Synthesis and biological evaluation of 2-benzoylpyridine thiosemicarbazones in a dimeric system: Structure-activity relationship studies on their anti-proliferative and iron chelation efficacy

Article abstract of DOI:10.1016/j.jinorgbio.2014.07.020

Thiosemicarbazone chelators represent an exciting class of biologically active compounds that show great potential as anti-tumor agents. Our previous studies demonstrated the potent anti-tumor activity of the 2'-benzoylpyridine thiosemicarbazone series. W

Full text of DOI:10.1016/j.jinorgbio.2014.07.020

Journal of Inorganic Biochemistry 141 (2014) 43–54
Contents lists available at ScienceDirect
Journal of Inorganic Biochemistry
journal homepage: www.elsevier.com/locate/jinorgbio
Synthesis and biological evaluation of 2-benzoylpyridine
thiosemicarbazones in a dimeric system: Structure–activity
relationship studies on their anti-proliferative and iron chelation efficacy
b
a,
Adeline Y. Lukmantara a, , Danuta S. Kalinowski , Naresh Kumar , Des R. Richardson
⁎
⁎
b,⁎⁎
a
School of Chemistry, University of New South Wales, Sydney, NSW 2052, Australia
b
Molecular Pharmacology and Pathology Program, Department of Pathology and Bosch Institute, University of Sydney, Sydney, NSW, 2006, Australia
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 1 July 2014
Received in revised form 29 July 2014
Accepted 29 July 2014
Available online 7 August 2014
Thiosemicarbazone chelators represent an exciting class of biologically active compounds that show great
potential as anti-tumor agents. Our previous studies demonstrated the potent anti-tumor activity of the
2′-benzoylpyridine thiosemicarbazone series. While extensive studies have been performed on monomeric
thiosemicarbazone compounds, dimeric thiosemicarbazone chelators have received comparatively less
attention. Thus, it was of interest to investigate the anti-proliferative activity and iron chelation efficacy
of dimeric thiosemicarbazones. Two classes of dimeric thiosemicarbazones were designed and synthesized.
The first class consisted of two benzoylpyridine-based thiosemicarbazone units connected via a hexane or
dodecane alkyl bridge, while the second class of dimer consisted of two thiosemicarbazones attached to a
2,6-dibenzoylpyridine core. These dimeric ligands demonstrated greater anti-proliferative activity than
the clinically used iron chelator, desferrioxamine. This study highlights the importance of optimal lipophi-
licity as a factor influencing the cytotoxicity and iron chelation efficacy of these chelators.
Keywords:
Thiosemicarbazone
Dimer
Lipophilicity
Iron
© 2014 Elsevier Inc. All rights reserved.
1. Introduction
Iron is an essential element for many cellular processes, including
energy production, electron transport and DNA synthesis due to its
Thiosemicarbazones have received significant attention from
academia and the pharmaceutical industry over the past 50 years
due to their rich biological activity [1]. These properties include
potent anti-viral [2,3], anti-bacterial [4–6] and anti-cancer [7,8] effi-
cacy. The α-(N)-heterocyclic class of thiosemicarbazones are derived
from the Schiff base condensation reaction of thiosemicarbazides
with aldehydes or ketones [1]. These ligands possess a conjugated
N,N,S tridentate system that has been shown to be particularly im-
portant for their cytotoxic activity [9]. This effect is due to the fact
that the N and S atoms are able to act as “soft” electron donors and
chelate transition metal ions such as iron (Fe), copper and zinc to
form cytotoxic metal complexes [5,9–11]. Such complexes can
catalyze the formation of reactive oxygen species (ROS), including
the hydroxyl radical, that can damage DNA and inhibit cellular
proliferation [12–14].
role as a co-factor for proteins such as oxidases, cytochromes and ribo-
nucleotide reductase [15–17]. While tumor cells and normal cells share
highly similar biochemical processes, the higher requirement for Fe in
cancerous tissues highlights the potential to target Fe and develop
novel and selective chemotherapeutics [1,18–21]. The enhanced de-
mand for Fe in tumor cells arises, at least in part, from the increased
Fe requirement for enzymes that play critical roles in metabolism.
These include ribonucleotide reductase (RR), an Fe-containing enzyme
that catalyzes the conversion of ribonucleotides to deoxyribonucleo-
tides, which is the rate-limiting step of DNA synthesis [1,15]. Hence,
the inactivation of RR via Fe deprivation can inhibit DNA synthesis
and cellular proliferation of tumor cells [22,23]. Furthermore, Fe chela-
tion up-regulates the potent growth and metastasis suppressor, N-myc
downstream regulated gene-1 (NDRG1), which suppresses multiple
signaling pathways involved in tumorigenesis and metastasis [24–27].
Due to the importance of Fe in cancer cell proliferation, numerous
Fe-chelating compounds have been investigated as potential anti-can-
cer agents [1]. For example, the thiosemicarbazone chelator, 3-
aminopyridine 2-carboxaldehyde thiosemicarbazone (known as 3-AP
or Triapine®), exhibited inhibition of L1210 leukemia cells in vitro and
in vivo [15] and suppressed the growth of murine M109 lung carcinoma
and human A2780 ovarian carcinoma xenografts in mice [20,28–31]. In
fact, Triapine® was assessed in over 20 Phase I and II clinical trials and
⁎
Corresponding authors at: Department of Chemistry, University of New South Wales,
Australia. Tel.: +61 2 9385 4698.
⁎⁎ Correspondence to: D.R. Richardson, Department of Pathology and Bosch Institute,
University of Sydney, Sydney 2006, Australia. Tel.: +61 2 9036 6548; fax: +61 2 9351
3429.
E-mail addresses: adeline.lukmantara@gmail.com (A.Y. Lukmantara),
n.kumar@unsw.edu.au (N. Kumar), d.richardson@med.usyd.edu.au (D.R. Richardson).
http://dx.doi.org/10.1016/j.jinorgbio.2014.07.020
0162-0134/© 2014 Elsevier Inc. All rights reserved.

44
A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
recently has shown promising activity against cervical and vaginal can-
cers in combination with cisplatin and radiotherapy [32].
to connect iron chelator moieties, ranging from 2 to 11 atoms in length [1,
40,41]. Thus, we synthesized dithiosemicarbazones using both a short
(hexyl) and long (dodecyl) alkyl bridge to investigate the effect of the
linker length on anti-proliferative activity and iron chelation efficacy.
The second strategy employed 2,6-dibenzoylpyridine (6) as a core scaf-
fold to connect together two thiosemicarbazide “tails”. We herein report
the synthesis of two novel dithiosemicarbazones and five 2,6-
dibenzoylpyridine thiosemicarbazones (2,6-diBpT), and their ability to
mobilize cellular Fe and inhibit Fe uptake from the Fe transport protein,
transferrin (Tf).
Notably, this investigation represents the first attempt to combine two
thiosemicarbazide “tails” into a single molecule using dibenzoylpyridine.
In addition, it is also the first study to investigate the anti-proliferative ac-
tivity of dimeric dibenzoylpyridine thiosemicarbazones. The current re-
search highlights important structure–activity relationships regarding
the structural requirements necessary for dimeric thiosemicarbazone-
based chelators with potent anti-cancer activity.
Other thiosemicarbazone-based chelators, including the 2′-
benzoylpyridine thiosemicarbazone (BpT; 1; Fig. 1), 2′-(3-nitrobenzoyl)
pyridine thiosemicarbazone (NBpT; 2; Fig. 1) and halogenated 2′-
benzoylpyridine thiosemicarbazone (XBpT; 3; Fig. 1) series have also
shown potent and selective anti-tumor activity in vitro and in vivo
(Fig. 1) [33,34]. In particular, 2′-benzoylpyridine 4,4-dimethyl-3-
thiosemicarbazone (Bp44mT) showed potent anti-tumor activity upon
oral administration to nude mice bearing human DMS-53 lung cancer xe-
nografts and was very well tolerated [35]. Recently, we have also devel-
oped substituted 2′-benzoyl-6-methylpyridine thiosemicarbazones and
4-phenyl-substituted 2′-benzoylpyridine thiosemicarbazones that dem-
onstrated selective anti-proliferative activity towards cancer cells
in vitro [36,37].
While extensive studies have been performed on monomeric
thiosemicarbazones compounds [1,33,36,37], dimeric thiosemicarbazone
chelators have received comparatively less attention. Previous studies on
the dimerization of thiazolones, quinones and aminopyridines, produced
compounds with improved potency compared to the original monomers
[38,39]. One of the most studied groups of dimeric thiosemicarbazones
were the bis(thiosemicarbazone) series (4; Fig. 2). These ligands were
composed of two thiosemicarbazone moieties connected by their imine
nitrogens via a two-carbon bridge [40–42]. Earlier studies showed that
bis(thiosemicarbazones) derived from dialdehydes, ketoaldehydes and
diketones demonstrated anti-viral effects and anti-tumor activity against
HeLa cells [42–45]. The copper(II), zinc(II), platinum(II) and palladium(II)
complexes of bis(thiosemicarbazone) also possessed anti-neoplastic
properties [43,45–54].
2. Experimental
2.1. Chemicals
All commercially available reagents were purchased from Fluka,
Sigma Aldrich, Alfa Aesar and Lancaster, and used without further puri-
fication. Desferrioxamine (DFO) was purchased from Novartis, Basel,
Switzerland. All reactions requiring anhydrous conditions were per-
formed under an argon/nitrogen atmosphere. Anhydrous solvents
were obtained using a PureSolv MD Solvent Purification System.
Due to the potential observed with bis(thiosemicarbazones), other
dimeric structures were also investigated. Dithiosemicarbazones (5) are
2.2. Physical measurements
a
class of dimeric thiosemicarbazones that consist of two
thiosemicarbazone units connected by their amide nitrogen N(4) atoms
via an aliphatic or aromatic spacer [41,42]. Notably, studies by Gingras
et al. [55] have demonstrated that the dithiosemicarbazones (5) exhibited
effective anti-fungal activity and in vitro anti-tumor activity [41,56].
Furthermore, the copper(II), platinum(II) and palladium(II) complexes
of dithiosemicarbazones also showed anti-fungal, anti-bacterial and
anti-tumor activities [40,41,56]. It was demonstrated that the
dithiosemicarbazones formed 1:1 complexes with copper, in which the
two thiocarbonyl groups of the symmetrical dithiosemicarbazone acted
as donor atoms to a single copper ion [56].
Considering the promising anti-proliferative properties exhibited by
our monomeric thiosemicarbazone chelators [33,34], it was of interest
to investigate the effect of dimerization of these compounds on their bio-
logical activity. In fact, chelators of higher denticity generally form more
stable Fe complexes than those chelators of lower denticity [1]. Addition-
ally, dimers have demonstrated greater Fe chelation efficacy in vivo than
their corresponding monomer [1]. Thus, it was important to further ex-
plore the biological activity of dimeric thiosemicarbazones derived from
the potent BpT series [33]. In this study, two types of dimerization strate-
gies were explored. In the first approach, we investigated the synthesis of
dithiosemicarbazones containing two benzoylpyridine moieties connect-
ed via an alkyl bridge. Previous studies have employed a variety of linkers
1H and 13C NMR spectra were obtained in the selected solvent on a
Bruker DPX 300 spectrometer at the designated frequency and were in-
ternally referenced to the solvent peaks. Chemical shifts (δ) are in parts
per million (ppm) downfield from tetramethylsilane (TMS) and the ob-
served coupling constant (J) is in Hertz (Hz). Multiplicities are recorded
as singlet (s), doublet (d), doublet of doublet (dd), doublet of triplet
(dt), triplet (t), quarter (q), multiplet (m) and broad singlet (bs),
where appropriate. Melting points were measured using a Mel-Temp
melting point apparatus and are uncorrected. Microanalysis was per-
formed on a Carlo Erba Elemental Analyzer EA 1108 at the Campbell Mi-
croanalytical Laboratory, University of Otago, New Zealand. Infrared
spectra were recorded with a Thermo Nicolet 370 FTIR spectrometer
as KBr disks. Ultraviolet visible spectra were recorded using a Varian
Cary 100 Scan spectrometer in the designated solvents and data report-
ed as wavelength (λ) in nm and extinction coefficient (ε) in cm−1 M−1
.
Gravity column chromatography was carried out using Grace Davison
LC60A 40–63 μm silica gel. Flash chromatography was done
implementing Grace Davison LC60A 6–35 μm silica gel. Reactions were
monitored using thin layer chromatography, performed on Merck DC
aluminum plates coated with silica gel GF254. Compounds were detect-
ed by short and long wavelength ultraviolet light. The log Pcalc values
were calculated using ChemBio Draw Ultra 13.0.
Fig. 1. General structures of the BpT, NBpT and XBpT series of thiosemicarbazone chelators.

