Anticancer copper pyridine benzimidazole complexes: ROS generation, biomolecule interactions, and cytotoxicity

Article abstract of DOI:10.1016/j.jinorgbio.2016.11.006

The Cu(II) complex CuCl₂(pbzH), pbzH = 2-(2-pyridyl)benzimidazole, and derivatives modified at the non-coordinated nitrogen of the benzimidazole fragment, have been studied as anticancer agents. These compounds show promising cytotoxicity against A549 adenocarcinomic alveolar basal epithelial cells with IC₅₀ values in the range of 5–10 μM. Importantly, this activity is higher than either CuCl₂·2H₂O or the individual ligands, demonstrating that ligand coordination to the Cu(II) centres of the complexes is required for full activity. Electron paramagnetic resonance (EPR) and UV–Vis spectroscopies were used to characterize the solution behaviour of the complexes. These studies demonstrate: (i) two types of solvated species in buffer, (ii) both coordinate and non-coordinate interactions with albumin, and (iii) weak interactions with DNA. Further DNA studies using agarose gel electrophoresis demonstrate strand cleavage by the complexes in the presence of ascorbate, which is mediated by reactive oxygen species (ROS). Through a fluorescence-based in vitro assay, intracellular ROS generation in the A549 cell line was observed; indicating that damage by ROS is responsible for the observed activity of the complexes.

Full text of DOI:10.1016/j.jinorgbio.2016.11.006

Anticancer Copper Pyridine Benzimidazole Complexes: ROS Generation,
Biomolecule Interactions, and Cytotoxicity
Kathleen E. Prosser, Stephanie W. Chang, Felix Saraci, Ph u´ c H. L eˆ ,
Charles J. Walsby
PII:
DOI:
Reference:
S0162-0134(16)30377-4
doi:10.1016/j.jinorgbio.2016.11.006
JIB 10106
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
12 August 2016
12 October 2016
3 November 2016
Please cite this article as: Kathleen E. Prosser, Stephanie W. Chang, Felix Saraci, Ph u´ c
H. L ˆe , Charles J. Walsby, Anticancer Copper Pyridine Benzimidazole Complexes: ROS
Generation, Biomolecule Interactions, and Cytotoxicity, Journal of Inorganic Biochemistry
(2016), doi:10.1016/j.jinorgbio.2016.11.006
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ACCEPTED MANUSCRIPT
Anticancer Copper Pyridine Benzimidazole Complexes: ROS Generation, Biomolecule
Interactions, and Cytotoxicity
Kathleen E. Prosser, Stephanie W. Chang, Felix Saraci, Phúc H. Lê, and Charles J. Walsby*
Department of Chemistry, Simon Fraser University, 8888 University Dr, Burnaby, British
Columbia, V5A 1S6, Canada
Keywords: copper; anticancer; reactive oxygen species; albumin; DNA
*
Corresponding Author.
Email address: cwalsby@sfu.ca
TEL: 778-782-4607
FAX: 778-782-3765
1

ACCEPTED MANUSCRIPT
ABSTRACT: The Cu(II) complex CuCl (pbzH), pbzH = 2-(2-pyridinyl)benzimidazole, and
2
derivatives modified at the non-coordinated nitrogen of the benzimidazole fragment, have been
studied as anticancer agents. These compounds show promising cytotoxicity against A549
adenocarcinomic alveolar basal epithelial cells with IC50 values in the range of 5-10 μM.
Importantly, this activity is higher than either CuCl ·H O or the individual ligands,
2
2
demonstrating that ligand coordination to the Cu(II) centres of the complexes is required for full
activity. Electron paramagnetic resonance (EPR) and UV-Vis spectroscopies were used to
characterize the solution behaviour of the complexes. These studies demonstrate: (i) two types of
solvated species in buffer, (ii) both coordinate and non-coordinate interactions with albumin, and
(iii) weak interactions with DNA. Further DNA studies using agarose gel electrophoresis
demonstrate strand cleavage by the complexes in the presence of ascorbate, which is mediated by
reactive oxygen species (ROS). Through a fluorescence-based in vitro assay, intracellular ROS
generation in the A549 cell line was observed; indicating that damage by ROS is responsible for
the observed activity of the complexes.
2

ACCEPTED MANUSCRIPT
INTRODUCTION
[
1-4]
Copper-based chemotherapeutics
are attracting increasing interest as alternatives to non-
[
5]
[6, 7]
[8]
essential metal-based drugs, such as those containing platinum, ruthenium,
and gold.
[
9]
Copper is an endogenous metal that is vital to the function of all organisms, but is highly toxic
[
10]
if not properly trafficked.
Toxicity originates primarily from damage to biological
macromolecules through redox-reactions, especially via formation of reactive oxygen species
[
11]
(
ROS). The Cu(II)/Cu(I) redox-couple is the major source of these reactions and is influenced
[
12]
by the ligand environment of copper centres.
Reports of copper chemotherapeutics are dominated by complexes of the 2+ oxidation
state, primarily due to the diversity of structures afforded to this oxidation state derived from
[
1-
diverse donor atoms (N, O, S and halides), coordination numbers (four to six), and geometries.
4
, 13]
An array of copper compounds has been evaluated using both in vitro testing and in vivo
[
1, 2]
animal studies.
Antitumor activity from Cu complexes with diverse ligand systems, including
thiosemicarbazones, mixed chelates, tetradentate ligands, and Schiff bases, have been reported in
[
1, 2]
vivo.
To date, none of these complexes have progressed to human clinical trials as
chemotherapeutics in their own right. However, copper gluconate coadministered with the
dithiocarbamate compound disulfiram is currently under clinical development for the treatment
[
14]
[15-17]
of solid liver tumors,
and glioblastomas.
In this case, anticancer activity may involve
generation of a bis(N,N-diethyl dithiocarbamato)copper(II) complex and induction of oxidative
[
18, 19]
stress.
Cu complexes with bidentate N-N donors derived from heterocyclic ring systems have
been widely reported, in many cases showing promising anticancer activity. Notable ligand types
include phenanthroline and bipyridine derivatives, pyrazole-pyridine ligands, and benzimidazole
3

ACCEPTED MANUSCRIPT
[
1, 3]
based ligands.
The origin of the anticancer activity of these types of Cu complexes has been
[3]
linked to DNA interactions, enzyme inhibition, and redox-mediated generation of ROS.
The ligands used in the work described herein are based on 2-(2-pyridinyl)benzimidazole
[
20]
[21]
(pbzH) (Scheme 1), which inhibits hepatic enzymes,
and exhibits antiplasmodial
and
[
22]
[23, 24]
anticancer
activities. The synthesis of Cu(II)(pbzH)Cl has been described,
2
but the
complex has not previously been studied for medicinal properties. However, the anticancer
[
25]
[26]
activity of metal-pbzH complexes has been explored using ruthenium, gold, palladium, and
[27-29]
platinum.
Furthermore, several bis-pbzH Cu(II) complexes have been structurally
[
30-32]
[33]
characterized,
with one such compound demonstrating in vitro anticancer activity.
A
number of related mixed-ligand complexes with other ancillary ligands have also been
structurally characterized and examined for their DNA cleaving abilities and for antibacterial
[
34-36]
activity.
These studies suggest the viability of these types of complexes for use in a
therapeutic setting.
In this study, we have synthesized and characterized Cu(pbzH)Cl and five derivatives
2
with functionalization at the non-coordinated pbz benzimidazole nitrogen (Scheme 1). This
study is a rare example of a chemical and biological investigation of copper complexes that have
[
1, 2, 13, 37]
both labile coordination sites and N,N-donor ligands.
We describe a variety of
approaches to characterize these Cu(II) complexes and their biological behaviour, including
electron paramagnetic resonance (EPR) spectroscopy, electrochemistry, DNA gel
electrophoresis, and in vitro assays, providing insight into their cytotoxic activity. Overall, these
studies demonstrate that Cu(II)(pbzH)Cl is a promising chemotherapeutic scaffold, with well-
2
defined biological interactions and activity derived from the redox-active copper center.
4

