Role of Metalation in the Topoisomerase IIα inhibition and antiproliferation activity of a series of α-heterocyclic-N 4-substituted thiosemicarbazones and their Cu(II) complexes

Article abstract of DOI:10.1021/jm101532u

The topoisomerase-IIα inhibition and antiproliferative activity of α-heterocyclic thiosemicarbazones and their corresponding copper(II) complexes have been investigated. The Cu^II(thiosemicarbazonato)Cl complexes were shown to catalytically inhi

Full text of DOI:10.1021/jm101532u

ARTICLE
pubs.acs.org/jmc
Role of Metalation in the Topoisomerase IIr Inhibition and
Antiproliferation Activity of a Series of r-Heterocyclic-N4-Substituted
Thiosemicarbazones and Their Cu(II) Complexes
Brian M. Zeglis, Vadim Divilov, and Jason S. Lewis*
Department of Radiology and the Molecular Pharmacology and Chemistry Program, Memorial Sloan-Kettering Cancer Center,
New York, New York 10065, United States
ABSTRACT: The topoisomerase-IIR inhibition and antipro-
liferative activity of R-heterocyclic thiosemicarbazones and
their corresponding copper(II) complexes have been investi-
gated. The CuII(thiosemicarbazonato)Cl complexes were
shown to catalytically inhibit topoisomerase-IIR at concentra-
tions (0.3-7.2 μM) over an order of magnitude lower than
their corresponding thiosemicarbazone ligands alone. The
copper complexes were also shown to inhibit the proliferation
of breast cancer cells expressing high levels of topoisomerase-
IIR (SK-BR-3) at lower concentrations than cells expressing
lower levels of the enzyme (MCF-7).
’ INTRODUCTION
reductase and the compound’s potent cytotoxicity.9 For example,
detailed studies suggest that the enzymatic inhibition by the
Fe(TSC) complex stems from the reactive oxygen species (ROS)
mediated quenching of an important tyrosyl radical in the R2
subunit of RR and the more widespread deleterious effects of the
intracellular production of ROS.10
For over 30 years, thiosemicarbazones (TSC) have been a
focus of chemists and biologists because of their wide range of
pharmacological effects; these compounds and their chemical
relatives have been shown to have marked antibacterial, antiviral,
antifungal, and, most intriguingly, antineoplastic activity.1 Soon
after the discovery of the potency of organic thiosemicarbazones
alone, it was determined that many of these molecules are often
excellent chelators of transition metals, particularly Fe(II),
Cu(II), and Zn(II), because of their inherent N-N-S tridentate
coordination scaffold. Moreover, it was quickly shown that these
metal complexes often possess potent pharmacological effects. A
very rich literature exists exploring the chemistry and biology of
metal-bound thiosemicarbazones, particularly CuII-thiosemicar-
bazonato and FeII-thiosemicarbazonato complexes.1-5
The antineoplastic activity of thiosemicarbazones is most
often attributed to the ability of the compounds to inhibit
mammalian ribonucleotide reductase (RR), an enzyme essential
in the de novo production of deoxyribonucleotides.1 One family
of thiosemicarbazones in particular (those with an R-pyridyl
moiety adjacent to the N1 position) has shown significant
potential as anticancer agents.6,7 Perhaps the best known mem-
ber of this family, 3-aminopyridine carboxaldehyde thiosemicar-
bazone (3-AP), is a potent ribonucleotide reductase inhibitor
that is currently in phase II clinical trials for the treatment of a
number of forms of cancer, including non-small-cell lung cancer
and renal carcinoma.8 Mechanistic studies of 3-AP and its
analogues suggest that the biological effects of the compounds
result predominantly from the intracellular chelation of Fe(II). It
has been shown that not only the simple sequestration of Fe(II)
but also the redox cycling properties of the resultant FeII(3-Ap)
complexes are responsible for the inhibition of ribonucleotide
The study of other R-pyridylthiosemicarbazones and their
metal complexes has yielded similarly promising results.11 The
ability of these compounds to inhibit critical enzymatic pathways
responsible for DNA synthesis and polymerization (e.g., ribonu-
cleotide reductase or DNA polymerase R) has been well-
established.2,3,11-14 More recently, however, other important
mechanisms for the cytotoxicity of TSCs and M(TSC) com-
plexes have been illuminated.1 For example, in elegant and
thorough studies by Bernhardt, Richardson, and co-workers,
di-2-pyridyl-N4-substituted thiosemicarbazones (DpT) and
2-acetylpyridine-N4-substituted thiosemicarbazones (ApT) have
been shown to have potent antiproliferative effects in a variety of
tumor cell lines, and convincing evidence suggests that this
activity is mediated at least in part by intracellular iron chelation
by the parent thiosemicarbazones and the subsequent redox
cycling of the Fe(TSC) complexes to produce ROS within the
cytoplasm.1,15,16 Even more relevant to the work at hand, related
studies exploring the biological activity and redox properties of
Cu complexes of ApT and DpT analogues have shown that these
compounds, particularly monovalent Cu(TSC) species, are
potent cytotoxic agents. Further, the CuI/II redox cycling of
these complexes, like their FeII cousins, plays a significant role in
their biological activity.15 Importantly, this work and that of
Received: November 30, 2010
Published: March 10, 2011
r
2011 American Chemical Society
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Scheme 1. General Synthetic Route to the TSCs and Corre-
sponding CuII(TSC)Cl Complexes
Chart 1. Structures of the Thiosemicarbazone Ligands and
Their CuII(TSC)Cl Complexesa
others strongly support the hypothesis that it is the copper
complexes rather than any dissociated ligands or cellular meta-
bolites that are responsible for the biological effects in vitro and
in vivo.13-15
Recent research into the ability of thiosemicarbazones and
their metal complexes to inhibit topoisomerase IIR (Topo-IIR)
has served not only to further reinforce the significant potential
of metal-thiosemicarbazonato complexes in cancer research but
also to expand the array of possible biochemical targets for the
molecules.12-14,17-19 Topo-IIR is a well-known biomarker that
is overexpressed in many forms of cancer and represents one of
the most important targets for modern chemotherapeutics, with
a wide variety of inhibitors (including etoposide, doxorubicin,
mitoxantrone, amsacrine, and idarubicin) employed in the clinic
against an array of malignancies.20,21 A small number of recent
publications indicate that R-heterocyclic thiosemicarbazones and
their Cu(II) complexes are capable of in vivo and in vitro
inhibition of Topo-IIR at IC50 below that of the widely employed
Topo-IIR poison etoposide (VP-16).12-14 Given the great
potential of TSCs and their metal complexes in the development
of chemotherapeutic agents and the importance of Topo-IIR in
many forms of cancer, these results suggest a promising new
avenue for study.12-14 Significantly, despite these exciting re-
sults, little has been reported on the role that metalation plays in
the ability of TSCs to inhibit Topo-IIR. Considering the well-
known complexity of the relationship between TSC metalation
and biological activity, such an investigation could aid the
progress of research in this field.
aWhen the ligand is discussed alone in the text, an “H” is placed before
the name (e.g., HFp4mT).