A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
45
Fig. 2. General structures of bis(thiosemicarbazone) 4, dithiosemicarbazone 5 and 2,6-dibenzoylpyridine 6.
2.3. Single crystal X-ray diffraction studies
56Fe2–Tf (56Fe–Tf) or 59Fe2–Tf (59Fe–Tf), using previously reported
methods [62,63].
Single crystals for X-ray diffraction of (16c) were crystallized from
EtOH or CH2Cl2/n-hexane. Suitable single crystals were selected under
a polarizing microscope (Leica M165Z). These crystals were then loaded
onto a MicroMount (MiTeGen, USA) consisting of a thin polymer tip
with a wicking aperture. The X-ray diffraction measurements were car-
ried out on a Bruker kappa-II CCD diffractometer at 150 K by using
graphite-monochromated Mo-Kα radiation (λ = 0.710723 Å). The sin-
gle crystals, mounted on the goniometer using cryo loops for intensity
measurements, were coated with paraffin oil and then quickly trans-
ferred to the cold stream using an Oxford Cryo stream attachment.
Symmetry-related absorption corrections using the program SADABS
[57] were applied and the data were corrected for Lorentz and polariza-
tion effects using Bruker APEX2 software [58]. All structures were solved
by direct methods and the full-matrix least-square refinements were
carried out using SHELXL [59]. The non-hydrogen atoms were refined
anisotropically. The molecular graphic was generated using mercury
[60].
2.5.3. Cellular proliferation assay
The effect of the chelators on cellular proliferation was determined
by the MTT [1-(4,5-dimethylthiazol-2-yl-)-2,5-diphenyl tetrazolium]
assay [33,61]. The SK-N-MC cell line was seeded in 96-well microtitre
plates at 1.5 × 104 cells/well in medium containing human 56Fe–Tf
(1.25 μM) and chelators or their Fe complexes (0–25 μM). Chelators
were freshly dissolved in DMSO as 10 mM stock solutions and diluted
in medium so that the final [DMSO] b 0.5% (v/v). At this final concentra-
tion, DMSO had no effect on proliferation. Control samples contained
medium with human 56Fe–Tf (1.25 μM) without the ligands or com-
plexes. After a 72 h incubation, 10 μL of MTT (5 mg/mL) was added to
each well and the cells incubated for a further 2 h/37 °C. The cells were
then lyzed with 100 μL of 10% SDS–50% isobutanol in 10 mM HCl. The
plates were then read at 570 nm using a scanning multi-well spectro-
photometer. The inhibitory concentration (IC50) was defined as the che-
lator concentration necessary to reduce the absorbance to 50% of the
untreated control.
2.4. Electrochemistry
2.5.4. Effect of chelators on 59Fe efflux from SK-N-MC cells
Cyclic voltammetry was performed using
a
BAS100B/W
Iron efflux experiments examining the ability of various chelators to
mobilize 59Fe from SK-N-MC cells were performed using established
techniques [33,61]. Briefly, following prelabeling of cells with 59Fe–Tf
(0.75 μM) for 3 h/37 °C, the cell cultures were washed four times with
ice-cold phosphate buffered saline (PBS). The cells were then subse-
quently incubated with either medium alone (control) or medium con-
taining the chelator (25 μM) for 3 h/37 °C. The overlying media
containing released 59Fe was then separated from the cells using a Pas-
teur pipette. Radioactivity was measured in both the cell pellet and su-
pernatant using a γ-scintillation counter (Wallace Wizard 3, Turku,
Finland). In this study, the novel ligands were compared to the well
characterized chelators, DFO, BpT, 2-benzoylpyridine 4-ethyl-3-
thiosemicarbazone (Bp4eT) and di-2-pyridylketone 4,4-dimethyl-3-
thiosemicarbazone (Dp44mT) [14,33,61].
potentiostat. A glassy carbon working electrode, an aqueous Ag/AgCl
reference, and a Pt wire auxiliary electrode were used. All complexes
were at ca. 1 mM concentration in MeCN:H2O (7:3) v/v. This solvent
combination was used to ensure solubility of all compounds and has
been utilized in our previous studies examining thiosemicarbazones
[33,34]. The supporting electrolyte was Et4NClO4 (0.1 M), and the solu-
tions were purged with nitrogen prior to measurement. All potentials
are cited versus the normal hydrogen electrode (NHE) by addition of
196 mV to the potentials measured relative to the Ag/AgCl reference
electrode.
2.5. Biological studies
2.5.1. Cell culture
2.5.5. Effect of chelators at preventing 59Fe uptake from 59Fe–Tf by SK-N-MC
cells
The human SK-N-MC neuroepithelioma and MRC-5 fibroblast cell
lines were obtained from the American Type Culture Collection (ATCC,
Manassas, VA, USA) and were grown at 37 °C in a humidified atmo-
sphere of 5% CO2/95% air in an incubator [61]. All cell lines were
grown in Eagle's minimum essential medium (MEM; Invitrogen,
Mulgrave, Victoria, Australia) supplemented with 10% (v/v) fetal calf
serum (Commonwealth Serum Laboratories, Melbourne, Victoria,
Australia), 1% (v/v) non-essential amino acids (Invitrogen), 1% (v/v) so-
dium pyruvate (Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μg/mL
of streptomycin (Invitrogen), 100 U/mL penicillin (Invitrogen), and
0.28 μg/mL of fungizone (Squibb Pharmaceuticals, Montreal, Canada).
The ability of the chelators to prevent cellular 59Fe uptake from 59Fe–
Tf in SK-N-MC cells was examined and performed using standard proce-
dures [14,33,61]. Briefly, cells were incubated with 59Fe–Tf (0.75 μM) for
3 h/37 °C in the presence of medium alone (control) or in medium con-
taining each of the chelators (25 μM). The cells were then washed four
times with ice-cold PBS and internalized 59Fe was determined by plac-
ing the culture plates on ice and incubating the cell monolayer with
the protease, Pronase (1 mg/mL, Sigma) for 30 min/4 °C. The cells
were then removed from the monolayer using a plastic spatula and cen-
trifuged at 12,000 rpm/1 min. The supernatant represents membrane-
bound, Pronase-sensitive 59Fe, while the Pronase-insensitive fraction
represents internalized 59Fe [33,62,63]. In this assay, the novel chelators
were compared to DFO and Dp44mT which are well characterized and
2.5.2. Preparation of Fe–Tf
Human serum Tf (Sigma-Aldrich, St. Louis, MO, USA) was labeled
with 56Fe or 59Fe (Perkin-Elmer, Waltham, MA, USA) to produce

