Synthesis and characterization of quinoline-based thiosemicarbazones and correlation of cellular iron-binding efficacy to anti-tumor efficacy

Article abstract of DOI:10.1016/j.bmcl.2012.07.030

Iron chelators have emerged as a potential anti-cancer treatment strategy. In this study, a series of novel thiosemicarbazone iron chelators containing a quinoline scaffold were synthesized and characterized. A number of analogs show markedly greater anti-cancer activity than the 'gold-standard' iron chelator, desferrioxamine. The anti-proliferative activity and iron chelation efficacy of several of these ligands (especially compound 1b), indicates that further investigation of this class of thiosemicarbazones is worthwhile.

Full text of DOI:10.1016/j.bmcl.2012.07.030

Bioorganic & Medicinal Chemistry Letters 22 (2012) 5527–5531
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and characterization of quinoline-based thiosemicarbazones and
correlation of cellular iron-binding efficacy to anti-tumor efficacy
Maciej Serda a, Danuta S. Kalinowski b, , Anna Mrozek-Wilczkiewicz a,c, Robert Musiol a,
⇑
Agnieszka Szurko c,d, Alicja Ratuszna c, Namfon Pantarat b, Zaklina Kovacevic b, Angelica M. Merlot b,
a,
Des R. Richardson b, , Jaroslaw Polanski
⇑
⇑
a Department of Organic Chemistry, Institute of Chemistry, University of Silesia, PL-40006 Katowice, Poland
b Department of Pathology and Bosch Institute, University of Sydney, Sydney, New South Wales 2006, Australia
c Department of Solid State Physics, Institute of Physics, University of Silesia, PL-40007 Katowice, Poland
d Department of Experimental and Clinical Radiobiology, Center of Oncology, M. Sklodowska-Curie Memorial Institute, PL-44101 Gliwice, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
Iron chelators have emerged as a potential anti-cancer treatment strategy. In this study, a series of novel
thiosemicarbazone iron chelators containing a quinoline scaffold were synthesized and characterized. A
number of analogs show markedly greater anti-cancer activity than the ‘gold-standard’ iron chelator, des-
ferrioxamine. The anti-proliferative activity and iron chelation efficacy of several of these ligands (espe-
cially compound 1b), indicates that further investigation of this class of thiosemicarbazones is
worthwhile.
Received 14 May 2012
Revised 5 July 2012
Accepted 6 July 2012
Available online 15 July 2012
Keywords:
Ó 2012 Elsevier Ltd. All rights reserved.
Anti-tumor activity
Desferrioxamine
Iron chelation
Iron (Fe) chelators are commonly used to treat diseases con-
nected with altered iron metabolism, for example, b-thalassaemia
major.1 However, considering the marked anti-proliferative activ-
ity of this group of agents, recent investigations have focused on
the anti-cancer efficacy of iron chelators.2,3 In fact, there are many
reports of the anti-proliferative activity of desferrioxamine (DFO),
TriapineÒ and other ligands based on the (thio)urea moiety.2
The cytotoxic mechanisms of chelators include: (1) the inhibi-
tion of cellular iron uptake from the iron-binding protein, transfer-
rin (Tf);4–7 (2) mobilization of iron from cells;4–7 (3) the inhibition
of the iron-containing enzyme involved in the rate-limiting step
of DNA synthesis, ribonucleotide reductase;8 and (4) the formation
of redox-active iron complexes that generate reactive oxygen spe-
cies (ROS).5,7 The latter mechanism is significant, especially in the
context of recent reports demonstrating the role of ROS generation
in increasing the anti-proliferative activity of chelators against
tumor cells.5,7,9
Alterations in the metabolism of iron10–13 and copper14,15 are
known to occur in cancer cells and may play a role in angiogene-
sis16 and metastasis.17 The rationale behind the potential applica-
tion of iron chelators for cancer treatment is due to the higher
demand for iron in rapidly proliferating tumor cells in comparison
to their normal counterparts.10–12 The greater requirement for iron
in tumor cells results in high levels of the transferrin receptor
(TfR1) on the cell surface which binds Tf.13 Furthermore, the
expression of ribonucleotide reductase is markedly higher in neo-
plastic cells relative to their normal counterparts.2 Hence, this also
increases the sensitivity of cancer cells to iron-depletion.
