Synthesis, catalase, superoxide dismutase and antitumour activities of copper(II) carboxylate complexes incorporating benzimidazole, 1,10-phenanthroline and bipyridine ligands: ...

Article abstract of DOI:10.1016/j.poly.2007.05.006

A series of simple Cu(II) carboxylate complexes {carboxylate = acetate (CH₃COO), salicylate (salH) or benzoate (BZA)}, some containing either one chelating {1,10-phenanthroline (phen) or 2,2′-bipyridine (bipy)} or two monodentate {benzimidazole (BZDH) or 5,6-dimethylbenzimidazole (5,6-DMBZDH)} nitrogen donor ligands, have been prepared and characterised. X-ray crystal structures have been determined for the square pyramidal complex [Cu(BZA)₂(bipy)(H₂O)] and the two square planar complexes [Cu(CH₃COO)₂ (5,6-DMBZDH)₂] and [Cu(salH)₂(BZDH)₂]. All of the complexes are poor catalase mimics in the presence of imidazole and totally inactive in its absence. The complexes all exhibit excellent superoxide dismutase (SOD) mimetic activity and the two phen derivatives [Cu(CH₃COO)₂(phen)] and [Cu(sal)(phen)] display potent in vitro cytotoxicity against human hepatic (Hep-G), renal (A-498) and lung (A-549) cancer cell lines.

Full text of DOI:10.1016/j.poly.2007.05.006

Polyhedron 26 (2007) 4073–4084
www.elsevier.com/locate/poly
Synthesis, catalase, superoxide dismutase and antitumour activities
of copper(II) carboxylate complexes incorporating benzimidazole,
1,10-phenanthroline and bipyridine ligands: X-ray crystal structures
of [Cu(BZA)2(bipy)(H2O)], [Cu(SalH)2(BZDH)2]
and [Cu(CH3COO)2(5,6-DMBZDH)2] (SalH2 = salicylic acid;
BZAH = benzoic acid; BZDH = benzimidazole and
5,6-DMBZDH = 5,6-dimethylbenzimidazole)
a,
a
a
a
Michael Devereux *, Denis O’Shea , Mark O’Connor , Helen Grehan ,
a
b
c
d
Gavin Connor , Malachy McCann , Georgina Rosair , Fiona Lyng ,
Andrew Kellett a,e, Maureen Walsh , Denise Egan , Bhumika Thati
e
e
e
a
The Inorganic Pharmaceutical and Biomimetic Research Laboratory, Dublin Institute of Technology, Cathal Brugha St., Dublin 1, Ireland
b
Chemistry Department, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland
School of Engineering and Physical Sciences, Heriot Watt University, Edinburgh EH14 4AS, UK
c
d
The Radiation and Environmental Science Centre, FOCAS Research Institute, Dublin Institute of Technology, Kevin St., Dublin 2, Ireland
e
Centre for Pharmaceutical R&D, School of Science, Institute of Technology, Tallaght, Dublin 24, Ireland
Received 7 January 2007; accepted 2 May 2007
Available online 22 May 2007
Abstract
A series of simple Cu(II) carboxylate complexes {carboxylate = acetate (CH3COO), salicylate (salH) or benzoate (BZA)}, some con-
taining either one chelating {1,10-phenanthroline (phen) or 2,20-bipyridine (bipy)} or two monodentate {benzimidazole (BZDH) or 5,6-
dimethylbenzimidazole (5,6-DMBZDH)} nitrogen donor ligands, have been prepared and characterised. X-ray crystal structures have
been determined for the square pyramidal complex [Cu(BZA)2(bipy)(H2O)] and the two square planar complexes [Cu(CH3COO)2
(5,6-DMBZDH)2] and [Cu(salH)2(BZDH)2]. All of the complexes are poor catalase mimics in the presence of imidazole and totally inac-
tive in its absence. The complexes all exhibit excellent superoxide dismutase (SOD) mimetic activity and the two phen derivatives
[Cu(CH3COO)2(phen)] and [Cu(sal)(phen)] display potent in vitro cytotoxicity against human hepatic (Hep-G), renal (A-498) and lung
(A-549) cancer cell lines.
ꢀ 2007 Elsevier Ltd. All rights reserved.
Keywords: Copper(II)carboxylates; Benzimidazole; 1,10-Phenanthroline; 2,20-Bipyridine; Catalase; SOD and anticancer activity
1. Introduction
In normal mammalian cells reactive oxygen species
(ROS) are produced through metabolic reactions resulting
*
from aerobic respiration. Low levels of ROS are essential
for proper cell function and a fine balance exists between
Corresponding author. Tel.: +353 1 4024486; fax: +353 1 4024495.
E-mail address: michael.devereux@dit.ie (M. Devereux).
0277-5387/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.poly.2007.05.006

4074
M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
the level of ROS produced during normal cellular metabo-
lism and the amount of endogenous antioxidants (such as
ROS scavengers and enzymes) present in the cells which
protect tissues from oxidative damage. If this balance is
disrupted a condition referred to as oxidative stress devel-
ops which is implicated in numerous pathophysiological
processes such as rheumatoid arthritis, aging, inflammation
and carcinogenesis [1]. Recently, it has been found that
ROS such as the superoxide radical (Oꢀ2ꢀ) or hydrogen per-
oxide (H2O2) are important regulators of cell death [2].
Particularly, H2O2 is implicated as a mediator of apoptosis
in cells [3]. The cellular damage caused by H2O2 is likely
produced in part through OHꢀ radical production formed
when H2O2 reacts with Fe2+ or Cu2+ [4]. Enzymatic antiox-
idants regulate superoxide concentration by dismutation of
Oꢀ2ꢀ to hydrogen peroxide (SOD activity) which is then con-
verted to water (peroxidase activity) or dismutated to water
and dioxygen (catalase activity).
of action significantly different to that of the clinically used
drug cisplatin [16,17]. Furthermore, a number of these cop-
per complexes are excellent SOD mimics [14,18] and have
been shown to induce apoptosis in cultured mammalian
cells [19].
In an effort to throw some light on the structure–activ-
ity-relationship of this class of simple copper complexes
we have studied systems in which only two nitrogen atoms
(either as chelates or monodentate ligands) are bound to
the copper centre. Herein, we detail the preparation, cata-
lase and SOD mimetic activities and the chemotherapeutic
potentials of copper acetate, salicylate and benzoate com-
plexes incorporating phen, bipy and simple benzimidazole
ligands. The X-ray crystal structures of [CuBZA2-
(bipy)(H2O)], [Cu(salH)2(BZDH)2] and [Cu(CH3CO2)2-
(5,6-DMBZDH)2] are also reported.
2. Results and discussion
Many tumour cells have increased rates of metabolism
compared with normal cells, which would typically lead
to increased numbers of ROS [5]. Manganese SOD
(MnSOD) is found in the matrix of mitochondria and
has been shown to be depleted in most cancers when com-
pared to normal tissue [6]. The activity of cytoplasmic SOD
(CuZnSOD) has also been shown to vary in cancer cells [6].
This suggests that a large amount of intracellular Oꢀ2ꢀ is
found in such cancerous cells and that in the absence of sig-
nificant SOD activity this Oꢀ2ꢀ can further metabolise to
peroxynitrite (OONOꢀ) and the perhydroxyl radical
ðHOꢀ2Þ which play a role in the tumour formation [7]. It
has been demonstrated that returning MnSOD and CuZn-
SOD enzyme activity to levels close to those found in non-
malignant cells has resulted in conversion of the excess O2ꢀꢀ
to H2O2 which leads to decreased tumour cell growth in a
number of model systems [8–10].
The use of SOD enzymes as pharmaceutical agents is
limited by cost and because of instability and low mem-
brane permeability resulting from their high molecular
weight. Low molecular weight copper SOD mimetic com-
plexes can dismutate excess intracellular Oꢀ2ꢀ to H2O2 and
O2, suggesting that they may increase the concentration
of H2O2 in cancer cells thus inducing cell death by apotosis.
Furthermore, compared to manganese systems copper
complexes are less likely to act as catalase mimics and cat-
alytically disproportionate the H2O2 to water and molecu-
lar oxygen but rather react with it to form the desirable
cytotoxic hydroxyl radical (Fenton chemistry). However,
a small number of copper carboxylate complexes have
recently been reported in the literature that show signifi-
cant catalase mimetic functionality [11,12].
2.1. Synthesis and characterisation of the complexes
[Cu2(CH3COO)4(H2O)2] (1) was obtained commercially
and was not further purified. [Cu(salH)2(H2O)2] (2)
(salH2 = salicylic acid) was generated using a method pre-
viously published [20]. {Cu(BZA)2(H2O)}n (3) was the
major product from a reaction between copper(II) hydrox-
ide and benzoic acid. Reaction of 1 with 1,10-phenanthro-
line (phen) in ethanol gave [Cu(CH3COO)2(phen)] (4) in
good yield and a similar reaction between 2 and phen
resulted in the generation of [Cu(sal)(phen)] (5). [Cu(B-
ZA)2(bipy)(H2O)] (6) was obtained in good yield when 1
reacted with 2,20-bipyridine (bipy) in the presence of
benzoic acid. [Cu(CH3COO)2 (5,6-DMBZDH)2] (7),
[Cu(salH)2(BZDH)2] (8) and [Cu(BZA)2(BZDH)2] (9) were
easily produced when 1, 2 and 3 were treated with the rel-
evant benzimidazoles in refluxing ethanol (where 5,6-
DMBZDH = 5,6-dimethylbenzimidazole and BZDH =
benzimidazole, respectively).
The physico-chemical properties for 2 are the same as a
compound that was previously reported for which the crys-
tal structure revealed a square planar geometry with the
salicylate ligands trans to one another and bound through
the carboxylate groups in a monodentate fashion [21]. In
the IR spectrum of 3, the asymmetric and symmetric
OCO bands of the benzoate ligands are split [m(OCO)assym
:
1597 cmꢀ1 and 1556 cmꢀ1; m(OCO)sym: 1408 cmꢀ1 and
1376 cmꢀ1] and thus two modes of interaction are likely
for the carboxylate ligands with the copper centres. The
microanalysis and the magnetic moment of 3 are similar
to those of an anhydrous copper benzoate complex previ-
ously reported which had an empirical formula of
Cu(BZA)2 [22]. The magnetic moment (1.56 BM) for 3 is
slightly lower than that expected for simple copper(II) spe-
cies (i.e. those lacking Cu–Cu interactions) [23]. Thus, it is
likely that at least one of the benzoate ligands is in some
type of bridging mode as reported recently for several cop-
per(II) complexes of substituted benzoates [24–26]. The
Recently, we have been studying the anticancer activities
of a range of simple copper complexes incorporating differ-
ent types of nitrogen donor ligands [13–17]. We have found
that the bis- or tris-chelate copper complexes incorporating
1,10-phenanthroline and 1,10-phenanthroline-5,6-dione
(which possess N4 or N6 ligation) are potent anticancer
agents in vitro and that they appear to have a mechanism

