Piperazinyl fragment improves anticancer activity of Triapine

Article abstract of DOI:10.1371/journal.pone.0188767

A new class of TSCs containing piperazine (piperazinylogs) of Triapine, was designed to fulfill the di-substitution pattern at the TSCs N4 position, which is a crucial prerequisite for the high activity of the previously obtained TSC compounds–DpC and Dp4

Full text of DOI:10.1371/journal.pone.0188767

RESEARCH ARTICLE
Piperazinyl fragment improves anticancer
activity of Triapine
Marta Rejmund1, Anna Mrozek-Wilczkiewicz2,3, Katarzyna Malarz1,3, Monika Pyrkosz-
Bulska1, Kamila Gajcy1, Mieczyslaw Sajewicz1, Robert Musiol1, Jaroslaw Polanski1*
1 Institute of Chemistry, University of Silesia, Katowice, Poland, 2 A. Chełkowski Institute of Physics,
University of Silesia, Katowice, Poland, 3 Silesian Center for Education and Interdisciplinary Research,
´
University of Silesia, Chorzow, Poland
* jaroslaw.polanski@us.edu.pl
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Abstract
A new class of TSCs containing piperazine (piperazinylogs) of Triapine, was designed to fulfill
the di-substitution pattern at the TSCs N4 position, which is a crucial prerequisite for the high
activity of the previously obtained TSC compounds–DpC and Dp44mT. We tested the impor-
tant physicochemical characteristics of the novel compounds L1-L12. The studied ligands are
neutral at physiological pH, which allows them to permeate cell membranes and bind cellular
Fe pools more readily than less lipid-soluble ligands, e.g. DFO. The selectivity and anti-can-
cer activity of the novel TSCs were examined in a variety of cancer cell types. In general,
the novel compounds demonstrated the greatest promise as anti-cancer agents with both a
potent and selective anti-proliferative activity. We investigated the mechanism of action more
deeply, and revealed that studied compounds inhibit the cell cycle (G1/S phase). Additionally
we detected apoptosis, which is dependent on cell line’s specific genetic profile. Accordingly,
structure-activity relationship studies suggest that the combination of the piperazine ring with
Triapine allows potent and selective anticancer chelators that warrant further in vivo examina-
tion to be identified. Significantly, this study proved the importance of the di-substitution pat-
tern of the amine N4 function.
OPEN ACCESS
Citation: Rejmund M, Mrozek-Wilczkiewicz A,
Malarz K, Pyrkosz-Bulska M, Gajcy K, Sajewicz M,
et al. (2018) Piperazinyl fragment improves
anticancer activity of Triapine. PLoS ONE 13(4):
e0188767. https://doi.org/10.1371/journal.
pone.0188767
Editor: Swati Palit Deb, Virginia Commonwealth
University, UNITED STATES
Received: June 1, 2017
Accepted: November 13, 2017
Published: April 13, 2018
Copyright: © 2018 Rejmund et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Introduction
Thiosemicarbazones (TSCs) have a broad range of biological activity including antitumor,
antimalarial and antimicrobial activity [1], and therefore, for many years, studies of α-(N)-het-
erocyclic TSCs have been attracting considerable interest. In particular, the antitumor proper-
ties of 2-formylpyridine thiosemicarbazone were reported over 50 years ago [2]. With regard
to potential pharmaceutical applications, Triapine (3-aminopyridine-2-carboxaldehyde thiose-
micarbazone; 3-AP) is the most prominent representative of this class, as it has already been
investigated in more than 30 clinical phase I/II trials [3–10]. Moreover, di-2-pyridylketone-
4-cyclohexyl-4methyl-3-thiosemicarbazone (DpC) is currently entering clinical phase I studies
as a potential anticancer agent. Although clinical studies have concluded that Triapine revealed
activity against hematological cancer types (e.g. advanced leukemia [8,11]), it also showed
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The financial support of the National
Center of Science grants 2014/13/D/NZ7/00322
(AMW), 2013/09/B/NZ7/00423 (RM) and NCBiR
Warsaw ORGANOMET No: PBS2/A5/40/2014 (JP)
is greatly appreciated.
Competing interests: The authors have declared
that no competing interests exist.