Although DFO was investigated as an anti-cancer agent, interest
in this compound was diverted in favor of more effective ligands
such as aroylhydrazones that show greater iron chelation efficacy
and cellular permeability.3 Some of these compounds were shown
to have moderate anti-tumor activity that was significantly greater
than that of DFO for example, 2-hydroxy-1 naphthylaldehyde
isonicotinoyl hydrazone (311; Fig. 1).18,19 Further structural modi-
fications of this series produced the 2-hydroxy-1-naphthylalde-
hyde thiosemicarbazone (NT)20 and di-2-pyridyl ketone
isonicotinoyl hydrazone (PKIH) series21 of chelators (Fig. 1), which
showed superior activity.
Abbreviations: Bp44mT, 2-benzoylpyridine-4,4-dimethyl-3-thiosemicarbazone;
BpT, 2-benzoylpyridine thiosemicarbazone; DFO, desferrioxamine; DpT, dipyridyl
thiosemicarbazone; Dp44mT, di-2-pyridyl ketone-4,4-dimethyl-3-thiosemicarba-
zone; NT, 2-hydroxy-1-naphthylaldehyde thiosemicarbazone; PKIH, di-2-pyridyl
ketone isonicotinoyl hydrazone; ROS, reactive oxygen species; Tf, transferrin; TfR1,
transferrin receptor 1; QCIH, 2-quinolinecarboxaldehyde isonicotinoyl hydrazone;
QT, quinoline thiosemicarbazone.
⇑
Corresponding authors. Tel.: +61 2 9036 6547; fax: +61 2 9351 3429 (D.S.K.);
tel.: +61 2 9036 6548; fax: +61 2 9351 3429 (D.R.R.); tel./fax: +48 322599978 (J.P.).
E-mail addresses: danuta.kalinowski@sydney.edu.au (D.S. Kalinowski), d.
richardson@med.usyd.edu.au (D.R. Richardson), polanski@us.edu.pl (J. Polanski).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.07.030

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M. Serda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5527–5531
Table 1
N
The quinoline-based thiosemicarbazones examined in this study
N
NH2
S
O
HN
N
HN
N
O
HN
N
N
N
R1
N
OH
OH
S
N
N
NH
OH
NH
N
N
R2
R2
N
S
R3
R3
311
PKIH
N
NT
Series 1
Series 2
S
H2N
S
N
N
S
N
Compd
R1
R2
R3
Compd
R2
R3
HN
HN
HN
1a
1b
1c
1d
1e
1f
8-OH
H
8-OH
H
8-OH
H
8-OH
H
Me
Me
Me
Me
Et
Et
Ph
Ph
Me
Me
H
H
H
H
H
H
2a
2b
2c
2d
Me
Et
Me
Ph
Me
N
H
H
H
N
N
N
N
N
Bp44mT
Dp44mT
DpT
1g
1h
NR2
S
NR2
S
HN
N
design of novel anti-cancer agents27 (e.g., the clinically used cam-
ptothecin derivatives, topotecan and irinotecan)28 and it was of
interest to assess its effect on the biological activity of thiosemicar-
bazones. In series 1, the four-atom moiety (N@C–C@N), which is
found in the DpT and BpT ligands is maintained (Fig. 1). In contrast,
in series 2, a five-atom fragmental construct (N@C–C@C–OH) sim-
ilar to 311 or NT (Fig. 1) was preserved, while the four-atom
moiety (N@C–C@N) of the DpT and BpT series was extended
(N@C–C–C@N). Of the two series of QTs described herein, several
analogs acted similarly to Dp44mT, markedly preventing cellular
iron uptake and promoting iron mobilization.5,7,22
HN
N
N
R1
N
OH
R1=H, OH,
1a-h
2a-d
R2=H, Me, Et, Ph
Figure 1. Representative structures of the hydrazones and thiosemicarbazones
used to design the quinoline thiosemicarbazone (QT) analogs, 1a–h and 2a–d.