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RESULTS AND DISCUSSION
. Synthesis and structures
The synthetic method used for the ligands pbzX (Scheme 1) is based on the literature report of
1
[
38, 39]
Huang et al.,
which gives the compounds in reasonable yields under mild conditions. One
particularly appealing feature of these ligands from a medicinal perspective is that they can be
functionalized readily. As we demonstrate here, modification at the non-coordinated nitrogen of
the benzimidazole fragment potentially enables inclusion of a variety of functional groups. The
Cu(pbzX) complexes were synthesized as shown in Scheme 1, based on literature reports for the
[37]
synthesis of dichloro copper benzimidazole compounds. The pure copper complexes can be
isolated without residual solvent following trituration with ether. The identity and purity of all
compounds was confirmed by elemental analysis, IR spectroscopy, mass spectrometry, and X-
ray crystallography where possible.
X-ray crystal structures for complexes Cu(pbzBz), Cu(pbzF), Cu(pbzF ) and Cu(pbzF )
2
5
were determined, shown in Figure 1. In each case, crystals suitable for X-ray analysis were
obtained from concentrated acetonitrile solutions at low temperature. However, despite attempts
under a range of conditions, Cu(pbzH) and Cu(pbzMe) could only be obtained as fine powders,
indicating that the benzyl groups of the other complexes play an important role in promoting
crystallization.
Each of the crystal structures demonstrates bidentate coordination of the pbzX ligands
through the nitrogens of the benzimidazole and pyridyl moieties. The structures of Cu(pbzF) and
Cu(pbzF ) show mononuclear complexes with distorted square-planar geometries around the
5
Cu(II) centers. In the case of Cu(pbzBz) and Cu(pbzF ), dimeric structures are observed with the
2
individual Cu(II) centers exhibiting distorted trigonal bipyramidal geometries, and two chloride
5

ACCEPTED MANUSCRIPT
2
ligands providing a  bridge between them. The dichloro bridge contains two distinct bond
lengths of 2.26 and 2.68 Å for Cu(pbzBz) and 2.26 and 2.76 Å for Cu(pbzF ) (see Table S2 for
2
all Cu centered bond lengths). These observations are similar to the Cu(II) phenanthroline (phen)
2
dimer [CuCl(phen)μ-Cl] , where the  -chloro bridge has two shorter Cu-Cl bonds (2.26 Å) and
2
[
40]
longer Cu-Cl bonds (2.68 Å), indicating that this is common structural feature in these types
of complexes. Given the asymmetry in the Cu-Cl bonds linking the Cu centers of Cu(pbzBz) and
Cu(pbzF ), the dimeric structures observed are assigned to a consequence of crystal packing
2
forces, with monomeric Cu(II) centres similar to Cu(pbzF) and Cu(pbzF ) expected once the
5
solid is dissolved in solution. This is demonstrated by EPR measurements (see below) where the
spectra of all of the complexes are similar and typical for mononuclear S = 1/2 Cu(II) complexes,
[
41]
with no evidence for exchange coupling between Cu centres for Cu(pbzBz) and Cu(pbzF2).
2
. Anticancer Activity
The in vitro anticancer activity of compounds Cu(pbzX) was assessed against A549
adenocarcinomic alveolar basal epithelial cells, a non-small cell lung cancer (NSCLC) cell line.
In the clinic, NSCLC is often treated with cisplatin, and has a 5-year survival rate that is below
[
42, 43]
5
0%.
In addition to testing the Cu(pbzX) complexes, cisplatin, and each of the six pbzX
ligands were also tested as controls for activity. Furthermore, CuCl 2H O was also tested, as a
2
2
model for the activity of free copper.
All of the compounds were incubated with A549 cells for 72 hours to assess their
cytotoxic effect at concentrations ranging from 0.625 to 100 M. In these experiments, all wells
contained 1% dimethyl sulfoxide (DMSO), which was also tested as the vehicle control. This
concentration of DMSO is tolerated by A549 cells and allows for sufficient solubility of all
6

ACCEPTED MANUSCRIPT
compounds tested. The solubility of the complexes was assessed at all concentrations by
checking for precipitation by examination under a microscope and during plate imaging.
Furthermore, UV-Vis spectra at 50 M concentrations also showed no evidence of precipitation.
The fraction of cells affected in each case was imaged using a fluorescent cell-permeable nuclear
marker (Hoescht 33342 nucleic acid stain) to determine the total cell count, and a cell
impermeable nuclear marker (ethidium homodimer I) to count the number of dead cells.
Statistical analysis of the resulting images gave sigmoidal dose-response curves (Figure 2) that
were modelled using a four-parameter logistic model to calculate the 50% inhibitory
concentration (IC ) values (Table 1).
5
0
Each of the Cu(pbzX) complexes demonstrated strong cytotoxic activity with IC values
5
0
ranging from 5.5 to 12 M. This compares favourably with cisplatin, which had an IC value of
5
0
3
.5 M against the A549 cell line (Figure S24). Cu(pbzMe) was the most active complex, with
activity that was the same as cisplatin at the p < 0.001 level of significance. The activity of
CuCl ·2H O was significantly lower (p < 0.001) than any of the Cu(pbzX) complexes with an
2
2
IC50 value of 32 M. Similarly, all of the pbzX ligands showed only moderate to low
cytotoxicity. The most active ligand pbzF had IC = 30 M and three of the ligands, pbzH,
2
50
pbzMe and pbzF , did not achieve 50% inhibition of cell growth at the highest tested
5
concentrations of 100 µM. The low level of activity of the ligands may be due in part to low
solubility in aqueous media or DMSO, although under the assay conditions no significant
precipitation was observed under a microscope or during image analysis. Importantly, there is no
correlation between the activity of the ligands and the corresponding Cu(II) complexes. Indeed,
two of the ligands with the lowest responses (pbzH and pbzMe) give the most active complexes.
Taken together, these data highlight the necessity of both the copper centre and the pbzX ligands
7

