(6) 1.95(6) 2.711(4) 172(6)

Symmetry transformations used to generate equivalent atoms: #1 �x + 1,�y + 1, �z + 1 #2 x � 1, y, z #3 �x + 1, �y + 2, �z.

Table 4Hydrogen bonds for [Cu(2-PyBZIMH)(2-PyBZIM)(BZA)] Æ 1.66EtOH (3)[A and �]

D–H� � �A d(D–H) d(H� � �A) d(D� � �A) \(DHA)

O(5S)–H(5OS)� � �N(18) 0.84 1.89 2.729(3) 173.9O(4S)–H(4OS)� � �O(4) 0.84 1.79 2.600(3) 161.0O(3S)–H(3OS)� � �O(1S)#1 0.84 1.93 2.770(3) 175.1O(2S)–H(2OS)� � �N(6) 0.84 2.00 2.786(3) 156.3O(1S)–H(1OS)� � �N(12) 0.84 1.91 2.750(3) 176.2N(15)–H(15N)� � �O(4S) 0.88 1.79 2.659(3) 168.7N(9)–H(9N)� � �O(2) 0.88 1.94 2.798(3) 165.1N(3)–H(3N)� � �O(6)#2 0.88 1.89 2.762(3) 173.1

Symmetry transformations used to generate equivalent atoms: #1 x,�y + 1, z + 1/2 #2 x + 1/2, �y + 1/2, z � 1/2.

Fig. 7. The structure of [Cu(BZA)2(phen)(H2O)] (4). Selected bondlengths [A] and angles [�]: Cu(1)–O(1): 1.9590(10). Cu(1)–O(5):2.0037(11). Cu(1)–N(2): 2.0309(11). Cu(1)–N(1): 2.0314(12). Cu(1)–O(4):2.2093(10). O(5)–Cu(1)–N(2): 97.31(4). N(1)–Cu(1)–N(2): 81.70(5). O(1)–Cu(1)–O(5): 89.63(4). O(1)–Cu(1)–O(4): 96.18(4). O(1)–Cu(1)–N(1):91.16(5). O(5)–Cu(1)–O(4):89.30(4).

Table 5Hydrogen bonds in [Cu(BZA)2(phen)(H2O)] (4) [A and �]

D–H� � �A d(D–H) d(H� � �A) d(D� � �A) <(DHA)

O(5)–H(5Q)� � �O(3) 0.846(14) 1.727(14) 2.5639(14) 169.9(18)O(5)–H(5Q)� � �O(4) 0.846(14) 2.563(18) 2.9643(15) 110.3(14)O(5)–H(5P)� � �O(2)#1 0.810(14) 2.026(14) 2.8270(15) 169.4(18)O(5)–H(5P)� � �O(5)#1 0.810(14) 2.527(18) 2.868(2) 106.8(15)C(4)–H(4)..O(3) #2 0.95 2.49 3.420(2) 165C(16)–H(16)..O(3) #3 0.95 2.58 3.5004(19) 163C(20)–H(20)..O(2) #4 0.95 2.55 3.223(2) 128C(24)–H(24)..O(2) #1 0.95 2.36 3.311(2) 179

Symmetry transformations used to generate equivalent atoms: #1 �x + 2,�y, �z + 2 #2: 2 � x, �1/2 + y, 3/2 � z #3: 1 � x, �y, 1 � z, #4: 1 � x,�y, 2 � z.

888 M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892

neighbour (Fig. 7 and Table 5). Furthermore, a short intra-molecular hydrogen bond (e.g. O3 is 2.5639(14) A fromO5) is formed between the second hydrogen atom on eachwater molecule and the uncoordinated carboxylate oxygenatom of the second benzoate ligand attached to each cop-per (Fig. 7 and Table 5). The other unbound carboxylateoxygen atom O2 makes a linear C–H� � �O hydrogen bondwith H24 with the C..O distance of 3.311(2) A (Fig. 7and Table 5). The two copper centres are 5.3361(4) A apartprecluding any possibility of direct Cu–Cu interaction.Each copper centre has square pyramidal geometry withthe basal plane composed of phen N atoms, N1 and N2,water (O5) and one of the monodentate carboxylate Oatoms, O1. The copper atom sits 0.156 A from this planein the direction of O4, the axially-bound carboxylate O,from the second carboxylate ligand. The plane of the phen