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A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
acted as positive controls [14,61]. Radioactivity was assessed as de-
scribed above.
2.6.4. General procedure for the synthesis of (12), (13) and (16a–e)
The ketones were reacted with the appropriate thiosemicarbazide in
ethanol (EtOH). A few drops of concentrated HCl (32%) were added as a
catalyst and the solution was heated at reflux for 6 h. The resulting mix-
ture was concentrated by rotary evaporation and the resulting precipi-
tate was collected by vacuum filtration. The precipitate formed upon
cooling was collected by vacuum filtration and recrystallized from EtOH.
2.5.6. Statistical analysis
Experimental data were compared using Student's t-test. Results
were expressed as mean SD (number of experiments).
2.6. Chemical synthesis
2.6.4.1. 2′-Benzoylpyridine-(1,6-hexane)-dithiosemicarbazone (12). Com-
pound (12) was synthesized following the general procedure above
using 2-benzoylpyridine (11; 0.543 g, 3 mol) and 4,4′-hexanediyl-
bis(thiosemicarbazide) (8; 0.413 g, 1.5 mol) in EtOH (25 mL) with 10
drops of conc. HCl. The compound was obtained as a yellow powder
(655 mg, 39%). M.p. 206–208 °C; 1H NMR (300 MHz, DMSO-d6): δ 1.33
(s, 4H, CH2), 1.63 (t, J = 6 Hz, 4H, CH2), 3.42 (t, J = 6 Hz, 4H, CH2), 7.35
(d, J = 9 Hz, 1H, ArH), 7.44–7.49 (m, 5H, ArH), 7.62–7.68 (m, 6H, ArH),
7.76–7.81 (m, 3H, ArH), 8.02 (dd, J = 9.0, 3.0 Hz, 1H, ArH), 8.24 (d, J =
9 Hz, 1H, ArH), 8.74–8.78 (m, 1H, ArH), 8.85 (s, 1H, NH), 9.06 (s, 1H,
NH), 9.81 (s, 1H, NH), and 12.68 (s, 1H, NH); 13C NMR (75.6 MHz,
DMSO-d6): δ 26.52 (2 × CH2), 28.93 (2 × CH2), 44.47 (2 × CH2), 116.64
(ArCH), 118.76 (ArCH), 124.61 (ArCH), 125.31 (ArCH), 126.49 (ArCH),
128.76 (4 × ArCH), 129.28 (ArCH), 129.55 (4 × ArCH), 130.12 (ArCH),
130.80 (ArCH), 131.27 (2 × ArC), 138.76 (ArCH), 143.13 (2 × ArC),
149.14 (ArCH), 151.70 (2 × C_N), and 177.52 (2 × C_S); IR (KBr):
2.6.1. 2,6-Dibenzoylpyridine (6)
Excess thionyl chloride (25 mL) and two drops of DMF were added to
2,6-pyridinedicarboxylic acid (14; 5.0 g, 0.03 mol) and the mixture was
heated at reflux for 2 h. The excess thionyl chloride was reduced and
the crude residue was dissolved in benzene (50 mL). Then AlCl3 (10 g,
0.07 mol) was added and the mixture was heated under reflux for
19 h. The resulting mixture was poured into a mixture of ice and concen-
trated HCl (32%, 50 mL), and extracted with diethyl ether (3 × 20 mL).
The organic layer was washed with 1 M NaOH, dried over anhydrous
Na2SO4 and evaporated under reduced pressure. The product was puri-
fied by flash chromatography using dichloromethane (DCM):n-hexane
(7:3) to give dibenzoylpyridine (6) as a flaky-white powder (1.14 g,
93%). M.p. 108–110 °C, lit. [64] 109–110 °C; 1H NMR (300 MHz,
CDCl3): δ 7.44–7.47 (m, 3H, ArH), 7.60–7.63 (m, 3H, ArH), 8.13–8.19
(m, 6H, ArH), and 8.32 (d, J = 8.1 Hz, 1H, ArH); 13C NMR (75.6 MHz,
CDCl3): δ 126.92 (ArCH), 128.04 (4 × ArCH), 131.17 (4 × ArCH),
133.00 (2 × ArCH), 135.97 (ArCH), 138.32 (2 × ArC), 153.84 (2 × ArC),
and 192.57 (2 × C_O).
υ
max 3346, 3236, 2925, 2859, 1615, 1561, 1472, 1359, 1316, 1159, 1132,
1079, 997, 974, 834, 793, 771, 733, 703, 649, 622, 606, and 494 cm−1
;
UV–visible (UV–vis; MeOH): λmax 260 nm (ε 12,800 cm−1 M−1); anal.
calcd. for C32H34N8S2·0.75CH2Cl2: C, 59.74; H, 5.43; N, 17.02. Found C,
59.58; H, 5.78; and N, 17.36; HRMS (+ESI): Found m/z 595.2347
[M + H]+; C32H25N8S2 required 595.2348.
2.6.2. 4,4′-Hexanediyl-bis(thiosemicarbazide) (8)
Carbon disulfide (CS2; 4.5 mL, 7.5 mmol) was added to a mixture of
1,6-diaminohexane (7; 2.87 g, 25 mmol) and NaOH (2.08 g, 50 mmol)
in water (25 mL). The mixture was stirred for 3.5 h at room tempera-
ture. Sodium chloroacetate (5.85 g, 50 mmol) was added and the
resulting mixture was left to stir overnight at room temperature. The
mixture was acidified with HCl (2 M, 8 mL), followed by the addition
of excess hydrazine hydrate (12 mL). The solution was heated at reflux
for 2 h and the precipitate that formed upon cooling was collected. The
product was obtained as white crystals (1.55 g, 24%). M.p. 141–143 °C,
lit. [65] 144–145 °C; 1H NMR (300 MHz, DMSO-d6): δ 1.24 (t, J = 6 Hz,
4H, CH2), 1.47–1.51 (m, 4H, CH2), 3.42 (t, J = 6 Hz, 4H, N–CH2), 4.40 (bs,
4H, NH2), 7.77 (s, 2H, NH), and 8.50 (s, 2H, NH); 13C NMR (75.6 MHz,
DMSO-d6): δ 26.54 (2 × CH2), 29.47 (2 × CH2), 43.19 (2 × CH2), and
181.48 (2 × C_S).
2.6.4.2. 2′-Benzoylpyridine-(1,12-dodecane)-dithiosemicarbazone (13).
Compound (13) was synthesized following the general procedure
using 2-benzoylpyridine (11; 0.548 g, 3 mol) and N,N′-(dodecane-
1,12-diyl)bis(hydrazinecarbothioamide) (10; 0.519 g, 1.5 mol) in EtOH
(25 mL) with 8 drops of conc. HCl. The compound was obtained as a yel-
low powder (160 mg, 8%). M.p. 273–275 °C; 1H NMR (300 MHz, DMSO-
d6): δ 1.22 (s, 16H, CH2), 1.53 (t, J = 6 Hz, 4H, CH2), 2.66–2.73 (m, 4H,
CH2), 7.22 (dd, J = 6.2 Hz, 2H, ArH), 7.38 (d, J = 3 Hz, 2H, ArH),
7.50–7.54 (m, 5H, ArH), 7.99 (d, J = 9 Hz, 2H, ArH), 8.09–8.12 (m, 5H,
ArH), 8.27 (d, J = 6 Hz, 2H, ArH), 9.10 (s, 1H, NH), 9.12 (s, 1H, NH),
12.70 (s, 1H, NH), and 12.72 (s, 1H, NH); 13C NMR (75.6 MHz,
DMSO-d6): δ 26.77 (2 × CH2), 29.03 (2 × CH2), 29.23 (4 × CH2),
29.44 (2 × CH2), 39.20 (2 × CH2), 125.35 (2 × ArCH), 126.52
(2 × ArCH), 128.84 (4 × ArCH), 129.08 (ArCH), 129.39 (4 × ArCH),
129.64 (ArCH), 129.64 (ArCH), 137.41 (2 × ArC), 138.67 (ArCH),
143.32 (2 × ArC), 149.33 (2 × ArCH), 151.92 (2 × C_N), and 177.59
(2 × C_S); IR (KBr): υmax 3399, 2924, 2849, 2522, 2361, 2046, 1604,
1527, 1494, 1471, 1397, 1299, 1257, 1225, 1207, 1164, 1147, 1047,
2.6.3. N,N′-(Dodecane-1,12-diyl)bis(hydrazinecarbothioamide) (10)
Carbon disulfide (CS2, 8 mL) was added to a mixture of 1,12-
diaminododecane (9; 3.32 g, 16.7 mmol) and NaOH (1.36 g,
33.3 mmol) in water (30 mL). The mixture was stirred for 6 h at
room temperature. Sodium chloroacetate (5.0 g, 50 mmol) was
added and the resulting mixture was left to stir overnight at room
temperature. The mixture was acidified with HCl (2 M, 15 mL). The
solution was heated at reflux for 2 h and the precipitate that formed
upon cooling was collected. The product was obtained as an off white
powder (2.30 g, 40%). M.p. 122–124 °C; 1H NMR (300 MHz, DMSO-
d6): δ 1.22 (s, 16H, CH2), 1.45–1.48 (m, 4H, CH2), 3.39 (t, J = 9 Hz,
4H, N–CH2), 4.39 (bs, 4H, NH2), 7.77 (s, 2H, NH), and 8.49 (s, 2H,
NH); 13C NMR (75.6 MHz, DMSO-d6): δ 26.32 (CH2), 26.71 (CH2),
29.21 (2 × 1CH2), 29.40 (4 × CH2), 29.48 (2 × CH2), 43.21 (2 × CH2),
and 181.45 (2 × C_S). IR (KBr): υmax 3243, 2915, 2849, 2366, 2062,
1604, 1566, 1501, 1470, 1413, 1364, 1288, 1260, 1216, 1148, 1095,
1058, 1048, 1028, 972, 934, 836, 775, 719, 632, 572, 499, 475, and
410; anal. calcd. for C14H32N6S20.2CH2Cl2: C 46.66; H, 8.93; and N,
22.99. Found C, 46.60; H, 8.61; N, 22.70; High-resolution mass
spectrometry (HRMS) (electrospray ionization; +ESI): Found m/z
371.2019 [M + Na]+; C14H32N6S2Na required 371.2028.
1036, 1014, 996, 939, 915, 825, 787, 767, 728, 699, 509, and 476 cm−1
;
UV–vis (MeOH): λmax 273 nm (ε 14,900 cm−1 M−1), and 326
(26,500); anal. calcd. for C38H46N8S2.0.5CH2Cl2: C, 64.10; H, 6.57; and
N, 15.53. Found C, 63.86; H, 6.15; N, 15.36; HRMS (+ESI): Found m/z
701.3478, [M + Na]+ C38H46N8S2Na required 701.3185.
2.6.4.3. 2,6-Dibenzoylpyridine thiosemicarbazone (16a). Compound (16a)
was synthesized following the general procedure using 2,6-
dibenzoylpyridine (6; 0.197 g, 0.696 mol) and thiosemicarbazide
(15a; 0.159 g, 1.74 mol) in EtOH (25 mL) with 8 drops of conc. HCl.
The compound was obtained as a yellow powder (253 mg, 85%).
M.p. 223–228 °C; 1H NMR (300 MHz, CDCl3): δ 7.22 (dd, J = 1.2,
7.8 Hz, 1H, ArH), 7.33 (dd, J = 1.0, 8.1 Hz, 1H, ArH), 7.42 (dd, J = 1.8,
6.3 Hz, 1H, ArH), 7.48–7.51 (m, 3H, ArH), 7.54–7.58 (m, 4H, ArH),
7.64–7.73 (m, 3H, ArH), 7.77 (s, 2H, NH), 8.49 (s, 1H, NH), 8.78 (s, 1H,
NH), and 13.88 (s, 2H, NH2); 13C NMR (75.6 MHz, CDCl3): δ 125.38
(ArCH), 126.68 (ArCH), 128.61 (4 × ArCH), 129.13 (4 × ArCH), 130.52