(TSC) and their corresponding CuII(thiosemicarbazonato)Cl
[Cu(TSC)Cl] complexes were synthesized. The thiosemicarba-
zone ligands were synthesized via the typical condensation route
from their parent thiosemicarbazides and the appropriate ketone
or aldehyde; subsequently, the Cu(TSC)Cl complexes were
prepared via heating of the TSC ligands with CuCl2
(Scheme 1).7,12-14,22 The identity of both the N1-imine and
N4-alkyl substituents were altered to provide a structurally
systematic series of compounds (Chart 1). Because of the
immense interest in their antineoplastic and antibacterial proper-
ties, a number of these complexes have been previously described
in the literature.2,7 The ligands were characterized with 1H NMR,
ESI-MS, and HRMS, and the Cu(TSC)Cl complexes were
characterized via ESI-MS, UV-vis, and HRMS. The TSCs and
Cu(TSC)Cl complexes were purified via reversed-phase HPLC
and recrystallization, respectively, and prior to experimentation,
the purity of all compounds was determined to be >98% by 1H
NMR (ligands) and RP-HPLC (ligands and metal complexes).
Topoisomerase IIr Inhibition. Agarose gel electrophoresis
experiments were performed to assay the Topo-IIR inhibition
activity of the Cu(TSC)Cl complexes and their corresponding
ligands. Importantly, a commercially available Topo-IIR drug
screening kit (TopoGen, Port Orange, FL) was employed for
these assays, providing a significant improvement in gel clarity
over previously published work using Topo-IIR isolated from
cultured L1210 cells.12-14 In the present experiments, a super-
coiled pHOT DNA substrate was incubated for 30 min at 37 °C
with Topo-IIR in the presence of various concentrations of
the agent of interest, and the resultant reaction solutions were
electrophoresed on a 1% agarose gel to separate the possible
products: supercoiled DNA, linear DNA, and relaxed DNA. The
presence of each type of DNA indicates a different behavior by
Herein, we report the evaluation of the Topo-IIR inhibition
activity of a series of R-heterocyclic Cu(II)-thiosemicarbazonato
complexes and their corresponding thiosemicarbazone ligands.
Further, we examine the ability of the metal complexes to inhibit
the proliferation of two breast cancer cell lines, SK-BR-3 and
MCF-7, that we have shown to express high and low levels of
Topo-IIR, respectively.
’ RESULTS AND DISCUSSION
Synthesis and Characterization. For the study at hand, a
series of R-heterocyclic-N4-substitued thiosemicarbazones
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the enzyme: relaxed DNA reveals that the enzyme’s isomerase
activity remains intact; supercoiled DNA suggests that the
enzyme’s action has been inhibited; linear DNA reveals the
formation of permanent double strand breaks during the catalytic
cycle. In the study, gel results clearly show that the Cu(TSC)Cl
complexes are potent Topo-IIR inhibitors, with all complexes
completely inhibiting the action of Topo-IIR at 10 μM
(Figure 1A). In contrast, almost all of the TSC ligands alone
(Figure 1B) fail to significantly inhibit the enzyme at 100 μM,
with only HFp4mT, HFp4eT, HAp4mT, and HAp4eT display-
ing any degree of inhibition at that concentration. Importantly,
control experiments revealed that CuCl2 alone does not inhibit
the enzyme at any concentrations below 500 μM, that the
addition of 5% DMSO as a drug carrier does not inhibit the
enzyme, and that neither the TSCs nor the Cu(TSC)Cl com-
plexes alter the migration of the DNA below 500 μM. Subse-
quent experiments elucidated IC50 ranging from 300 nM to 7 μM
for the Cu(TSC)Cl complexes, far below the corresponding
values for the TSCs alone, which are all greater than 75 μM and in
all but four cases exceed 100 μM (Chart 2). Easily the most
pronounced feature of these assays was the marked difference in
Topo-IIR inhibition activity of the Cu(TSC)Cl complexes
compared to the TSCs alone, though the causal mechanism of
this disparity remains unclear (vide supra). Importantly, a similar
discrepancy in the Topo-IIR inhibitory activity of metalated vs
free thiosemicarbazones has been reported for 1,2-naphthoqui-
none thiosemicarbazone and its CuII, PdII, and NiII complexes.23
In this case, the metallo-TSC complexes effectively inhibited
Topo-IIR while the ligands alone did not; however, no IC50
values were presented in this work to quantitate the effect.
Little structure-activity relationship for Topo-IIR inhibition
could be discerned for the Cu(TSC)Cl complexes or the TSCs
alone. In the former case, the complexes bearing ligands with
larger N4-allyl, N4-pyrrolidine, and N4-benzyl substituents
Figure 1. Agarose gel assay for Topo-IIR inhibition by Cu(TSC)Cl
complexes and TSC ligands alone. In the Cu(TSC)Cl gel (A), 1 mM
etoposide was employed as a positive control, and lanes 1-11 denote
the reaction of Topo-IIR with supercoiled (sc) DNA in the presence of
10 μM Cu(Fp4mT)Cl, Cu(Fp4eT)Cl, Cu(Fp4ipT)Cl, Cu(Fp4alT)Cl,
Cu(Ap4mT)Cl, Cu(Ap4eT)Cl, Cu(Fp4pyrrT)Cl, Cu(Fp4bzT)Cl, Cu-
(FpT)Cl, Cu(Ap4alT)Cl, and Cu(Apz4mT)Cl. In the TSC gel (B), 100
μM etoposide was employed as a positive control, and lanes 1-11
denote the reaction of Topo-IIR with supercoiled DNA in the presence
of 100 μM HFp4mT, HFp4eT, HFp4ipT, HFp4alT, HAp4mT, HAp4-
eT, HFp4pyrrT, HFp4bzT, HFpT, HAp4alT, and HApz4mT.