All the QTs herein were synthesized as shown in Figure 2 by
reacting the respective quinolinecarbaldehyde (3) and thiosemicar-
bazide (4) in a microwave reactor (experimental details for all pro-
cedures in this study are described in the Supplementary data). We
confirmed the iron chelating ability of these QTs by titrating the
two most biologically active analogs (see below), 1b and 2a with
Fe3+ to generate their Fe3+ complexes in situ at the following ligand
to Fe ratios: 2:1, 2.5:1, 3.3:1, 5:1, and 10:1 (Supplementary Fig. 1).
This was carried out in comparison to the well characterized iron
chelator, Dp44mT.5 As previously observed, the electronic spec-
trum of the Fe3+ complex of Dp44mT displayed characteristic
intense transitions (400 nm) that spanned into the visible region
(Supplementary Fig. 1A).5 Additionally, isosbestic points were ob-
served at 310 and 365 nm (Supplementary Fig. 1A). The Fe3+ com-
plexes of 1b and 2a exhibited a similar shift into the visible
region in comparision to that of the free ligands. The Fe3+ complex
of compound 1b displayed an intense band at 430 nm, with an isos-
bestic point found at 375 nm (Supplementary Fig. 1B). Similarly, the
Fe3+ complex of 2a showed a transition at 450 nm, while isosbestic
points were observed at 330 and 385 nm (data not shown). The
electronic spectra of the Fe3+ complexes of 1b and 2a display
characteristics that are typical of Fe3+ thiosemicarbazone com-
plexes5,9,29 and confirm their ability to act as iron chelators.
The anti-proliferative activity of the QT analogs was assessed
against cancer and non-neoplastic cells, including the human SK-
N-MC neuroepithelioma, HCT116 colon cancer cell lines, and
normal human dermal fibroblast (NHDF) cells by standard meth-
ods.5,9,19 The SK-N-MC cell line was chosen as the effects of iron
chelators are well characterized in this cell line.5,9 Additionally,
we examined whether the p53 status of HCT116 cells altered their
response to the QT analogs. The protein, p53, is an important tumor
The di-2-pyridyl ketone thiosemicarbazone (DpT; Fig. 1) class of
chelators are essentially hybrids of these two latter classes of che-
lators.7,22 The DpT series, and in particular the chelator, di-2-pyri-
dyl ketone-4,4-dimethyl-3-thiosemicarbazone (Dp44mT; Fig. 1),
have shown potent and selective anti-tumor activity both in vitro
and in vivo against a variety of murine and human xenografts.7,22,23
For example, Dp44mT has demonstrated marked activity in vivo,
reducing the growth of a murine M109 lung cancer by approxi-
mately 50% within 5 days of treatment, while at the same time
having little effect on normal hematological indices.7 In addition
to the ability of Dp44mT to effectively induce cellular iron-depriva-
tion in vitro, initial studies revealed that the iron complex was
redox-active within cells.5,7 Hence, it was proposed that the anti-
tumor activity of these compounds relates both to their ability to
bind intracellular iron and to form redox-active iron complexes
that generate cytotoxic ROS.5,24
More recent studies have led to the 2-benzoylpyridine thiosem-
icarbazone (BpT) series of ligands (e.g., 2-benzoylpyridine-4,4-di-
methyl-3-thiosemicarbazone; Bp44mT; Fig. 1) that show
selective anti-tumor activity in vitro and in vivo and which are
effective via the intravenous or oral routes.9,25 Additionally, thio-
semicarbazones are more stable in plasma and are advantageous
over aldehyde-derived aroylhydrazones (such as 311) which un-
dergo hydrolysis of the hydrazone bond.26
In the current study, a series of novel thiosemicarbazones were
synthesized as potential anti-cancer agents. These compounds
were designed to contain a quinoline scaffold (quinoline thiosemi-
carbazones; QTs; compounds 1a–h and 2a–d; Fig. 1 and Table 1).