ACCEPTED MANUSCRIPT
for the observed cytotoxic activity. When compared to other copper complexes tested against
A549 cells, the Cu(pbzX) complexes perform as well as or better than most. A few examples
[
44]
include a Cu-quercetin complex with an IC of 21.5 μM, a set of binuclear complexes with an
5
0
[45]
[46]
[47]
average IC of 10 μM and copper complexes with nitroimidazole and N, N, N donors
5
0
having average IC values of 15 and 67 μM, respectively.
5
0
3
. Electrochemical Properties.
Cyclic voltammograms from solutions of each complex in 2-(N-morpholino)ethanesulfonic acid
MES) buffer were collected (Figure S22) to probe the effect of the ligands pbzX on the
(
reduction potentials of the complexes, and the impact of ligand exchange processes. A scan rate
of 25 mV/s was chosen for these experiments due to slow electron transfer rates which have been
[
48]
reported for similar Cu complexes. Cyclic voltammograms were measured at pH 4.4 since at
higher pH (pH 6.5, 7.4 for example) significant peak broadening and irreversibility was
[
49]
observed, likely due to formation of insoluble copper hydroxide species. Even at pH 4.4 all of
the complexes exhibited relatively broad peaks, likely due to overlapping waves from different
species arising from ligand exchange in solution. This was particularly evident for Cu(pbzH) and
Cu(pbzMe), which showed wide peaks and evidence for irreversibility. By contrast, the benzyl
substituted complexes give rise to narrower and more reversible waves, likely reflecting greater
solution stability at pH 4.4 The E values of the complexes (Table 1) range from 295 mV for
Cu(pbzMe) to 370 mV for Cu(pbzF ). These reduction potentials are within a typical range for
5
[
50]
copper compounds with aromatic nitrogen-containing ligands. There is a trend of increasing
reduction potentials with the addition of fluorines to the pendant benzene ring (E: Cu(pbzBz) <
Cu(pbzF) < Cu(pbzF ) < Cu(pbzF )) which can be attributed to the electron-withdrawing effects
2
5
of the fluorine substituents. Similarly, the electron donating methyl group of Cu(pbzMe) results
8

ACCEPTED MANUSCRIPT
in the lowest reduction potential.
The principal motivation for characterizing the electrochemistry in aqueous solutions was
to evaluate whether the Cu(II)/Cu(I) couples were within the range of common biological
reductants. Based on reduction potentials for Cu(pbzX), in the range of 295-370 mV, all of these
complexes could undergo reduction by biological reducing agents such as glutathione (E =
[
51]
[52]

240 mV)
or ascorbate (E = 50 mV).
Thus, we conclude that the complexes will be
reduced in the in vitro experiments described below. This demonstrates the relevance of the gel
electrophoresis studies of DNA cleavage that implicate hydroxyl radical generation as the source
of cytotoxic activity. Furthermore, this is consistent with the observation of the generation of
ROS by Cu(pbzX) in A549 cells (see below).
4
. Solution Behaviour.
Ligand exchange processes are central to the activity of many metal-based chemotherapeutics
since they determine the pharmacologically important species in vivo. Consequently, anticancer
[53]
[54]
metallodrugs such as platinum
and ruthenium
complexes are considered to be pro-drugs
that exchange ligands to generate activated species. Following administration, the original
ligands of such complexes can by replaced by water and other prevalent small molecules, as well
[
54, 55]
as biomolecules such as DNA and proteins.
In the case of copper complexes, such as those
[56]
described here, ligand exchange is highly favoured under physiological conditions,
and so
characterization of these processes is required to understand their mechanisms. We have applied
EPR and UV-Vis spectroscopies to define changes to the coordination environment of the
Cu(pbzX) complexes.
9
EPR analysis. The paramagnetic Cu(II) centers (d , S = ½) of the Cu(pbzX) complexes enabled
9

ACCEPTED MANUSCRIPT
EPR studies of their solution behavior and interactions with biomolecules. Spectra were analysed
[
57]
using the MATLAB program EasySpin to determine g values and hyperfine interactions with
63
65
the copper centers of the complexes ( Cu, 69.17%, I = 3/2, g = +1.4824; Cu, 30.83%, I = 3/2,
N
1
4
g = +1.5878) and nitrogen nuclei ( N, I = 1, 99.63%) of the ligands. Simulations of Cu
N
hyperfine splittings include appropriately weighted contributions from both isotopes, and scaled
6
3
65
63
65
values of copper hyperfine interactions according to A( Cu)/A( Cu) = g ( Cu)/g ( Cu);
N
N
6
3
values of A(Cu) are quoted for the more abundant Cu isotope. For solutions containing
multiple Cu(II) species, spectra from individual species were identified and their respective
simulated spectra added together with appropriate weightings to reproduce the experimental data.
EPR measurements of Cu(pbzX) in frozen solutions showed uniaxial spectra (with one
exception, see below) with g > g . This is consistent with Cu(II) ions in square planar or axially
‖

elongated octahedral crystal fields, with the unpaired electron in the
ꢀ2
- ꢁ2
orbital, indicating that
this is the approximate geometry of the Cu(II) species generated from Cu(pbzX) in solution. To
8
ꢁ
2ꢁ
first-order in perturbation theory, the g values are given by g ꢀ 2 - and g ꢀ 2 - , where the
ꢀ
ꢁ
ꢂ1
ꢂ2
spin- orbit coupling coefficient (ꢁ) is estimated to be 85% that of the free ion (free Cu(II) ion:
-1
-1

828 cm , here: 700 cm ) and  is the energy difference between
ꢀ2
- ꢁ2
and the d , orbitals
1
xy
[58]
and  is the energy difference between
ꢀ2
- ꢁ2
and the degenerate d and d orbitals.
xz yz
2
Complexes in MES buffer. The EPR spectra of the complexes in MES buffer (pH 6.5) show
contributions from two Cu(II) species (Figure 3, Figures S17-18). With the exception of
Cu(pbzH), the Cu(pbzX) complexes showed similar spectra with both species having uniaxial g
tensors: MES-1 (g = 2.064-2.065, g = 2.256-2.258), MES-2 (g = 2.069-2.070, g = 2.315-

‖

‖
2
1
.320). Both species also exhibit copper hyperfine interactions around g : MES-1 (A (Cu) = 185-
‖ ‖
90 G), MES-2 (A (Cu) = 170-172 G). Complex Cu(pbzH) also shows the uniaxial spectrum
‖
1
0

ACCEPTED MANUSCRIPT
from species MES-2 detected from the other complexes. However, a uniaxial spectrum
corresponding to MES-1 was not observed from Cu(pbzH), but instead a rhombic spectrum with
g = [2.247, 2.100, 1.925] and A = 200 MHz was detected (Figures S17-18, Table S3). This is
1
likely due to the exchangeable proton of the benzimidazole of the pbzH ligand, which is
otherwise functionalized in the other complexes. Deprotonation of the ligand would result in
generation of an effective negative charge on the benzimidazole ring, perturbing the ligand field
of the Cu centre and leading to a loss of degeneracy of the d and d orbitals, resulting in
xz
yz
splitting of g into two distinct g values. Spectral deconvolution of each of the complexes

determined that MES-2 was the predominant species, comprising around 2/3 of the total spectral
intensity in each case.
By taking numerical derivatives of the EPR spectra, to give second-derivative line shapes,
additional hyperfine structure was resolved in the g region of each complex (Figure 3b, Figure