A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
47
(2 × ArCH), 130.90 (ArCH), 138.07 (2 × ArC), 148.40 (2 × ArC), 153.50
(2 × C_N), and 179.13 (2 × C_S); IR (KBr): υmax 3261, 1584, 1474,
1265, 1084, 835, 769, and 702 cm−1; UV–vis (MeOH): λmax 211 nm (ε
113,720 cm−1 M−1), 240 (88,488), 308 (105,526) and 313 (94,427);
anal. calcd. for C21H19N7S2·0.2H2O: C, 57.70; H, 4.47; and N, 22.43.
Found C, 57.76; H, 4.83; and N, 22.29; HRMS (+ESI): Found m/z
434.1213, [M + H]+; C21H20N7S2 required 434.1177.
(KBr): υmax 3286, 3069, 1637, 1524, 1274, 1189, 1109, 926, 827, 770,
and 696 cm−1; UV–vis (MeOH): λmax 215 nm (ε 101,000 cm−1 M−1),
237 (76,400), 308 (91,000) and 316 (81,000); anal. calcd. for
C27H27N7S2: C, 63.13; H, 5.30; and N, 19.09. Found C, 63.24; H, 5.11;
and N, 18.89; HRMS (+ESI): Found m/z 514.1829, [M + H]+
;
C27H28N7S2 required 514.1803.
2.6.4.7. 2,6-Dibenzoylpyridine 4-phenyl-3-thiosemicarbazone (16e). Com-
pound (16e) was synthesized following the general procedure using
2,6-dibenzoylpyridine (6; 0.198 g, 0.696 mmol) and 4-phenyl-3-
thiosemicarbazide (15e; 0.288 g, 1.74 mmol) in EtOH (20 mL) with 6
drops of conc. HCl and was obtained as a yellow powder (219 mg,
54%). M.p. 239–243 °C; 1H NMR (300 MHz, CDCl3): δ 7.18 (dd, J = 1.0,
8.1 Hz, 2H, ArH), 7.28 (dd, J = 1.0, 8.1 Hz, 2H, ArH), 7.42 (dd, J = 1.8,
8.1 Hz, 2H, ArH), 7.48–7.52 (m, 5H, ArH), 7.53–7.58 (m, 4H, ArH),
7.61–7.70 (m, 6H, ArH), 7.73 (d, J = 8.1 Hz, 2H, ArH), 7.80 (s, 1H,
NH), 8.72 (s, 1H, NH), and 13.70 (s, 1H, NH); 13C NMR (75.6 MHz,
CDCl3): δ 126.12 (4 × ArCH), 128.71 (4 × ArCH), 129.11 (4 × ArCH),
129.36 (4 × ArCH), 130.66 (2 × ArCH), 130.90 (2 × ArCH), 137.62
(ArCH), 137.83 (2 × ArC), 142.38 (2 × ArC), 146.48 (2 × ArC), 152.37
(C_N), 153.42 (C_N), 177.16 (C_S), and 177.89 (C_S); IR (KBr):
υmax 3288, 2973, 1529, 1459, 1277, 1199, 1107, 951, 811, and
699 cm−1; UV–vis (MeOH): λmax 319 nm (ε 57,800 cm−1 M−1);
anal. calcd. for C33H27N7S2·3H2O: C, 61.95; H, 5.20; and N, 15.32.
Found C, 61.69; H, 5.37; and N, 15.43; HRMS (+ESI): Found m/z
586.1805, [M + H]+; C33H28N7S2 required 586.1803.
2.6.4.4. 2,6-Dibenzoylpyridine 4-methyl-3-thiosemicarbazone (16b). Com-
pound (16b) was synthesized following the general procedure using 2,6-
dibenzoylpyridine (6; 0.199 g, 0.696 mmol) in EtOH (25 mL), 4-methyl-
3-thiosemicarbazide (15b; 0.181 g, 1.74 mmol) and 6 drops of conc. HCl
and was obtained as a yellow powder (300 mg, 93%). M.p. 226–229 °C;
1H NMR (300 MHz, CDCl3): δ 3.31 (d, J = 4.8 Hz, 3H, CH3), 3.35 (d, J =
4.8 Hz, 3H, CH3), 7.12 (dd, J = 1.0, 8.1 Hz, 1H, ArH), 7.28 (dd, J = 1.0,
8.0 Hz, 1H, ArH), 7.41–7.43 (m, 2H, ArH), 7.48–7.51 (m, 3H, ArH),
7.54–7.57 (m, 2H, ArH), 7.62–7.74 (m, 4H, ArH), 7.86 (s, 1H, NH), 8.61
(s, 1H, NH), 8.77 (s, 1H, NH), and 13.89 (s, 1H, NH); 13C NMR
(75.6 MHz, CDCl3): δ 31.13 (CH3), 32.20 (CH3), 125.18 (ArCH), 126.20
(ArCH), 128.69 (4 × ArCH), 129.13 (4 × ArCH), 130.46 (2 × ArCH),
130.91 (ArC), 137.59 (ArC), 137.87 (ArCH), 142.40 (ArC), 146.42 (ArC),
152.44 (C_N), 153.27 (C_N), 178.16 (C_S), and 178.91 (C_S); IR
(KBr): υmax 3473, 3361, 3281, 3041, 1541, 1475, 1218, 1105, 1041,
819, 772, and 699 cm−1
; UV–vis (MeOH): λmax 212 nm (ε
247,000 cm−1 M−1), 237 (166,000), 308 (215,000), and 315
(204,000); anal. calcd. for C23H23N7S2: C, 59.84; H, 5.02; and N, 21.24.
Found C, 60.09; H, 5.00; and N, 21.21; HRMS (+ESI): Found m/z
484.1338, [M + Na]+; C23H23N7S2Na required 484.1354.
2.6.5. General procedure of the synthesis of dimeric [FeIII(BpT)] complexes
The desired thiosemicarbazone chelator (3.5 mmol) was dissolved
in absolute EtOH (15 mL). A few drops of Et3N were added to the solu-
tion followed by the addition of Fe(ClO4)3·6H2O (1.7 mmol). The mix-
ture was gently refluxed overnight, and after cooling, the dark brown
precipitate was filtered and washed with EtOH.
2.6.4.5. 2,6-Dibenzoylpyridine 4-ethyl-3-thiosemicarbazone (16c). Com-
pound (16c) was synthesized following the general procedure using
2,6-dibenzoylpyridine (6; 0.151 g, 0.52 mmol) and 4-ethyl-3-
thiosemicarbazide (15c; 0.159 g, 1.31 mmol) in EtOH (20 mL) with 8
drops of conc. HCl and was obtained as a yellow powder (230 mg,
89%). M.p. 234–237 °C; 1H NMR (300 MHz, CDCl3): δ 1.34 (t, J =
6.3 Hz, 6H, 2 × CH3), 3.85 (q, J = 6.3 Hz, 4H, 2 × CH2), 7.17 (dd, J =
1.2, 7.8 Hz, 1H, ArH), 7.27 (dd, J = 1.0, 8.1 Hz, 1H, ArH), 7.40 (dd, J =
1.8, 9.3 Hz, 1H, ArH), 7.48–7.52 (m, 4H, ArH), 7.53–7.58 (m, 3H, ArH),
7.61–7.75 (m, 3H, ArH), 7.82 (s, 1H, NH), 8.64 (s, 1H, NH), 8.72 (s, 1H,
NH), and 13.69 (s, 1H, NH); 13C NMR (75.6 MHz, CDCl3): δ 14.45
(CH3), 14.49 (CH3), 39.29 (CH2), 40.01 (CH2), 124.97 (ArCH), 126.13
(ArCH), 128.71 (4 × ArCH), 129.10 (ArCH), 129.22 (2 × ArCH), 130.43
(2 × ArCH), 130.89 (ArC), 137.60 (ArC), 142.40 (ArC), 146.51 (ArC),
152.36 (C_N), 153.41 (C_N), 177.15 (C_S), and 177.88 (C_S); IR
(KBr): υmax 3290, 3054, 2973, 1525, 1460, 1279, 1199, 1107, 949, 813,
and 701 cm−1; UV–vis (MeOH): λmax 217 nm (ε 99,100 cm−1 M−1),
238 (89,700), 308 (92,500), 313 (77,000), 323 (76,000) and 352
(50,100); anal. calcd. for C25H27N7S2: C, 61.32; H, 5.56; and N, 20.02.
Found C, 61.38; H, 5.49; and N, 19.74; HRMS (+ESI): Found m/z
512.1653, [M + Na]+; C25H27N7S2Na required 512.1667.
2.6.5.1. [Fe(Bp-(1,6-hexane)diT)]ClO4·0.5H2O (17). Dark brown solid
(19.2 mg, 35%); anal. calcd. for C32H32FeClN8O4S2·0.5H2O: C, 50.77; H,
4.39; and N, 14.80. Found: C, 50.93; H, 4.67; and N, 14.68. HRMS
(+ESI): Found m/z 648.1520, [M]+; C32H32FeN8S2 required 648.1541.
2.6.5.2. [Fe(Bp-(1,12-dodecane)diT)]ClO4 (18). Dark brown solid (5.4 mg,
13%); anal. calcd. for C38H48FeClN8O4S2: C, 54.58; H, 5.79; and N, 13.40.
Found: C, 54.63; H, 6.01; and N, 13.46. HRMS (+ESI): Found m/z
736.2752, [M]+; C38H48FeN8S2 required 736.2793.
2.6.5.3. [Fe(2,6-diBpT)2]4H2O (19a). Dark brown solid (33.9 mg, 32%);
anal. calcd. for C42H36FeN14S4·4H2O: C, 50.80; H, 4.47; and N, 19.75.
Found: C, 51.18; H, 4.10; and N, 19.93. HRMS (+ESI): Found m/z
920.1458, [M]+; C42H36FeN14S4 required 920.1479.
2.6.5.4. [Fe(2,6-diBp4mT)2]ClO4 (19b). Dark brown solid (47 mg, 44%);
anal. calcd. for C46H44FeClN14O4S4: C, 51.32; H, 4.12; and N, 18.22.
Found: C, 51.03; H, 4.62; and N, 18.10. HRMS (+ESI): Found m/z
976.2316, [M]+; C46H44FeN14S4 required 976.2105.
2.6.4.6. 2,6-Dibenzoylpyridine 4-allyl-3-thiosemicarbazone (16d). Com-
pound (16d) was synthesized following the general procedure using
2,6-dibenzoylpyridine (6; 0.102 g, 0.348 mmol) and 4-allyl-3-
thiosemicarbazide (15d; 0.119 g, 0.912 mmol) in EtOH (20 mL) with 6
drops of conc. HCl and was obtained as a yellow solid (153 mg, 84%).
M.p. 177–180 °C; 1H NMR (300 MHz, CDCl3): δ 4.41–4.50 (m, 4H, CH2),
5.20–5.39 (m, 4H, CH2), 5.80–6.10 (m, 2H, CH), 7.23 (dd, J = 8.1,
2.2 Hz, 1H, ArH), 7.43 (dd, J = 1.8, 8.1 Hz, 1H, ArH), 7.46–7.52 (m, 4H,
ArH), 7.53–7.57 (m, 3H, ArH), 7.61–7.71 (m, 4H, ArH), 7.75 (s, 1H,
NH), 7.89 (s, 1H, NH), 8.79 (s, 1H, NH), and 13.72 (s, 1H, NH); 13C
NMR (75.6 MHz, CDCl3): δ 46.71 (CH2), 47.19 (CH2), 116.71 (CH2),
116.94 (CH2), 124.92 (ArCH), 126.23 (ArCH), 128.70 (4 × ArCH),
129.20 (4 × ArCH), 129.42 (ArCH), 130.71 (2 × ArCH), 133.46 (CH),
133.60 (CH), 137.50 (ArC), 137.89 (ArC), 142.61 (ArC), 146.81 (ArC),
152.29 (C_N), 153.40 (C_N), 177.78 (C_S), and 178.24 (C_S); IR
2.6.5.5. [Fe(2,6-diBp4eT)2]ClO4·7H2O (19c). Dark brown solid (24.3 mg,
23%); anal. calcd. for C50H52ClFeN14O4S4·7H2O: C, 47.63; H, 5.44; and
N, 15.55. Found: C, 47.96; H, 5.47; and N, 15.37. HRMS (+ESI): Found
m/z 1033.3249, [M]+; C50H52FeN14S4 required 1033.2765.
2.6.5.6. [Fe(2,6-diBp4aT)2]ClO4·5H2O (19d). Dark brown solid (13.2 mg,
13%); anal. calcd. for C53H52ClFeN14O4S4·5H2O: C, 50.96; H, 5.07; and
N, 15.41. Found: C, 50.79; H, 5.14; and N, 15.03. HRMS (+ESI): Found
m/z 1080.2581, [M]+; C53H52FeN14S4 required 1080.2731.
2.6.5.7. [Fe(2,6-diBp4pT)2]ClO4·8H2O (19e). Dark brown solid (8.2 mg,
11%); anal. calcd. for C66H58ClFeN14O4S4·8H2O: C, 53.75; H, 5.06; and

