Molecular mechanisms of the biological activity of the anticancer drug elesclomol and its complexes with Cu(II), Ni(II) and Pt(II)

Article abstract of DOI:10.1016/j.jinorgbio.2013.04.013

The bis(thiohydrazide) amide elesclomol has extremely potent antiproliferative activity and is currently in clinical trials as an anticancer agent. Elesclomol strongly binds copper and may be exerting its cell growth inhibitory effects by generating copper-mediated oxidative stress. Nickel(II) and platinum(II) complexes of elesclomol were synthesized and characterized in order to investigate if these biologically redox inactive metal complexes could also inhibit cell growth. The nickel(II)-elesclomol and platinum(II) elesclomol complexes were 34- and 1040-fold less potent than the copper(II)-elesclomol complex towards human leukemia K562 cells. These results support the conclusion that a redox active metal is required for elesclomol to exert its cell growth inhibitory activity. Copper(II)-elesclomol was also shownto efficiently oxidize ascorbic acid at physiological ascorbic acid concentrations. Reoxidation of the copper(I) thus produced would lead to production of damaging reactive oxygen species. An X-ray crystallographic structure determination of copper(II)-elesclomol showed that it formed a 1:1 neutral complex with a distorted square planar structure. The kinetics and equilibria of the competition reaction of the strong copper(II) chelator TRIEN with copper(II)-elesclomol were studied spectrophotometrically under physiological conditions. These results showed elesclomol bound copper(II) with a conditional stability constant 24-fold larger than TRIEN. A log stability constant of 24.2 was thus indirectly determined for the copper(II)-elesclomol complex.

Full text of DOI:10.1016/j.jinorgbio.2013.04.013

Journal of Inorganic Biochemistry 126 (2013) 1–6
Contents lists available at SciVerse ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Molecular mechanisms of the biological activity of the anticancer drug
elesclomol and its complexes with Cu(II), Ni(II) and Pt(II)
⁎
Arun A. Yadav, Daywin Patel, Xing Wu, Brian B. Hasinoff
Faculty of Pharmacy, Apotex Centre, 750 McDermot Avenue, University of Manitoba, Winnipeg, Manitoba R3E 0T5, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
The bis(thiohydrazide) amide elesclomol has extremely potent antiproliferative activity and is currently in
clinical trials as an anticancer agent. Elesclomol strongly binds copper and may be exerting its cell growth
inhibitory effects by generating copper-mediated oxidative stress. Nickel(II) and platinum(II) complexes of
elesclomol were synthesized and characterized in order to investigate if these biologically redox inactive metal
complexes could also inhibit cell growth. The nickel(II)-elesclomol and platinum(II) elesclomol complexes were
34- and 1040-fold less potent than the copper(II)-elesclomol complex towards human leukemia K562 cells.
These results support the conclusion that a redox active metal is required for elesclomol to exert its cell growth
inhibitory activity. Copper(II)-elesclomol was also shown to efficiently oxidize ascorbic acid at physiological ascorbic
acid concentrations. Reoxidation of the copper(I) thus produced would lead to production of damaging reactive
oxygen species. An X-ray crystallographic structure determination of copper(II)-elesclomol showed that it formed
a 1:1 neutral complex with a distorted square planar structure. The kinetics and equilibria of the competition
reaction of the strong copper(II) chelator TRIEN with copper(II)-elesclomol were studied spectrophotometrically
under physiological conditions. These results showed elesclomol bound copper(II) with a conditional stability
constant 24-fold larger than TRIEN. A log stability constant of 24.2 was thus indirectly determined for the
copper(II)-elesclomol complex.
Received 6 February 2013
Received in revised form 23 April 2013
Accepted 23 April 2013
Available online 28 April 2013
Keywords:
Elesclomol
Copper
Nickel
Platinum
Oxidative stress
Bis(thiohydrazide) amide
© 2013 Elsevier Inc. All rights reserved.