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Piperazinyl fragment improves anticancer activity of Triapine
disappointing results against a variety of solid tumor types such as advanced adenocarcinoma
of the pancreas [12], non-small-cell lung cancer [3] and renal cell carcinoma [13]. Further-
more, some side effects include the formation of methemoglobin and hypoxia [3,8,9], which
were observed after administration. Therefore, the development of new Triapine analogs with
a potent anticancer activity would be significant.
The broad range of the biological activity of TSCs corresponds to their versatile binding
modes with the transition and main group metal ions [14]. Moreover, it has been observed
that, generally, the biological activity of the complexes of TSCs is often higher than that of cor-
responding metal-free ligands. To gain further insight into the coordination chemistry of
TSCs, thermodynamic data such as the stability constants of metal complexes, which help in
optimizing the chemical or biological properties that are essential for potential medicinal
applications, are needed. In particular, copper complexes have a considerably higher antican-
cer activity than the uncomplexed ligands that also have lower IC50 values against cancer cells
than other described topoisomerase-II inhibitors [15]. In contrast, Triapine complexation to
iron resulted in the reduced cytotoxicity compared to the metal free ligand.
Although Triapine, similar to the recently developed Dp44mT (di-2-pyridylketone-4,4-
dimethyl-3-thiosemicarbazone), has been evaluated as a potential anticancer agent, the molecu-
lar mechanisms of its action have not been fully elucidated. Several paradigms have been pro-
posed to explain the activity of these compounds [16,17] including blocking cellular iron uptake
from transferrin [18]; mobilizing iron from cells; inhibiting ribonucleotide reductase, the iron-
containing enzyme that is involved in the rate-limiting step of DNA synthesis [19] or forming
reactive oxygen species (ROS) [20]. The effect of metal chelation suggested among potential
determinants of the mechanism origins are among the most important determiners deciding
that the mechanism(s) of action of these compounds are incompletely understood.
In this research, we designed and obtained new Triapine analogs for the first time through
incorporating the piperazine ring as a promising new pharmacophore group to replace the N
terminal amino group (Fig 1). While the substitution pattern at the N4 atom of TSCs appears
to be critical for the activity of Dp44mT, there were no similar studies for Triapine. Therefore,
we tested di-substitution at the N4 atom by constructing an N4-based piperazine, which is a
fragment that is present in several active TSCs [21–23]. First, we hoped that this could modu-
late the antiproliferative activity of the new analogs because the piperazine heterocycle is
found in a wide variety of biologically active compounds, some of which are currently being
used in clinical therapy [24–27]. In particular, new derivatives could have a significant impact
on pharmacokinetics and pharmacodynamics, while the replacement of the unsubstituted
NH2 function with the piperazine fragment should increase the lipophilicity of the new ana-
logs. Second, the modification of the substitution pattern of piperazine is a standard drug
design scheme that has often resulted in an increased medicinal potential of the analogs [28].
Accordingly, we describe the synthesis of a series of new N4- piperazinylogs of Triapine
that were tested for their anticancer activity vs. a broad spectrum of cancer cell lines. As the
metal complexation of the piperazine ring has been reported as an important issue in the
biological activity for these compounds [29,30], we tested L1-L12 as potential chelators for tran-
sition metals. Solution equilibria of the all the ligands and their copper(II) and iron(III) com-
plexes were studied using UV-Vis titration.
Results and discussion
Chemistry
Design and synthesis. Identifying the functional fragments for drug design is a complex
problem that involves different approaches including those that have an experimental and
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Piperazinyl fragment improves anticancer activity of Triapine
Fig 1. Design strategy for novel TSCs (L1-L12). All designed ligands are based on the Triapine skeleton, which is present in the active analogs Dp44mT,
DpC and 1b, 1d that have been described as highly active analogs [21].
https://doi.org/10.1371/journal.pone.0188767.g001
theoretical basis. The latter consist of a variety of methods, among which are those to identify
advantageous sub-structures, scaffolds and/or linkers on the basis of previously reported com-
pounds. Alternatively, the fragmentation of organic molecules into smaller moieties is an
important method in retrosynthetic analysis and has inspired various pseudo-retrosynthetic
approaches [31]. This has identified fragments that may be useful for drug design. For exam-
ple, the di-2-pyridyl [32–35], quinolinyl [36], piperazinyl [37,38], morpholinyl [39] and qui-
noxalinyl [40] motifs, which are common fragments in other anti-cancer agents, have been
incorporated into the design of the novel TSCs reported herein (Fig 1).