S18). This was simulated by including copper hyperfine coupling (A (Cu) = 2-9 G) and

hyperfine interactions from two nitrogen nuclei with approximately equal coupling constants
1
4
(
A ( N) = 14-15 G) in the simulation of MES-2. Spectral overlap in the g region meant that


resolution of such splittings from the secondary species MES-1 was not possible. Nonetheless,
observation of hyperfine structure from two nitrogen atoms is consistent with coordinated pbzX
ligands.
The EPR spectra on their own do not enable unequivocal assignment of the species
present when Cu(pbzX) are dissolved in MES buffer. However, the rapid ligand-exchange rates
[
56]
of Cu(II) complexes
suggests that the chloride ligands will be replaced by water ligands in
aqueous solution. Furthermore, comparison of the EPR spectra with previous studies of Cu(II)
phenanthroline in aqueous solution are instructive. Indeed, the EPR spectra of the substituted
1
1

ACCEPTED MANUSCRIPT
2
+
Cu(pbzX) complexes are remarkably similar to those reported for [Cu(phen)] in aqueous
[
59]
solution, which shows two species with g = 2.314, A (Cu) = 179 G and g = 2.259, A (Cu) =
‖
‖
‖
‖
1
84 G. In this earlier study, these species were identified as the di-aquo complex and its
deprotonated aquo-hydroxy derivative respectively. Based on the striking similarities in spectral
+
parameters we provisionally assign MES-1 as [Cu(pbzX)(OH )(OH)] and MES-2 as
2
2+
−
[
Cu(pbzX)(OH ) ] . This implies that exchange of both Cl ligands from Cu(pbzX) is completed
2 2
in the course of preparing the EPR samples; attempts to minimize ligand exchange by freezing
samples immediately after mixing with the buffer solution did not change the appearance of the
spectra. In the case of the unsubstituted complex Cu(pbzH), deprotonation of
2
+
[
[
Cu(pbzH)(OH ) ]
(MES-2) at the ligand, rather than coordinated water, to give
2
2
+
Cu(pbz)(OH ) ] is consistent with the observation of a species with a rhombic EPR spectrum,
2
2
rather than the uniaxial MES-1 signal observed with the other complexes.
UV-Vis spectra of the Cu(pbzX) complexes in MES buffer show a strong absorbance at
~320 nm from a ligand centered transition. Time dependent UV-Vis spectra (Figure S13) of
each complex show little change over the course of one hour at 37 °C. This is again consistent
with chloride ligand exchange occurring on a time scale faster than sample preparation. Due to
limited aqueous solubility, it was not possible to monitor the more diagnostic Cu(II) d-d
absorption bands at longer wavelengths.
5
. Interactions with Human Serum Albumin.
Serum proteins, such as human serum albumin (HSA), have been implicated in the transport and
[
60]
bioaccumulation of many inorganic anticancer agents.
HSA binds Cu(II) with high affinity,
with the primary binding site located at the N-terminus involving coordination by histidine, two
1
2

ACCEPTED MANUSCRIPT
[
61, 62]
peptide nitrogens, and the terminal aspartic acid -NH2.
HSA has several exposed surface
histidines, as well as hydrophobic binding sites, each of which have been implicated in the
[
63-66]
transport of medicinal metal complexes.
To establish whether HSA could be a transporter of Cu(pbzX), the complexes were
incubated with the protein, and protein-bound fractions were isolated by ultrafiltration. The EPR
spectra of each complex with HSA show the presence of two species (Figure 4, and Figure
S19). The first of these species, HSA-1, has g values (g = 2.033-2.043, g = 2.180-2.189) that

‖
are distinct from either of the species in MES buffer. Furthermore, the copper hyperfine
interaction of HSA-1 (A (Cu) = 207-215 G) is significantly larger than either of the buffer
‖
species. The hyperfine splitting pattern around g of HSA-1 is comprised of unique contributions

from both nitrogen and copper interactions, which are partially resolved in the experimental EPR
data and well defined in the second-derivative spectra (Figure 4b, and Figure S20). Simulations
demonstrate that the hyperfine structure is derived from contributions of both the copper centre
1
4
(
A (65Cu) = 17-18 G) and three approximately equivalent nitrogens (A( N) = 14.5 G.) The

relatively large value of A (65Cu), as compared to MES-1, further demonstrates that HSA-1 is

distinct from either of the species in MES buffer. However, even more significant is the
observation of contributions from three nitrogens. Since only two coordinated nitrogens are
provided by the pbzX ligands, this demonstrates an additional nitrogen atom coordinated to the
copper centre that arises from HSA. This species is likely from coordination to the imidazole
side-chains of histidines.
The second species observed in the presence of HSA, HSA-2, has g values and copper
hyperfine couplings (g = 2.052-2.063, g = 2.267-2.295, A (Cu) = 180-185 G) that are similar to

‖
‖
MES-2. This suggests that a fraction of the complexes are not coordinated to the protein.
1
3

ACCEPTED MANUSCRIPT
However, since the samples studied are isolated protein-bound fractions, this may reflect non-
coordinate interactions with HSA, as has been reported for a variety of metal complexes,
[
65-69]
particularly with hydrophobic sites.
Small differences in g values and hyperfine couplings
[
70]
may be ascribed to the influence of second coordination sphere effects.
Interestingly, while
Cu(pbzH) shows a rhombic spectrum in MES, the complex shows a uniaxial spectrum with
similar parameters to the other complexes with HSA. This suggests that the uncoordinated
benzimidazole is protonated in the presence of the HSA, and may reflect the effect of the local
environment in the protein. Overall, the EPR data show that the Cu(pbzX) complexes readily
form both coordinate and non-coordinate interactions with HSA. This is promising since such
interactions are associated with metallodrug transport to tumour sites in vivo, and could provide a
[
71]
route for bioaccumulation.
6
. Interactions with DNA
The ability to intercalate or cleave DNA has been linked to the activity of several Cu-based
[
72-77]
anticancer agents.
To determine if this behaviour was involved in the cytotoxicity of
Cu(pbzX), the interactions of the complexes with calf-thymus DNA (ctDNA) and bacterial
plasmid DNA were characterized using UV-Vis titrations, EPR measurements, and gel
electrophoresis.
UV-Vis titrations. The reaction of the Cu(pbzX) complexes with ctDNA was characterized by
concentration-dependent changes in the absorption spectrum. To quantify the DNA interactions
of Cu(pbzX), each complex (50 µM) was incubated with ctDNA (50 - 350 μM molar base-pair
concentration) for 40 minutes. Titrating the complexes with increasing concentrations of DNA
resulted a red shift and hypochromism of the ꢁmax (Figure S14), changes which are indicative of
1
4