1. Introduction
conclusion breast cancer MCF7 cells with a compromised ability to
repair oxidative DNA damage displayed increased sensitivity to
The anticancer drug elesclomol (4) (Fig. 1) has undergone
randomized Phase 2 and 3 clinical trials for the treatment of a variety
of cancers [1–3]. Currently it is in phase 2 and 3 clinical trials for
the treatment of acute myeloid leukemia, gynecological and other
types of cancers (http://www.clinicaltrials.gov). In addition a study
with elesclomol plus paclitaxel versus paclitaxel alone has just been
published [4]. This study showed that patients on the elesclomol plus
paclitaxel arm with normal serum lactate dehydrogenase levels had
improved outcomes versus patients with high lactate dehydrogenase
levels [4]. It was speculated that high lactate dehydrogenase levels
may be a reflection of the state of tumor hypoxia. Because elesclomol
may target metabolically active mitochondria it may be most active in
well-oxygenated non-hypoxic cells dependent on oxidative phosphory-
lation rather than on glycolysis [4]. Elesclomol is an extremely potent
inhibitor of the growth of cancer cells in culture and typically inhibits
cell growth at low nanomolar concentrations [1,2,5]. Like the structurally
related thiosemicarbazones [6,7], elesclomol is a strong copper(II)
chelator [1].
elesclomol [5]. It has been shown [1,2] that elesclomol scavenges
copper from the culture medium and selectively transports copper
to the mitochondria which induces oxidative stress. Thus, elesclomol
may be inhibiting cell growth by specifically targeting the mitochondria
[1,2]. Cu(II)-bis(thiosemicarbazone) complexes can be reduced by bio-
logical reductants and have been shown to transfer the Cu+ produced
to the copper-binding proteins Atx1 and Ctr1c, thus suggesting a mech-
anism by which these complexes cause cellular retention of copper
[10]. It has also been shown that the Cu(II)-bis(thiosemicarbazone)
complexes are readily taken up by cells and that this uptake is driven
by intracellular aggregation of the highly insoluble complex [11]. The
sensitivity of yeast with homozygous gene deletions to treatment
with elesclomol suggests that it exerts its cytotoxicity by a distinct
mechanism of action which is unlike that of any other anticancer
drug [2]. Thus given that elesclomol may represent a whole new class
of anticancer drugs whose activity may be mediated through complex
formation with Cu2+, it is important that its mechanism of action be
elucidated.
It has been proposed that elesclomol induces cancer cell apoptosis
through the induction of oxidative stress [5,8,9]. In support of this
In order to test whether elesclomol exerted its growth inhibitory
effects by inducing oxidative stress mediated through its Cu2+ complex
we decided to prepare and characterize biologically redox inactive
Ni2+ and Pt2+ complexes of elesclomol in order to compare their cell
growth inhibitory activity with that of Cu(II)-elesclomol. Additionally,
⁎
Corresponding author. Tel.: +1 204 474 8325; fax: +1 204 474 7617.
E-mail address: B_Hasinoff@UManitoba.ca (B.B. Hasinoff).
0162-0134/$ – see front matter © 2013 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.jinorgbio.2013.04.013

2
A.A. Yadav et al. / Journal of Inorganic Biochemistry 126 (2013) 1–6
Fig. 1. Structure of elesclomol (4) and the reaction scheme for its synthesis and intermediates for the preparation of its complexes with Cu2+, Ni2+ and Pt2+ (5a, 5b and 5c, respectively).
the synthesis and characterization of elesclomol (4) and the Cu(II)-
elesclomol (5a) complex have only been incompletely described in
the patent literature [12]. Thus, elesclomol (4), Cu(II)-elesclomol (5a),
Ni(II)-elesclomol (5b), and Pt(II)-elesclomol (5c) (Fig. 1) were synthe-
sized and fully characterized by NMR, electrospray ionization high
resolution mass spectrometry HRMS (ESI) and for Cu(II)-elesclomol
by X-ray structural analysis. The results are discussed in light of mech-
anisms demonstrated for structurally related copper thiosemicarbazone
complexes and are put into perspective as putative inducers of oxida-
tive stress.
compounds were visualized by quenching of fluorescence by UV light
(254 nm).
2.2.2. Synthesis of N-methylbenzothiohydrazide (2)
NaOH (0.58 g, 14.7 mmol) was dissolved in H2O (5 ml) and then
cooled to 0 °C. S-(thiobenzoyl)thioglycolic acid 1 (1.2 g, 5.65 mmol)
was added and the mixture was stirred until all solids dissolved
(Fig. 1). The temperature was maintained between 0 and 10 °C. In a
separate flask, the methylhydrazine trifluoroacetic acid salt (1.12 g,
7.35 mmol) was dissolved in H2O (5 ml). The solution was cooled
to 0–5 °C and then the solution containing 1 was slowly added.