In fact, the quinoline moiety is often a fragmental motif in the

M. Serda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5527–5531
5529
R
S
N
S
R
R1
MW
NH
NH
+
R1
R2
R2
N
H2N
N
O
R3
Y
Z
R3
Y
Z
1a-h Y=N, Z=C, R=H, R1=OH
2a-d Y=C, Z=N, R=OH, R1=H
3
4
Figure 2. Synthesis of quinoline-based thiosemicarbazones.
suppressor involved in various mechanisms that prevent tumori-
genesis.30–32 In fact, it is well known that p53 acts as a molecular
guardian of the cell cycle and induces a G1/S arrest upon the induc-
tion of DNA damage and results in a repair response that prevents
propagation of mutations.30 Lack of p53 expression in some cancers
prevents repair, aids tumor cell progression and promotes resis-
tance against chemotherapeutics.33 The development of agents that
demonstrate activity against tumors with and without p53 is
vital,22 particularly considering the high prevalence of p53 muta-
tions in advanced cancer.32 Thus, we examined the anti-prolifera-
tive activity of the novel QT analogs against HCT116 cell lines
with (p53+/+) and without the p53 protein (p53À/À). The quinoline
analogs were compared to a number of chelators with well charac-
terized activity which acted as positive controls, namely DFO,19
31119 and Dp44mT.5
series,5,9,29 and may be explained by the increased lipophilicity of
1b in comparison to the more hydrophilic ligand, 1d. Due to its
increased lipophilicity, 1b may target different intracellular iron
pools to that of 1d, which are critical for cellular proliferation
(e.g., iron pools required by RR).5 However, no clear overall relation-
ship (r = 0.16) was found between the logPcalc value of the QT
ligands and their anti-proliferative efficacy, suggesting that factors
other than lipophilicity influenced their activity.
The QT analogs showed varying anti-proliferative efficacy when
comparing the SK-N-MC and HCT116 cell lines (Table 2). Most of
the active QT series (i.e., those with an IC50 <12.5 lM) had more
potent anti-cancer effects on SK-N-MC cells when compared to
the HCT116 cell lines, which is in good agreement with the general
sensitivity of these cell-types, as shown in previous studies in our
laboratories.34–36 Compounds 1a and 1b were the only analogs that
displayed significantly higher anti-proliferative activity against the
p53À/À HCT116 cell line than SK-N-MC and p53+/+ HCT116 cells.
Notably, the activity profile of 2a–2d appeared to be different in
the SK-N-MC and HCT116 cell lines. In particular, compounds 2a
and 2b that possessed the most potent anti-cancer activity of the
QT analogs in SK-N-MC cells, showed poor or very low anti-prolif-
The control chelators, DFO (IC50: 12.50 1.00
1.30 0.19 M), demonstrated moderate anti-proliferative effects
against SK-N-MC cells (Table 2).19 In contrast, Dp44mT possessed
potent anti-cancer activity in the SK-N-MC (IC50 0.014
0.016 M) and HCT116
M), HCT116 p53À/À (IC50: 0.005 0.002
p53+/+ (IC50: 0.002 0.001
M) cell-types. The most effective QT
analogs, namely 1b and 2a–2d, had moderate anti-proliferative
activity (IC50: 0.11–1.63 M) against SK-N-MC cells. In contrast,
the remaining compounds were relatively inactive against this cell
line (IC50: 9.69 to >12.50 M; Table 2). In fact, all the QT series 2
lM) and 311 (IC50:
l
:
l
l
l
erative activity (IC50 >20.75–25
l
M) in both the HCT116 p53+/+ and
l
p53À/À cell-types.