M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
4075
crystal structure of copper benzoate trihydrate has been
reported and the complex incorporates both coordinated
and uncoordinated benzoate anions [27]. The structure
comprises linear chains of {Cu(BZA)(l-H2O)2}n with
uncoordinated H2O molecules and BZAꢀ anions interven-
ing between them. The room temperature magnetic
moment for the trihydrate was reported as being unusual
for a (l-H2O)(l-BZA) bridged complex (1.87 BM) and
therefore it seems that the structure of 3 is not quite the
same as this trihydrate. Indeed it is significant that three
magnetically different modifications of anhydrous cop-
per(II) benzoate have been previously reported [28].
the metals through the nitrogen atom. For all three com-
plexes the m(OCO)asym and m(OCO)sym bands are not split
and are in similar positions. The D(OCO) values of
161 cmꢀ1, 173 cmꢀ1 and 177 cmꢀ1 for 7–9, respectively,
are all lower than the values expected for monodentate car-
boxylate ligands (>200 cmꢀ1) but the relative reduction in
the D(OCO) values are typical where such carboxylate
groups are involved in hydrogen bonding [32].
The room temperature magnetic susceptibility values for
4–9 (1.65–2.11 BM) are in the range expected for the
observed or proposed mononuclear structures in which
there are no significant Cu–Cu interactions [23]. Further-
more, the electronic spectra of the complexes obtained in
the solid state as Nujol mulls all exhibit broad bands
in the region 531–662 nm due to d–d transitions and are
in the range expected for mononuclear copper(II) com-
plexes that contain either a CuN2O2 or a CuN2O3 chromo-
phore and have square planar or related geometries [33].
When the electronic spectra are recorded in DMSO solu-
tion these bands all shift to higher energy indicative of
changes in geometry as a possible consequence of DMSO
solvation. The molar conductivities of complexes 2–9 in
DMSO were found to be in the range 0.9 and
7.9 S cm2 molꢀ1 indicating that they are essentially non-
electrolytes and thus do not dissociate in that solvent.
When compared to those of 1, 2 and 3 the spectra of 4, 5
an 6 are far more complex and contain new absorption
bands which are characteristic of the presence of chelating
phen {723 and 851 cmꢀ1 (4); 721 and 847 cmꢀ1 (5)} [29]
and bipy {775 cmꢀ1 and 729 cmꢀ1 (6)} [30]. As well as
the bands that have been assigned to the chelating phen
and bipy ligands the spectra of 4–6 also contain bands that
are characteristic of carboxylate anions with m(OCO)asym
{1631 cmꢀ1 (4), 1598 cmꢀ1 (5) and 1599 cmꢀ1 (6)} and
m(OCO)sym {1431 cmꢀ1 (4), 1360 cmꢀ1 (5) and
1379 cmꢀ1(6)} values. The calculated DOCO of 200 cmꢀ1
(4), 238 cmꢀ1 (5) and 220 cmꢀ1 (6) are quite similar and
in the region expected for monodentate carboxylate ligands
[30]. Complex 4 has very similar physico-chemical proper-
ties to those of the copper complex [Cu(N-baa)2(phen)]
(N-baaH = N-benzoylanthanilic acid) [12]. The X-ray crys-
tal structure of [Cu(N-baa)2(phen)] has revealed it to have a
mononuclear structure with two asymmetrical bidentate
chelating carboxylates which both have one very long
2.2. Crystal structures of 6, 7 and 8
The X-ray crystal structures of [Cu(BZA)2(bipy)(H2O)]
(6), [Cu(CH3COO)2 (5,6-DMBZDH)2] (7) and [Cu(salH)2-
(BZDH)2] (8) are shown in Figs. 1–6 and selected bond
lengths and angles are listed in Table 1.
˚
Cu–O bond (2.626 A). The overall geometry around what
is effectively a four-coordinate copper ion is described as
a distorted tetragonal motif which contains a trans-
N2O2 + O2 chromophore. It is likely that 4 has a similar
structure to this complex. Complex 5 clearly contains a
doubly deprotonated chelating sal2ꢀ ligand and its phys-
ico-chemical data fit for a square planar geometry similar
to that of the structurally characterized bipy analogue
[Cu(sal)(bipy)] Æ EtOH Æ H2O [20]. A peak at 844 cmꢀ1 in
the spectrum of 6, which does not appear in the spectra
of either 4 or 5 is characteristic of the presence of a coordi-
nated water molecule supporting its inclusion in the formu-
lation of 6 (also observed in the crystal structure) [31].
Complexes 7–9 formulate as having the same
metal:benzimidazole:carboxylate ratio of 1:2:2 and their
IR spectra are very similar suggesting that the three com-
plexes share a common structure. The N–H stretching
and bending bands in the spectra of the BZDH and 5,6-
DMBZDH free ligands also appear (although shifted
slightly) in the spectra of the three complexes indicative
of the fact that the ligands are present in their neutral state.
Furthermore, the bands associated with the imidazolic
The crystal structure determination of 6 showed that the
square pyramidal copper centre has an axially coordinated
water molecule. An aqua H forms an intramolecular
hydrogen bond with unbound carboxylate oxygen O2
(Fig. 1 and Table 2). Consequently, this carboxylate group
t(C@N) in the free ligands (1587 cmꢀ1 and 1553 cmꢀ1
,
respectively) have shifted significantly in the spectra of 7,
8 and 9 (1499 cmꢀ1, 1581 cmꢀ1 and 1615 cmꢀ1, respec-
tively) indicating that the benzimidazoles are bound to
Fig. 1. The structure of [Cu(BZA)2(bipy)(H2O)] (6).