ACCEPTED MANUSCRIPT
intercalation. This has been shown, for example, in studies of Cu(II) phenanthroline
[78]
complexes. These data were analyzed for each complex using Equation 1 to calculate binding
[
79]
constants Kb:
ꢂꢃꢄꢅꢆ
ꢂꢃꢄꢅꢆ
ꢐ
ꢍ ꢈ
ꢏ
ꢑ ꢇꢈ ꢊ ꢈꢋꢌ
ꢒ
ꢎ
(1)
ꢇꢈꢉ ꢊ ꢈꢋꢌ
ꢊ ꢈꢋ
ꢎ
where [DNA] is the molar concentration of base pairs, ε is the extinction coefficient of the
B
complex fully bound to DNA, ε is the apparent extinction coefficient of the complex bound to
A
DNA and ε is the extinction coefficient of the complex free in solution. A plot of [DNA] vs.
f
[
DNA]/(ε – ε ) gives a linear relationship with K calculated by dividing the slope by the
A f b
intercept. Such plots for each Cu(pbzX) complex are shown in Figure S15, with corresponding
values of K in Table 1. Overall, the magnitude of the binding constants for all of the complexes
b
4
-1
5-6
(
10 M ) is well below that of classic intercalators, which typically have K values in the 10
b
-1
[80]
M
range.
These relatively low binding constant agree with the results from gel
electrophoresis studies (see below) where significant intercalation was also not observed, and
demonstrate that while there is an interaction between the Cu(pbzX) complexes and DNA, it is
unlikely to be by a strong intercalative mechanism.
EPR. To further characterize interactions between Cu(pbzX) and DNA, EPR spectra were
collected from each complex with ctDNA in MES buffer (Figure S21 and Table S5). The
resulting spectra show two distinct species with uniaxial EPR signals: DNA-1 (g = 2.055-2.076,

g = 2.326-2.330, A (Cu) = 154-160 G), DNA-2 (g = 2.057-2.064, g = 2.285-2.287, A (Cu) =
‖
‖

‖
‖
1
67-170 G). The g values of these species are close to those of the uniaxial spectra from the
buffer species MES-1 and MES-2, respectively, indicating a similar primary coordination sphere
in each case. However, the differences in the g values although small are still significant, as
1
5

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determined from the spectral simulations, particularly for g . This is assigned to secondary
‖
[70]
coordination sphere effects, resulting from interaction with DNA.
The uniaxial species DNA-1 and DNA-2, when compared to MES-1 and MES-2, have
values of A (Cu) that are reduced by 12-15 and 16-22 G, respectively. This observation, and the
‖
2+
changes in g values, are similar to literature reports of [Cu(phen)(H O) ] interacting with
2
3
[
59]
DNA. Using DNA-fiber EPR, it has been shown that these signals from the phenanthroline
[
81]
complex are assignable to a combination of intercalative and minor-groove binding. Based on
the UV-Vis studies described above, the Cu(pbzX) complexes only weakly intercalate with
ctDNA, thus the EPR signals are more likely to reflect groove binding.
Additional evidence for DNA interactions is provided by the EPR spectrum from
Cu(pbzH) (Figure S21a). In this case a contribution from the rhombic species previously
observed from the MES buffer sample (MES-2) is observed in addition to the uniaxial signals
DNA-1 and DNA-2. Based on relative signal intensities determined by simulation, this indicates
~1/3 of Cu(pbzH) is found in solution, rather than interacting with DNA, which is consistent
with weak binding.
Gel Electrophoresis and ROS effects. To further examine interactions with DNA,
electrophoresis experiments using agarose gels and pet22b plasmid DNA were performed.
Plasmids are a useful tool for these studies because they are found primarily in three forms that
migrate at different rates on the gel. From fastest to slowest these forms are: i) supercoiled (SC),
ii) linear (L), which is the result of a double-strand break, and iii) open circular (OC), which is
[
82]
seen as a consequence of single-strand breaks or nicks. In addition, intercalation between base
[83, 84]
pairs, or kinking of the strands, can induce changes to the migration rate of SC plasmids.
SC is the naturally occurring form of plasmid DNA and is therefore the most prominent band.
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However, the process through which the plasmids were isolated in this work resulted in small
amounts of the L and OC forms in all samples.
The Cu(pbzX) complexes were first incubated with plasmid DNA only. As shown in
Figure 5a, this did not result in any change to the rate of migration or the relative concentration
of the three DNA forms, as compared to the control. Thus, we conclude that the Cu complexes
alone do not substantially intercalate or otherwise modify the DNA. To explore ROS generation
as the origin of anticancer activity, the Cu(pbzX) complexes were incubated with plasmid DNA
and sodium ascorbate. Addition of ascorbate generates Cu(I) species, which can be concluded
from the reduction potentials of the complexes (see above), and these can initiate Fenton-like
[85, 86]
chemistry to produce ROS.
The combined action of the complexes and ascorbate is evident
in Figure 5b, where there is a clear transformation of the plasmids from the SC to the L form.
This transformation indicates a double-strand cleavage of the DNA through an oxidative damage
[
87, 88]
pathway involving ROS.
These experiments were repeated with each of the pbzX ligands
alone (Figure S23), which showed no change from the control. This is consistent with the
cleavage process being driven by the copper centres of the complexes.
To probe the DNA-cleavage mechanism, superoxide dismutase (SOD) and catalase were
used as enzymatic scavengers of superoxide and peroxide respectively. Incubation of the
Cu(pbzX) complexes with plasmid DNA, sodium ascorbate, and 1 unit of SOD (Figure 5c) gave
results that were indistinguishable from experiments without the enzyme. For every complex
there was still a clear transformation of the SC for of the plasmid to the L form. However,
incubation of the plasmids with sodium ascorbate, 1 Unit of catalase, and each of the Cu(pbzX)
complexes, inhibited DNA cleavage (Figure 5d), demonstrating that hydrogen peroxide is
important to the DNA cleavage process.
1
7

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We propose the step-wise mechanism of ROS generation shown in Scheme 2. Initial
[
89]
reduction of Cu(pbzX) by ascorbate enables generation of superoxide from molecular oxygen.
The reduced form of Cu(pbzX) then catalyzes reduction of superoxide to hydrogen peroxide. In
our gel experiments, it is following this step that catalase inhibited further catalysis to the active
species. In the absence of catalase, H O is further reduced to give a hydroxyl radical and
2
2
[
89]
hydroxide.
Incubation with SOD fails to inhibit DNA cleavage because SOD catalyzes the
[90, 91]
reduction of superoxide to hydrogen peroxide,
which ultimately promotes the formation of
hydroxyl radicals (Scheme 2). While hydroxyl radicals were not directly detected in these
experiments, previous studies of Cu(II) complexes with reducing agents have established
hydrogen peroxide as an intermediate step in their generation, as well as their role in cleaving
[
87, 92, 93]
DNA.
7
. In Vitro ROS Measurements
Fluorescence-based assays of ROS generated by the Cu(pbzX) complexes with A549 cells were
performed using the non-polar cell-permeable dye, 2,7-dichlorodihydrofluorescein diacetate
[
94-97]
(DCFH-DA).
This non-fluorescent molecule readily diffuses across cell membranes, after
which its acetate groups are enzymatically hydrolyzed by intracellular esterases to give 2,7-
[
95, 97]
dichlorodihydrofluorescein (DCFH).
Oxidation of DCFH produces 2,7-dichlorofluorescein
(DCF), which is fluorescent due to its extended  system, enabling quantification of ROS species
in vitro. The A549 cells were exposed to the Cu(pbzX) complexes and imaged after 1.5 hours of
incubation. The fluorescence of each sample was normalized to a media control, with positive
controls provided by 1 and 10 μM H O , and statistical significance was assessed using Student’s
2
2
t test. The results of this assay are summarized in Figure 6.
All of the complexes produce elevated levels of ROS versus the media control, with at
1
8