The temperature of the reaction mixture was maintained between
0–10 °C. After 30 min the reaction was monitored by TLC to determine
completeness. Upon completion, dichloromethane (100 ml) was added
to the reaction mixture. The organic layer was separated from the aque-
ous layer and then washed with 0.1 M aqueous NaOH. The organic ex-
tract was concentrated by half. Heptane was then added to precipitate
out the desired product which was then filtered and dried to constant
weight to give the title compound 2 (0.8 g, 86%). 1H NMR (CDCl3,
300 MHz): δ 7.33–7.37(m, 5H), 5.99 (s, 2H), 3.33 (s, 3H); 13C NMR
(CDCl3): δ 189.9, 141.2, 129.0, 128.4, 126.2, 42.8.
2. Experimental
2.1. Materials, spectrophotometry, cell culture and growth inhibition assays
Unless specified, all reagents were obtained from Sigma-Aldrich
(Oakville, Canada). The spectrophotometric experiments were carried
out in 1 cm or 0.1 cm quartz cells on a Cary 300 double beam
spectrophotometer with a thermostated cell holder. Human leukemia
K562 cells, obtained from the American Type Culture Collection,
were maintained as suspension cultures in αMEM (minimal essential
medium alpha) (Invitrogen, Burlington, Canada) containing 10% fetal
calf serum. The spectrophotometric 96-well plate cell (5 × 104 cell/ml,
0.1 ml/well) growth inhibition 3-(4,5-dimethylthiazol-2-yl)-5(3-
carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS)
CellTiter 96 AQueous One Solution Cell Proliferation assay (Promega,
Madison, WI), which measures the ability of the cells to enzymatically
reduce MTS after drug treatment, has been described previously [13].
The compounds tested were dissolved in DMSO and the final concentra-
tion of DMSO did not exceed an amount (typically 0.5% or less) that
had any detectable effect on cell growth. The cells were incubated
with the drugs for 72 h and then assayed with MTS. The IC50 values
for cell growth inhibition were measured by fitting the absorbance-
drug concentration data to a four-parameter logistic equation as we
described [14]. The errors quoted are the S.E.M.s.
2.2.3. Synthesis of elesclomol (4)
N-methylbenzothiohydrazide (2) (0.8 g, 4.82 mmol) was dissolved
in ethyl acetate (5 ml) and triethylamine (1.34 ml, 9.64 mmol). The
solution was cooled to maintain a temperature of 0–10 °C. Malonyl
chloride (0.34 g, 2.41 mmol) was dissolved in ethyl acetate (1 ml)
and was added to the solution containing the hydrazide at a rate that
maintained the temperature between 0 and 10 °C. The reaction mixture
was stirred for about 30 min and then quenched with H2O. The organic
layer was separated and concentrated under vacuum to give the crude
product which was purified by column chromatography on silica gel
to afford the title compound 4 (1.25 g, 65%); mp: 192–195 °C. 1H
NMR (CDCl3): δ 11.03 (s, 2H), 7.21–7.43 (m, 10H), 3.63 (s, 6H), 2.51
(s, 2H); 13C NMR (CDCl3): δ 202.5, 163.1, 141.7, 128.7, 128.2, 127.3,
125.9, 125.8, 42.9, 38.7.
2.2. Chemistry
2.2.4. Synthesis of the Cu(II)-elesclomol complex (5a)
To a stirred solution of elesclomol (4) (200 mg, 0.5 mmol) in
ethanol (5.0 ml), CuCl2·2H2O was added in one portion. The mixture
was stirred at room temperature for 20 min. Water was then added,
and the precipitated solid was collected by filtration. The solid was
taken up in methylene chloride. The resulting solution was washed
with water (2×), dried (Na2SO4), filtered and concentrated to
give the crude solid. The solid was washed with acetone to give
the dark red title compound 5a; (185 mg, 90%); mp: 198–202 °C
(decomp); lit. 198–202 °C [15]. Single crystals for X-ray analysis
2.2.1. General
1H and 13C NMR spectra were recorded on a Bruker Avance NMR
spectrometer operating at 300 MHz for 1H NMR and 75 MHz for 13
C
NMR, respectively, in CDCl3 or DMSO-d6. Melting points were
uncorrected. The high resolution mass spectra were run on a Bruker
microTOF Focus mass spectrometer (Fremont, CA) using electrospray
ionization. TLC was performed on plastic-backed plates bearing 200 μm
silica gel 60 F254 (Silicycle, Quebec City, Canada). Where applicable

A.A. Yadav et al. / Journal of Inorganic Biochemistry 126 (2013) 1–6
3
were obtained by recrystallization from acetonitrile at room tem-
perature overnight. HRMS (ESI) C19H18CuN4O2S2 m/z (M)+: calcd
461.0162, obsd 461.0141.
before, the synthesis of compounds 4, 5a and 5b have been briefly de-
scribed in the patent literature, albeit with only minimal characteriza-
tion [12]. Elesclomol and its metal complexes 5a, 5b and 5c were fully
characterized by NMR, HRMS (ESI) and for 5a by X-ray structural
analysis.