Chart 2. Topo-IIR Inhibition Activity (IC50) and Antiproliferative Activity (GI50) of the TSCs and CuII(TSC)Cl Complexesb
aThe Topo-IIR inhibition IC50 for etoposide, merbarone, and doxorubicin vary somewhat in the literature; in this chart, a range of concentrations and
representative publications are presented. bTopo-IIR inhibition IC50 values were determined via the described agarose gel supercoiled DNA relaxation
assay employing a range of drug concentrations from 500 μM to 10 nM. Cell proliferation GI50 values were determined via MTT assay with drug
concentrations ranging from 500 μM to 10 nM. All experiments were performed in triplicate, and all values are shown with (1 SD.
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[Cu(Fp4alT)Cl (300 ( 200 nM), Cu(Fp4pyrrT)Cl (600 (
300 nM), and Cu(Fp4bzT)Cl (800 ( 300 nM)] generally had
greater activity, though the least active complex, Cu(Ap4alT)Cl
(7.2 ( 2.4 μM), also bears an N4-allyl group. Likewise, though a
first glance suggests that the compounds with the lowest IC50 all
bear a hydrogen rather than a methyl group in the R1 position,
some of the complexes with the highest IC50 values [e.g.,
Cu(Fp4mT)Cl and Cu(Fp4eT)Cl] also possess a hydrogen at
the R1 position. Among the TSCs alone, the four compounds
with IC50 under 100 μM (HFp4mT, HFp4eT, HAp4mT, and
HAp4eT) bear relatively small N4-substituents. However, the
IC50 values of two other N4-methyl and N4-hydrogen-bearing
TSCs, HApz4mT and HFpT, were both >100 μM. Clearly, the
range of structures will have to be widened before a straightfor-
ward structure-activity relationship for these compounds be-
comes evident.
Notably, the IC50 values for the Cu(TSC)Cl complexes are
comparable to those of the well-characterized Topo-IIR inhibitor
doxorubicin (1-5 μM) and lie well below those of the Topo-IIR
inhibitors merbarone (40-50 μM) and etoposide (50-90
μM).24-27 Further, these values generally agree with the in vitro
IC50 and the in vivo concentrations at which Topo-IIR inhibition
was observed in the studies of Miller et al.12-14 Similarly, the in vitro
Topo-IIR IC50 values for the TSCs generally conform to those
found for R-heterocyclic TSCs in Miller’s study of acetylpyridyl-N4-
substituted thiosemicarbazones and the recently published work of
Huang and co-workers.13,17 Further still, a recent study with di-2-
pyridylketone-4,4-dimethylthiosemicarbazone (Dp44mT) found
evidence of in vivo Topo-IIR inhibition at very low concentrations
(<1 μM); this finding, however, is not inconsistent with the data
described herein because of the significant structural difference of
Dp44mT compared to the compounds at hand and because of the
in vivo nature of the experiments.19
Topoisomerase IIr Inhibition Mechanism. To further char-
acterize the inhibitory action of the Cu(TSC)Cl complexes, we
next determined the mechanistic class of Topo-IIR inhibitors to
which the complexes belong. Two distinct classes of Topo-IIR
inhibitors exist: (1) those, commonly referred to as poisons, that
bind to and stabilize the DNA-Topo-IIR cleavage complex,
ultimately promoting the formation of extremely toxic double
strand breaks (e.g., etoposide) and (2) those, commonly referred
to as catalytic inhibitors, that antagonize the ability of the enzyme
to perform catalysis (e.g., merbarone).20 Further gel-based
experiments aimed at the detection of double strand breaks were
performed to discriminate between those two types of inhibitors.
In a representative gel (Figure 2), it is clear that while the well-
known Topo-IIR poisons etoposide, daunorubicin, genistein,
ellipticine, and m-AMSA all produce discrete bands of linear
DNA (thus indicating poison behavior), Cu(Fp4alT)Cl com-
pletely inhibits Topo-IIR without promoting the formation of
linear DNA products. Similar results were observed with the
other Cu(TSC)Cl complexes in the study. Thus, Cu(Fp4alT)Cl
and its family of Cu(TSC)Cl complexes are catalytic inhibitors of
Topo-IIR rather than poisons of the enzyme.
Figure 2. Representative gel-based topoisomerase IIR inhibition assay
illustrating the mechanism of Topo-IIR inhibition by Cu(Fp4alT)Cl.
The top gel (A) was poststained with 5 μg/mL ethidium bromide in 1ꢀ
TAE buffer, while the bottom gel (B) was electrophoresed with 5 μg/mL
ethidium bromide within the gel. High concentrations of enzyme (4
units) and inhibitors (500 μM) were used to maximize the intensity of
linear DNA bands (where present). Arrows mark representative linear
DNA bands.
Cu(thiosemicarbazonato) complexes;15 however, we believe that
it is unlikely that the metal-mediated production of ROS plays a
role in the observed Topo-IIR inhibition. The production of ROS
would almost certainly result in the formation of single and double
strand breaks in the DNA during the Topo-IIR incubation
experiments, and no significant single- or double-strand break
products were observed in the resultant gels. Further experimenta-
tion is currently underway to confirm this hypothesis.
A more likely mechanistic explanation lies perhaps in a convin-
cing recent study by Huang et al. in which the authors propose that
an R-heterocyclic-N4-substituted thiosemicarbazone in their study
(2-quinoneline carboxaldehyde-4,4-dimethyl thiosemicarbazone)
inhibits Topo-IIR by binding to the ATP hydrolysis domain of
Topo-IIR and thus interfering with the ATP hydrolysis of the
enzyme.17 Further, molecular docking experiments performed in
the above investigation show the thiosemicarbazone in question
bound to the ATPase binding site in a planar conformation with
the heterocylic nitrogen, imine nitrogen, and sulfur forming a
meridinal tridentate coordination environment.17 On the basis of
our experimental observations and this modeled conformation of
the thiosemicarbazone at the ATPase binding site, we believe it is
possible that the Cu(TSC)Cl complexes in this study inhibit
Topo-IIR via a similar ATP binding site-based mechanism. Inter-
estingly, the binding of the Cu(TSC)Cl complex to the ATPase
domain in a manner similar to that described in the Huang, et al.
study may also present an explanation for the disparity between
the inhibitory effects of the metal complexes and ligands alone.