ligand is almost co-planar (8�) with the basal coordinationplane above whereas it is far closer to perpendicular to thetwo carboxylate ligands. This dimeric arrangement is alsoseen in the manganese analogue which is practicallyiostructural with 4 [32]. This Mn complex and 4 are exam-ples of mono-chelates of 1,10-phenanthroline, a structuralmotif which is quite rare as phen tends mainly to formbis- and tris-chelate complexes. Indeed another similarMn structure [33], with two monodentate phenyl carboxy-lates binds a second phen rather than water and sobecomes a six coordinate bis-chelate complex with a watermolecule sitting in the crystal lattice unbound to Mn.Beyond the carboxylate-water dimer, there are C–H� � �O

Fig. 8. The Packing diagram for [Cu(BZA)2(phen)(H2O)] (4).

Table 7Molecules of H2O2 disproportionated by the complexes (in the presence ofadded imidazole) at standard temperature and pressure (S.T.P.) conditions

Complex Total no. ofmolecules of H2O2

disproportionatedby one moleculeof complex

Molecules of H2O2

disproportionatedduring first min byone molecule of thecomplex

(1) 327 277(2) 595 141(3) 1351 143(4) 800 149[Cu(mal)(phen)2] 1330 475[Cu(phendione)2](ClO4)2 1109 325{[Mn2(oda)(phen)4(H2O)2]

[Mn2(oda)3(phen)4]}24,570 19,500

M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892 889

interactions involving the unbound carboxylate oxygenatoms with both phen and phenyl H atoms and extensivep–p interactions involving the phen ligands are also presentin the crystal structure (Fig. 8).

3.2. In vitro catalase activity

The catalytic activity of complexes 1–4, [Cu(mal)(phen)2]and [Cu(phendione)3](ClO4)2 towards the disproportion-ation of hydrogen peroxide was investigated by measuringthe volume of evolved oxygen during the course of the reac-tion. None of the 6 complexes exhibited catalytic activity ontheir own. We have previously shown that the catalasemimetic activity of manganese complexes is significantlyimproved in the presence of the base imidazole [34–36], aphenomenon also observed by others [37]. Consequentlywe examined the catalase mimetic properties of the six com-plexes in the presence of this base. The results from thesereactions are summarised in Tables 6 and 7. The catalyticactivity of the complexes is very poor when compared tothe double salt {[Mn2(oda)(phen)4(H2O)2] [Mn2(oda)3

(phen)4]} (odaH2 = octanedioic acid) which had beenshown to be the best manganese catalase mimic previously

Table 6Time course of oxygen evolution in H2O2 disproportionation by com{[Mn2(oda)(phen)4(H2O)2][Mn2(oda)3(phen)4]} [25,34] (malH2 = malonic acid;

Time (min) Imidazole (1) (2) (3) (4) [Cu(mal)(phen)2]

1 18 94 19 24 87 1012 22 105 25 36 171 1323 26 108 33 54 206 1904 27 109 39 69 217 2385 30 110 44 88 223 2706 33 110 50 106 227 2757 45 110 56 123 229 2798 52 110 61 147 231 2819 55 110 65 191 233 283

10 59 110 68 221 233 28315 59 110 80 227 233 28320 59 110 80 227 233 283

produced in this laboratory [25,34]. Table 6 shows the rateof evolution of oxygen from the respective reactions forthe complexes over the first 20 min. Examination of Table7 shows that over the first minute of reaction [Cu(mal)-(phen)2] appears to be the most efficient catalyst with 475molecules of peroxide disproportionated by one moleculeof the complex. Complex 2 appears to be the least efficientcatalyst over the first minute with one molecule of the com-pound knocking down just 141 molecules of the peroxide.However, it should be noted that imidazole itself causes onlya very slight disproportionation of the peroxide (Table 6).