4076
M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
Fig. 2. The packing diagram for [Cu(BZA)2(bipy)(H2O)] (6) showing
stacking and hydrogen bonding.
Fig. 5. The structure of [Cu(salH)2(BZDH)2] (8).
Fig. 3. The structure of [Cu(CH3COO)2 (5,6-DMBZDH)2] (7).
Fig. 6. The packing diagram for [Cu(salH)2(BZDH)2] (8) where suffixes
on labels correspond to the symmetry operations A: 1 + x, y, z; B: ꢀx + 3/
2, y + 1/2, ꢀz + 1/2; C: ꢀx + 3/2, y + 3/2, ꢀz + 1/2.
nyl C2–C7 (bound to the intramolecular hydrogen bonding
carboxylate) and 77.04(6)ꢁ to the other phenyl group (C9–
C14). There are no close p-stacking interactions (Fig. 2) but
˚
several close H. . .H distances, the shortest being 2.43 A
between H4A and H7A in neighbouring molecules.
In contrast both 7 and 8 have square planar copper cen-
tres (Figs. 3 and 5, respectively) which contain two trans
benzimidazole and two acetate (7) or two salicylate (8)
ligands. Whereas the copper atom is situated on an inver-
sion centre in 7, it is not so in 8 where the hydrogen bond-
ing motif differs between the two carboxylate groups. The
protonated benzimidazole N is capable of hydrogen bond-
ing and makes intermolecular hydrogen bonds with car-
boxylate O atoms in both cases (Fig. 4; Table 3 and
Fig. 5; Table 4, respectively) thus forming a double linked
chain which is propagated along the shortest axis (b) in 7 so
that the hydrogen bonding pattern is similar to 6 apart
Fig. 4. The packing diagram for [Cu(CH3COO)2(5,6-DMBZDH)2] (7)
showing the hydrogen bonding where suffixes on labels correspond to the
symmetry operations A: x + 1, ꢀy + 2, ꢀz; B ꢀx + 1, ꢀy + 1, ꢀz.
is significantly twisted, by 36.31(9)ꢁ, with respect to the
phenyl ring. This is in contrast to the other chemically iden-
tical benzoate which takes part in intermolecular hydrogen
bonding with water bound to another molecule. In this case
the phenyl-carboxylate twist angle is 6.56(9)ꢁ. Thus, this
hydrogen bonding motif generates a chain parallel to the
a-axis. The bipyridyl ligand is inclined at 85.18(7)ꢁ to phe-

M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
4077
Table 1
˚
Selected bond lengths (A) and angles (ꢁ) around the copper centres for [Cu(BZA)2(bipy)(H2O)] (6), [Cu(CH3COO)2(5,6-DMBZDH)2] (7) and
[Cu(salH)2(BZDH)2] (8)
6
7
8
Cu(1)–O(1)
Cu(1)–O(3)
Cu(1)–O(5)r
Cu(1)–N(1)
Cu(1)–N(2)
1.9551(12)
1.9633(11)
2.2272(12)
2.0111(13)
2.0064(14)
Cu(1)–O(1)
Cu(1)–O(1a)
Cu(1)–N(1A)
1.9477(8)
1.9477(8)
1.9805(10)
Cu(1)–O(1)
Cu(1)–O(4)
Cu(1)–N(1)
Cu(1)–N(3)
1.9601(9)
2.0115(9)
1.9951(10)
1.9827(10)
N(1)–Cu(1)–N(2)
O(1)–Cu(1)–O(3)
O(5)–Cu(1)–N(1)
O(5)–Cu(1)–N(2)
O(5)–Cu(1)–O(1)
O(5)–Cu(1)–O(3)
80.53(5)
90.22(5)
96.43(5)
101.96(5)
91.53(5)
96.81(5)
O(1)–Cu(1)–O(1A)
O(1)–Cu(1)–N(1)
O(1A)–Cu(1)–N(1)
O(1)–Cu(1)–N(1A)
180.00(3)
N(1)–Cu(1)–N(3)
O(1)–Cu(1)–O(4)
O(1)–Cu(1)–N(1)
O(1)–Cu(1)–N(3)
O(4)–Cu(1)–N(1)
O(4)–Cu(1)–N(3)
179.47(4)
90.82(4)
89.18(4)
90.82(4)
169.42(3)
89.86(4)
90.52(4)
87.90(4)
91.65(4)
The suffix A denotes the symmetry operation x + 1, ꢀy + 2, ꢀz applied to generate equivalent atoms.
Table 2
seen in 6 and 7, 8 forms hydrogen bonded chains parallel to
the shortest axis, although there is an extra NH. . .O bond
in 7 and 8. However, in 8 these chains interdigitate such
that the benzimidazoles form a stack and the distance
between the imidazole centroids in these stacks alternates
˚
Hydrogen bonds for [Cu(BZA)2(bipy)(H2O)] (6) (A) and (ꢁ)
D–H. . .A
d(D–H)
d(H. . .A)
d(D. . .A)
\(DHA)
O(5)–H(52). . .O(2)
O(5)–H(51). . .O(4A)
0.823(16)
0.808(16)
1.879(17)
1.925(17)
2.6720(17)
2.7237(17)
161(2)
170(3)
˚
between 3.6622(7) and 3.8424(7) A, albeit rather too long
The suffix A denotes the symmetry operation: x + 1, y, z applied to gen-
erate equivalent atoms.
for significant p–p stacking interactions. In the structure
of 7 neighbouring molecules are link by significant p–p
stacking involving the benzimidazole ligands (Fig. 4).
Despite these differences, the major hydrogen bonding
motif, a chain propagated by OH. . .O (6, 8) or NH. . .O
(7) bonds along the shortest axis, is common to all three
crystal structures.
Table 3
˚
Hydrogen bonds for [Cu(CH3COO)2(5,6–DMBZDH)2] (7) (A) and (ꢁ)
D–H. . .A
d(D–H)
d(H. . .A)
d(D. . .A)
\(DHA)
N(2)–H(2). . .O(2A)
0.81(2)
1.92(2)
2.7169(14)
168.3(19)
The suffix A denotes the symmetry operation: A: ꢀx + 1, ꢀy + 1, ꢀz
applied to generate equivalent.
2.3. Catalase activity
The catalytic activity of complexes 1–9 towards the dis-
proportionation of hydrogen peroxide was investigated by
measuring the volume of evolved oxygen during the course
of the reaction. None of the nine complexes exhibited cat-
alytic activity on their own. We have previously shown that
the catalase mimetic activity of manganese complexes is
significantly improved in the presence of the base imidazole
[29,34,35], a phenomenon also observed by others [36].
Consequently, we examined the catalase mimetic properties
of complexes 1–9 in the presence of this base. The results
from these reactions are summarised in Tables 5 and 6.
The catalytic activity of the complexes is relatively poor
when compared to the double salt [{Mn2(oda)(phen)4-
(H2O)2][Mn2(oda)3(phen)4}] (odaH2 = octanedioic acid)
which had been shown to be the best manganese catalase
mimic previously produced in this laboratory [29,37]. Table
5 shows the rate of evolution of oxygen from the respective
reactions for the complexes over the first 20 min. Examina-
tion of Table 6 shows that over the first minute of reaction
5 appears to be the most efficient catalyst with 1012 mole-
cules of peroxide disproportionated by one molecule of the
complex. Complex 8 appears to be the least efficient cata-
lyst over the first minute with one molecule of the com-
pound knocking down just 216 molecules of the peroxide.
Table 4
˚
Hydrogen bonds for [Cu(salH)2(BZDH)2] (8) (A) and (ꢁ)
D–H. . .A
d(D–H)
d(H. . .A)
d(D. . .A)
\(DHA)
O(3)–H(3O). . .O(2)
O(6)–H(6O). . .O(5)
O(6)–H(6O). . .O(2A)
N(2)–H(2). . .O(2B)
N(4)–H(4A). . .O(5C)
N(4)–H(4A). . .O(6C)
0.82
0.82
0.82
0.86
0.86
0.86
1.85
1.88
2.28
2.04
2.11
2.85
2.5681(15)
2.5974(13)
2.8682(13)
2.8220(14)
2.9627(13)
3.4523(14)
146.3
145.2
129.5
150.9
168.7
128.8
Symmetry transformations used to generate equivalent atoms:
A: x ꢀ 1, y, z B: ꢀx + 3/2, y + 1/2, ꢀz + 1/2 C: ꢀx + 1/2, y ꢀ 1/2,
ꢀz + 1/2.
from two NHꢁ ꢁ ꢁO hydrogen bonds forming the link rather
than the single OH. . .O seen in 6. In 8, the ortho hydroxy
group makes hydrogen bonds to one carboxylate O atom
and so would be expected to keep the carboxylate co-pla-
nar with the phenyl ring which is the case for the carboxyl-
ate containing O1, O2. However, for the other benzoate
ligand the carboxylate is twisted by 18.33(6)ꢁ with respect
to the plane made by the phenyl ring and O6. In this case,
the hydroxyl O makes intermolecular hydrogen bonds to a
carboxyl O and a longer NH. . .O link (Table 4). In contrast
O3 makes just an intramolecular hydrogen bond to O2. As