We have previously examined a variety of TSCs that demonstrate in vitro anti-proliferative
activity [21,41,42]. Earlier studies indicated that di-substitution at the terminal (N4) nitrogen
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Piperazinyl fragment improves anticancer activity of Triapine
Fig 2. Synthesis of the thiosemicarbazides K1-K12 and thiosemicarbazones L1-L12
.
https://doi.org/10.1371/journal.pone.0188767.g002
is crucial for effective anti-cancer activity [21,32,34,42]. Therefore, in the present study, we
transformed the amine function in the southern part of the Triapine molecule into the form of
a piperazine ring. This formed a di-substitution at the N4 position that was discovered to be of
crucial importance for the activity of DpC and Dp44mT, which has never been tested for Tria-
pine. In particular, there is a fragment that is present in several active TSCs in this piperazine
ring [23,43].
The synthetic protocol that was used to produce the target molecules is outlined in Fig 2.
The precursors that are required to obtain the desired Triapine derivatives, the thiosemicarba-
zides K1-K12, were synthesized from commercially available reagents in a two-step process that
mostly produced high yields (69–98%). The treatment of (1,1’-thiocarbonyl)bis-1H-imidazole
with the appropriate derivative of piperazine, followed by the reaction with hydrazine hydrate,
produced the N-substituted piperazine-based thiosemicarbazides in a high yield. The final
TSC series, L1-L12 (Fig 2), were synthesized in a moderate to high yield (20–83%) using the
Schiff-based condensation of the 3-aminopyridine-2-carboxaldehyde with the prepared thiose-
micarbazides K1-K12 in a microwave reactor, which produced novel Triapine-based ligands
after crystallization with methanol. All the synthesized compounds were confirmed using 1H,
13C NMR and MS spectroscopic techniques.
Chelating properties
Protonation constants of the Triapine-derivative ligands. Spectrophotometric titrations
were performed in order to probe the acid-based equilibria that were associated with each
ligand and to determine the pH range over which the chelator was in its charge neutral form.
This property is important in understanding the passage of a molecule through the cell mem-
branes, as charged chelators have poor access [33,44]. These studies were performed in an 80%
(w/w) MeOH/H2O solvent mixture due to the low solubility of these compounds in pure
water. The fully protonated forms of the ligands have four L1-L7 or five L8-L12 dissociable pro-
tons, respectively. All the studied ligands possess one dissociable proton at the hydrazanic
group of the thiosemicarbazone moiety, one at pyridine ring and two at the piperazine moiety.
An additional proton in L8-L12 derives from the additional pyridine, pyrimidine or pyrazine
ring, respectively; however, not all of them could be determined under these experimental
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Piperazinyl fragment improves anticancer activity of Triapine
conditions. The protonation constants that were obtained are given in Table 1 and the species
distribution diagram of the protonated species of the selected ligand is presented in Fig 3.
The UV/Vis spectrophotometric titrations revealed characteristic spectral changes in the
240–450 nm wavelength range. All studied ligands displayed intense absorption bands in
this range, which were dependent on the protonated state of the molecule. The bands with a
maximum absorption λmax ꢀ 380 nm-404 nm were assigned to the n!πà transitions of the
pyridine ring. The less intense bands at ꢀ 290 nm originated mainly from the π!πà transi-
tions of the azomethine chromophore [45–47]. During the first deprotonation step ([H3L]2+
! [H2L]+, we observed a blueshift and a decrease in the intensity of the absorption maxi-
mum in the visible region. The next two deprotonation steps were accompanied by a red-
shift and an increase in intensity (Fig 1B and S1 Fig). Following the deprotonation of the
N2-H group, the negative charge is transferred mainly to the S atom via the thione-thiol tau-
tomeric equilibrium.
The proton dissociation constants and the spectra of the individual ligand species (Table 1)
were calculated based on the deconvolution of the pH-dependent UV-Vis spectra. The
obtained logKa values are in a reasonably good agreement with previously reported thiosemi-
carbazones ligands [45–47]. The concentration distribution curves, together with the elec-
tronic spectra as a function of pH, are reported in the Supporting Information (S1 Fig).