2.2.5. Synthesis of the Ni(II)-elesclomol complex (5b)
Compound 5b was prepared as described for compound 5a by
reaction with 4 and NiCl2·6H2O and afforded a green solid; mp:
321–325 °C. 1H NMR (CDCl3): δ 7.59–7.44 (m, 10H), 3.61 (s, 6H), 3.60
(s, 2H); 13C NMR (CDCl3): δ 183.9, 172.6, 133.1, 131.9, 128.9, 128.5,
47.2, 44.6; HRMS (ESI) for C19H18N4NaNiO2S2 m/z (M + Na)+: calcd
479.0117, obsd 479.0117.
3.2. X-ray structure analysis of Cu(II)-elesclomol
The ORTEP structure of Cu(II)-elesclomol is shown in Fig. 2. Coordi-
nation of the nitrogen and sulfur atoms about the Cu2+ atom gave a
structure that was only approximately square planar. For example,
the S\Cu\N and S\Cu\S bond angles were 86.66(6) and 97.10(3)°,
respectively. Similarly, the S\Cu\N and S\Cu\N bond angles were
160.47(6) and 164.93(6)°, respectively. The Cu\S and Cu\N bond
distances of 2.248(7) and 1.9548(19) Å were similar to those observed
for [Cu(ATSM)], a Cu(II)-bis(thiosemicarbazone) complex (2.237 and
1.959 Å, respectively) [16]. These values can also be compared to
[Cu(AATSM)], the acetylacetonate bis(thiosemicarbazone) complex
of Cu2+, with a Cu\S and Cu\N bond distances of 2.2469(5) and
1.9737(16), respectively, and with S\Cu\N and S\Cu\S bond angles
of 86.79(5) and 91.728(19)°, respectively [17].
2.2.6. Synthesis of the Pt(II)-elesclomol complex (5c)
Compound 5c was prepared as described for compound 5a by reac-
tion with 4 and potassium tetrachloroplatinate(II) using ethanol:H2O
(1:1) as solvent and afforded a yellow-orange solid; mp: 314–317 °C.
1H NMR (CDCl3): δ 7.62–7.48 (m, 10H), 3.83 (s, 2H), 3.70 (s, 6H); 13C
NMR (CDCl3): δ 180.6, 174.4, 134.0, 131.7, 129.0, 128.3, 47.9, 46.9;
HRMS (ESI) for C19H18N4NaO2PtS2 m/z (M + Na)+: calcd 616.0411,
obsd 616.0440.
2.2.7. X-ray structure determination of the Cu(II)-elesclomol complex (5a)
X-ray data and crystal structure analysis for the Cu(II)-elesclomol
complex were performed by the X-ray Laboratory, Department
of Chemistry, University of Toronto. Crystal data were collected on
a Nonius Kappa-CCD diffractometer using monochromated Mo Kα
radiation. Crystallographic data for the Cu(II)-elesclomol complex
has been deposited in the Cambridge Crystallographic Data Centre
as supplementary publication CCDC-915646. A copy of the structure
can be obtained free of charge by application to CCDC, 12 Union
Road, Cambridge CB2 1EZ, UK.
3.3. Spectrophotometric titration of elesclomol with Cu2+
Elesclomol was titrated with CuCl2 and the titration monitored
by spectrophotometry in Tris buffer (10 mM, pH 7.4, 37 °C) that
contained 25% (v/v) DMSO in order to characterize complex forma-
tion under conditions that approximated those found biologically.
It was found necessary to carry out the titration in 25% (v/v) DMSO
due to the low aqueous solubility of elesclomol and Cu(II)-elesclomol.
As shown in Fig. 3A the absorbance at the 355 nm shoulder systemati-
cally increased with the addition of Cu2+. A tight isosbestic point was
seen at 285 nm, at least until Cu2+ was in molar excess. These results
were consistent with only two absorbing species in solution, elesclomol
and Cu(II)-elesclomol. The least square fits of the two linear segments
of the plot of absorbance vs. Cu2+ concentration (Fig. 3B) intersected
at 21 μM or at a Cu2+:elesclomol ratio of 1:1.05. The fact that the plot
had no significant curvature in the equivalence region was consistent
with the formation of a very strong complex. Also, in accord with the
X-ray structure (Fig. 2) these results were consistent with the formation
of a 1:1 complex.