The planar molecular geometry strongly favored by the d9 Cu(II)
center forces the thiosemicarbazone ligand to adopt a more rigidly
planar structure than the unmetalated TSC would assume; if a
planar conformation is the biologically active conformation, as is
suggested by Huang and co-workers, then the Cu(TSC)Cl com-
plexes would bind to the inhibition site more strongly than the
ligands alone. Biochemical and structural investigations into the
While it is clear that the Cu(TSC)Cl complexes at hand are
catalytic inhibitors of Topo-IIR, a number of different mechanisms
for this type of antagonist have been identified. Thus, the precise
molecular mechanism of inhibition by the Cu(TSC)Cl complexes
remains unknown. It has been well-established that the redox
cycling of Fe(thiosemicarbazonato) complexes plays a significant
role in their ribonucleotide reductase inhibition and cytotoxicity.1
And indeed, similar results have recently been published for
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tide reductase, purine biosynthesis, and DNA polymerase R.13
More recently, the important role of redox chemistry in the
antiproliferative effects of mono- and divalent Cu-thiosemicar-
bazonato complexes has come to light.15 In this work, we have
investigated yet another possible mechanism for the cytotoxicity
of these compounds. The mild nature of the trends correlating
IC50 and GI50 in SK-BR-3 cells and the relatively low GI50 of the
complexes in the MCF-7 cells illustrate that Topo-IIR inhibition
is clearly not the whole story. Yet taken together, the trends in the
data described herein and the previous research of others into the
Topo-IIR inhibitory activity of monovalent Cu(TSC) complexes
suggest that Topo-IIR inhibition by Cu(TSC) complexes con-
tributes to their antiproliferative activity.
Figure 3. Western blot (left) of relative Topo-IIR expression in SK-BR-
3 and MCF-7 cells. Quantiation (right) was normalized using actin
loading control. Error bars are (1 standard deviation and are the result
of six independent experiments.
’ CONCLUSIONS
precise mechanism of Topo-IIR inhibition are currently underway
to test this hypothesis; hopefully, these will also illuminate the
cause of the discrepancy between TSC and Cu(TSC)Cl Topo-IIR
inhibition activity.
We have synthesized a series of R-heterocyclic thiosemicar-
bazones and their corresponding copper(II) complexes and
evaluated their in vitro Topo-IIR inhibition and antiproliferative
activity in SK-BR-3 and MCF-7 breast cancer cells. Importantly,
the Cu(TSC)Cl complexes are more potent Topo-IIR inhibitors
than the thiosemicarbazone ligands alone, with IC50 for the
complexes in most cases lower by well over an order of
magnitude. Gel electrophoresis experiments revealed that the
Cu(TSC)Cl complexes are catalytic inhibitors, rather than
poisons, of Topo-IIR, though a molecular mechanism remains
elusive. Finally, antiproliferation experiments with breast cancer
cell lines expressing high and low levels of Topo-IIR (SK-BR-3
and MCF-7, respectively) revealed micromolar GI50 and suggest
a role for Topo-IIR inhibition in the complex antiproliferative
effects of Cu(TSC)Cl compounds. Further in vivo and in vitro
mechanistic experiments are underway, as well as efforts to
develop 64Cu-based positron emission tomography radiotracers
for the noninvasive delineation of Topo-IIR expression in vivo. It
is hoped that the investigation described herein will contribute to
the development of more potent and effective Topo-IIR inhibi-
tors and an enhanced understanding of the role of metalation in
the biological activity of thiosemicarbazones.
Antiproliferative Activity of the TSCs and Cu(TSC)Cl Com-
plexes. While Topo-IIR expression is often associated with
malignancy, particularly strong links exist between Topo-IIR
levels and breast cancer. Topo-IIR expression levels in breast
cancer have been found to provide accurate and clinically helpful
information on the number of cycling cancer cells, the likelihood
of treatment response, the potential for remission after che-
motherapy, and the possibility of disease-related death.28 Be-
cause of these well-established links, we endeavored to assay the
antiproliferative effects of the Cu(TSC)Cl complexes and their
parent ligands in two breast cancer cells lines, SK-BR-3 and
MCF-7, that express high and low levels of Topo-IIR, respec-
tively. To confirm the relative expression levels of Topo-IIR in
SK-BR-3 and MCF-7, Western blots were performed, and it was
determined that SK-BR-3 cells express approximately 10-fold
more Topo-IIR than their MCF-7 counterparts (Figure 3).
Subsequently, MTT assays were performed using 24 h drug
incubations. Experiments with the Cu(TSC)Cl complexes re-
vealed that all of the complexes are active antiproliferative agents,
with GI50 of <1 to 6 μM with SK-BR-3 cells and 2 to 12 μM with
MCF-7 cells (Chart 2). These values are in general agreement
with those previously reported for Cu(TSC)Cl complexes and
human cancer cells.12-14,29 Again, few structure-activity rela-
tionships are evident. However, two trends suggest that Topo-
IIR inhibition may play a role in the antiproliferative effects of
these complexes. First, in the SK-BR-3 cells, the GI50 indicate a
positive correlation with the Topo-IIR inhibition IC50; that is,
the Cu(TSC)Cl complexes that are more potent Topo-IIR
inhibitors are also generally more active with respect to anti-
proliferation. Second, the GI50 for the complexes are typically
lower in the Topo-IIR overexpressing SK-BR-3 cells than in the
MCF-7 cells. Further, in SK-BR-3 cells, the GI50 of the Cu-
(TSC)Cl complexes are lower than those of their corresponding
TSC ligands alone (4-9 μM). Differences in cellular uptake and
the possibility of intracellular metal chelation make quantitative
comparisons between the activity of metal complexes and ligands
somewhat suspect, but these data nevertheless suggest a role for
Topo-IIR inhibition in the complex antiproliferative effects of
these metal compounds.