For the studied ligands, the first protonation constant corresponded to the protonation of
the hydrazanic = N-NH group. A decreased value, about one order of magnitude lower than in
the other reported N-pyridyl thiosemicarbazones ligands (logK* 10.2–11), can most probably
be attributed to the substitution of the piperazine ring by an aromatic ring (L1-L7), pyridine
(L8-L10) or the pyrimidine and pyrazine moiety in L11 and L12, respectively. This effect is the
result of the electron-withdrawing effect of these substituents. On other hand, the hydrogen
bond between the pyridyl nitrogen and the = N-NH hydrazanic moiety is most probably
responsible for the marked differences in the value of logK. The next protonation constant,
logK with a value of 5.7–7.2 was assigned to the piperazine moiety. The obtained values of
logK of the piperazine functional group of the studied ligands are comparable to the reported
Table 1. Protonation constants (logβ H) of the L1-L12 ligands in the MeOH/H2O mixed solutiona.
logbH1
9.67(1)
9.73 (1)
9.25(1)
9.58(1)
9.47(1)
9.43(1)
9.94(1)
9.96(1)
9.70 (1)
9.66 (1)
9.55(1)
9.63 (1)
10.64 (1)
10.86 (1)[45]
logbH2
logbH3
logbH4
logK1
logK2
logK3
logK4
L1
L2
L3
L4
L5
L6
L7
L8
L9
16.17(1)
15.40 (2)
15. 83(2)
13.35(2)
15.15(2)
15.65(1)
16.93(2)
17.34 (1)
17.26 (2)
17.23 (1)
15.93 (2)
16.37 (2)
14.12 (1)
19.49(1)
18.70(2)
19.05(2)
19.76(3)
18.31(2)
18.91(1)
20.21(3)
22.07(3)
22.41(4)
22.74(2)
19.33(2)
19.60(2)
9.67
9.73
9.25
9.58
9.47
9.43
9.94
9.96
9.70
9.66
9.55
9.63
10.64
10.86
6.50
5.77
6.58
6.77
5.68
6.22
6.99
7.38
7.56
7.57
6.38
6.74
3.48
3.79
3.32
3.20
3.22
3.41
3.16
3.26
3.28
4.73
5.15
5.51
3.40
3.23
25.39(3)
25.58(4)
25.96(3)
3.32
3.17
3.22
L10
L11
L12
Triapine
Triapine
14.65 (1)[45]
a Solvent MeOH/H2O 80/20 by weight
I = 0.1 M KCl, T = 25.0˚C. The reported errors on logβ are given as 1σ.
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Piperazinyl fragment improves anticancer activity of Triapine
Fig 3. (a) Absorption spectrophotometric titration vs. pH of the free L2 ligand; (b) electronic spectra of the protonated
species of L2; (c) concentration distribution curves for the L2 species. (I = 0.1 M (KCl) in 80% (w/w) MeOH/H2O;
T = 25.0˚C; [L2] = 5x10-5M; pH 1.6–11.02).
https://doi.org/10.1371/journal.pone.0188767.g003
values [48–50] or logK values determined for the other TSC-based hybrids, mPip-FTSC and
mPip-dm-FTSC (logK 7.28) [46]. The logK with value 3.1–3.4 can presumable be attributed to
protonation of the pyridinium nitrogen. These values are in good agreement with the most
popular TSC as is Triapine and other published α-N- pyridyl thiosemicarbazones [45,51]. For
ligands L8-L10 were observed additional logK with value 4.73; 5.15 and 5.51 respectively. These
values most probably corresponded to protonation of additional pyridinium unit, which is in
agreement with the tabulated logK values of pyridine (5.23) [52].
The next acidic logKs occurred well below pH 2.5 and the constants corresponding to these
processes were not determined under the experimental conditions used in this study.
It should be noted that all of the measured logKs are macroscopic constants and that further
constants cannot be ascribed to the protonation of either of the donor groups without detailed
NMR titrations of the ligand [53]. However, taking into account the solvent conditions (vide
supra) and comparing the observed data with those of a series of previously investigated
ligands [45,46], the basicity of the substituents included in the studied ligand most probably
follows the trend–hydrazine N2H group > piperazine > additional function group (e.g. pyri-
dine, pyrimidine or pyrazine) ꢀ pyridine.
For the studied ligands, the neutral uncharged form dominated at a physiological pH of
7–8.5, thus enabling a facile passage across the cell membranes. This would explain, at least in
part, the high biological activity of these chelators by mobilizing intracellular Fe; preventing Fe
uptake from the serum Fe transport protein, transferrin (Tf) and also inhibiting cellular prolif-
eration [20]. The protonated form (Fig 3) became dominant below pH 5, while the deproto-
nated form was only important above pH 11 (Fig 3 and S1 Fig). Hence, if these agents are ever
given as drugs via the oral route, the low pH of the stomach (pH 1–2) would prevent the
absorption of the drug as the molecule would be charged [54]. More facile absorption would
occur in the small intestine where the higher pH (pH 5–7) would result in a neutral ligand and
a greater uptake [54].