3. Results
3.1. Synthesis of elesclomol and its complexes with Cu2+, Ni2+ and Pt2+
Elesclomol was synthesized in two steps (Fig. 1) using commercially
available S-(thiobenzoyl)thioglycolic acid 1 as a starting material.
Because methylhydrazine was not commercially available in Canada
it was prepared from the acid hydrolysis of 1-boc-1-methylhydrazine
using trifluoroacetic acid. Condensation of 1 with methylhydrazine
under basic conditions smoothly produced the advanced intermediate
N-methylbenzothiohydrazide 2. The hydrazide was then reacted with
malonyl chloride 3 to give elesclomol 4. The transition metal complexes
were formed by reacting equimolar amounts of the divalent metal salts
with 4 to give 5a, 5b and 5c (Fig. 1). While 5c has not been described
A spectrophotometric experiment to test whether Cu2+ could
displace Ni2+ from the Ni(II)-elesclomol complex was also carried
out by recording UV–vis spectra for 20 h of a 20 μM solution of
Ni(II)-elesclomol to which 200 μM CuCl2 had been added. The lack
of any measurable increase in absorbance at 355 nm corresponding
Fig. 2. ORTEP view of the molecular structure of the Cu(II)-elesclomol complex (ellipsoids at 30%) obtained from an X-ray crystallographic determination. No solvent molecules
were detected in the structure. Selected bond lengths (Å): Cu1\S1 2.248(7), Cu1\N1 1.9548(19). Selected bond angles (°): S1\Cu1\N1 86.66(6), S1\Cu1\S2 97.10(3),
S1\Cu1\N2 160.47(6) and S2\Cu1\N1 164.93(6).

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A.A. Yadav et al. / Journal of Inorganic Biochemistry 126 (2013) 1–6
Fig. 3. Spectrophotometric titration of elesclomol by Cu2+. A. UV–vis spectral changes observed when microliter amounts of CuCl2 were added to 20 μM elesclomol in Tris buffer
(10 mM, pH 7.4, 25% DMSO) at 37 °C in a 1 cm spectrophotometer cell. As indicated by the arrow, the lowest trace at the 355 nm shoulder increased in absorbance with the
successive total concentrations of Cu2+ indicated. B. Spectrophotometric titration of elesclomol by Cu2+ at 355 nm. The intersection of the least squares calculated straight lines
occurred at 21 μM which was consistent with the formation of a 1:1 complex between Cu2+ and elesclomol.
to that seen in Fig. 3A indicated that Cu2+ did not displace Ni2+ from
Ni(II)-elesclomol to produce active Cu(II)-elesclomol.
TRIEN with Cu(II)-elesclomol proceeded relatively quickly, (e.g. t1/2 of
3.3 min at 100 μM TRIEN). Even when TRIEN was in 100-fold excess
(2000 μM), it still only partially displaced Cu2+ from Cu(II)-elesclomol.
The stability of the Cu(II)-elesclomol complex relative to that of
Cu(II)-TRIEN can be obtained from the concentrations of the various
species at equilibrium using a spectrophotometric method that has
been described for a copper binding peptide [20]. The small contribu-
tions to the absorbance of free elesclomol and Cu(II)-TRIEN are indicated
by the broken horizontal line on Fig. 4.
Ligands that strongly bind Cu2+ often also bind Fe3+ or Fe2+. It
had been reported in an ESI mass spectrometry study that elesclomol
may be binding to Fe2+ [18]. However, no evidence for binding in solu-
tion phase was seen during chromatographic separation [18]. Thus spec-
trophotometric experiments were carried out to determine if elesclomol
bound to either Fe2+ or Fe3+. The lack of any significant UV–vis spectral
changes upon the addition of either ferrous ammonium sulfate or FeCl3
in 3-fold excess over 10 μM elesclomol in Tris buffer (pH 7.4) was
consistent with Fe3+ and Fe2+ having low affinity for elesclomol.