4078
M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
Table 5
Time course of oxygen evolution in H2O2 disproportionation by complexes 1–9 and {[Mn2(oda)(phen)4(H2O)2][Mn2(oda)3(phen)4]} (odaH2 = octanedioic
acid) [29,37] with added imidazole (50 mg) at 25 ꢁC
Time (min)
Imidazole
1
2
3
4
5
6
7
8
9
{[Mn2(oda)(phen)4(H2O)2] [Mn2(oda)3(phen)4]}
1
2
3
4
5
6
7
8
9
18
22
26
27
30
33
45
52
55
59
123
136
106
117
119
94
87
295
346
369
376
379
381
186
261
281
295
303
308
314
316
320
323
334
341
93
41
72
88
98
104
108
112
114
115
116
121
122
84
800
900
930
970
991
1021
1070
1082
1099
1110
1122
1141
105
108
109
110
145
271
297
305
312
317
319
102
104
105
107
117
120
122
10
15
20
Table 6
Molecules of H2O2 disproportionated by the complexes (in the presence of added imidazole) at STP conditions
Complex
Total number of molecules of H2O2
Number of molecules of H2O2 disproportionated
disproportionated by one molecule of complex during first min by one molecule of the complex
1
2
3
4
5
6
7
8
9
486
409
317
1017
1308
1450
446
440
364
270
278
1012
790
396
216
416
640
606
{[Mn2(oda)(phen)4(H2O)2][Mn2(oda)3(phen)4]} 24570
19500
It should be noted that imidazole itself causes only a very
slight disproportionation of the peroxide (Table 5).
DMBZDH)2] (7) > [Cu(CH3COO)2(phen)] (4) where it
appears that the inclusion of the nitrogen donor ligands
reduce the SOD activity. Whereas 4 and 7 are monomeric
complexes 1 is dimeric and has a similar ‘‘paddle wheel’’
structure to that of [Cu2(indo)4(DMF)2] [42]. The cop-
per(II) complexes [Cu2(valp)4(H2O)2] and [Cu(valp)2(phen)]
[valpH = valproic acid {(CH3CH2CH2)2CHCOOH}] were
recently reported and are iso-structural to 1 and 4, respec-
tively [43]. Their SOD mimetic activities were much lower
relative to 1 and 4 (10.4 and 4.5 lM) with the phen deriv-
ative being the superior catalyst. Clearly there is no corre-
lation between solid state nuclearity (monomeric versus
dimeric structures) and SOD mimetic activity but the type
of carboxylate present appears to be important. The three
monomeric copper(II) salicylates exhibit SOD activity in
the order [Cu(salH)2(BZDH)2] (8) > [Cu(sal)(phen)] (5) >
[Cu(salH)2(H2O)2] (2) where the activity increases upon
inclusion of the nitrogen bases in the inner sphere of the
complexes. The activities of complexes 2 and 5 fall in the
lower end of the range (0.17–29 lM) previously reported
for copper(II) salicylate derivatives, a number of which
are used therapeutically as anti-inflammatory agents in
human and veterinary medicine [40,44]. The benzoate
complex {Cu(BZA)2(H2O)}n (3) displays potent SOD
activity (0.9 lM) and is approximately twice as active
as its bipy derivative [Cu(BZA)2(bipy)(H2O)] (6).
2.4. Superoxide dismutase activity
The SOD mimetic activities of the complexes 1–9 were
examined with an indirect method in which the xanthine/
xanthine-oxidase system served as the source for superox-
ide radicals [38]. The activities of the complexes were com-
pared to that of [Cu2(indo)4(H2O)2] {indoH = indomethacin
[1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-ace-
tic acid]} which is considered to be an excellent SOD
mimetic [39] and which is used therapeutically as an oral
anti-inflammatory drug in veterinary medicine [40,41].
The results are given in Table 7 as concentrations equiva-
lent to one unit of SOD activity (IC50 values).
Significant activities were seen for all compounds tested
with one unit of SOD activity arising from the range of
0.63–1.98 lM aqueous solutions. Interestingly, the com-
plexes all have activities comparable with that (1.31 lM
in this study) of [Cu2(indo)4L2] (L = DMSO or DMF)
for which the IC50 values vary significantly in the literature
and appear to be solvent dependent (the reported value in
DMSO is 2 lM [39]). It is interesting that the SOD activity
of the copper(II) acetate complexes decreases in the
order [Cu2(CH3COO)4(H2O)2] (1) > [Cu(CH3COO)2 (5,6-

M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
4079
Table 7
human derived cancer cells was investigated using the
hepatocellular carcinoma (Hep-G2), kidney adenocarci-
noma (A-498) and lung cancer (A-549) cell lines and was
determined by calculation of IC50 (the drug concentration
causing a 50% reduction in cellular viability). Cells were
continuously exposed to test agent for 96 h, and their
effects on cellular viability was evaluated. It was intended
that the results from these studies would allow the identifi-
cation of those derivatives with cancer chemotherapeutic
potential. Therefore, profiles of cell viability against com-
plex concentration were established and used to calculate
the IC50 values for each derivative (Table 8). Comparison
of IC50 values, allowed the relative potency of each of the
test compounds to be determined and ranked.
Superoxide dismutase (SOD) activities of complexes 1–8, [Cu2(indo)4-
(H2O)2] [39], CuSO4 and native CuZnSOD as assessed by the NBT assay
Complex
Concentration (lM) equivalent to 1 U
SOD (IC50
)
[Cu2(CH3COO)4(H2O)2] (1)
[Cu(salH2)(H2O)2] Æ 0.5H2O
(2)
{Cu(BZA)2(H2O)}n (3)
[Cu(CH3COO)2(phen)] (4)
[Cu(sal)(phen)] (5)
[Cu(BZA)2(bipy)(H2O)] (6)
[Cu(CH3COO)2(5,6-
DMBZDH)2] (7)
[Cu(salH)2(BZDH)2] (8)
[Cu2(indo)4(H2O)2] [39,40]
CuSO4 [41]
0.63
1.23
0.90
1.18
1.01
1.98
0.91
0.74
1.31
30
With the exception of phen all of the free ligands are
essentially inactive. A number of the compounds screened
displayed a concentration-dependent cytotoxic profile
across the three cell lines studied here. The most sensitive
cell line was the A-549 lung cancer with seven of the com-
pounds showing significant improvements in activity when
compared to the uncoordinated free ligands. The overall
order of the observed cytotoxicity was seen as
(4) > (5) > (6) > (7) > (8) > (1) > (2) o (3) with (4) appear-
ing as the most potent and (3) as the least potent effectively
displaying no activity. The results presented in Table 8 also
illustrates that the CuSO4 (free Cu2+) was incapable of elic-
iting a cytotoxic response. The inclusion of the N,N-donor
ligands 1,10-phenanthroline (phen) and 2,20-bipyridine
(bipy) in the simple Cu(II) complexes of the acetic, salicylic
and benzoic acids significantly increased the potency of the
system. However, it is also noteworthy that the metal free
phenanthroline is itself significantly cytotoxic and that
the best copper complex containing it (4) is approximately
2, 6 and 2 times more potent for the respective cell lines.
Complexes 4 and 5 were capable of killing all three cancer
Native CuZnSOD [39]
0.04
Where salH2 = salicylic acid; BZAH = benzoic acid; phen = 1,10-phe-
nanthroline; 5,6-DMBZDH = 5,6-dimethyl-
bipy = 2,20-bipyridine;
benzimidazole; BZDH = benzimidazole; indoH = indomethacin {1-(4-
Chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3-acetic acid}.
[Cu(BZA)2(BZDH)2] (9) was insoluble in DMSO and its
SOD activity could not be determined. Attempts to dis-
solve 9 in hot DMSO caused an immediate precipitation
of the known red polymeric Copper(II) complex
{Cu(BZD)2}n [45]. All of the other complexes were stable
in hot DMSO (no change in their UV spectra) again dem-
onstrating how changing the type of carboxylate ligand
may influence the reactivity of this class of simple cop-
per(II) complexes.
2.5. Cytotoxicity studies
To evaluate their cancer chemotherapeutic potential the
ability of the DMSO soluble copper complexes 1–8 to kill
Table 8
The anti-cancer activity of the free ligands, complexes 1–8 and cisplatin against human hepatic (Hep-G2), renal (A-498) and lung (A-549) cancer cell lines
expressed as IC50 (lM)a
Test compound
Toxicities (IC50 lM)
Hep-G2 Mean SD
A-498 Mean SD
A-549 Mean SD
salH2
>500
>500
>500
BZAH
>500
>500
>500
Phen
Bipy
4.1 0.5
>500
5.8 0.3
>500
2.94 0.1
>500
BZDH
>500
>500
>500
5,6-DMBZDH
>500
>500
>500
CuSO4
>500
>500
>500
[Cu2(CH3COO)4(H2O)2] (1)
[Cu(salH)2(H2O)2] Æ 0.5H2O (2)
{Cu(BZA)2(H2O)}n (3)
[Cu(CH3COO)2(phen)] (4)
[Cu(sal)(phen)] (5)
[Cu(BZA)2(bipy)(H2O)] (6)
[Cu(CH3COO)2(5,6-DMBZDH)2] (7)
[Cu(salH)2(BZDH)2] (8)
Cisplatin
320 2.5
350 2.8
>500
1.8 0.3
2.5 0.5
26 6.1
105 2.6
138 2.5
15 2.7
134 2.2
142 1.8
>500
1.0 0.2
1.6 0.3
16 6.7
58 1.9
65 1.4
>500
59.81 13.7
>500
1.19 0.2
1.42 0.3
82.99 7.8
23.44 4.9
58.15
14
1
11.0 4.7
a
IC50 (lM), indicates the compound concentration that inhibits the proliferation rate of tumour cells by 50% as compared to the control untreated cells.
The values are means S.D. of 9 independent experiments.