Comparing the activity of the QT analogs in HCT116 p53+/+ and
p53À/À cells, the anti-proliferative activity was either similar, or
the IC50 was greater in the p53+/+ cell-type (i.e., for 1a, 1b, 2a).
Hence, loss of p53 does not lead to a general increase in resistance
against the QT analogs, which is similar to previous observations
using other chelators.22 This is an important characteristic of these
compounds especially considering that approximately half of all
cancers have non-functional p53, which can lead to resistance to
some chemotherapeutics.33
l
analogs demonstrated moderate anti-cancer activity in SK-N-MC
cells, while only one member of series 1 (1b) showed appreciable
anti-proliferative effects (Table 2). Interestingly, a marked differ-
ence in the anti-cancer activity was observed between compound
1b (IC50: 0.81 0.30 lM; Table 2) and 1d (IC50: >12.5 lM; Table
2) that differ by a single methyl group at the terminal N4 atom. This
is a common structure–activity relationship that is observed in a
number of thiosemicarbazone classes, including the DpT and BpT
For the QT series to be considered as anti-tumor agents, they
must demonstrate selective anti-proliferative activity againts neo-
plastic cells and leave non-neoplastic cells unaffected. Thus, a selec-
tion of the most potent analogues were screened against NHDF cells
(Table 2). Importantly, the anti-proliferative effects of the QT series
were generally markedly decreased against NHDF cells. For exam-
ple, the anti-proliferative effects of the most potent analogs, 1b
and 2a, were decreased in NHDF cells in comparison to SK-N-MC
cells by 14- and 131-fold, respectively. This suggests that an appre-
ciable therapeutic index exists, allowing these analogs to selec-
tively target cancer cells over normal cells, a feature that has been
observed with other thiosemicarbazone iron chelators.7,9,22
The clonogenicity assay37 was used to evaluate the survival frac-
tion and was assessed for a representative QT analog (2c) and two
positive control chelators, namely Dp44mT and Bp44mT (see
Supplementary Tables 1 and 2), in HCT116 cells. The latter two che-
lators are members of the DpT and BpT series, respectively, and pos-
sess pronounced anti-tumor efficacy.7,9,22,25 In these studies, 2c
demonstrated similar activity in both the HCT116 p53+/+ and p53À/
À cell lines, although the cells without p53 appeared slightly more
sensitive to the agent in terms of the survival fraction (Supplemen-
tary Table 1). As suggested by their marked anti-tumor activity
in vitro and in vivo,5,24,38 Dp44mT and Bp44mT (Supplementary
Table 2
Anti-proliferative activity of the QT analogs in comparison to the positive control
chelators, DFO, 311 and Dp44mT, after a 72 h incubation
SK-N-MC
HCT116
HCT116
NHDF
(p53À/À
)
(p53+/+
)
IC50 (mean SD) (lM)
DFO
311
Dp44mT
12.50 1.00
1.3 0.19
0.014 0.016
—
—
—
—
—
—
0.005 0.002
0.002 0.001
15.38 5.06
1a
1b
1c
1d
1e
1f
1g
1h
2a
2b
2c
2d
9.69 1.50
0.81 0.30
>12.50
>12.50
>12.50
>12.50
>12.50
>12.50
0.11 0.04
0.19 0.07
0.62 0.09
1.63 0.34
0.27 0.01
0.37 0.02
2.17 0.47
>25.00
3.15 0.68
19.56 3.92
20.87 3.22
21.39 5.69
>25.00
>15.00
15.84 2.65
11.66 2.63
>25.00
—
>25.00
—
4.86 1.48
1.71 0.34
>25.00
2.62 0.59
23.25 4.02
24.97 4.29
22.34 5.18
20.75 5.34
>25.00
—
—
>25.00
>25.00
>25.00
14.94 1.61
>25.00
8.06 1.03
18.62 2.79
>25.00
16.28 1.69
Results are mean SD (three experiments).