3.3. In vitro Superoxide dismutase (SOD) activity

The SOD mimetic activities of the complexes 1–4,[Cu(mal)(phen)2] and [Cu(phendione)3](ClO4)2 were exam-ined with an indirect method in which the xanthine/xan-thine-oxidase system served as the source for superoxideradicals [26]. The activities of the complexes were comparedto that of [Cu2(indo)4(H2O)2] which is considered to be aexcellent SOD mimetic [38] and which is used therapeuti-cally as an oral anti-inflammatory drug in veterinarymedicine [38,39]. The results are given in Table 8 as concen-trations equivalent to one unit of SOD activity (IC50 values).Significant activities were seen for all compounds tested with

plexes (1)–(4), [Cu(mal)(phen)2] [4], [Cu(phendione)3](ClO4)2 [5] andodaH2 = octanedioic acid) with added imidazole (50 mg) at 25 �C

[Cu(phendione)2](ClO4)2 {[Mn(oda)(phen)4(H2O)2] [Mn(oda)3(phen)4]}

40 800120 900126 930130 970132 991133 1021134 1070135 1082135 1099135 1110135 1122135 1141

Table 8Superoxide dismutase (SOD) activities of complexes 1–4, [Cu(mal)(phen)2][4], [Cu(phendione)2](ClO4)2 [5], [Cu2(indo)4(H2O)2] [21] (indoH = indo-methacin), CuSO4 and Native CuZnSOD as assessed by the NBT assay

Complex Concentration (lM) equivalentto 1U SOD (IC50)

(1) 0.90(2) 0.83(3) 0.95(4) 2.83[Cu(mal)(phen)2] 0.35[Cu(phendione)2](ClO4)2 0.40[Cu2(indo)4(H2O)2] 1.31CuSO4 [41] 30Native CuZnSOD [39] 0.04

890 M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892

one unit of SOD activity arising from the range of 0.35 to2.83 lM aqueous solutions. Interestingly, [Cu(mal)(phen)2]and [Cu(phendione)3](ClO4)2 are the best catalysts withactivities of 0.35 and 0.40 lM, respectively. It is however sig-nificant that the complexes all have activities comparablewith that (1.31lM in this study) of [Cu2(indo)4L2](L = DMSO or DMF) for which the IC50 values vary signif-icantly in the literature and appear to be solvent dependent(the reported value in DMSO is 2 lM [37]).

Conc (μM)1 10 100 1000 10000

Viab

ility

as %

con

trol

0

20

40

60

80

100

120

TBZH2-PyBDZH(1)(2)(3)(4)

Fig. 9. Effects of TBZH, PyBZIMH and 1–4 on the viability of Hep-G2

cells (human heptacellular carcinoma) following continuous incubationwith increasing drug concentration (1–1000 lM) for 96 h. Bars indicatestandard error of the mean (SEM) and results were statistically significantfrom control at p < 0.05. Results are representative of three independentexperiments (n = 3).

3.4. In vitro anticancer activity

Recently several reports have appeared in the literaturedescribing the anti-cancer activity of copper(II) derivativesof several classes of nitrogen donors including purine [40],thiosemicarbazone [41], imidazole [42], benzohydroxamicacid [43] and amino acid [44] ligands. Some mixed chelatecopper-based drugs have exhibited greater antineoplasticpotency than cisplatin in in vitro and in vivo studies[45,46]. We have recently demonstrated the chemothera-peutic potential of 1,10-phenanthroline [4,5] and their cop-per chelates against renal and hepatic cancer derived celllines and demonstrated that they may have a mechanismof action which appears to be different to that of cis-platin.The complex [Cu(mal)(phen)2] was shown to induce apop-tosis in cultured mammalian cells [47] and is known tomediate significant cellular oxidative stress, promote mem-brane lipid peroxidation and interfere with mitochondriarespiratory activity in fungal cells [48]. Similar copper che-lates of phen were studied by other workers and alsoshowed high antineoplastic activity by inhibiting respira-tion and ATP synthesis [49]. Furthermore, [Cu(phen)]2+