’ EXPERIMENTAL SECTION
General Remarks. All chemicals, unless otherwise noted, were
acquired from Sigma-Aldrich (St. Louis, MO) and were used as received
without further purification. All water employed was ultrapure (>18.2
MΩ cm-1 at 25 °C, Milli-Q, Millipore, Billerica, MA). All DMSO was of
molecular biology grade (>99.9%; Sigma, D8418), and all other solvents
were of the highest grade commercially available. All MTT assay kit
materials were purchased from ATCC (Manassas, VA). All Topo-IIR
Western blot and inhibition assay materials were purchased from
TopoGEN, Inc. (Port Orange, FL). All instruments were calibrated
and maintained in accordance with standard quality-control procedures.
UV-vis measurements were taken on a Cary 100 Bio UV-vis spectro-
photometer. NMR spectroscopy was performed on a Bruker 500 MHz
NMR with Topsin 2.1 software for spectrum analysis. HPLC was
performed using a Shimadzu HPLC equipped with a C-18 reversed-
phase column (Phenomenex Luna analytical 4.6 mm ꢀ 250 mm or
SemiPrep 21.2 mm ꢀ 100 mm, 5 μm, 1.0 or 6.0 mL/min), 2 LC-10AT
pumps, and a SPD-M10AVP photodiode array detector. Data analysis
was performed using Microsoft Excel, version 12.2.3, and OriginPro,
version 8.1. All compounds employed were >98% pure, as purified via
preparative reversed phase HPLC or recrystallization and as determined
Over a decade ago, a well-designed study by Miller et al. on the
cytotoxicity of copper complexes of R-pyridylthiosemicarba-
zones proposed a variety of mechanisms for the antiproliferative
effects of the compounds, including the inhibition of ribonucleo-
1
by H NMR and analytical RP-HPLC. Thiosemicarbazide precursors
were synthesized according to previously reported protocols.7
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General Synthetic Procedure for the Synthesis of Thiose-
micarbazones. The general protocol of Klayman et al. was followed
for the synthesis of all thiosemicarbazones.7 The relevant thiosemicar-
abazide (4.75 mmol) and ketone or aldehyde (4.75 mmol) were
combined in methanol (9 mL) with acetic acid (1-2 drops). The
mixture was refluxed for 4 h, during which a white or off-white
precipitate appeared. After 4 h, the mixture was allowed to cool to room
temperature, and water (40 mL) was added to further aid precipitation.
The mixture was subsequently filtered, washed with cold water, and
dried in vacuo. Ligands were further purified via semipreparative RP-
HPLC with a gradient of 0:100 MeCN/H2O (both with 0.1% TFA) to
DMSO-d6), δ, ppm: 11.01 (s, 1H), 8.34 (d, 1H), 7.83 (s, 1H), 7.64 (m,
2H), 7.12 (m, 1H), 1.8-1.6 (m, 10H). ESI-MS: 233.1 [M - H]-, 235.2
[M þ H]þ. HRMS found (calculated, [M þ H]þ): 235.1026
(235.1017). HPLC tR = 10.2 min.
(E)-N-Benzyl-2-(pyridin-2-ylmethylene)hydrazinecarbothio-
amide (HFp4bzT). Yield: 4.7 mmol (99%). 1H NMR (500 MHz,
DMSO-d6), δ, ppm: 11.82 (s, 1H), 9.25 (m, 1H), 8.57 (d, 1H), 8.30 (d,
2H), 8.13 (s, 1H), 7.83 (s, 1H), 7.40-7.25 (m, 6H), 4.87 (s, 2H). ESI-MS:
269.2 [M -H]-, 271.3 [M þ H]þ. HRMS found (calculated, [M þ H]þ):
271.1008 (271.1017). HPLC tR = 11.3 min.
(E)-2-(Pyridin-2-ylmethylene)hydrazinecarbothioamide
(HFpT). Yield: 4.6 mmol (96%). 1H NMR (500 MHz, DMSO-d6), δ,
ppm: 11.62 (s, 1H), 8.56 (d, 1H), 8.34 (s, 1H), 8.26 (d, 1H), 8.16 (s,
1H), 8.08 (s, 1H), 7.83 (dd, 1H), 7.38 (dd, 1H). ESI-MS: 178.9 [M -
H]-. HRMS found (calculated, [M þ H]þ): 181.0559 (181.0548)
HPLC tR = 8.2 min.
(E)-N-Allyl-2-(1-(pyridin-2-yl)ethylidene)hydrazinecarbothio-
amide (HAp4alT). Yield: 4.55 mmol (96%). 1H NMR (500 MHz,
DMSO-d6), δ, ppm: 10.38 (s, 1H), 8.84 (s, 1H), 8.58 (d, 1H), 8.41 (d,
1H), 7.83 (dd, 1H), 7.40 (dd, 1H), 5.93 (m, 1H), 5.18 (m, 2H), 4.26 (dd,
2H), 2.36 (s, 3H). ESI-MS: 233.0 [M - H]-, 235.0 [M þ H]þ. HRMS
found (calculated, [M þ H]þ): 235.1010 (235.1017). HPLC tR = 10.3 min.
(E)-N-Methyl-2-(1-(pyrazin-2-yl)ethylidene)hydrazinecar-
bothioamide (HApz4mT). Yield: 3.97 mmol (83%). 1H NMR (500
MHz, DMSO-d6), δ, ppm: 10.54 (s, 1H), 9.67 (s, 1H), 8.78 (s, 1H), 8.60
(s, 2H), 3.05 (s, 3H), 2.51 (s, 3H). ESI-MS: 210.0 [M þ H]þ. HRMS
found (calculated, [M þ H]þ): 210.0825 (210.0813). HPLC tR = 8.0
min.
1
100:0 MeCN/H2O over 15 min. ESI-MS, HRMS, and H NMR data
were collected for all ligands.
General Synthetic Procedure for the Synthesis of Cu(II)-
(thiosemicarbazonato)Cl Complexes. The general protocols
published by West et al. were employed for the metalation of the
thiosemicarbazone ligands.30 Thiosemicarbazone (0.5 mmol) and
CuCl2 (0.48 mmol) were combined in EtOH (10 mL). The resultant
mixture was stirred and refluxed for 1 h, during which a bright green
precipitate appeared. After 1 h, the mixture was allowed to cool to room
temperature. The mixture was then filtered, and the bright green product
was washed with EtOH and Et2O and dried in vacuo. All metal
complexes were subsequently purified by recrystallization from EtOH.