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M. Serda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5527–5531
59Fe mobilization (Fig. 3). In fact, their activity was similar or
40
30
20
10
0
slightly less than 311 and Dp44mT, resulting in the release of
33–37% of cellular 59Fe. Of interest, limited correlation (r = 0.40)
was identified between the extent of iron release from SK-N-MC
cells and moderate anti-proliferative activity (i.e., IC50 <12.5 lM)
against this cell line (Table 2). For example, compounds 1a,
2a–2c that showed marked iron mobilization efficacy (Fig. 3), dem-
onstrated moderate anti-proliferative effects against SK-N-MC cells
(IC50: 0.11–9.69 lM; Table 2). In contrast, 1b and 2d that displayed
significantly (p <0.01) decreased 59Fe mobilization efficacy relative
to 1a and 2a–2c, had similar or greater anti-proliferative activity
(IC50: 0.81–1.63 lM) against SK-N-MC cells. Compounds 1c–1h
were significantly (p <0.01) less effective than 1a and 2a–2c at
inducing cellular 59Fe release (Fig. 3) and had low anti-proliferative
effects against SK-N-MC cells (IC50 >12.5 lM; Table 2). Notably
ligands 1c, 1e, 1g and 1h showed reduced ability to promote cellu-
lar 59Fe mobilization (Fig. 3), which may be due to their increased
hydrophilicity relative to the other analogs of the QT series. Lim-
ited correlation (r = À0.60) was observed between the logPcalc
value of the QT ligands and their ability to mobilize cellular 59Fe,
suggesting that other factors, apart from lipophilicity, are also
important in this process.
Figure 3. Effect of the chelators on 59Fe mobilization from prelabeled SK-N-MC
cells. Cells were incubated for 3 h/37 °C with 59Fe-transferrin (0.75
M), washed 4
times with ice-cold PBS and then reincubated for 3 h/37 °C in the presence or
absence of the chelators (25
M). Release of 59Fe was then assessed using a
scintillation counter. Results are mean SD (three experiments).
l
l
c-
Table 2) were highly effective, with anti-proliferative activity
evident at nanomolar levels.
Another important factor to consider was the ability of the
chelators to prevent 59Fe uptake from 59Fe-Tf19,21 (Fig. 4). As previ-
ously demonstrated,9,19,39 the positive controls, Dp44mT and 311,
effectively prevented 59Fe uptake, reducing it to <10% of the con-
trol. In contrast, DFO only slightly influenced 59Fe uptake, reducing
it to 81% of the control.9,39 As found in 59Fe mobilization experi-
ments (Fig. 3), compounds 1a, 2a–2c were the four most active
amongst the QT analogs, decreasing 59Fe uptake to 14%, 18%, 24%
and 45% of the control, respectively. Other compounds of the QT
series had variable effects on the inhibition of 59Fe uptake from
59Fe-Tf, reducing it to 49–98% of the control. Of interest, 1c, 1e
and 1h did not have greater activity than DFO at reducing 59Fe up-
take (Fig. 4). Importantly, we previously demonstrated that a num-
ber of series of thiosemicarbazones, including the DpT and BpT
series, cannot directly remove 59Fe from 59Fe-Tf.29 Hence, it is
likely that these structurally related thiosemicarbazones prevent
59Fe uptake by directly chelating 59Fe intracellularly after its
release from 59Fe-Tf. As was evident from 59Fe mobilization exper-
iments (Fig. 3), limited correlation (r = 0.58) was observed between
the logPcalc of the compounds and their ability to inhibit 59Fe up-
take from 59Fe-Tf in the SK-N-MC cell line.