In the expressions below Cu–E and E are Cu(II)-elesclomol and
elesclomol, respectively, and Cu-TRIEN and TRIEN are Cu(II)-TRIEN
and TRIEN, respectively. K′
and K′
are the pH-dependent
Cu-E
Cu-TRIEN
conditional stability constants in equilibrium expressions (3) and
(4), respectively. In the equilibrium expressions the concentrations
refer to the total concentration of all protonated and non-protonated
form of each species. The expressions for the competing equilibria
(1) and (2) and the conditional stability constant expressions (3)
and (4) are:
3.4. Kinetic and equilibrium competition reactions of TRIEN with
Cu(II)-elesclomol
The competition reaction of TRIEN (triethylenetetramine) with
Cu(II)-elesclomol (20 μM) was followed spectrophotometrically in
Tris buffer (10 mM, pH 7.4, 37 °C, 25% (v/v) DMSO) both in order to
examine the lability of the Cu(II)-elesclomol complex in competition
with this nitrogen-containing ligand, and to obtain a measure of
the conditional stability constant for Cu(II)-elesclomol, relative to
that of TRIEN. TRIEN binds Cu2+ very strongly with a stability constant
KCu-TRIEN (ß) value of 1020.0 M−1 [19]. As shown in Fig. 4 the reaction of
Cu−E⇋Cu þ E
ð1Þ
ð2Þ
Cu−TRIEN⇋Cu þ TRIEN
K′Cu−E ¼ ½Cu−Eꢀ=½Cuꢀ½Eꢀ
ð3Þ
ð4Þ
K′Cu−TRIEN ¼ ½Cu−TRIENꢀ=½Cuꢀ½TRIENꢀ:
Because both TRIEN and elesclomol bind Cu2+ strongly, at equilib-
rium the free Cu2+ concentration is very low relative to the other
equilibrium concentrations. Thus the ratio of the two conditional sta-
bility constants from Eqs. (3) and (4) gives:
K′Cu−E=K′Cu−TRIEN ¼ ½Cu−Eꢀ½TRIENꢀ=½Cu−TRIENꢀ½Eꢀ:
ð5Þ
From the stoichiometry of reactions (1) and (2) and the starting
and equilibrium concentrations calculated from the data in Fig. 4
it was possible to calculate values for the ratio K′Cu-E/K′Cu-TRIEN. The
average K′Cu-E/K′
trations was 24
ratio calculated from the four TRIEN concen-
Cu-TRIEN
1. Thus, at pH 7.4 elesclomol binds Cu2+ 24-fold
more strongly than does TRIEN.
The pH-dependent conditional stability constant K′
reaction (2) at pH 7.4 can be calculated from the successive pKa's
of protonated TRIEN of 3.59, 6.77, 9.22 and 9.81 [19] and the fraction
for
Cu-TRIEN
Fig. 4. Kinetics of the reaction of various concentrations of TRIEN with Cu(II)-elesclomol
(20 μM) in Tris buffer (10 mM, pH 7.4, 25% DMSO) at 37 °C in a 1 cm spectrophotometer
cell followed at 355 nm. Increasing concentrations of TRIEN displaced an increasing amount
of Cu2+ from the Cu(II)-elesclomol complex at equilibrium. The broken dashed line is the
absorbance that would be achieved for 100% removal of Cu2+ from Cu(II)-elesclomol.
α
TRIEN of TRIEN present in its neutral bound form. The fraction αTRIEN
at pH 7.4 is 4.7 × 10−5 which yielded a conditional stability constant

A.A. Yadav et al. / Journal of Inorganic Biochemistry 126 (2013) 1–6
5
K′
Cu-TRIEN
= αTRIEN × KCu-TRIEN = 1015.7 M−1. The conditional stability
MDA-MB345 cell growth with an IC50 of 30 nM [1]. The lack of activity
constant for K′Cu-E for Cu(II)-elesclomol was 24-fold higher, and
thus it was calculated from the average K′Cu-E/K′ ratio to have
of the Ni(II)-elesclomol complex was also noted in an abstract [22].
Cu-TRIEN
a value of K′
= 24 × K′
= 1017.1 M−1. This value compares
3.6. Comparison of the oxidation of ascorbic acid by Cu(II)-elesclomol
and Cu2+
Cu-E
Cu-TRIEN
to a conditional stability constant of 1018.8 M−1 at pH 7.4 determined
for a 3-ethoxy-2-oxobutyraldehyde bis(thiosemicarbazato) copper(II)
complex [21].
Cu2+ and Cu2+ chelates are well known to be able to efficiently
catalyze the oxidation of ascorbic acid [23]. The first step in the
copper-catalyzed oxidation would initially yield the ascorbate radical
which would in turn react with O2 to produce dehydroascorbic acid.