4080
M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
derived cell lines at very low concentrations (IC50 values:
1.8, 1.0 and 1.55 lM for 4 and 2.5, 1.6 and 1.44 lM for 5
for the liver, kidney and lung cell lines, respectively). The
activities of 4 and 5 fall well within the accepted activity
parameters adopted for in vitro screening of potential che-
motherapeutic drugs [46]. Furthermore, the IC50 values for
them are superior to those of the clinically used drug cis-
platin [47]. Significantly, no correlation was found between
the potencies of SOD mimic capabilities and cell growth
inhibitory activity for the eight complexes. However, with
the exception of 3 all of the complexes do show significant
activity against the renal and lung cell lines. It is of course
possible that the complexes act on the cancer cells through
mechanism other than (or in conjunction with) SOD activ-
ity. Indeed the bis-1,10-phenanthroline copper(II) complex
[Cu(phen)2]2+ is known as a potent nuclease agent which
causes oxidative cleavage of DNA in the presence of reduc-
ing agents [48].
eter. Magnetic susceptibility measurements were made
using a Johnson Matthey Magnetic Susceptibility balance.
[HgCo(SCN)4] was used as a reference. Conductivity
readings were obtained using an Orion model 150 plus
conductivity meter. Satisfactory microanalytical data for
the complexes were reported by the Microanalytical Lab-
oratory, University College Cork, Ireland. [Cu2(indo)4-
(H2O)2] (indoH = indomethacin) was synthesised and
characterised using the method previously published [39].
4.1.1. [Cu2(CH3COO)4(H2O)2] (1)
[Cu2(CH3COO)4(H2O)2] was obtained commercially
from the Aldrich Chemical Company and used without
further purification.
4.1.2. [Cu(salH)2(H2O)2] (2)
[Cu(salH)2(H2O)2] (2) was synthesised using a method
previously published [20] as follows: Cu(OH)2(0.97 g,
9.94 mmol) and salicylic acid (salH2) (2.76 g, 19.98 mmol)
were refluxed in ethanol (50 cm3) for 4 h giving a brown
suspension. On cooling to room temperature the brown
product was filtered off, washed with two portions of
water and ethanol, and then air-dried. Yield: 1.42 g,
56%. Anal. Calc. C, 40.3; H, 2.4. Found: C, 41.09; H,
2.3%. IR (KBr): 3438, 1603, 1568, 1529, 1485, 1455,
1410, 1303, 1232, 1149, 1099, 1037, 891, 838, 808, 775,
763, 711, 673, 621, 595, 512, 451, 428, 415, 406 cmꢀ1. leff:
1.81 BM. Solubility: insoluble in water, ethanol, metha-
nol, acetone, trichloromethane. Soluble in DMSO. kmax
3. Conclusion
The carboxylate complexes 1–8 exhibit potent SOD
mimicking activity and are inactive as catalase mimics in
the absence of added imidazole. Upon addition of the base
they display only poor catalytic potential for the dispropor-
tionation of hydrogen peroxide. Complexes 4–8 exhibit sig-
nificant cytotoxicity with the mono-phen derivatives 4 and
5 being the most potent displaying activity approximately
seven times that of the clinical anticancer drug cisplatin.
Significantly, 4 and 5 are relatively soluble compared to
the other six complexes and they have cytotoxicity compa-
rable with the copper(II) bis-phenanthroline complexes
previously studied in this laboratory [17] but are one quar-
ter as active as the copper(II) tris-phenanthroline-diones
[16]. The lack of correlation between the SOD and cytotox-
icy IC50 values supports the notion that mechanisms other
than SOD mimicking may also be responsible for the anti-
cancer properties of these complexes. Indeed complexes 4
and 5 are structurally related to the novel anticancer agent
casiopeina II {[Cu(1,4-dimethyl-1,10-phenanthroline)-
(glycine)]NO3} which has been shown to have a very com-
plex in vivo cytotoxicity [49]. An interesting compound is
the benzoate complex 3 which is an excellent SOD mimic
but has little or no cytotoxicity. Such a non-toxic com-
pound might have potential use as a therapeutic agent
for treatment of other disorders where SOD activity is
pathologically reduced.
(Nujol Mull):
754 nm, e = 169.5 Mꢀ1 cmꢀ1
cm2 molꢀ1
k
d–d = 742 nm. kmax (DMSO): kd–d
=
.
KM (DMSO): 4.2 S
.
4.1.3. {Cu(BZA)2(H2O)}n (3)
To a solution of freshly prepared Cu(OH)2 (1.0 g,
10.25 mmol) in ethanol (50 cm3) was added an ethanolic
solution (25 cm3) of benzoic acid (2.5 g, 20.5 mmol) and
the resulting suspension was refluxed for 3 h. The blue-
green powder that deposited was filtered off, washed with
a small volume of ethanol, and then air-dried. Yield:
1.6 g, 49%. Anal. Calc. C, 51.93; H, 3.73. Found: C,
53.3; H, 3.3%. IR (KBr): 3061, 2970, 1597, 1561, 1520,
1509, 1445, 1402, 1309, 1179, 1146, 1071, 1026, 929,
845, 718, 685, 510, 481 cmꢀ1 leff: 1.57 BM. Solubility:
insoluble in water, ethanol, methanol, acetone, and tri-
chloromethane. Soluble in DMSO. kd–d (Nujol Mull) =
686 nm. kd–d (DMSO) = 734 nm, e = 125 Mꢀ1 cmꢀ1. KM
(DMSO) = 1.17 S cm2 molꢀ1
.
4. Experimental
4.1.4. [Cu(CH3COO)2(phen)] (4)
4.1. Chemistry
To a solution of phen (1 g, 5.6 mmol) in EtOH
(50 cm3) was added [Cu2(CH3COO)4(H2O)2] (1) (0.5 g,
1.25 mmol) and the resulting blue solution refluxed for
1 h. The solution was reduced by rotary evaporation to
5 cm3 and acetone (20 cm3) then added. This solution
was further reduced to 10 cm3. The resulting blue powder
which deposited was filtered off washed with two por-
Chemicals were purchased from commercial sources
and used without further purification. IR spectra were
recorded in the region 4000–400 cmꢀ1 on a Nicolet-400
Impact spectrometer. UV spectra were recorded in the
region of 400–1000 nm on a Specord 200 spectrophotom-