Considering that thiosemicarbazones avidly bind cellular iron
and that this plays a role in their anti-cancer activity,7,9 the ability
of the studied compounds to induce cellular 59Fe mobilization and
prevent iron uptake from Tf2,3 was crucial to assess. Thus, this was
examined in the SK-N-MC neuroepithelioma cell line as the effects
of other chelators on inducing 59Fe mobilization and preventing
59Fe uptake in these cells have been well characterized.38–40 The
ability of the compounds to affect iron metabolism was compared
to that of the control compounds, DFO, 311 and Dp44mT, that have
been extensively assessed in this cell line.7,9,19,40
As is shown in Figure 3, the highly hydrophilic chelator, DFO,
exhibited limited ability to promote 59Fe mobilization, as previously
described,9,38 releasing only 12 2% of intracellular 59Fe. The highly
efficient iron chelators, 31119 and Dp44mT,7 increased 59Fe mobili-
zation resulting in the release of 38% of total cellular 59Fe (i.e., 59Fe
efflux was approximately 8-fold higher than the control).2,7,41
In the context of the QT analogs studied in this investigation,
compounds 1a and 2a–2c were the most effective at inducing
Considering structure activity relationships, in general, the QT
series were less efficient as anti-proliferative agents than their pre-
cursors, namely the BpT and DpT series.7,9,22,25 Interestingly, the
anti-proliferative activity of these analogs is dependent on the par-
ticular cell line examined (Table 2).
Interestingly, in series 1, where the quinoline nitrogen acts as a
donor atom, only 1a and 1b demonstrated moderate anti-prolifer-
ative activity against the SK-N-MC cell line, respectively. These
results are consistent with our previous studies examining the
2-quinolinecarboxaldehyde isonicotinoyl hydrazone (QCIH) series
of aroylhydrazone ligands.21 Importantly, the QCIH series were
found to have poor anti-proliferative activity and iron mobilizing
efficacy in the SK-N-MC cell line.21 This suggests that the quinoline
motif, where the quinoline nitrogen acts as a donor atom, confers
low iron chelation efficacy.
120
100
80
60
40
20
0
The activity of series 2 was much more pronounced than that of
series 1 (Table 2), which reveals the importance of the quinoline
OH group. As mentioned previously, in the QT series, the five-atom
fragmental construct (N@C–C@C–OH) that is similar to 31118 or
the NT analogs20 was preserved, while the four-atom moiety
(N@C–C@N) of the DpT series was extended (N@C–C–C@N). This
indicates that the oxygen of the quinoline OH (in alpha position)
group acts as a donor atom for chelation, as observed for 311
Figure 4. Effect of the chelators on 59Fe uptake from 59Fe-transferrin by SK-N-MC
cells. Cells were incubated for 3 h/37 °C with 59Fe-transferrin (0.75
lM) in the
presence or absence of the chelators (25 lM). At the end of this incubation, cells
were washed 4 times with ice-cold PBS. Internalization of 59Fe was assessed by
incubation for 30 min/4 °C with the protease, Pronase (1 mg/mL).19 Cellular 59Fe
was then assessed using a
c-scintillation counter. Results are mean SD (three
experiments).

M. Serda et al. / Bioorg. Med. Chem. Lett. 22 (2012) 5527–5531
5531
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and the NT series, leading to increased potency. It is of interest that
in series 1 and 2, the N,N-dimethyl derivatives, in general, had
more potent anti-proliferative activity and iron mobilizing efficacy
than their parent or Et-substituted analogs, in accordance with
previous findings.42
The anti-tumor efficacy of thiosemicarbazones has been shown
to involve iron chelation.5,7,9 In previous investigations, we tested a
number of thiosemicarbazones and quinoline analogs as potential
anti-cancer drugs.7,22 In this study, a series of novel thiosemicarba-
zones were designed to contain a quinoline scaffold in order to
investigate their effect on iron uptake, iron mobilization and
anti-proliferative activity against tumor cell lines. Several QT ana-
logs demonstrated marked chelation efficiency in terms of mobiliz-
ing cellular iron and preventing iron uptake from Tf. The anti-
proliferative effect of this series of agents showed limited correla-
tion with their ability to promote cellular iron efflux or inhibit iron
uptake. Thus, their iron chelating ability only partially explains
their anti-proliferative efficacy and suggests that other properties
such as their redox activity may be important. The anti-prolifera-
tive activity and iron chelation efficacy of several of these agents,
in particular 1b, indicates that further investigation of this class
of thiosemicarbazones is certainly worthwhile.