At longer times dehydroascorbic acid is oxidized further to other
more highly oxidized species [24]. In this redox cycling system the
reaction of O2 with Cu+ would directly yield superoxide anion, which
would in turn dismutate to H2O2. The kinetics of the oxidation of
1 mM ascorbic acid was followed by monitoring the first few percent
of the reaction in Tris buffer (10 mM, pH 7.4) in 25% DMSO at 25 °C
in a 0.1 cm spectrophotometer cell at the peak maximum of 268 nm.
A 0.1 cm cell was used to reduce the background absorbance of the
25% DMSO present in the buffer. The initial velocities were calculated
from the rate of decrease in absorbance and the change in extinction
coefficient observed under our reaction conditions. A linear least squares
fit of the initial velocity data of Fig. 6 showed that Cu2+ oxidized ascorbic
acid 14.6-fold faster than did Cu(II)-elesclomol.
From a distribution of the protonated species for elesclomol it is
possible to calculate a stability constant KCu-E (ß) for reaction (1).
The pKa's for elesclomol have not been measured experimentally
and likely would be difficult to measure given its low micromolar
solubility in aqueous media. Thus MarvinSketch and its associated
calculator plugins were used to calculate the elesclomol pKa values
and the major protonated microspecies present at pH 7.4 (Marvin
version 5.4, 2010, ChemAxon, http://www.chemaxon.com). The pKa
values for elesclomol were thus calculated to be 10.45 and 11.41.
From these values the fraction αE of the dianionic elesclomol species
present at pH 7.4 was calculated to be 7.8 × 10−8. Therefore the
stability constant KCu-E can be calculated from the conditional sta-
bility constant and αE to yield a value of KCu-E = K′Cu-E/αE
=
1017.1 M−1/7.8 × 10−8 = 1024.2 M−1
.
3.5. Comparison of the cell growth inhibitory properties of elesclomol
and its complexes with Cu2+, Ni2+ and Pt2+
4. Discussion
Cu2+ complexes may be reduced under biological conditions both
enzymatically and by non-enzymatic reductants such as ascorbic acid,
glutathione or NADH. However, complexes of Ni2+ or Pt2+ would
typically not be expected to be reduced under biological conditions.
Thus, a comparison of the K562 cell growth inhibitory properties
of elesclomol and its complexes with Cu2+, Ni2+ and Pt2+ should
shed light on whether a biologically reducible redox active metal ion
is required for the cell growth inhibitory effects of elesclomol and its
complexes. The cell growth inhibitory IC50 values for elesclomol,
Cu(II)-elesclomol, Ni(II)-elesclomol and Pt(II)-elesclomol were deter-
mined from the data shown in Fig. 5. K562 cell growth was inhibited
with IC50 values of 8.2, 5.4, 185 and 5600 nM for elesclomol, Cu(II)-
elesclomol, Ni(II)-elesclomol and Pt(II)-elesclomol, respectively. Both
elesclomol and Cu(II)-elesclomol inhibited K562 cell growth with
similar IC50 values in the low nM range. However, in contrast, both
Ni(II)-elesclomol and Pt(II)-elesclomol inhibited cell growth much
less potently with IC50 values that were 34- and 1040-fold larger
than for Cu(II)-elesclomol. For comparison a previous study showed
that a 48 h treatment with Cu(II)-elesclomol inhibited breast cancer
The results of this study showed that elesclomol formed complexes
with Cu2+, Ni2+ and Pt2+. The X-ray structure of Cu(II)-elesclomol
(Fig. 2) showed that binding of Cu2+ occurred with the loss of two
protons from the N1 and N2 atoms (Fig. 2) of elesclomol to produce a
neutral Cu(II)-elesclomol complex. Cu(II)-elesclomol had bond lengths
and bond angles similar to Cu(II) thiosemicarbazone complexes [7,16].
A spectrophotometric titration of elesclomol by Cu2+ at pH 7.4 also
showed the formation of a strongly bound 1:1 complex in pH 7.4 Tris
buffer.
The copper(II) binding ligand TRIEN was shown to be able to
only partially displace Cu2+ from its complex with elesclomol even
in large excess. This indicated that at pH 7.4 elesclomol bound Cu2+
much more strongly than did TRIEN. From the K′Cu-E/K′
ratios
Cu-TRIEN
calculated elesclomol bound Cu2+ 24-fold more strongly than did
TRIEN at pH 7.4. The stability constant of Cu(II)-elesclomol was thus
determined to be 1024.2 M−1 compared to 1020.0 M−1 for Cu(II)-TRIEN
[19]. Because elesclomol bound Cu2+ so strongly it would be expected
to dissociate extremely slowly from its complex. Thus, the fact that
Fig. 5. Comparison of the growth inhibitory effects of elesclomol (○), solid line;
Cu(II)-elesclomol (●), short dashed line; Ni(II)-elesclomol (Δ), long dashed line; and
Pt(II)-elesclomol (■), dotted line on K562 cells. The cells were treated with the
compounds for 72 h prior to the assessment of growth inhibition by an MTS assay.