48
A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
N, 13.30. Found: C, 53.97; H, 5.13; and N, 13.19. HRMS (+ESI): Found m/
z 1230.3221, [M]+; C53H52FeN14S4 required 1230.3201.
2,6-dibenzoylpyridine in EtOH using concentrated HCl as a catalyst
(Scheme 4). The desired 2,6-diBpT analogs (16a–e) were obtained in
54–93% yields.
Identification of compounds (16a–e) was performed using 1H NMR
and 13C NMR spectroscopy. The 1H NMR spectrum of compound (16c)
in CDCl3 was characteristic for all the symmetrical dimeric
thiosemicarbazones (16a–e). A triplet at δ 1.34 and a quartet at δ 3.85
corresponded to the presence of the CH3 and CH2 protons of the ethyl
group, respectively. The aromatic protons H2′/H2″, H3′/H3″, H4′/H4″,
H5′/H5″ and H6′/H6″ (see Scheme 4 for numbering) appeared between
δ 7.48 and 7.75. The characteristic hydrogen-bonded NH protons ap-
peared as a singlet at δ 13.69. Further structural confirmation of (16c)
was obtained from the 13C NMR spectrum, where the disappearance of
the peak at δ 192.6 along with the appearance of peaks at δ 152.4 and
δ 153.4 indicated that the C_O bonds in the molecule had been replaced
with C_N bonds. Furthermore, the new peaks at δ 177.2 and δ 177.9
corresponded to the presence of C_S carbon atoms in the structure.
The presence of two C_N and two C_S peaks at very close values
was attributed to the formation of both E and Z isomers at the imine
bond.
3. Results and discussion
3.1. Synthesis and characterization
The synthesis of dithiosemicarbazides was performed using a litera-
ture method [47] in which CS2 was reacted with 1,6-diaminohexane (7)
or 1,12-diaminododecane (9) in the presence of aqueous NaOH. Sodium
chloroacetate was then added and the reaction was stirred overnight.
This was followed by the addition of H2NNH2·H2O to yield the
dithiosemicarbazides (8) and (10) (Scheme 1).
The structures of the products were confirmed by NMR spectroscopy.
In the 1H NMR spectrum of the symmetrical molecule (8) in d6-DMSO,
triplets at δ 1.24, 1.47 and 3.39 corresponded to the –CH2– groups of the
hexane bridge, while a broad singlet at δ 4.40 corresponded to the –NH2
protons. The NH protons adjacent to the C_S group appeared at δ 7.77
and δ 8.50. In the 13C NMR spectrum, the appearance of a peak at δ
181.5 indicated the presence of the C_S carbon atoms. In comparison
to the monomeric thiosemicarbazides, the dithiosemicarbazides showed
lower solubility and could not be dissolved in CDCl3.
3.2. Synthesis of iron complexes of dimeric thiosemicarbazone
The synthesis of the dithiosemicarbazones was conducted using
established Schiff base condensation methodology [33,36,37], in
which 1 equivalent of dithiosemicarbazide (8) or (10) was reacted
with 2 equivalents of benzoylpyridine (11) in EtOH using HCl as cata-
lyst. The reaction was heated at reflux overnight and yielded the novel
dithiosemicarbazones (12) and (13) as yellow crystalline solids
(Scheme 2).
Previous studies on the complexation of thiosemicarbazones and
aroylhydrazones with metal ions have demonstrated marked changes
in the biological activity of the ligands [13,33,34,66]. Therefore, the Fe
complexes of the dimeric thiosemicarbazones were synthesized in
order to investigate the anti-proliferative activity in comparison with
the free ligands. The FeIII complexes of the dimeric thiosemicarbazones
were synthesized and characterized. The dimeric thiosemicarbazones
were dissolved in EtOH with the addition of few drops of Et3N as a cat-
alyst, followed by the addition of Fe(ClO4)3·6H2O. The reactions were
heated and the resulting precipitate was filtered and washed with EtOH.
All the synthesized complexes were brown in color and air stable,
signifying the formation of FeIII complexes. Moreover, these compounds
were only soluble in polar solvents such as dimethylformamide (DMF)
or DMSO. The analytical data showed that the 2,6-diBpT ligands (16a–
e) formed 2:1 ligand:metal complexes, while the dithiosemicarbazones
(12 and 13) formed 1:1 ligand:metal complexes. This result suggests
that the dithiosemicarbazones (12 and 13) act as hexadentate ligands,
using their pyridyl nitrogens, imine nitrogens and thiocarbonyl sulfurs
as donor atoms.
In the 1H NMR spectrum of (12) in d6-DMSO, broad triplets at δ 1.04,
1.33 and 3.57 corresponded to the CH2 groups of the hexane linker. The
successful incorporation of the benzoylpyridine rings was indicated by
the appearance of aromatic doublets and multiplet peaks at δ 7.22–8.86.
Moreover, the NH protons of the hydrazide proton that are known to
hydrogen bond to the pyridine nitrogen [13,33], appeared downfield
at δ 12.68. Finally, the 13C NMR spectrum of (12) revealed the appear-
ance of C_N carbon atom at 154.88 and the C_S carbon atom reso-
nance at δ 177.5.
To generate the 2,6-diBpT analogs, 2,6-dibenzoylpyridine (6) was
first synthesized using a method reported by Lash et al. [64] in which
pyridine dicarboxylic acid (14) was treated with excess thionyl chloride
and catalytic DMF to afford a diacyl chloride intermediate. The diacyl
chloride was then reacted with benzene under Friedel–Crafts acylation
conditions to give 2,6-dibenzoylpyridine (6) as
a
white solid
3.3. Crystal structure of ligand 16c
(Scheme 3). The crude solid was purified by flash chromatography
using n-hexane:DCM (3:7) to yield the target compound in 93% yield.
The structure of 2,6-dibenzoylpyridine (6) was confirmed by 1H
NMR spectroscopy, which was consistent with reported literature
values [64]. The presence of additional doublets and multiplets in the
aromatic region δ 7.29–8.92 were indicative of the two phenyl rings
that had been incorporated into the molecule. In the 13C NMR spectrum,
the presence of a peak at δ 192.6 confirmed the presence of the two
C_O carbon atoms.
Final confirmation of the structure of the 2,6-diBpT analogs was pro-
vided through X-ray crystallography (Fig. 3). In the ORTEP view of the
ligand (16c), there are three intra-molecular N\H…N hydrogen
bonds, as well as three intra-molecular C\H…S contacts which have
also been observed with the monomer ligands [33,36]. The most signif-
icant interactions are N3–H4N…S, C15\H15A…S and C24\H24A…S,
which maintain the overall planarity of the molecule. Hydrogen bond-
ing S…H distances are in the range of 2.68–2.86 Å, while hydrogen
bonding H…N distances are in the range of 0.98–2.31 Å. The H
(N\H…N) angle varies from 107 to 134°, which are similar to the
values observed in the previously synthesized BpT ligands [33].
Similarly, the 2,6-diBpT series of ligands were synthesized through
Schiff base condensation methodology [33,36,37] by reacting 2 equiva-
lents of the thiosemicarbazide analogs (15a–e) with 1 equivalent of
Scheme 1. Reagents and conditions: i) NaOH/H2O, CS2, ClCH2COONa, ii) NH2NH2·H2O.