M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
4081
tions of water and ethanol, and then air-dried. Yield:
0.28 g, 62%. Anal. Calc. C, 50.6; H, 4.3; N, 7.4. Found:
C, 51.0; H, 4.1, N, 7.9%. IR (KBr): 3442, 3012, 2925,
2854, 1632, 1608, 1587, 1544, 1524, 1448, 1431, 1401,
1341, 1255, 1220, 1143, 1107, 1054, 1020, 962, 942,
875, 851, 785, 723, 683, 648, 627, 482, 432 cmꢀ1. leff:
1.89 BM. Solubility: soluble in water, EtOH, MeOH
and DMSO. kmax (Nujol Mull): kd–d = 630 nm. kmax
(DMSO): kd–d = 682 nm, e = 51.6 Mꢀ1 cmꢀ1. KM (DMSO):
the solution upon standing for one week. Yield: 0.25 g,
42%. Anal. Calc. C, 55.7; H, 5.5; N, 11.8. Found: C,
55.9; H, 5.6; N, 11.4%. IR (KBr): 3432, 3102, 3062, 2966,
2934, 2879, 2815, 2656, 1772, 1706, 1559, 1500, 1474,
1448, 1408, 1337, 1307, 1271, 1251, 1212, 1191, 1159,
1087, 1047, 1006, 968, 931, 882, 856, 841, 823, 784, 732,
675, 647, 618, 575, 486, 452, 435 cmꢀ1. leff: 1.65 BM. Solu-
bility: insoluble in water, ethanol, methanol, acetone,
trichloromethane. Soluble in DMSO. kmax (Nujol Mull):
7.9 S cm2 molꢀ1
.
k
d–d = 531 nm. kmax (DMSO):
kd–d = 726 nm, e =
123.4 Mꢀ1 cmꢀ1. KM (DMSO): 0.9 S cm2 molꢀ1
.
4.1.5. [Cu(sal)(phen)] (5)
[Cu(sal)(phen)] (5) was synthesised using a method pre-
viously published [20] as follows: [Cu(salH)2(H2O)2] (2)
(0.4 g, 1.92 mmol) and 1,10-phenanthroline (0.427 g,
2.37 mmol) were refluxed in EtOH (50 cm3) for 2 h giving
a green suspension. On cooling to room temperature the
green product was filtered, washed with two portions of
water and ethanol, and then air-dried. Yield: 0.73 g, 72%.
Anal. Calc. C, 60.1; H, 3.2; N, 7.4. Found: C, 58.9; H,
3.3, N, 7.0%. IR (KBr): 3416, 3089, 3059, 2360, 2342,
1629, 1599, 1574, 1543, 1519, 1492, 1461, 1450, 1429,
1360, 1342, 1320, 1243, 1223, 1136, 1107, 1032, 911.884,
874, 847, 809, 772, 741, 721, 710, 667, 651, 628, 585, 543,
432 cmꢀ1. leff: 2.11. Solubility: insoluble in water acetone
and trichloromethane. Soluble in EtOH, MeOH and
DMSO. kmax (Nujol Mull): kd–d = 589 nm. kmax (DMSO):
4.1.8. [Cu(salH)2(BZDH)2] (8)
[Cu(salH)2(H2O)2] (2) (0.5 g, 2.39 mmol) and benzimid-
azole (0.846 g, 7.17 mmol) were refluxed in ethanol
(100 cm3) for 3 h giving a red suspension. On cooling to
room temperature
a small amount of red powder
({Cu(bzd)2}n) was filtered off, and the blue filtrate allowed
to stand for several days where a quantity of blue crystals
of 8 which were suitable for X-ray structural analysis depos-
ited. Yield: 0.51 g, 37%. Anal. Calc. C, 58.6; H, 3.9; N, 9.8%.
Found: C, 58.9; H, 3.8; N, 9.8%. IR (KBr): 3238, 3145,
3112, 3064, 2990, 2916, 2839, 1760, 1741, 1619, 1595,
1582, 1568, 1509, 1497, 1479, 1454, 1395, 1355, 1323,
1305, 1272, 1224, 1195, 1141, 1113, 1086, 1031, 1010, 977,
971, 925, 864, 849, 832, 817, 777, 756, 733, 716, 705, 669,
634, 606, 560, 548, 536, 443, 420 cmꢀ1. leff: 1.83 BM. Solu-
bility: insoluble in water, ethanol, methanol, acetone,
trichloromethane. Soluble in DMSO. kmax (Nujol Mull):
k
d–d = 635 nm, e = 134.3 Mꢀ1 cmꢀ1. KM (DMSO): 1.8 S
cm2 molꢀ1
.
k
M
d–d = 579 nm. kmax (DMSO): kd–d = 738 nm, e = 84.1
4.1.6. [Cu(BZA)2(bipy)(H2O)] (6)
ꢀ1 cmꢀ1. KM (DMSO): 13.2 S cm2 molꢀ1
.
To a heated solution of [Cu2(CH3COO)4(H2O)2] (1)
(0.5 g, 1.25 mmol) and benzoic acid (0.31 g, 2.5 mmol) in
ethanol (50 cm3) was added 2,2-bipyridine (0.39 g,
2.5 mmol). The resulting dark blue solution was stirred
for 30 min and allowed to cool and stand. After 48 h blue
needle-like crystals formed that were suitable for X-ray
crystallography. Yield: 0.37 g, 61.67%. Anal. Calc. C,
60.0 ; H, 4.2; N, 5.83. Found: C, 59.40; H, 4.18; N,
5.66%. IR (KBr): 3357.11, 3111.63, 3081.23, 3030.09,
1599.59, 1555.06, 1493.96, 1473.36, 1445.49, 1397.01,
1379.35, 1313.09, 1285.66, 1260.50, 1254.75, 1170.95,
1156.30, 1142.41, 1105.07, 1070.15, 1054.39, 1031.28,
1019.61, 977.99, 930.12, 913.08, 846.41, 820.11, 775.38,
729.24, 678.25, 665.38, 650.65, 639.83, 548.40, 455.46,
432.92, 416.63 cmꢀ1. leff: 1.87 BM. Solubility: insoluble in
water, ethanol, methanol, acetone, and trichloromethane.
Soluble in DMSO. kmax (Nujol): kd–d = 662 nm. kmax
(DMSO): kd–d = 677 nm, e = 117.77 dm3 molꢀ1 cmꢀ1. KM
4.1.9. [Cu(BZA)2(BZDH)2] (9)
To solution of {Cu(BZA)2(H2O)}n (3) (1.0 g,
a
3.0 mmol) in ethanol/water (100 cm3, 1:1) was added benz-
imidazole (1.25 g, 10.0 mmol) and the resulting suspension
was stirred for 24 h. The purple powder that deposited was
filtered off, washed with a small volume of ethanol and
water, and then air-dried. Yield: 1.53 g, 94.09%. Anal. Calc.
C, 62.04; H, 4.09; N, 10.33. Found: C, 61.33; H, 4.03; N,
10.05%. IR (KBr): 3326.11, 3139.56, 3056.98, 2985.24,
2915.51, 2887.72, 2845.62, 2754.33, 1964.64, 1911.00,
1783.54, 1615.42, 1597.55, 1557.91, 1487.96, 1464.43,
1446.28, 1428.34, 1405.57, 1381.42, 1361.48, 1315.83,
1302.93, 1270.64, 1252.56, 1176.14, 1150.84, 1125.03,
1107.73, 1068.13, 1024.20, 1010.63, 973.86, 933.40,
906.24, 886.97, 846.44, 840.40, 827.51, 788.77, 776.50,
764.82, 742.46, 714.43, 685.37, 631.93, 613.62, 577.07,
548.59, 447.32, 425.24 cmꢀ1. leff: 1.86 BM. Solubility:
(DMSO): 7.2 S cm2 molꢀ1
.
insoluble in all common solvents. kmax (Nujol): kd–d =
593 nm.
4.1.7. [Cu(CH3COO)2 (5,6-DMBZDH)2] (7)
To a solution of Cu2(CH3COO)4(H2O)2] (1) (0.5 g,
1.25 mmol ) in ethanol (100 cm3) was added 5,6-dimethyl-
benzimidazole (0.73 g, 5 mmol) and the resulting dark
green solution refluxed for 4 h. A quantity of blue crystals
suitable for X-ray structural analysis, were obtained from
4.2. X-ray crystallography
Intensity data were collected in single crystals of 6, 7 and
8 at 100 K on a Bruker Nonius X8 Apex2 diffractometer
with monochromatic Mo Ka radiation. Solution and