type complexes are known to bind to DNA both intercala-tively and non-intercalatively and are known as potent oxi-dative nucleases but the exact structure of [Cu(phen)]2+

when bound to DNA has not been fully characterised[50]. Recently we have been examining alternative chelatingligands such as TBZH and 2-PyBZIMH. The anti-canceractivity of TBZH is enhanced greatly when it is bound toa copper centre [9,51] but none of the complexes reportedso far have exhibited activity close to that of cis-platin.Reports of the biological activity of 2-PyBZIMH are scarce

with no papers published describing the anti-cancer activityof its copper complexes. Its use as a ligand in the prepara-tion of novel Pt(II) and Pd(II) anti-cancer agents has beenreported [17]. These workers did not test the free 2-PyB-ZIMH ligand but found that [Pt(2-PyBZIMH)Cl2] wasthe most efficient anti-cancer agent against eight braintumour cell lines, but the activity was significantly less thanthat of cisplatin.

The chemotherapeutic potential of TBZH, 2-PyBZIMHand the DMSO soluble complexes 1–4 against the hepato-cellular carcinoma (Hep-G2) and kidney adenocarcinoma(A-498) cell lines was determined by calculation of IC50.Calculation of this value allows a direct comparison ofthe cytotoxicity of each of the test agents. The IC50 valueswere calculated using the data presented in Figs. 9 and 10.The values were obtained for each compound, and in eachcell line (Table 9). The IC50 values for benzoic acid, cop-per(II) perchlorate, cisplatin and phen were calculated sim-ilarly (Table 9). TBZH and 2-PyBZIMH were capable ofkilling both cancer derived cell lines only at higher concen-trations with an IC50 value greater than 200 lM. This is incontrast to the case for ‘metal free’ phen and phendionewhich are found to exhibit potent anti-cancer activity(Table 9) [4,5]. Whereas metal free benzoic acid and cop-per(II) perchlorate are essentially inactive (Table 9) thecopper benzoate complex 1 is active at relatively lower con-centrations. When the TBZH and 2-PyBZDH ligands arereacted with the copper benzoate the resulting complexes2 and 3 exhibit anti-cancer activity, for both the liver andkidney cell lines, comparable to that of cisplatin. Whenthe TBZH and 2-PyBZIMH ligands are replaced with phenin complex 4 we do not get any significant increase in thecytotoxicity but it is worth noting that the activity of 4 issignificantly less than that of the metal free phen (Table

Table 9The anti-cancer activity

Test compound Toxicities (IC50 lM)

Hep-G2

Mean ± SDA-498Mean ± SD

BZAH >1000 >1000Cu(ClO4)2 Æ 6H2O >1000 >1000TBZH >200 >2002-PyBZDH >200 >200Phen 4.1 ± 0.5 5.8 ± 0.3Phendione 2.8 ± 1.34 4.2 ±0.36[Cu(phen)2(mal)] Æ 2H2O 0.8 ± 0.02 3.8 ± 0.41[Cu(phendione)3(ClO4) Æ 4H2O 0.4 ± 0.09 0.6 ± 0.06Complex (1) >200 130 ± 16.7Complex (2) 32 ± 0.3 23.32 ± 1.8Complex (3) 21.2 ± 7.7 13.2 ± 4.4Complex (4) 9.5 ± 2.1 21.3 ± 6.7Cisplatin 15 ± 2.7 14 ± 1

Cancer chemotherapeutic potential of complexes 1–4, Cisplatin, the metalfree ligands and a simple copper(II) salt in Hep-G2 and A-498, followingcontinuous incubation for 96 h in the concentration range of 0.1–1000 lM, using MTT assay. A graph of viability as % of solvent treatedcontrol verses drug concentration was used to calculate IC50 values (lM),(Mean ± SD; n = 5). The cytotoxic activity of Phen [4], [Cu(phen)2-(mal)] Æ 2H2O (malH2 = malic acid) [4], Phendione and [Cu(phendi-one)3(ClO4) Æ 4H2O [5] are also included.