A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
49
Scheme 2. Reagents and conditions: EtOH, HCl, reflux, 8 h and 90 °C.
3.4. Electrochemistry
with the clinically used positive control chelator, DFO, and the monomeric
thiosemicarbazones of the BpT series [33].
The electrochemical properties of the Fe complexes of the dimeric
thiosemicarbazones were examined via cyclic voltammetry to determine
the potential of the complexes to undergo redox cycling in a physiological
context [13,33,34]. Previous studies on thiosemicarbazone-based chela-
tors have shown a correlation between anti-proliferative activity and
the capability to undergo Fenton chemistry upon complexation with in-
tracellular Fe [13,33]. Hence, it was important to investigate the electro-
chemical properties of the Fe complexes formed by these dimeric
systems.
The Fe complex of 2′-benzoylpyridine-(1,6-hexane)-dithiosemi
carbazone (17) underwent reversible FeIII/II redox coupling in MeCN:
H2O (7:3) at +131 mV vs the NHE (Table 1). This represented an anodic
shift of 32 mV compared with the monomeric 2′-benzoylpyridine 4-
ethyl-3-thiosemicarbazone [Fe(Bp4eT)2] complex, which had a redox
potential of +99 mV vs. the NHE [33]. Investigation of the redox potential
of the Fe complex 2′-benzoylpyridine-(1,12-dodecane)-dithiosemi
carbazone (18) proved to be problematic due to its low solubility in the
solvent combination used. Notably, the solvent combination imple-
mented herein (i.e., MeCN:H2O; 7:3) was utilized as it has shown to
be optimal for dissolving other thiosemicarbazones that our group has
studied [33,34].
The redox potential of the Fe complexes of the 2,6-diBpT analogs
(19a–d) were measured and exhibited totally reversible FeIII/II couples
in MeCN:H2O (7:3), with significant cathodic shifts of 57–184 mV rela-
tive to the parent BpT chelators [33] (Table 1). Of the Fe complexes of
the novel chelators 19a–d, [Fe(2,6-diBpT)2] (19a) had the highest
redox potential of +123 mV vs. the NHE. In contrast, [Fe(2,6-
diBp4mT)2] (19b), [Fe(2,6-diBp4eT)2] (19c) and [Fe(2,6-diBp4aT)2]
(19d) had lower redox potential values of +56, −4.5 and +18 mV
vs. the NHE, respectively (Table 1). Again, the redox potential of
[Fe(2,6-diBp4pT)2] (19e) could not be measured due to its low solubil-
ity. When comparing the redox potentials of the two classes of dimeric
thiosemicarbazones, the Fe complex of dithiosemicarbazone (17)
showed an anodic shift of 11–135.5 mV compared to the Fe complexes
of the 2,6-diBpT series (19a–d) (Table 1).
The 7 novel dimeric chelators displayed moderate anti-proliferative
activity against SK-N-MC cells, with IC50 values ranging from 0.31 to
10.64 μM, and were more potent than DFO (IC50: 21.06 μM; Table 2).
The most potent dimeric chelators, namely 2,6-diBpT (16a) and the
dithiosemicarbazone (12), exhibited IC50 values of 0.31
0.009 and
0.33 0.049 μM, respectively, and were significantly (p b 0.001)
more potent than DFO. On the other hand, 2,6-diBp4pT (16e) was the
least active of all the dimeric thiosemicarbazones (IC50 N 6.25 μM). In
general, in comparison to the dimeric thiosemicarbazones, their corre-
sponding monomeric chelators showed far more potent activity in SK-
N-MC cells, with IC50 values of 0.002–4.66 μM [33] (Table 2). A notable
exception to this observation was 2,6-diBpT (16a), which showed ap-
proximately 15-fold greater anti-proliferative activity than its mono-
meric counterpart, BpT (Table 2).
It has been demonstrated that lipophilicity of chelators plays an im-
portant role in determining their anti-proliferative activity as its influ-
ences membrane permeability [61]. Optimal lipophilicity enables
chelators to permeate the membrane lipid bilayer without retention.
In this study, the less potent chelators 13 and 16b–d had log Pcalc values
of 4.84–6.69 (Table 2). Notably, the least active compounds, namely 13
and 16e (R = Ph), possessed the highest log Pcalc values of 9.19 and 8.17,
respectively. The correlation between lipophilicity and anti-
proliferative efficacy (Fig. 4) is consistent with previous studies, where
thiosemicarbazones with the most potent anti-proliferative activity
had log Pcalc values between 3.5 and 4.5 [13,33,34,69]. In light of these
observations, the more hydrophobic chelators, 13 and 16e, could be-
come trapped in the cell membrane, preventing entrance into the cell
and their ability to target Fe [34,69]. This indicates that optimal lipophi-
licity is crucial for enhancing the anti-proliferative activity of these
ligands.
3.5.2. Anti-proliferative activity of Fe complexes of dimeric
thiosemicarbazones against tumor cells
Previous studies have shown that complexation of the chelators
with Fe can result in changes in the anti-proliferative activi-
ty [33,66,70]. Thus, the effect of Fe complexation of the dimeric chela-
tors on anti-proliferative activity was examined using SK-N-MC
neuroepithelioma cells (Table 2). The results showed the Fe complexes
of compounds 12, 13, and 16a–16d, namely the Fe complexes 17, 18 and
19a–d, respectively, were significantly (p b 0.005) less potent than their
corresponding free ligands, with the Fe complexes 17 and 18 showing
IC50 values greater than 6.25 μM. In fact, most of the Fe complexes, espe-
cially 19a–d, showed a 2- to 20-fold decrease in anti-proliferative activ-
ity compared to the free chelators, which was consistent with previous
findings [33,66,70]. Both the ligand 16e and its Fe complex 19e did not
The reversible electrochemical behavior of the Fe complexes sug-
gests that the novel chelators could be able to assist the generation of in-
tracellular ROS and undergo reversible redox cycling, which is known to
play a key role in the anti-proliferative activity of thiosemicarbazones
against tumor cells [13,14,67,68].
3.5. Biological studies
3.5.1. Anti-proliferative activity against tumor cells
The anti-proliferative activity of the dimeric thiosemicarbazones was
examined in SK-N-MC neuroepithelioma cells using the MTT assay
(Table 2). This cell-type was utilized as it has been widely used in our lab-
oratory to examine the anti-proliferative activity of a range of related
monomeric thiosemicarbazones [33,34]. Hence, the results obtained
could be readily compared to these previous data. The anti-proliferative
activity of 2′-benzoylpyridine-(1,6-hexane)-dithiosemicarbazone (12),
2′-benzoylpyridine-(1,12-dodecane)-dithiosemicarbazone (13) and the
2,6-dibenzoylpyridine thiosemicarbazones (16a–e) were compared
Scheme 3. Reagents and conditions: i) SOCl2, DMF, ii) benzene, AlCl3.