Ability to chelate Cu(II) and Fe(III). To evaluate the complex formation ability for the
studied Triapine-derivative ligands, we performed the spectroscopic titration of the solutions
of all the L1-L12 compounds with copper and iron ions. We titrated the studied ligands with
the above-mentioned metal ions to generate their metal-complexes in situ at the following
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Piperazinyl fragment improves anticancer activity of Triapine
metal to ligand ratios 1:1–1:5. The isosbestic curves for the selected ligands are presented in
Fig 4.
In general, the addition of a metal ion to the ligand solution caused distinguishable changes
in the visible region of the ligand spectra. This behavior suggests an instantaneous complex
formation in the solution from the reaction of each of the studied ligands with the metal ions
used. All the ligands displayed the characteristic intense transitions in the range 400–500 nm
Fig 4. Electronic absorption spectra of the Cu(II) and Fe(III)-L2; L4; L8 system recorded at various metal to ligand ratios. I = 0.1
M (KCl) in 80% (w/w) MeOH/H2O; T = 25.0˚C; [L] = 5x10-5M.
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Piperazinyl fragment improves anticancer activity of Triapine
for Cu(II) and 380–510 nm for Fe(III) that spanned into the visible region. Fig 4 shows the
electronic spectral change that was observed in the MeOH/H2O 80/20 w/w solution when Cu
(II) or Fe(III) was added to the selected ligands L2, L4 and L8. In the absence of metal ions, the
light yellow solution showed intensive absorption bands at λmax ꢀ 250 nm, λmax ꢀ 290 nm
and λmax ꢀ 380 nm—404 nm. As the Cu(II) or Fe(III) ion solution was added, the signals of
the ligand alone decreased and those of the complexes increased in intensity at 430–450 nm
with some isosbestic points at ꢀ 337 nm and 395 nm (S1 Table). The presence of this band
indicates a LMCT (ligand to metal charge transfer), which is typical of the Cu(II) L complex.
This transition band corresponds to the S!Cu(II) and Npy!Cu(II) transition. Similar ligand
to metal charge transfer bands have also been observed in the complexes of other TSC ligands
[45,46,51]. No d-d bands were observed due to the low concentration (~ 10-5M range) of the
Cu(II) complexes in the solution. These bands should have a low intensity in the region of
600–700 nm.
In the studied ligands, the formation of mono complexes for Fe(III) resulted in a shoulder
in the interval 450–620 nm. As metal-free ligands do not absorb in this region, these are most
probably charge-transfer (CT) bands of the Fe-L complexes [46,51]. Additionally, we devel-
oped the characteristic CT bands λmax ꢀ 639 nm of the Fe(III)-L complexes in the wavelength
of 580–680 nm, which was also detected in the other Fe(III)-TSC systems [46,51]. In conclu-
sion, the analysis of the results showed that the presented ligands can act as effective copper or
iron chelators.
Anticancer activity
Cytotoxicity of novel Triapine analogs. All the newly synthesized compounds were eval-
uated against a panel of cancer and normal cell lines for their cytotoxic activity (Table 2).
Among the cancer cells, we analyzed the cytotoxicity of the tested compounds against colon
cancer with a normal (HCT116 p53+/+) and deleted TP53 suppressor gene (HCT116 p53-/-),
which encodes the p53 protein. This was designed to allow the assayed activities to be com-
pared with the literature data as a number of previous reports on the antiproliferative potency
of TSCs presented results from these cells [21,41,42,55–58]. Moreover, colon cancers are
among those that are particularly associated with a higher iron uptake and metabolism [59,60].
Table 2. Anti-proliferative activity (IC50 values) of the novel Triapine analogs compared to Triapine in several tumor cell-types and normal human dermal fibro-
blast (NHDF) cells. Individual IC50 values IC50 < 1μM, IC50 1–10 μM, IC50 > 10 μM are coded by red, yellow and grey, respectively.