ACCEPTED MANUSCRIPT
least as much ROS activity as the 1 μM H O positive control. While DCFH is a well-established
2
2
[
97, 98]
probe for cellular peroxides, H O alone does not directly oxidize DCFH.
By contrast,
2
2
hydroxyl radicals do readily oxidize DCFH, so that detection of H O requires decomposition
2
2
[
97, 98]
into radicals.
This may occur either by Fenton chemistry in the presence of reduced metal
[
99]
ions such as Cu(I) or Fe(II), or by cellular peroxidases. Additionally, it has been reported that
[
97, 99]
superoxide is not capable of oxidizing DCFH.
Thus, the observation of DCFH oxidation
when the cells are exposed to Cu(pbzX) is consistent with the mechanism determined in the
DNA electrophoresis studies (see above). In the cell studies, endogenous reducing agents likely
reduce the Cu(pbzX) complexes to Cu(I) species, initiating a redox cascade analogous to that in
Scheme 2, where molecular oxygen is reduced to H O and ultimately converted to hydroxyl
2
2
radicals.
Cu(pbzMe) showed the greatest increase in ROS, reaching the same level as the 10 μM
H O positive control. Interestingly, this complex also has highest cytotoxic activity against
2
2
A549 cells (see above), further implicating ROS in the mechanism of action of these types of
complexes. Overall, the observation of elevated ROS in the A549 cells in the presence of
Cu(pbzX) is further evidence for a mechanism of action involving oxidative DNA damage.
CONCLUSIONS
The ongoing development of copper complexes as potential chemotherapeutics is dependent on
characterization of active species and mechanisms of anticancer activity. In this study we have
used a variety of physical and biological methods to characterize the Cu(pbzX) complexes.
These compounds are useful models for the medicinal application of copper complexes with
chelating nitrogen donors and exchangeable ligands. The latter are less common features of
1
9

ACCEPTED MANUSCRIPT
reported Cu anticancer candidates, but as we show here, they can play an important role in
biological speciation. Another feature of the pbz ligands is functionalization at the non-
coordinated nitrogen of the benzimidazole moiety, which provides a mode for modification of
their properties. This suggests that these complexes could be a useful scaffold for development
of multifunctional Cu(II) metallotherapeutics.
The Cu(pbzX) complexes are comprised of three components that influence their
pharmacological behaviour: exchangeable chlorides, a non-exchangeable hydrophobic pbzX
ligand, and a redox-active Cu(II) centre. As we show here, each one of these components plays a
key role in the cytotoxicity and pharmacological behaviour of the compounds.
From EPR and UV-Vis studies we have shown that the chloride ligands are likely rapidly
exchanged under physiological conditions to give aquo- and hydroxo-coordinated species. Thus,
Cu(pbzX) complexes can be considered to be potential pro-drugs. An important consequence of
the ligand-exchange processes of metallodrug candidates is coordination to biomolecules. In the
case of the Cu(pbzX) complexes, EPR spectra demonstrate coordination to HSA, likely though a
histidine imidazole. Analysis of the nitrogen hyperfine structure from these species shows that
this process leaves the pbzX ligands coordinated, and introduces an additional nitrogen-donor
ligand from the protein.
The hydrophobic non-exchangeable pbzX ligands also influence interactions with
biomolecules. This is shown by a second type of EPR signal from the Cu(pbzX) complexes in
the presence of HSA, that is similar, though distinct from, species observed in buffer. Since
isolated protein fractions were characterized in these studies, these signals indicate non-
coordinate interactions with HSA, possibly with hydrophobic binding domains. This observation,
and detection of HSA-coordinated species, indicates that if complexes of this type were
2
0

ACCEPTED MANUSCRIPT
administered intravenously that they would be found significantly associated with HSA. By
contrast, EPR measurements and UV-Vis titrations indicate that the pbzX ligands mediate only
relatively weak interactions with DNA. These techniques show respectively that new species are
formed upon incubation with DNA and that one possible mode of interaction is weak
intercalation.
The central role that the copper centres of the Cu(pbzX) complexes play in their
anticancer activity is shown by the electrochemical, gel electrophoresis, and in vitro studies
reported here. The reduction potentials of all of the complexes are within the range of biological
reducing agents. Generation of Cu(I) species and redox cycling can then lead to generation of
ROS. As we show by gel studies with ascorbate and SOD or catalase, hydroxide radicals are
likely the ultimate result of this process. The relevance of these results to the mechanism of
anticancer activity is shown by detection of elevated ROS levels in A549 cells in the presence of
Cu(pbzX). Notably, the compound with the highest activity, Cu(pbzMe), also generates the
highest level of ROS in vitro.
Perhaps the most important observation in this work is that the cytotoxicity of the
Cu(pbzX) complexes is greater than either the pbzX ligands on their own or free Cu(II). This
may indicate that the ligands play a role in enabling the copper centres to effectively generate
elevated levels of toxic ROS, leading to cell death. We suggest that this could be the result of
enhanced transport into cells, subsequent intracellular targeting, or perhaps a consequence of
altered chemical properties such as modulated reduction potentials. Work is currently being
undertaken to further examine the enhanced biological activity of Cu(pbzX) complexes over
copper chloride and the implications for the rational design of new Cu(II) anticancer agents.
2
1

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EXPERIMENTAL
Synthesis. The starting compounds CuCl ·2H O, o-phenylenediamine, 2-picolinic acid,
2
2
iodomethane, benzyl bromide (Fisher Scientific), 4-fluorobenzyl bromide, 3,5-fluorobenzyl
bromide and pentafluorobenzyl bromide (Sigma-Aldrich) were used without further purification.
All of the ligands pbzX were prepared according to the general procedures reported by Huang et
al. for 2-(2-pyridinyl)benzimidazole (pbzH), 1-methyl-2-(2-pyridinyl)benzimidazole (pbzMe),
[
38, 39]
and 1-(phenylmethyl)-2-(2-pyridinyl)benzimidazole (pbzBz).
Syntheses of 1-[(4-
and 1-[(2,3,4,5,6-
have also been reported
using other methods, whereas 1-[(3, 5-difluorphenyl)methyl)-2-(2-pyridinyl)benzimidazole
pbzF ) is, to our knowledge, a new compound. The synthetic procedure is outlined in Scheme 1,
[
100]
fluorphenyl)methyl)-2-(2-pyridinyl)benzimidazole
(pbzF)
[
101]
pentafluorphenyl)methyl)-2-(2-pyridinyl)benzimidazole (pbzF5)
(
2
where in the first step pbzH was produced by the reaction of 2-picolinic acid and o-
phenalinediamine in polyphosphoric acid (PPA). The derivatized ligands were then produced by
reaction of pbzH in a dimethylformamide (DMF) solution of K CO with either iodomethane to
2
3
give pbzMe, or with the appropriate benzyl bromide to give pbzBz, pbzF, pbzF , and pbzF .
2
5
1
Details of synthetic methods and H NMR (Figures S1-6) are provided in Supporting
Information.
All Cu complexes were synthesized using the same general procedure, based on the literature
[37]
synthesis of dichloro copper benzimidazole complexes, and outlined in Scheme 1. The pbzX
ligands (0.5 mmol) were dissolved in minimal DMF and added to a solution of CuCl ·2H O (8.5
2
2
mg, 0.5 mmol) dissolved in 0.5 mL of DMF. The combined solutions were stirred at room
temperature, during which time a precipitate formed. The complexes were isolated via suction
filtration and thoroughly dried under reduced pressure. Structure and purity were verified using
2
2