4082
M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
Table 9
refinement were performed using the BRUKER suite of pro-
grams [50]. All H atom positions were calculated (except
N-bound H2 in 7) and all H atom displacement parameters
were treated as riding on the bound atom. Further details
are given in Table 9.
Crystal data and structure refinement for complexes 6, 7 and 8
Compound
6
7
8
Empirical
formula
Formula
weight
Temperature
(K)
C24H20CuN2O5
C22H26N4O4Cu
C28H22N4O6Cu
479.96
100(2)
474.01
100(2)
574.04
100(2)
4.3. Catalase activity
The ability of the complexes to disproportionate hydro-
gen peroxide was assayed using a method previously pub-
lished by this laboratory [29] as follows: To a solid
sample (ca. 10 mg) of the copper complex was added aque-
ous H2O2 (35% w/w, 10 cm3, 114 mmol). The mixture was
stirred and thermostated at 25 ꢁC and the evolved O2 was
measured volumetrically (volume 0.10 cm3). In cases
where imidazole (50 mg) was added, this was introduced
into the reaction vessel before the addition of H2O2.
Crystal system monoclinic
monoclinic
P21/n
10.6616(6)
.0458(5)
14.8903(9)
90
100.950(3)
90
1098.19(12)
2
monoclinic
P21/n
7.2216(6)
13.9991(11)
23.9378(18)
90
91.502(4)
90
2419.2(3)
4
Space group
P21/c
˚
a (A)
6.8631(4)
18.7829(12)
16.8774(9)
90
97.573(4)
90
2156.7(2)
4
1.478
˚
b (A)
˚
c (A)
a (ꢁ)
b (ꢁ)
c (ꢁ)
3
˚
V (A )
Z
Dcalc (Mg/m3)
Absorption
coefficient
(mmꢀ1
F(00,0)
1.433
1.030
1.576
0.957
1.052
4.4. Superoxide dismutase (SOD) activity
)
The Oꢀ2ꢀ dismutase activities of the metal complexes
were assessed using a modified Nitro-Blue-Tetrazolium
(NBT) assay with xanthine–xanthine oxidase system as
the source of Oꢀ2ꢀ [38]. The quantitative reduction of
NBT to blue Formazan by the Oꢀ2ꢀ was followed spectro-
photometrically at 550 nm. All reagents were obtained
from Sigma–Aldrich Chemical Co. Ltd. and assays were
run in 3 ml of solution. Results are graphed as the % inhi-
bition of NBT reduction for three concentrations. Tabu-
lated results were derived from linear regression analyses
and are given as the concentration (lM) equivalent to
1 U of bovine erythrocyte SOD activity. A unit SOD
activity is the concentration of the complex or enzyme
which causes 50% inhibition in the reduction of NBT
(referred to as the IC50 value).
988
494
1180
Crystal size
(mm3)
0.22 · 0.18 · 0.08 0.42 · 0.40 · 0.22 0.5 · 0.20 · 0.15
Crystal
blue needle
blue block
blue blocks
description
h Range (ꢁ)
Reflections
collected
2.17–30.64
32564
2.60–30.45
13637
1.67–28.52
35163
Independent
reflections
6522 [0.0505]
(25.00) 99.2%
3214 [0.0183]
(25.00) 98.3%
10562 [ 0.0309]
[Rint
]
Completeness
(25.00) 99.4%
to h (ꢁ)
Maximum and 0.919 and 0.715
minimum
transmission
0.8051 and
0.6715
0.866 and 0.654
Data/
restraints/
parameters
Goodness-of-
fit on F2
6522/2/297
0.996
3214/0/194
1.041
10562/0/354
0.850
4.5. Cytotoxicity testing
Final R indices R1 = 0.0334
R1 = 0.0254
wR2 = 0.0687
R1 = 0.0303
wR2 = 0.0719
R1 = 0.0366
wR2 = 0.1070
R1 = 0.0582
wR2 = 0.1219
Dimethyl sulphoxide (DMSO) and all cell culture
reagents and media were purchased from Signa–Aldrich
Ireland, Ltd., unless otherwise stated. Cytotoxicity assays
were performed using three human malignant model cell
lines in order to assess the cancer chemotherapeutic
potential of the Cu complexes. Hepatocellular carcinoma
(Hep-G2), kidney adenocarcinoma (A-496) and lung can-
cer (A-549) cell lines were purchased from the ATCC.
The hepatocellular carcinoma (Hep-G2) and kidney
adenocarcinoma (A-496) cell lines were grown as monolay-
ers in Eagle’s minimum essential medium, supplemented
with 2 mM L-glutamine and Earle’s balanced salt solution,
containing 1.5 g dmꢀ3 sodium bicarbonate, 0.1 mM non-
essential amino acids, 1.0 mM sodium pyruvate,
100 U cmꢀ3 penicillin and 100 lg cmꢀ3 streptomycin sup-
plemented to contain 10% (v/v) foetal bovine serum (Flow
Laboratories, Herts, UK). All cells were grown at 37 ꢁC in
a humidified atmosphere, in the presence of 5% CO2 and
were in the exponential phase of growth at the time of
[I > 2r(I)]
R indices (all
data)
wR2 = 0.0733
R1 = 0.0510
wR2 = 0.0800
Largest
0.438 and ꢀ0.400 0.418 and ꢀ0.236 0.952 and
difference
peak andꢀ3
ꢀ0.939
˚
hole (e A
)
assay. Cytotoxicity was assessed using MTT assay. Each
of the two cell lines (100 ll) were seeded at a density of
5 · 104 cells cmꢀ3 into sterile 96 well flat-bottomed plates
(Falcon Plastics, Becton Dickinson) and grown in 5%
CO2 at 37 ꢁC. Test compounds were dissolved in DMSO
and diluted with culture media. The maximum percentage
of DMSO present in all wells was 0.2% (v/v). Each drug
solution (100 ll) was added to replicate wells in the concen-
tration range of 0.1–1000 lM and incubated for 96 h A
miniaturised viability assay using 3-(4,5-dimethylthiazol-
2-yl)-2,5-diphenyl tetrazolium bromide (MTT) was carried