Conc (μM)1 10 100 1000 10000

Viab

ility

as %

con

trol

0

20

40

60

80

100

120

TBZH2-PyBZDH(1)(2)(3)(4)

Fig. 10. Effects of TBZH, PyBZIMH and 1–4 on the viability of A-498cells (human renal cell adenocarcinoma)following continuous incubationwith increasing drug concentration (1–1 lM) for 96 h. Bars indicatestandard error of the mean (SEM) and results were statistically significantfrom control at p < 0.05. Results are representative of three independentexperiments (n = 3).

M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892 891

9) and the copper(II) phen bis-chelates previously studiedby this group [4]. Furthermore, the cytotoxic activity ofall three compounds is concentration dependent for bothcell lines (Figs. 9 and 10).

4. Conclusions

The carboxylate complexes 1–4 exhibit potent SODmimicking activity and are inactive as catalase mimics inthe absence of added imidazole. Upon addition of the base

they display only poor catalytic potential for the dispropor-tionation of hydrogen peroxide. The known complexes[Cu(mal)(phen)2] and [Cu(phendione)3](ClO4)2 exhibitgreater SOD activity than complexes 1–4. The four com-plexes 1–4 exhibit significant cytotoxicity with the mono-phen derivative 4 being the most potent but it displayssignificantly less activity than [Cu(mal)(phen)2] and[Cu(phendione)3](ClO4)2. It is probably significant that[Cu(mal)(phen)2] and [Cu(phendione)3](ClO4)2 are watersoluble and complexes 1–4 are only soluble in DMSO.The lack of correlation between the SOD and cytotoxicyIC50 values supports the notion that this type of coppercomplex may be multimodal whereby mechanisms otherthan SOD mimicking may also be responsible for theiranticancer properties. Indeed complex 4 is structurallyrelated to the novel anticancer agent casiopeina II{[Cu(1,4-dimethyl-1,10-phenanthroline)(glycine)]NO3} whichhas been shown to have a very complex in vivo cytoxicity[52]. The cytoxicity results for complexes 2–4 are encourag-ing and this type of complex warrants further study withwater solubility being desirable (possibly through modifica-tion of the ligands and use of alternative counterions).Furthermore, a study of how copper complexes of TBZHand 2-PyBZIMH interact with DNA compared to the phenand phendione systems would further elucidate the modeof action of these potential anticancer agents. An interest-ing compound is the benzoate complex 1 which is an excel-lent SOD mimic but has little or no cytotoxicity, a factwhich supports the hypothesis that the nitrogen donorligands enable the copper complexes to interact with bio-molecules such as DNA. Such a non-toxic compound as1 might have potential use as a therapeutic agent fortreatment of other disorders where SOD activity is patho-logically reduced.

5. Abbreviations

ANOVA analysis of variance

BZAH benzoic acid CAT catalase MalH2 malonic acid MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl

tetrazolium bromide

NBT nitro-blue-tetrazolium OdaH2 octanedioic acid Phen 1,10-phenanthroline Phendione 1,10-phenanthroline-5,6-dione 2-PyBZIMH 2-(2-pyridyl)benzimidazole SOD superoxide dismutase TBZH 2-(4 0-thiazole)benzimidazole (thiaben-

dazole)

Acknowledgements

We wish to acknowledge financial support from the Ir-ish Technological Sector Research Strand III programme

892 M. Devereux et al. / Journal of Inorganic Biochemistry 101 (2007) 881–892

2002 (Project No. CRS02-TA01). This work has been car-ried out (in part) within the structures of the Facility forOptical Characterisation and Spectroscopy (now the FO-CAS Institute, DIT), funded under The Irish NationalDevelopment Plan 2000–2006 with assistance from theEuropean Regional Development Fund.

Appendix A. Supplementary data

Crystallographic data have been deposited with theCCDC (12 Union Road, Cambridge, CB2 1EZ, UK) andare available on request quoting the deposition numbers294709, 294710 and 294708, respectively. Supplementarydata associated with this article can be found, in the onlineversion, at doi:10.1016/j.jinorgbio.2007.02.002.

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