ACCEPTED MANUSCRIPT
elemental analyses, Fourier transform infrared spectroscopy (FT-IR) (Figures S7-12), X-ray
crystallography and matrix-assisted laser desorption/ionization - time of flight (MALDI-TOF)
mass spectrometry. Detailed physical characterization and synthetic procedures for each complex
can be found in the Supporting Information.
Crystallographic Structure Determination. Single crystal X-ray diffraction analysis was
performed on a Bruker SMART diffractometer equipped with an APEX II CCD area detector, a
Mo Kα fine focus sealed tube (ꢁ ꢀ 0.71073 nm) and a Cu Kα fine focus sealed tube (ꢁ ꢀ 1.54178
nm) operating at 1.5 kW (50 kV, 30 mA), and filtered with a graphite monochromator. The
temperature was regulated using an Oxford Cryosystems Cryostream to 150 K when necessary.
The structures were solved by direct methods, and subsequent refinements were performed using
SHELXL and shelXle. For Cu(pbzBz), reflections (-4 0 8, 0 0 6, 1 1 5, 1 2 4, 2 2 2) were omitted
due to systematic error. Structure diagrams were generated by ORTEP-3(v. 2.00) and rendered
using Adobe Illustrator (CS6). Crystal data, data collection parameters, and details of structure
refinement are listed in Table S1. CCDC-1496889-1496892 contains the supplementary
crystallographic data for this paper. These data can be obtained free of charge from The
Cambridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data%5Frequest/cif.
Electrochemical Measurements. Stock solutions (10 mM) of each compound were initially
prepared in DMSO to improve solubility in aqueous media. Cyclic voltammograms were
recorded on a CH Instruments 660 potentiostat, equipped with a Ag/AgCl (1 M KCl) reference
electrode, a platinum wire counter electrode and a basal plane graphite working electrode. The
basal plane graphite electrode was prepared using a modified literature preparation using
[
102]
electrochemically inert Loctite 9460 Hysol Epoxy to seal the carbon blocks.
All data were
collected in an aqueous buffer solution at pH 4.4 consisting of 10 mM 2-(N-
2
3

ACCEPTED MANUSCRIPT
morpholino)ethanesulfonic acid (MES) and 150 mM NaCl. K [Fe(CN) ] was used to calibrate
3
6
the electrode potential. Measurements were performed using 150 µM concentrations of each
complex with final concentrations of 1.5% DMSO in 3 mL of MES buffer using a 25 mV/s scan
rate.
Cell Studies. A549 cells were acquired directly from Dr. Marcel Bally’s lab (Vancouver, BC) at
the British Columbia Cancer Research Agency. Cells were cultured at 37 °C under a 5% CO2
atmosphere in RPMI 1640 media (Gibco) supplemented with 2 mM L-glutamine (Gibco) and
1
0% fetal bovine serum (Gibco). All of the Cu compounds were diluted from 10 mM stocks in
DMSO to give the desired concentrations in complete cellular media for cytotoxicity testing. To
verify that the compounds would remain soluble under assay conditions, each compound was
incubated in complete cell media at the maximum testing concentrations for 72 hours at 37 C
and in each case there was no evidence of precipitation.
For in vitro cytotoxicity assays, cells were seeded in quadruplet at 2000 cells/well in 384-
well plates (Greiner Bio-One). Following a 24 hr incubation period, 20 µL aliquots of each
complex in media were added to each well to give the desired complex concentration with a final
DMSO content of 1%. Wells containing a media control and a vehicle (DMSO) control also were
prepared. The cells were stained using 5 µL of a 10 mg/mL stock of Hoescht 33342 nucleic acid
stain (Life Technologies) and 3 µL of a 1 mM stock of ethidium homodimer I (Biotium) per mL
of media. These stains were added 72 hrs after treatment by each complex, generating a total cell
count and a dead cell count respectively. After a 20-minute incubation period the plates were
then imaged using an IN Cell Analyzer 1000 (GE Healthcare), which is an automated fluorescent
microscopy platform that enables high content screening. Cell counts were determined via the IN
Cell Developer Toolbox software. Cells were classified as “dead” if they showed >30% overlap
2
4

ACCEPTED MANUSCRIPT
of the two stains. Statistical analyses to determine IC50 were performed using GraphPad
software.
For in vitro ROS assays, cells were seeded at 50,000 cells/well in a 96-well plate (Grener
Bio-One). Following a 24 hr incubation period, the culture medium was aspirated off and 100 µL
of 10 µM 2′,7′-dichlorofluorescin diacetate (Sigma Aldrich) in sterile phosphate buffered saline
(PBS) was added to each well and subsequently incubated for 45 minutes. The PBS was removed
via aspiration and fresh media with 10 µM of the respective Cu complexes was added to each
well. Wells containing 1 and 10 µM H O were used as positive controls to establish
2
2
concentration dependence. After 1.5 hours the plates were imaged using the IN Cell Analyzer
000 with five images and 10× magnification. Cell counts were then determined via the IN Cell
1
Developer Toolbox software. Due to scatter in the data associated with incomplete well coverage
by the images, outliers were eliminated before calculation of the average number of fluorescent
cells and statistical significance was determined by Student’s t-test.
DNA electrophoresis gels. Plasmid DNA (pet22b, Novagen, 5493bp) was isolated and purified
using a Qiagen MaxiPrep Kit. A 20 µM stock solution of plasmid in water was used as the
starting material for all successive solutions. Stock solutions of each ligand and copper complex
in DMSO (10 mM) were prepared to facilitate solubility in aqueous media. Fresh stock solutions
(8 µM) of each ligand and complex were prepared in 10 mM MES buffer, with 150 mM NaCl,
immediately prior to sample preparation. Using these aqueous stock solutions, all samples
consisted of 10 µM DNA and 2 µM ligand or copper complex. In addition, buffer, sodium
ascorbate (20 µM), superoxide dismutase (SOD) (10 Units), and/or catalase (1 Unit) were added
to the appropriate samples such that the final volume was 20 µL.
2
5