The curved lines were calculated from non-linear least squares fits to 4-parameter logistic
Fig. 6. Comparison of the initial velocities for the oxidation of 1 mM ascorbic acid by
Cu(II)-elesclomol (○) and CuCl2 (●). The initial velocities were measured by following
the decrease in absorbance at 268 nm in Tris buffer (10 mM, pH 7.4, 25% DMSO at
25 °C) in a 0.1 cm spectrophotometer cell. The straight lines were linear least squares
calculated. A comparison of the slopes indicated that Cu2+ oxidized ascorbic acid at a
rate that was 14.6-fold faster than did Cu(II)-elesclomol.
equations and yielded IC50 values of 8.2
0.9, 5.4
0.6, 185
48 and 5600
1900 nM,
for elesclomol, Cu(II)-elesclomol, Ni(II)-elesclomol and Pt(II)-elesclomol, respectively.

6
A.A. Yadav et al. / Journal of Inorganic Biochemistry 126 (2013) 1–6
the reaction with 100 μM TRIEN occurred with a t1/2 of 3.3 min suggests
that TRIEN may have displaced Cu2+ from Cu(II)-elesclomol through
an associative displacement mechanism. An associative displacement
reaction could also provide a mechanism by which copper binding
proteins could remove copper from Cu(II)-elesclomol.
potential targets such as DNA damage and inhibition/inactivation of
redox sensitive enzymes critical for cell growth.
Acknowledgments
It has been proposed [1,2] that elesclomol exerts its in vitro
cell growth inhibitory activity by scavenging copper from the culture
medium. The fact that elesclomol and Cu(II)-elesclomol potently
inhibited the growth of K562 cells in the low nanomolar range to
about the same extent is consistent with this proposal. This result
also indicated that our culture medium contained sufficient adventitious
copper in the medium and/or copper binding proteins in the added
serum for elesclomol to exert nearly its full potency by complexing
with this available Cu2+. Because Ni(II)-elesclomol and Pt(II)-elesclomol
were 34- and 1040-fold less potent at inhibiting K562 cell growth,
respectively, than Cu(II)-elesclomol, these results suggest that the
redox inactive Ni2+ and Pt2+ complexes of elesclomol were almost inac-
tive compared to Cu(II)-elesclomol. Given the fact that elesclomol itself
displays cell growth inhibitory activity it was possible that what activity
the Ni2+ and Pt2+ complexes did possess was due to partial dissociation
of these complexes to produce free elesclomol. This free elesclomol could
then bind Cu2+ from the media and exert its cell growth inhibitory
activity through its copper complex. In a liquid chromatography–mass
spectrometry study elesclomol was shown to bind Cu2+ much more
strongly than Ni2+ [18] consistent with the Irving–Williams series [25]
for ligand binding to divalent metals. This would be consistent with
the Ni(II)-elesclomol complex exerting its cell growth inhibitory effects
through partial dissociation to produce elesclomol. No spectrophoto-
metric evidence was seen for Cu2+ being able to directly replace Ni2+
from its complex with elesclomol.
This work was supported by the Canadian Institutes of Health
Research, the Canada Research Chairs program, and a Canada Research
Chair in Drug Development for B.B.H.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.jinorgbio.2013.04.013.
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In conclusion, the results of this study have shown that Cu2+ binds
elesclomol to form a 1:1 neutral complex that has a distorted square
planar structure. The Ni(II)-elesclomol and Pt(II)-elesclomol complexes
that were synthesized were much less potent than Cu(II)-elesclomol in
inhibiting K562 cell growth. This result suggests that the inhibition of
cell growth and the anticancer activity of elesclomol are dependent
upon formation of its complex with Cu2+, and possibly through the
formation of damaging reactive oxygen species mediated through
the reduction of Cu2+. The Cu(II)-elesclomol complex was reduced by
physiological concentrations of ascorbic acid. Thus, our results using a
variety of assays, indicate that coordination with redox active Cu2+
was likely necessary for elesclomol to exert its activity. Our subsequent
investigations of elesclomol will proceed with a focus on other specific