HRMS and analytical HPLC [gradient of 0:100 MeCN/H2O (both with
0.1% TFA) to 100:0 MeCN/H2O over 15 min] were performed for all
metal complexes.
Characterization Data. (E)-N-Methyl-2-(pyridin-2-ylmethy-
lene)hydrazinecarbothioamide (HFp4mT). Yield: 4.61 mmol
1
(97%). H NMR (500 MHz, DMSO-d6), δ, ppm: 11.69 (s, 1H), 8.66
2-Formylpyridine-N4-methylthiosemicarbazonatochloro-
copper(II) [Cu(Fp4mT)Cl]. Yield: 0.46 mmol (96%). HPLC tR = 6.3
min. ESI-MS: 294.2, 296.3 [M þ H]þ, 257.9 [M - Cl]þ. HRMS found
(calculated, [M - Cl]þ): 258.0007 (258.0000). UV-vis (PBS, pH 7.4):
221 (λmax), 295, 328, 385 (ε385 = 11 800 mol-1 L-3 cm-1).
2-Formylpyridine-N4-ethylthiosemicarbazonatochloro-
(s, 1H), 8.55 (d, 1H), 8.25 (d, 1H), 8.08 (s, 1H), 7.86 (dd, 1H), 7.38 (dd,
1H), 3.03 (s, 3H). ESI-MS: 192.9 [M - H]-. HRMS found (calculated,
[M þ H]þ): 195.0713 (195.0704). HPLC tR = 6.9 min.
(E)-N-Ethyl-2-(pyridin-2-ylmethylene)hydrazinecarbothio-
1
amide (HFp4eT). Yield: 4.70 mmol (99%). H NMR (500 MHz,
DMSO-d6), δ, ppm: 11.64 (s, 1H), 8.71 (s, 1H), 8.56 (d, 1H), 8.27 (d,
1H), 8.09 (s, 1H), 7.86 (dd, 1H), 7.38 (dd, 1H), 3.61 (q, 2H), 1.17 (t,
3H). ESI-MS: 207.0 [M - H]-, 231 [M þ Na]þ. HRMS found
(calculated, [M þ H]þ): 209.0866 (209.0861). HPLC tR = 8.1 min.
(E)-N-Isopropyl-2-(pyridin-2-ylmethylene)hydrazinecar-
bothioamide (HFp4ipT). Yield: 4.42 mmol (93%). 1H NMR (500
MHz, DMSO-d6), δ, ppm: 11.61 (s, 1H), 8.56 (s, 1H), 8.25 (d, 1H), 8.17
(d, 1H), 8.10 (1, 1H), 7.84 (dd, 1H), 7.38 (dd, 1H), 4.55 (m, 1H), 1.24
(d, 6H). ESI-MS: 221.1 [M - H]-, 245 [M þ Na]þ. HRMS found
(calculated, [M þ H]þ): 223.1012 (223.1017). HPLC tR = 9.3 min.
(E)-N-Allyl-2-(pyridin-2-ylmethylene)hydrazinecarbothio-
copper(II) [Cu(Fp4eT)Cl]. Yield: 0.45 mmol (94%). HPLC tR
=
7.8 min. ESI-MS: 308.1, 310.1 [M þ H]þ, 271.8 [M - Cl]þ. HRMS
found (calculated, [M - Cl]þ): 272.0169 (272.0157). UV-vis (PBS,
pH 7.4): 220 (λmax), 295, 335, 389 (ε389 = 12 200 mol-1 L-3 cm-1).
2-Formylpyridine-N4-isopropylthiosemicarbazonatochloro-
copper(II) [Cu(Fp4ipT)Cl]. Yield: 0.47 mmol (98%). HPLC tR = 8.4
min. ESI-MS: 322.4, 324.3 [M þ H]þ, 286.2 [M - Cl]þ. HRMS found
(calculated, [M - Cl]þ): 286.0308 (286.0313). UV-vis (PBS, pH 7.4):
219 (λmax), 296, 338, 395 (ε395 = 11 800 mol-1 L-3 cm-1).
2-Formylpyridine-N4-allylthiosemicarbazonatochlorocopper
(II) [Cu(Fp4alT)Cl]. Yield: 0.40 mmol (83%). HPLC tR = 8.2 min. ESI-MS:
319.7, 321.9 [M þ H]þ, 284.2 [M - Cl]þ. HRMS found (calculated, [M -
Cl]þ): 284.0166 (284.0157). UV-vis (PBS, pH 7.4): 222 (λmax), 295, 337,
393 (ε393 = 12100 mol-1 L-3 cm-1).
1
amide (HFp4alT). Yield: 4.65 mmol (98%). H NMR (500 MHz,
DMSO-d6), δ, ppm: 11.39 (s, 1H), 8.82 (s, 1H), 8.59 (d, 1H), 8.42 (d,
1H), 8.10 (s, 1H), 7.84 (dd, 1H), 7.38 (dd, 1H), 5.91 (m, 1H), 5.15 (m,
2H), 4.26 (dd, 2H). ESI-MS: 219.2 [M - H]-. HRMS found
(calculated, [M þ H]þ): 221.0878 (221.0861). HPLC tR = 8.9 min.
(E)-N-Methyl-2-(1-(pyridin-2-yl)ethylidene)hydrazinecar-
2-Acetylpyridine-N4-methylthiosemicarbazonatochloro-
copper(II) [Cu(Ap4mT)Cl]. Yield: 0.41 mmol (85%). HPLC tR = 8.5
min. ESI-MS: 308.1, 310.4 [M þ H]þ, 272.1 [M - Cl]þ. HRMS found
(calculated, [M -Cl]þ): 272.0153 (272.0157). UV-vis (PBS, pH 7.4):
220 (λmax), 295, 331 (sh), 389 (ε389 = 11 900 mol-1 L-3 cm-1).
2-Acetylpyridine-N4-ethylthiosemicarbazonatochlorocopper
(II) Cu(Ap4eT)Cl]. Yield: 0.35 mmol (73%). HPLC tR = 9.1 min. ESI-MS:
286.0 [M - Cl]þ. HRMS found (calculated, [M - Cl]þ): 286.0320
(286.0313). UV-vis (PBS, pH 7.4): 220 (λmax), 296, 337 (sh), 389 (ε389
= 11 200 mol-1 L-3 cm-1).