ACCEPTED MANUSCRIPT
Samples were incubated at 37 °C for 30 min, after which time the reactions were
quenched with 2.2 µL of a 10× loading buffer containing 50% (v/v) glycerol, 100 mM
Na EDTA•2H O (EDTA = ethylenediaminetetraacetic acid), 1% (v/v) sodium dodecyl sulfate
2
2
and 0.1% bromophenol blue (v/v) in deionized water. Samples were then stored at −4 °C until
immediately prior to loading onto agarose gel. Electrophoresis was performed on an agarose gel
(0.8%) in a Life Technologies Horizon 58 apparatus for 60 min at 90 V in 1X tris-acetate-EDTA
buffer. The completed gels were incubated for 45 min in a 5 µg/mL solution of ethidium
bromide, washed for in distilled water, and imaged under UV light.
UV-Vis Experiments. Measurements were conducted on a Cary 100 Bio UV-Visible
spectrophotometer with a water temperature controlled thermostat 66 Multicell Block Peltier
cooling module. Prior to the preparation of the final samples, stock solutions of each compound
were prepared in DMSO to enhance solubility in aqueous media. All spectra were collected at 37
°
C.
Complexes in Buffer. To 746.25 µL of MES buffer (pH 6.5) was added 3.75 µL of the 10 mM
stock solution of each complex in DMSO to give 50 µM solutions of each compound. UV-Vis
spectra were collected every 5 min over the course of one hour.
DNA Titrations. Calf-thymus DNA (ctDNA, Invitrogen, Thermo Fisher Scientific) was supplied
as 10 mg/mL solutions. A series of solutions were prepared from DMSO stock solutions of the
copper complexes (5 mM) to give samples containing copper complex (50 µM) and ctDNA (0
µM, 50 µM, 150 µM, 250 µM, 350 µM). Cu(pbzF ) required the preparation of a 100 µM
2
sample because the absorbance of the 350 µM significantly overlapped with that of the ctDNA
2
6

ACCEPTED MANUSCRIPT
and could not be determined. Samples were incubated for 40 min at 37 °C and the absorption
spectrum collected.
EPR Measurements and Simulations. EPR spectra were collected at X-band (9.3−9.4 GHz)
using a Bruker EMXplus spectrometer with a PremiumX microwave bridge and HS resonator.
Using a Bruker ER 4112HV helium temperature-control system and continuous-flow cryostat
with liquid nitrogen, spectra were collect at 100 K. To facilitate the comparison of samples, the
concentration of complexes and spectroscopic parameters were unchanged for each experiment.
A quartz-insert tube holder, which is a part of the Bruker cryostat system, ensures reproducible
sample placement within the EPR resonator. Automatic tuning of the spectrometer gave a Q-
factor of 6500 ± 10%, indicating limited variation in instrument sensitivity.
Preparations of EPR Samples. For all samples, 10 mM stock solutions of each compound were
prepared in DMSO, to facilitate subsequent dissolution in aqueous buffered solutions.
Complexes in Buffer. A 500 µM solution of each complex was prepared in MES buffer pH 6.5
using the DMSO stocks. Aliquots of 210 µL were promptly mixed with 90 µL of glycerol, and
immediately frozen in liquid nitrogen.
Complexes with human serum albumin. A 600 µL solution of 250 µM HSA with 500 µM of each
complex was prepared in MES buffer. These solutions were diluted to a final volume of 4 mL
then incubated for one hour at 37 °C. Following incubation, samples were concentrated down to
a volume of 200 µL using an Amicon centrifugal filter unit (30 kDa molecular-weight cutoff) by
centrifuging at 4500 rpm for 30 min at 4 °C. The filtered product was collected and mixed with
9
0 µL of glycerol and 10 µL of MES buffer to give a final volume of 300 µL. Samples were then
transferred to EPR tubes and frozen in liquid nitrogen.
2
7

ACCEPTED MANUSCRIPT
Complexes with ctDNA. A 210 µL solution containing 500 µM of each complex in MES buffer,
and 2 mM of ctDNA, was incubated for one hour at 37 °C. These solutions were then mixed with
9
0 µL of glycerol, transferred to EPR tubes, and frozen in liquid nitrogen.
ABBREVIATIONS
ctDNA
DCF
Calf-thymus DNA
Dichlorofluorescein
DCFH
DCFH-DA
DMF
DMSO
EDTA
EPR
Dichlorodihydrofluorescein
2′,7′-dichlorofluorescin diacetate
Dimethylformamide
Dimethyl sulfoxide
Ethylenediaminetetraacetic acid
Electron paramagnetic resonance
Fourier transform infrared spectroscopy
Human serum albumin
FT-IR
HSA
IC50
L
Inhibitory concentration 50%
Linear
MALDI-TOF
MES
NSCLC
OC
Matrix-assisted laser desorption/ionization - time of flight
2-(N-morpholino)ethanesulfonic acid
Non-small cell lung cancer
Open circular
pbzBz
pbzF
1-(phenylmethyl)-2-(2-pyridinyl)benzimidazole
1-[(4-fluorphenyl)methyl)-2-(2-pyridinyl)benzimidazole
1-[(3, 5-difluorphenyl)methyl)-2-(2-pyridinyl)benzimidazole
1-[(2,3,4,5,6-pentafluorphenyl)methyl)-2-(2-pyridinyl)benzimidazole
2-(2-pyridinyl)benzimidazole
1-methyl-2-(2-pyridinyl)benzimidazole
Phenanthroline
pbzF2
pbzF5
pbzH
pbzMe
phen
PPA
Polyphosphoric acid
ROS
Reactive oxygen species
SC
Supercoiled
SOD
Superoxide dismutase
ACKNOWLEDGEMENTS
Financial support was provided by The Natural Sciences and Engineering Research Council of
Canada (NSERC) and Simon Fraser University (SFU). K.E.P. acknowledges additional support
2
8

ACCEPTED MANUSCRIPT
from NSERC through a Vanier Canada Graduate Scholarship. P.H.L. acknowledges support
from Mitacs Canada through a Globalink Research Award. The authors are extremely grateful to
Marcel Bally at the BC Cancer Research Agency (Vancouver, BC, Canada) for providing the
A549 cells, and for access to tissue culture and cell imaging facilities. The authors also wish to
thank Robert Young (SFU) for access to GraphPad software, and Jeffrey Warren (SFU) for use
of electrochemical facilities.
REFERENCES
[
[
1]
2]
C. Marzano, M. Pellei, F. Tisato, C. Santini, Anticancer Agents Med. Chem. 2009, 9,
85-211.
C. Santini, M. Pellei, V. Gandin, M. Porchia, F. Tisato, C. Marzano, Chem. Rev. 2014,
14, 815-862.
1
1
[
[
3]
4]
S. Tardito, L. Marchiò, Curr. Med. Chem. 2009, 16, 1325-1348.
F. Tisato, C. Marzano, M. Porchia, M. Pellei, C. Santini, Med. Res. Rev. 2010, 30, 708-
7
49.
I. Ali, W. A. Wani, K. Saleem, A. Haque, Anticancer Agents Med. Chem. 2013, 13, 296-
06.
[
5]
3
[
[
[
[
[
[
6]
7]
8]
9]
A. Bergamo, G. Sava, Dalton Trans. 2011, 40, 7817-7823.
I. Kostova, Curr. Med. Chem. 2006, 13, 1085-1107.
T. Zou, C. T. Lum, C.-N. Lok, J.-J. Zhang, C.-M. Che, Chem. Soc. Rev. 2015.
W. Mertz, Science 1981, 213, 1332-1338.
10] L. M. Gaetke, C. K. Chow, Toxicology 2003, 189, 147-163.
11] B. Halliwell, J. M. C. Gutteridge, in Methods in Enzymology, Vol. 86 (Eds.: L. Packer,
A. N. Glazer), Academic Press, 1990, pp. 1-85.
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9