50
A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
Scheme 4. Reagents and conditions: EtOH, conc. HCl, reflux, 8 h and 95 °C.
show any marked anti-proliferative effects (IC50 N 6.25 μM; Table 2).
The lower potency of the Fe complexes may be attributed to their inabil-
ity to chelate intracellular Fe that is required for DNA synthesis and pro-
liferation [13,33]. In addition, due to the enhanced lipophilicity of the
complexes relative to the ligands [71], they may partition into mem-
branes to a greater extent and prevent entrance into the cytosol,
which could reduce their efficacy.
3.5.4. Mobilization of cellular 59Fe from cells
The ability of chelators to effectively mobilize intracellular Fe plays a
crucial role in their anti-proliferative activity [61]. Therefore, the ability
of the dimeric thiosemicarbazone-based chelators to mobilize intracel-
lular 59Fe from prelabeled SK-N-MC neuroepithelioma cells was evalu-
ated. The release of intracellular 59Fe by the ligands (25 μM) was
compared to the positive controls, DFO, Dp44mT, BpT and Bp4eT,
which have been previously characterized in this cell-type [33].
As shown previously, DFO exhibited limited ability to efflux cellular
59Fe [61,68], releasing only 14 1% of intracellular 59Fe, compared to
3.5.3. Anti-proliferative activity against mortal cells
The selectivity of the novel dimeric thiosemicarbazones was evalu-
ated by assessing their toxicity towards mortal human MRC-5 fibro-
blasts relative to the neoplastic SK-N-MC cell-type (Table 2).
Interestingly, the mortal cells were significantly (p b 0.001) less sensi-
tive to the novel chelators, 12 and 16a–d, compared to neoplastic
cells. The dimeric chelators exhibited a 3- to 20-fold decrease in anti-
proliferative activity against mortal cells, with all compounds showing
IC50 values greater than 5.32 μM. Notably, the dimer 16a, was the
most potent chelator against SK-N-MC cells and also showed the
highest selectivity when compared to the anti-proliferative activity ob-
served with MRC-5 cells. It is well known that cancer cells require
higher levels of Fe than normal cells due to their rapid proliferation
[1]. This arises from the increased Fe requirement for metabolic en-
zymes, such as RR, that is up-regulated in cancer cells [1]. Hence, the se-
lective anti-proliferative activity of the chelators towards neoplastic
cells suggests the presence of a “therapeutic window” in which tumor
cells can be selectively targeted with less effect against mortal cells.
7
1% of intracellular 59Fe mobilized by control medium alone
(Fig. 5a). On the other hand, the ligands used as positive controls, name-
ly Dp44mT, BpT and Bp4eT, mediated the release of 36 1%, 35 1%
and 33
1% of intracellular 59Fe, respectively. The high activity of
these latter ligands at mobilizing 59Fe was in good agreement with pre-
vious studies [33]. Of the 2,6-diBpT chelators, compound 16a showed
the greatest ability to mediate intracellular 59Fe efflux (28
1%),
while compounds 16b–e released 15–24% of cellular 59Fe. Among the
dithiosemicarbazones, compound 12 mediated the release of 18 1%
of intracellular 59Fe, while compound 13 released only 4 1% of intra-
cellular 59Fe (Fig. 5a). This latter result suggested that because of its
very high lipophilicity (log Pcalc 9.19), 13 became trapped within the
cell membrane, restricting efflux of intracellular 59Fe to a lesser extent
than the control.
The Fe mobilization efficacy of the novel dimeric thiosemicarbazones
was found to correlate with their anti-proliferative activity (R2 = 0.803,
Fig. 5b), with the most cytotoxic chelator 16a having the highest 59Fe
mobilization activity, while the least cytotoxic chelator 13, resulted in
limited 59Fe mobilization. Plotting 59Fe mobilization against log Pcalc
values showed a linear relationship (R2 = 0.813; Fig. 5c), indicating
that chelators with increased lipophilicity are least efficient in mediating
59Fe mobilization. The relatively low Fe mobilization efficacy of the di-
meric thiosemicarbazones compared to monomeric chelators such as
Table 1
The FeIII/II redox potentials of the dimeric thiosemicarbazones in 7:3, MeCN:H2O.
Ligand
FeIII/II redox potential (mV vs NHE)
[Fe(BpT)2]
[Fe(Bp4mT)2]
[Fe(Bp4eT)2]
[Fe(Bp4aT)2]
+120a
+108a
+99a
+117a
+180a
+131
ND
+123
+56
−4.5
+18
[Fe(Bp4pT)2]
[Fe(Bp-1,6-hexanediT)] (17)
[Fe(Bp-1,12-dodecanediT)] (18)
[Fe(2,6-diBpT)2] (19a)
[Fe(2,6-diBp4mT)2] (19b)
[Fe(2,6-diBp4eT)2] (19c)
[Fe(2,6-diBp4aT)2] (19d)
[Fe(2,6-diBp4pT)2] (19e)
ND
a
Fig. 3. ORTEP representation of 16c (thermal ellipsoids are drawn at 50% probability level).
FeIII/II redox potentials previously reported [33].

A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
51
Table 2
thiosemicarbazones, a correlation was evident where the most effective
chelators in the 59Fe efflux assay were also the most active at inhibiting
59Fe uptake (R2 = 0.884; Fig. 6b). For example, 2,6-diBpT, 16a, which
was the best of the dimeric ligands to induce 59Fe efflux (Fig. 5a), was
also the most effective dimeric ligand at reducing 59Fe uptake to 33
1% of the control (Fig. 6a).
The dithiosemicarbazones, 12 and 13, were relatively less effective
than the 2,6-diBpT series of ligands, and reduced 59Fe uptake to 62
1% and 81 1% of the control, respectively. In addition, a linear relation-
ship between inhibition of 59Fe uptake and lipophilicity was observed
(R2 = 0.716, Fig. 6c), indicating that high lipophilicity led to decreased
59Fe uptake efficacy. Again, this could be due to the highly lipophilic li-
gands becoming trapped in the cell membrane which would decrease
their ability to enter the cell and reduce the incorporation of Fe
IC50 values of the dimeric thiosemicarbazones and their FeIII complexes at inhibiting the
growth of SK-N-MC neuroepithelioma cells and mortal MRC-5 fibroblasts as determined
by the MTT assay. Log Pcalc values of the ligands were calculated using ChemBio Draw
software.
Average IC50 values (μM)
Free ligands
SK-N-MC
FeIII complex
SK-N-MC
Chelators
Log
MRC-5
Pcalc
DFO
21.74
2.58
1.59
0.002 N6.25
0.001 N6.25
0.004 N6.25
0.002 1.86
N25a
N6.25
N25b
1.17
0.013
0.40
0.34
BpTc
1.92 4.66
0.74 0.004
4.01 0.002
2.69 0.004
1.92 0.005
6.69 0.33
0.17
0.001
0.06
0.06
0.01
Bp4mTc
Bp4eTc
Bp4aTc
Bp4pTc
1.77 0.17
1.59 N6.25
N12.5
from Tf.
A relationship was also observed between the anti-
Bp-1,6-hexanediT (12)
Bp-1,12-dodecanediT (13) 9.19 10.64
2,6-diBpT (16a)
2,6-diBp4mT (16b)
2,6-diBp4eT (16c)
2,6-diBp4aT (16d)
2,6-diBp4pT (16e)
0.049
1.374 N12.5
5.32
proliferative activity (IC50) and the ability of the ligands to inhibit 59Fe
uptake (R2 = 0.918; Fig. 6d). In this latter case, the chelators with the
greatest efficacy at decreasing 59Fe uptake were also the most effective
at inhibiting proliferation.
3.8
0.31
0.009
0.39
0.24
0.76
N6.25
N6.25
N6.25
N6.25
N6.25
0.77
4.79
N6.25
3.83
0.15
1.90
4.84 1.98
5.51 1.65
6.51 1.50
8.17 N6.25
1.78
N6.25
a
Denotes data previously obtained in our laboratory using MRC-5 cells and DFO [14].
Denotes data previously obtained in our laboratory using SK-N-MC cells and the DFO
b
Fe complex [13].
c
Denotes data previously obtained in our laboratory using SK-N-MC and MRC-5 cells
and the appropriate monomeric thiosemicarbazones [33].
Dp44mT, BpT and Bp4eT, could be explained by their high lipophilicity
and molecular size, which could inhibit their ability to transverse the
cell membrane resulting in membrane sequestration [13,33].
3.5.5. Inhibition of cellular 59Fe uptake from 59Fe–Tf
The anti-proliferative activity of chelators is known to involve both
the release of intracellular Fe from cells, as well as the inhibition of Fe
uptake from Tf [13,33,61]. Thus, the ability of the novel dimeric
thiosemicarbazones (25 μM) to inhibit internalized 59Fe uptake from
59Fe–Tf was assessed using SK-N-MC neuroepithelioma cells, and the
results were compared to the well characterized positive controls,
DFO, Dp44mT, BpT and Bp4eT [33].
As shown in previous studies [61], the ability of DFO to inhibit 59Fe
uptake was poor, with uptake reduced to only 96 1% of the control,
while Dp44mT, BpT and Bp4eT decreased 59Fe uptake to 4 1%, 11
1% and 16 1% of the control, respectively (Fig. 6a). All of the dimeric
thiosemicarbazones were significantly (p b 0.05) more effective than
DFO at inhibiting 59Fe uptake from 59Fe–Tf. The 2,6-diBpT chelators,
16a–e, inhibited 59Fe uptake to 33–58% of the control, which was signif-
icantly (p b 0.01) more effective than DFO, but markedly less effective
than the monomeric thiosemicarbazones (Fig. 6a). Among the dimeric
Fig. 5. (A) The effect of the chelators on 59Fe mobilization from prelabeled SK-N-MC
neuroepithelioma cells. Results are mean SD of 3 experiments with 3 determinations
in each experiment. (B) The relationship between 59Fe mobilization efficacy of the novel
dimeric thiosemicarbazones and their anti-proliferative activity. (C) The relationship be-
tween 59Fe mobilization in SK-N-MC cells and lipophilicity (log Pcalc) of the dimeric
chelators.
Fig. 4. Relationship between the anti-proliferative activity (IC50) and lipophilicity (log
Pcalc) of the dimeric thiosemicarbazones.