1
bothioamide (HAp4mT). Yield: 3.9 mmol (82%). H NMR (500
MHz, DMSO-d6), δ, ppm: 10.34 (s, 1H), 8.63 (s, 1H), 8.57 (d, 1H),
8.42 (d, 1H), 7.83 (dd, 1H), 7.38 (dd, 1H), 3.06 (s, 3H), 2.36 (s, 3H).
ESI-MS: 209.2 [M þ H]þ. HRMS found (calculated, [M þ H]þ):
209.0851 (209.0861). HPLC tR = 8.3 min.
(E)-N-Ethyl-2-(1-(pyridin-2-yl)ethylidene)hydrazinecarbothio-
amide (HAp4eT). Yield: 4.1 mmol (86%). 1H NMR (500 MHz, DMSO-
d6), δ, ppm: 10.25 (s, 1H), 8.67 (s, 1H), 8.58 (d, 1H), 8.41 (d, 1H), 7.81 (dd,
1H), 7.40 (dd, 1H), 3.66 (q, 2H), 2.36 (s, 3H), 1.16 (t, 3H). ESI-MS: 221.9
[M - H]-, 223.1 [M þ H]þ. HRMS found (calculated, [M þ H]þ):
223.1021 (223.1017). HPLC tR = 9.2 min.
2-Formylpyridine-N4-pyrrolidinylthiosemicarbazonatochloro-
copper(II) [Cu(Fp4pyrrT)Cl]. Yield: 0.42 mmol (88%). HPLC tR =
8.7 min. ESI-MS: 333.9, 335.8 [M þ H]þ, 297.9 [M - Cl]þ. HRMS
found (calculated, [M - Cl]þ): 298.0299 (298.0313). UV-vis (PBS,
pH 7.4): 219 (λmax), 302, 346, 399 (ε399 = 13 700 mol-1 L-3 cm-1).
(E)-N0-(Pyridin-2-ylmethylene)pyrrolidine-1-carbothiohy-
drazide (HFp4pyrrT). Yield: 4.2 mmol (88%). 1H NMR (500 MHz,
2396
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Journal of Medicinal Chemistry
ARTICLE
2-Formylpyridine-N4-benzylthiosemicarbazonatochlor-
ocopper(II) [Cu(Fp4bzT)Cl]. Yield: 0.46 mmol (96%). HPLC tR =
11.8 min. ESI-MS: 370.2, 372.1 [M þ H]þ, 333.9 [M - Cl]þ. HRMS
found (calculated, [M - Cl]þ): 334.0324 (334.0313). UV-vis (PBS,
pH 7.4): 220 (λmax), 395, 334, 396 (ε396 = 11 600 mol-1 L-3 cm-1).
2-Formylpyridinethiosemicarbazonatochlorocopper(II)
[Cu(FpT)Cl]. Yield: 0.43 mmol (89%). HPLC tR = 5.8 min. ESI-MS:
279.8 [M þ H]þ, 243.7 [M - Cl]þ. HRMS found (calculated, [M -
Cl]þ): 243.9846 (243.9844). UV-vis (PBS, pH 7.4): 221 (λmax),
285, 324, 392 (ε392 = 9100 mol-1 L-3 cm-1).
acids, and 100 units/mL penicillin and streptomycin. MCF-7 cells were
grown in minimum essential medium, supplemented with 10% fetal calf
serum, 0.01 mg/mL bovine insulin (Sigma Aldrich, St. Louis, MO),
nonessential amino acids, 2 mM L-glutamine, 1 mM sodium pyruvate, 1.5
g/L sodium bicarbonate, and 100 units/mL penicillin and streptomycin.
Topoisomerase IIr Western Blots. SK-BR-3 and MCF-7 lysates
were prepared using a lysis buffer of 100 mM NaCl, 40 mM KCl, 0.1 mM
EDTA, 20 mM Tris-Cl (pH 7.5), 0.1 mM PMSF, 10% glycerol, 0.2%
NP-40, 0.1% Triton-X 100, and 1% SDS in conjunction with sonication.
Protein concentrations were calculated using the bicinchoninic acid
(BCA) protein assay kit (Pierce, Rockford, IL) using the manufacturer’s
protocol. Lysates were resolved by SDS-PAGE electrophoresis and
transferred to a nitrocellulose membrane. Human topoisomerase IIR
was probed using a rabbit polyclonal antibody against Topo-IIR
(TopoGEN, Port Orange, FL) at a 1:2500 dilution and a goat anti-
rabbit IgG antibody conjugated to horseradish peroxidase (HRP) (Santa
Cruz Biotechnology, Santa Cruz, CA) at a 1:3000 dilution. The R-
tubulin loading control was probed using mouse monoclonal antibody
(Abcam, Cambridge, MA) at a 1:5000 dilution and sheep anti-mouse
IgG antibody conjugated to HRP (General Electric Healthcare Life
Sciences, Piscataway, NJ) at a 1:4000 dilution. Bands were detected by
incubating membranes with enhanced chemiluminescent substrate
(Pierce, Rockford, IL). Detection was performed using the ECL-
enhanced chemiluminescence detection system (Amersham Bio-
sciences, Fairfield, CT) according to the manufacturer's instructions.
Blots were visualized by autoradiography. Gels were scanned using
Adobe Photoshop 7.0.1, and densitometric analysis was performed using
Un-Scan-It software (version 5.3, Silk Scientific, Orem, UT).
Cell Proliferation Assays. Cell proliferation of SK-BR-3 and
MCF-7 cells was quantified using an MTT assay kit obtained from
American Tissue Culture Collection (ATCC, Manassas, VA) and
performed according to the manufacturer’s specifications. To this end,
SK-BR-3 (20 000 cells/well) and MCF-7 (50 000 cells/well) cells were
seeded in 96-well plates (Costar 3596, Lowell, MA) overnight (18-24
h) (the number of cells/well that were seeded in each case was varied as
described to compensate for cell growth rates and thus provide the same
number of cells/well at the point of drug incubation). After overnight
incubation, the medium was aspirated and discarded. Fresh medium
mixed (via 1:20 dilution) with different drug concentrations was added
to each well in 100 μL aliquots. The plates were subsequently incubated
for 24 h at 37 °C/5% CO2. After 24 h, an amount of 10 μL of MTT
reagent was added to each well, and the plates were further incubated at
37 °C/5% CO2 for 3 h. Cells were solubilized with the provided
detergent reagent overnight (18-24 h) at room temperature. After this
solubilization step, the absorbance of each well was measured using a
SpectraMax M5 plate reader (Molecular Devices, New Orleans, LA).