M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
4083
out according to the method described by Mosman [51].
The IC50 value, defined as the drug concentration causing
a 50% reduction in cellular viability, was calculated for
each drug. Each assay was carried out using five replicates
and repeated on at least three separate occasions. Viability
was calculated as a percentage of solvent-treated control
cells, and expressed as a % of the control. The significance
of any reduction in cellular viability was determined using
one-way ANOVA (analysis of variance). A probability of
0.05 or less was deemed statistically significant.
The lung (A-549) cells were cultured in Kaighn’s med-
ium (F12K) with 2 mM L-glutamine supplemented with
10% foetal calf serum (FCS) and 45 IU mlꢀ1 penicillin
and 45 lg mlꢀ1 streptomycin at 37 ꢁC in 5% CO2 humidi-
fied incubator. For subcultivation, the cells were detached
using a Versene/trypsin solution (1 mM EDTA/0.25%
trypsin) in Ca2+- and Mg2+-free Hanks Balanced Salts
Solution (HBSS). For cytotoxicity assays cells were seeded
1EZ, UK; fax: (+44) 1223-336-033; or e-mail: deposit@
ccdc.cam.ac.uk.
Acknowledgements
M.O.C. acknowledges Irish Technological Sector Re-
search Strand I programme 2002 (Project No. PT03147).
The authors also acknowledge funding received through
the Irish Technological Sector Research Strand III pro-
gramme 2002 (Project No. CRS02-TA01) and DIT Team
Research Scheme (TERS) funding for Interdisciplinary
Cancer Research. This work has been carried out (in part)
within the structures of the Facility for Optical Character-
isation and Spectroscopy (now the FOCAS Institute, DIT),
funded under The Irish National Development Plan 2000–
2006 with assistance from the European Regional Develop-
ment Fund.
in 96-well microplates (Sarstedt) at
a density of
References
2 · 104 cells/ml. This density was found to be optimal to
achieve the desired confluence at the end of the exposure
period. After 24 h of cell attachment, plates were washed
with 100 ll/well phosphate buffered saline (PBS) and cells
treated with each compound for 96 h. Six replicate wells
were used for each control, and test concentration per
microplate. Cytotoxicity was assessed using the MTT
assay. Following 96 h of exposure, control or test expo-
sures were removed, cells rinsed with PBS and 100 ll of
fresh media (without phenol-red sodium salt, FCS or sup-
plements) was added to each well. Ten microlitres of MTT
(5 mg/ml) prepared in PBS was then added to each well
and plates incubated for 3 h at 37 ꢁC in a 5% CO2 humid-
ified incubator. After this incubation period the medium
was discarded, cells washed with 100 ll of PBS and
100 ll of DMSO was added to each well to extract the
dye. The plate was shaken at 240 rpm for 10 min and
absorbance measured at 570 nm. Experiments were con-
ducted in triplicate (three independent experiments). Raw
data from cell cytotoxicity assays were collated and ana-
lysed using Microsoft Excelꢂ (Microsoft Corporation,
Redmond, WA). Cytotoxicity is expressed as mean per-
centage survival relative to the unexposed control stan-
dard error of the mean (SEM). Control values were set at
100%. Coefficient of variation (CV) for the controls of each
test was calculated to ascertain reproducibility. Cytotoxic-
ity data were fitted to a sigmoidal curve and a four param-
[1] J.D. Scandalis (Ed.), Oxidative Stress and the Molecular Biology of
Antioxidant Defenses, Cold Spring Harbor Laboratory Press,
Raleigh, USA, 1997.
[2] S.M. Cardoso, C. Pereira, C.R. Oliveira, Biochem. Biophys. Res.
Commun. 246 (1998) 83.
[3] M. Mates, Toxicology 153 (2000) 83.
[4] D.M. Miller, G.R. Buettner, S.D. Aust, Free, Radic. Biol. Med. 8
(1990) 95.
[5] P. Huang, L. Feng, E.A. Oldam, M.J. Keating, W. Plunkett, Nature
407 (2000) 390.
[6] L.W. Oberley, Antioxid. Redox Signal. 3 (2001).
[7] C. Muscoli, S. Cuzzocrea, D. Riley, J.L. Zweier, C. Thiemerman-
Salvemini, Br. J. Pharmacol. 140 (2003) 445.
[8] C.J. Darby Weydert, B.B. Smith, L. Xu, K.C. Kregel, J.M.
Ritchie, C.S. Davis, L.W. Oberley, Free Radic. Biol. Med. 34
(2003) 316.
[9] E.W.N. Lam, H.M. Hammand, R. Zwacka, C.J. Darby, K.R.
Baumgardner, B.L. Davidson, T.D. Oberley, J.F. Engelhardt, L.W.
Oberley, Cancer Res. 57 (1997) 5550.
[10] C.J. Weydert, T.A. Waugh, J.M. Ritchie, K.S. Iyer, J.L. Smith, L. Li,
D.R. Spitz, L.W. Oberley, Free Radic. Biol. Med. 41 (2006) 226.
[11] J. Kaiser, R. Csonka, G. Speier, M. Giorgi, M. Reglier, J. Mol. Catal.
A: Chem. 236 (2005) 12.
[12] J. Kaiser, T. Csay, G. Speier, M. Reglier, M. Giorgi, Inorg. Chem.
Commun. 9 (2006) 1037.
[13] M. Devereux, M. McCann, D. O Shea, R. Kelly, D. Egan, C. Deegan,
V. McKee, J. Inorg. Biochem. 98 (2004) 1023.
[14] M. Devereux, M. McCann, D.O Shea, M.O Connor, E. Kiely, V.
McKee, D. Naughton, A. Fisher, A. Kellett, M. Walsh, D. Egan, C.
Deegan, Bioinorg. Chem. Appl. 2006, Article ID: 80283.
[15] B. Thati, A. Noble, B.S. Creavan, M. Walsh, M. McCann, K.
Kavanagh, M. Devereux, D.A. Egan, Cancer Lett. in press, 2006,
doi:10.1016/j.canlet.2006.08.009.
[16] C. Deegan, M. McCann, M. Devereux, B. Coyle, D. Egan. Chemico-
Biol. Interact., in press, 2006, doi:10.1016/j.cbi.2006.08.025.
[17] C. Deegan, M. McCann, M. Devereux, B. Coyle, D. Egan, Cancer
Lett., in press, 2006, doi:10.1016/j.canlet.2006.04.006.
[18] A. Kellett, Ph.D. thesis, The Institute of Technology, Tallaght
Dublin, Ireland, 2006.
eter logistic model used to calculate IC50 values. This
TM
analysis was performed using XLFIT3
a curve fitting
add-in for Microsoftꢂ Excel (ID Business Solutions, UK).
5. Supplementary material
CCDC 634440, 634441 and 634442 contain the supple-
mentary crystallographic data for 6, 7 and 8. These data
can be obtained free of charge via http://www.ccdc.cam.a-
c.uk/conts/retrieving.html, or from the Cambridge Crystal-
lographic Data Centre, 12 Union Road, Cambridge CB2
[19] B. Coyle, P. Kinsella, M. McCann, M. Devereux, R. O’Connor, M.
Clynes, K. Kavanagh, Toxicol. In Vitro 18 (2004) 63.
[20] M. Geraghty, V. Sheridan, M. McCann, M. Devereux, V. McKee,
Polyhedron 18 (1999) 2931.
[21] V.F. Hanic, J. Michalov, Acta Crystallogr. 13 (1960) 299.

4084
M. Devereux et al. / Polyhedron 26 (2007) 4073–4084
[22] R. Whyman, W.E. Hatfield, C.S. Fountain, Inorg. Chim. Acta 1
(1964) 429.
[36] Y. Naruta, K. Maruyama, J. Chem. Soc., Chem. Commun. (1994)
2667.
[23] F.A. Cotton, G. Wilkinson (Eds.), Advanced Inorganic Chemistry,
6th edn., Wiley, New York, 1999, p. 867.
[24] U. Schatzschneider, T. Weyhermuller, E. Rentschler, Inorg. Chim.
¨
[37] M. Curan, Ph.D. Thesis, National University of Ireland, 1996.
[38] S. Goldstein, G. Czapski, in: Free Radicals – A Practical Approach,
Oxford University Press, UK, 1996, p. 52.
Acta 337 (2002) 122.
[25] S. Takamizawa, E. Nakata, H. Yokoyama, Inorg. Chem. Comm. 6
[39] U. Weser, K.H. Sellenger, E. Lengfelder, W. Werner, J. Stra¨hle,
Biochim. Biophys. Acta 631 (1980) 232.
(2003) 763.
[40] J.F. Weder, C.T. Dillon, T.W. Hambley, B.J. Kennedy, P.A. Lay,
J.R. Biffin, H.L. Regtop, N.M. Davies, Coord. Chem. Rev. 232 (2002)
95.
[26] K. Deka, R.J. Sarma, J.B. Baruah, Inorg. Chem. Commun. 9 (2006)
931.
´
[27] H. Koizumi, K. Osaki, T. Watanabe, J. Phys. Soc. Jpn. 18 (1963)
[41] IVS Annual; MIMS Publishing, Crows Nest, NSW, Sydney, 1997, p.
145, 276.
117.
[28] M. Inoue, M. Kishita, M. Kubo, Inorg. Chem. 3 (1964) 239.
[29] M.T. Casey, M. McCann, M. Devereux, M. Curran, C. Cardin, M.
Convery, V. Quillet, C. Harding, J. Chem. Soc., Chem. Commun.
(1994) 2643.
[30] K. Nakamoto, Infrared and Raman Spectra of Inorganic and
Coordination Compounds, 5th ed., John Wiley and Sons,
1997.
[31] M.S. Refat, I.M. El-Deen, H.K. Ibrahim, S. El-Ghool, Spectrochi-
mica Acta, Part A 65 (2006) 1191.
[32] G.B. Deacon, R.J. Philips, Coord. Chem. Rev. 33 (1980) 227.
[33] B.N. Figgis, Introduction to Ligand Fields, Interscience, New York,
1966.
[42] J.E. Weder, T.W. Hambley, B.J. Kennedy, P.A. Lay, D. Maclachlan,
R. Brambley, C.D. Delfs, K.S. Murray, B. Moubaraki, B. Warick,
J.R. Biffin, H.L. Regtop, Inorg. Chem. 38 (1999) 1736.
[43] A.L. Abuhijleb, J. Inorg. Biochem. 68 (1997) 167.
[44] A.L. Abuhijleh, C. Woods, Inorg. Chem. Commun. 5 (2002) 269.
[45] M. Goodman, L.I.B. Haing, J. Chem. Soc., A (1966) 174.
[46] R.S. So, A.A. Adjei, J. Clin. Oncol. 17 (1999) 409.
[47] B. Rosenberg, Metal Ions Biol. Syst. 11 (1980) 127.
[48] T. Hirohama, Y. Kuranuki, E. Ebina, T. Sugizaki, H. Arii, M.
Chikira, P.T. Selvi, M. Palaniandavar, J. Inorg. Biochem. 99 (2005)
1205.
[49] A. Rivero-Muller, A. De Vizcaya-Ruiz, N. Plant, L. Ruiz, M.
¨
[34] M. Devereux, M. McCann, V. Leon, V. McKee, R.J. Ball, Polyhe-
dron 21 (2002) 1063.
[35] M. Devereux, M. McCann, M.T. Casey, M. Curran, International
Patent (1994) IPA No. S940892.
Dobrota, Chemico-Biol. Interact., 2006 doi:10.1016/j.cbi.2006.12.002.
[50] Bruker AXS. APEX2 and SHELXTL, Bruker AXS Inc., Madison, WI,
USA, 2004.
[51] T. Mosmann, J. Immunol. 65 (1983) 55.