52
A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
Fig. 6. (A) The effect of the dimeric chelators at inhibiting 59Fe uptake from 59Fe–Tf by SK-N-MC neuroepithelioma cells. Results are mean SD of 3 experiments with 3 determinations in
each experiment. (B) A correlation was evident where the most effective dimeric thiosemicarbazones in the 59Fe efflux assay were also the most active at inhibiting internalized 59Fe up-
take by SK-N-MC cells. (C) The relationship between 59Fe uptake by SK-N-MC cells and lipophilicity (log Pcalc) of the dimeric thiosemicarbazones. (D) The relationship between anti-
proliferative activity (IC50) and the ability of the dimeric ligands to inhibit 59Fe uptake from 59Fe–Tf by SK-N-MC cells.
Taken together, the 59Fe efflux and 59Fe uptake results above indicat-
ed that the dimeric chelators with a shorter hydrocarbon linker or lower
molecular weight exhibited higher 59Fe mobilization efficacy, as well as
higher inhibition of 59Fe uptake from 59Fe–Tf. This correlation is demon-
strated by the observation that chelator 16a (R_H) was more active in
terms of both 59Fe mobilization (Fig. 5a) and inhibition of 59Fe uptake
(Fig. 6a) compared to analogs 16b–e that contain larger alkyl, alkenyl
or phenyl substituents. Similarly, dithiosemicarbazone 12 containing a
hexyl linker showed higher 59Fe chelation efficiency compared to
dithiosemicarbazone 13, which possesses a dodecyl bridge. The correla-
tion between molecular weight and ability to inhibit 59Fe uptake is
shown in Fig. 7a, and demonstrates that as the molecular weight of
the dimeric ligand increases, the ability to inhibit 59Fe uptake decreases
(R2 = 0.763). In addition, Fig. 7b demonstrates that there is a decrease
in 59Fe efflux from SK-N-MC cells as the molecular weight of the dimeric
thiosemicarbazone increases (R2 = 0.745). This observation can be ex-
plained by the fact that when the chelators are too large or hydrophobic,
they do not permeate the cell membrane effectively, or become trapped
within the lipophilic environment of the membrane. Hence, molecular
size and lipophilicity are important factors that play crucial roles in facil-
itating chelator entrance into cells and chelation of intracellular Fe.
4. Conclusions
The current investigation examined the effect of dimerization of
thiosemicarbazones on their Fe chelation and anti-proliferative activity.
The results have also highlighted important structure–activity
relationships towards understanding the potential of dimeric
thiosemicarbazones to act as effective anti-proliferative agents for the
treatment of cancer. In this study, two new classes of dimeric
thiosemicarbazones were synthesized and characterized. The first
type of dimer was composed of two benzoylpyridine-based
thiosemicarbazone units joined via a hexane or dodecane alkyl bridge,
while the second class of dimer consisted of two thiosemicarbazone
tails attached to a 2,6-dibenzoylpyridine core. To the best of our
Fig. 7. The relationship between the molecular weight (MW) of the dimeric thiosemicarbazones and (A) 59Fe uptake from 59Fe–Tf by SK-N-MC cells and (B) 59Fe release from SK-N-MC
cells.

A.Y. Lukmantara et al. / Journal of Inorganic Biochemistry 141 (2014) 43–54
53
knowledge, these types of dimeric thiosemicarbazone have not yet
been reported in the literature.
DCM
DFO
dichloromethane
desferrioxamine
2′-Benzoylpyridine-(1,6-hexane)-dithiosemicarbazone (12) and the
2,6-diBpT analogs (16a–e), demonstrated moderate anti-proliferative
activity against SK-N-MC cells, being more potent than the well-
known chelator, DFO. The dimeric chelators also showed marked selec-
tivity for tumor cells over normal cells. Additionally, the 2,6-diBpT ana-
logs, 16a–e, and dithiosemicarbazone 12 were able to mobilize
intracellular 59Fe and inhibit 59Fe uptake with equal or greater efficacy
than DFO. Significantly, the most cytotoxic chelator 16a was also the
most effective at mobilizing 59Fe and preventing 59Fe uptake (reduced
to 33% of control). Moreover, the reversible electrochemical behavior
of the Fe complexes of most of the dimeric thiosemicarbazones occurred
in a range accessible to intracellular oxidants and reductants. These
studies suggested the Fe(III) complexes may participate in redox cycling
and ROS generation. The analytical data indicated the formation of 1:1
ligand:Fe complexes for the dithiosemicarbazones (12 and 13), while
the dibenzoylpyridine ligands (16a–e) formed 2:1 ligand:Fe complexes.
Therefore, together with their ability to mobilize intracellular Fe and
prevent Fe uptake from Tf, the anti-proliferative activity of the novel
chelators could be due, at least in part, to the formation of redox-active
Fe complexes that generate cytotoxic ROS.
2,6-diBpT 2,6-dibenzoylpyridine thiosemicarbazone
2,6-diBp4aT 2,6-dibenzoylpyridine 4-allyl-3-thiosemicarbazone
2,6-diBp4eT 2,6-dibenzoylpyridine 4-ethyl-3-thiosemicarbazone
2,6-diBp4mT 2,6-dibenzoylpyridine 4-methyl-3-thiosemicarbazone
2,6-diBp4pT 2,6-dibenzoylpyridine 4-phenyl-3-thiosemicarbazone
Dp44mT di-2-pyridylketone 4,4-dimethyl-3-thiosemicarbazone
ESI
electrospray ionization
HRMS
NBpT
high-resolution mass spectrometry
2′-(3-nitrobenzoyl)pyridine thiosemicarbazone
NDRG1 N-myc downstream regulated gene-1
NHE
PBS
ROS
RR
normal hydrogen electrode
phosphate buffered saline
reactive oxygen species
ribonucleotide reductase
transferrin
Tf
TMS
XBpT
tetramethylsilane
halogenated 2′-benzoylpyridine thiosemicarbazone
Acknowledgments
Structure–activity relationship analysis was performed in order to
better understand the structural requirements needed for the dimeric
thiosemicarbazones to exhibit biological activity. Of the novel chelators,
the dithiosemicarbazone 12 and the 2,6-diBpT derivative 16a showed
the highest anti-proliferative activity. By examining the calculated par-
tition coefficients of the synthesized compounds, it was shown that the
most active chelator 16a (R_H) had the lowest lipophilicity (log Pcalc
3.8). On the other hand, the larger and more lipophilic chelators, namely
16e (log Pcalc = 8.17; 586 Da) and 13 (log Pcalc = 9.19; 678 Da), showed
reduced potency. When comparing the two dithiosemicarbazones,
compound 13 (bearing a dodecane linker) was less effective than chela-
tor 12 (which possessed a shorter hexane linker) in terms of chelation
efficacy and inhibiting proliferation. Ligands with a high molecular
weight and lipophilicity may not effectively permeate the cell mem-
brane or may become trapped within the lipophilic environment of
the membrane. This effect will prevent intracellular Fe chelation and de-
crease the formation of redox-active metal complexes within the cell
that are necessary for their cytotoxic efficacy.
Overall, this investigation has demonstrated that the length of the hy-
drocarbon linker in dithiosemicarbazones and the nature of the substitu-
ents on dibenzoylpyridine-based thiosemicarbazones has a strong
influence on their biological activity. Furthermore, the study has, for the
first time, highlighted the potential of dimeric thiosemicarbazone com-
pounds to act as selective Fe chelators for the treatment of cancer. Given
that higher lipophilicity was detrimental towards dimer activity, future
work could be directed towards synthesizing dithiosemicarbazones
with shorter or more polar linkers and 2,6-diBpT analogs containing less
hydrophobic substituents. Hence, this study highlights structure–activity
relationships involved in the anti-proliferative activity and Fe chelation
efficacy of thiosemicarbazone dimers and provides greater understanding
towards the structural requirements for effective dimer design.
This work was supported by a Project Grant from the National
Health and Medical Research Council (NHMRC) Australia to D.R.R.
[Grant 632778]; and D.S.K. [Grant 1048972]; a NHMRC Senior Principal
Research Fellowship to D.R.R. [Grant 571123]; a Cancer Institute New
South Wales Early Career Development Fellowship to D.S.K. [Grant 08/
ECF/1-30]; a Cancer Institute Research Innovation Grant to D.R.R. and
D.S.K. [Grant 10/RFG/2-50]; and a Helen and Robert Ellis Fellowship to
D.S.K. from the Sydney Medical School Foundation of The University of
Sydney. The research was additionally supported by an ARC Discovery
Grant (DP1095159) and Linkage Project Grant (LP110200635) to
N.K. We kindly thank Dr. Mohan Bhadhade (Crystallography Laboratory,
Analytical Centre, The University of New South Wales, Sydney,
Australia) for collecting X-ray crystal structure data.
Appendix A. Supplementary data
Supplementary data to this article can be found online at http://dx.
doi.org/10.1016/j.jinorgbio.2014.07.020. These data include MOL files
and InChiKeys of the most important compounds described in this
article.
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Abbreviations
Bp-1,6-hexanediT 2′-benzoylpyridine-(1,6-hexane)-
dithiosemicarbazone
Bp-1,12-dodecanediT 2′-benzoylpyridine-(1,12-dodecane)-
dithiosemicarbazone
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V. Bernhardt, J. Med. Chem. 49 (2006) 6510–6521.
Bp4aT
Bp4eT
2′-benzoylpyridine 4-allyl-3-thiosemicarbazone
2′-benzoylpyridine 4-ethyl-3-thiosemicarbazone
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Bp44mT 2′-benzoylpyridine 4,4-dimethyl-3-thiosemicarbazone
Bp4mT 2′-benzoylpyridine 4-methyl-3-thiosemicarbazone
Bp4pT
BpT
2′-benzoylpyridine 4-phenyl-3-thiosemicarbazone
2′-benzoylpyridine thiosemicarbazone

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