24. Richardson, D. R.; Kalinowski, D. S.; Lau, S.; Jansson, P. J.; Lovejoy, D. B. Biochim.
Biophys. Acta 2009, 1790, 702.
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27. Solomon, V. R.; Lee, H. Curr. Med. Chem. 2011, 18, 1488.
28. Li, Q. Y.; Zu, Y. G.; Shi, R. Z.; Yao, L. P. Curr. Med. Chem. 2006, 13, 2021.
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Islam, M.; Bernhardt, P. V. J. Med. Chem. 2009, 52, 1459.
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Acknowledgments
M.S. was supported by a TWING fellowship and NCN grant
(DEC-2011/01/N/NZ4/01166). A.M.-W. appreciates the support of
UPGOW fellowship, NCN grant N405/068440 and Dokto-RIS stu-
dentship. This study was also supported by a grant from the Polish
Ministry of Science (R 0504303) to J.P. D.R.R. was supported by a
NHMRC Senior Principal Research Fellowship and Project grants
and an ARC Discovery Grant. A.M.M. was supported by the Bob
and Nancy Edwards Ph.D. Scholarship from the Sydney Medical
School of the University of Sydney. D.S.K. was supported by a Can-
cer Institute New South Wales Early Career Development Fellow-
ship and Innovation Grant.
34. Mrozek-Wilczkiewicz, A.; Kalinowski, D. S.; Musiol, R.; Finster, J.; Szurko, A.;
Serafin, K.; Knas, M.; Kamalapuram, S. K.; Kovacevic, Z.; Jampilek, J.; Ratuszna,
A.; Rzeszowska-Wolny, J.; Richardson, D. R.; Polanski, J. Bioorg. Med. Chem.
2010, 18, 2664.
35. Musiol, R.; Jampilek, J.; Kralova, K.; Richardson, D. R.; Kalinowski, D.; Podeszwa,
B.; Finster, J.; Niedbala, H.; Palka, A.; Polanski, J. Bioorg. Med. Chem. 2007, 15,
1280.
Supplementary data
36. Podeszwa, B.; Niedbala, H.; Polanski, J.; Musiol, R.; Tabak, D.; Finster, J.; Serafin,
K.; Milczarek, M.; Wietrzyk, J.; Boryczka, S.; Mol, W.; Jampilek, J.; Dohnal, J.;
Kalinowski, D. S.; Richardson, D. R. Bioorg. Med. Chem. Lett. 2007, 17, 6138.
37. Carmichael, J.; DeGraff, W. G.; Gazdar, A. F.; Minna, J. D.; Mitchell, J. B. Cancer
Res. 1987, 47, 936.
38. Richardson, D. R.; Ponka, P. J. Lab. Clin. Med. 1994, 124, 660.
39. Becker, E.; Richardson, D. R. J. Lab. Clin. Med. 1999, 134, 510.
40. Bernhardt, P. V.; Caldwell, L. M.; Chaston, T. B.; Chin, P.; Richardson, D. R. J. Biol.
Inorg. Chem. 2003, 8, 866.
Supplementary data (the details of experimental procedures
and biological activity data) associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/
j.bmcl.2012.07.030.
References and Notes
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42. DeNicola, G. M.; Karreth, F. A.; Humpton, T. J.; Gopinathan, A.; Wei, C.; Frese, K.;
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Hruban, R. H.; Iacobuzio-Donahue, C.; Kern, S. E.; Blair, I. A.; Tuveson, D. A.
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