Proliferation values were normalized to 1 using the untreated, control
well. All experiments were performed in triplicate. GI50 values were
obtained by plotting % proliferation vs Cu(TSC)Cl (or TSC) concen-
tration and determining the midpoint of the resultant hysteresis curve
using sigmoidal fitting in Origin (version 8.1).
2-Acetylpyridine-N4-allylthiosemicarbazonatochlorocop-
per(II) [Cu(Ap4alT)Cl]. Yield: 0.47 mmol (99%). HPLC tR = 10.1
min. ESI-MS: 334.1, 336.1 [M þ H]þ, 297.9 [M - Cl]þ. HRMS found
(calculated, [M - Cl]þ): 298.0307 (298.0313). UV-vis (PBS, pH 7.4):
220 (λmax), 297, 331, 398 (ε398) = 11 300 mol-1 L-3 cm-1).
2-Acetylpyrazine-N4-methylthiosemicarbazonatochloro-
copper(II) [Cu(Apz4mT)Cl]. Yield: 0.47 mmol (99%). HPLC tR =
10.1 min. ESI-MS: 308.7, 310.9 [M þ H]þ, 273.1 [M - Cl]þ. HRMS
found (calculated, [M - Cl]þ): 273.0112 (273.0109). UV-vis (PBS,
pH 7.4): 220 (λmax), 296, 330, 391.
Topoisomerase IIr Inhibition Assays. Topoisomerase inhibi-
tion assays were performed using a eukaryotic topoisomerase IIR drug
screening kit purchased from TopoGen (Port Orange, FL). All comple-
mentary materials (including various topoisomerase inhibitors) were also
purchased from TopoGen, and all experiments were performed according
to the manufacturer’s instructions and specifications. In a representative
inhibition experiment, 14 μL of H2O, 4 μL of topoisomerase reaction
buffer, 1 μL of supercoiled DNA subtrate, and 1 μL of eukaryotic
topoisomerase IIR (∼2 units of activity, as described by the manufacturer)
were combined in a 1.7 mL microcentrifuge tube. In experiments involving
added drug, 2 μL of DMSO stock solution of compound were added, and
the initial volume of water was adjusted to allow for a final reaction volume
of 20 μL. The reaction mixture was vortexed gently, centrifuged briefly, and
incubated at 37 °C for 30 min. After 30 min, 2 μL of 10% SDS was added to
quench the reaction, 2 μL of proteinase K was added to digest remaining
proteins, and the reaction mixture was again incubated at 37 °C for 15 min.
After this incubation, 2.5 μL of 10ꢀ loading dye was added to the reaction
mixture, and the tube was subsequently vortexed lightly and centrifuged.
The sample was then loaded onto a 1% agarose gel and electrophoresed for
5 h at 5 V/cm. Both prestained (5 μg/mL ethidium bromide in the 1%
agarose gel) and poststained (incubatedin a 1ꢀ TAE solution containing 5
μg/mL ethidium bromide) gels were performed in order to glean
maximum information from the experiments. In the mechanism elucida-
tion gels, higher amounts of enzyme (4 units) and higher concentrations of
inhibitor (500 μM) were used in order to maximize the intensity of the
linear DNA bands where present. Gels were visualized with a BioRad
chemiluminescence detector, and the data were processed with Quanti-
tyOne software (Bio-Rad, version 4.3.0), Un-Scan-It software (version 5.3,
Silk Scientific, Orem, UT), and Origin (version 8.1). IC50 values were
obtained by plotting % inhibition (as defined by the ratio of supercoiled
DNA to total DNA in each lane) vs drug concentration and determining
the midpoint of the resultant hysteresis curve using sigmoidal fitting in
Origin. In some cases (especially with the TSC ligands alone), high
background in the gel images made quantification somewhat difficult
and resulted in slightly larger errors for the IC50 measurements; this is
reflected in the data in Chart 2.
’ AUTHOR INFORMATION
Corresponding Author
*Phone: 1-646-888-3039. Fax: 1-646-888-3059. E-mail: lewisj2@
mskcc.org.
Cell Culture. Human breast cancer cell lines SK-BR-3 and MCF-7
were obtained from the American Tissue Culture Collection (ATCC,
Manassas, VA) and maintained by weekly serial passage in a 5% CO2(g)
atmosphere at 37 °C. Cells were harvested using a formulation of 0.25%
trypsin and 0.53 mM EDTA in Hank’s buffered salt solution (HBSS)
without calcium or magnesium. SK-BR-3 cells were grown in a 1:1
mixture of Dulbecco’s modified Eagle medium: F-12 medium, supple-
mented with 10% fetal calf serum, 2 mM L-glutamine, nonessential amino
’ ACKNOWLEDGMENT
The authors thank Drs. Pierre Daumar, Jason Holland,
Athanasios Glekas, and Darren Veach for helpful discussions.
This work was supported by an NSF NRSA postdoctoral grant
for B.M.Z. (Grant 1F32CA144138-01).
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Journal of Medicinal Chemistry
ARTICLE
’ ABBREVIATIONS USED
(16) Bernhardt, P. V.; Sharpe, P. C.; Islam, M.; Lovejoy, D. B.;
Kalinowski, D. S.; Richardson, D. R. Iron chelators of the dipyridyl-
ketone thiosemicarbazone class: precomplexation and transmetallation
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cytotoxic activity of nickel(II) complexes of thiosemicarbazones.
Met.-Based Drugs 1997, 4 (2), 89–95.
TSC, thiosemicarbazone; Topo-IIR, topoisomerase IIR; RR, ri-
bonucleotide reductase; ROS, reactive oxygen species; 3-AP,
3-aminopyridine carboxaldehyde thiosemicarbazone; DpT,
di-2-pyridyl-N4-substituted thiosemicarbazones; ApT, 2-acetyl-
pyridine-N4-substituted thiosemicarbazones
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