Name
IC50 [μM]
MCF-7
HCT116 p53+/+
1.524 0.445
0.526 0.077
0.120 0.005
1.328 0.209
1.450 0.452
0.139 0.016
0.762 0.238
1.900 0.292
0.422 0.113
0.170 0.015
0.668 0.045
0.435 0.108
1.121 0.277
HCT116 p53-/-
0.128 0.012
0.152 0.074
0.167 0.018
0.185 0.088
0.7461 0.354
0.270 0.007
1.133 0.049
0.139 0.020
0.123 0.061
0.159 0.011
0.650 0.055
0.342 0.042
1.336 0.338
U-251
Hs683
NHDF
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
L1
L2
L3
L4
L5
L6
L7
L8
L9
0.532 0.145
0.360 0.111
0.204 0.049
0.424 0.149
3.807 1.023
0.470 0.097
2.536 0.400
1.120 0.113
0.204 0.024
0.258 0.040
1.730 0.388
2.218 0.638
2.328 0.431
0.403 0.090
0.887 0.158
0.128 0.012
0.649 0.170
2.569 0.675
0.381 0.042
3.084 1.273
1.070 0.280
0.277 0.058
0.137 0.016
1.268 0.374
0.743 0.109
1.476 0.558
1.436 0.358
2.487 0.512
1.483 0.335
4.457 0.954
10.050 2.643
1.894 0.812
4.624 1.031
1.162 0.307
0.844 0.238
0.195 0.021
2.838 0.581
2.595 0.237
1.763 0.292
L10
L11
L12
L13/3-AP
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Piperazinyl fragment improves anticancer activity of Triapine
This makes them potentially vulnerable to treatments that target iron homeostasis. We also
tested the cell lines of the brain tumor (U-251, Hs683) and breast cancer (MCF-7) cell lines.
Breast cancers are among the most frequent cancers and their aggressiveness and treatment
prognosis is strongly connected with iron metabolism [61–63]. Namely, it has been confirmed
that the regulation pattern of the iron regulatory genes determined the metastasis potency and
may be used as a predictive factor in the therapeutic strategy planning [62,64]. Glioblastomas,
on the other hand, are one of the most dangerous cancers because no effective therapeutic
regimes exist to treat them. The prognoses in these types of cancer are generally poor and the
mean survival time does not exceed two years even under combination therapy [65,66]. Inter-
estingly, those cancers also express an altered metabolism of iron and remain susceptible to
treatments with iron chelators [67]. Both desferioxamine (DFO) and deferiprone have shown
promising results in the U-251 cell line [68]. However, despite these encouraging promises,
there are very few reports concerning the activity of TSCs in glioblastomas. Importantly, for
the compounds to be useful as anti-cancer drugs, the selectivity between the tumor cells and
normal, mortal cell types must be revealed. Therefore, we additionally tested the new com-
pounds on the NHDF cell line. In general, all the examined analogs showed a significantly
higher anti-proliferative activity than the reference Triapine. The most active compound was
compound L3 with an IC50 value equal to 0.12 μM in the HCT116 p53+/+ cell line. The activity
of this compound was very similar for all the cancer lines and fluctuated around 0.12–0.2 μM.
This derivative also has a promising therapeutic index (208 in HCT116 p53+/+ see S2 Table) as
its cytotoxicity against normal cell lines is relatively low. However, the glioma cell line Hs683 is
an exception here as it has an IC50 with a ten-fold higher value for L3. In fact, the glioblastoma
Hs683 exhibited resistance to almost all the tested analogs except for the L9 and L10 derivatives.
Those two analogs were active against all the tested cancer cell lines. In general, ligands that
are halogenated at the terminal aromatic ring exerted a higher activity than their unsubstituted
non-halogenated counterparts (compare L1 vs. L2, L3). The compounds with diazine rings
appeared to be less active than phenol (L1 vs. L11 and L12) and the pyridine ring produced a
derivative that was even less active (L8). This may have been the result of the unfavorable pKa
of the molecule and is in contrast with previous reports [21]. The trifluoromethyl substitution
was very active against all the cancer cell lines that were tested. It also appeared to be less toxic
against normal fibroblasts (L6, L9, L10).
The effect of the TP53 status on a cell’s susceptibility to the some of the Triapine analogs is
interesting. The derivatives L1, L4, L5 and L8 were approximately 2–14 times more effective
against HCT116 (p53-/-) than against the wild-type cells. In turn, this scheme was inverted for
compound L7, which exhibited a reversed effect, as these compounds appeared to be slightly
less active against the p53-/- cell line. In addition to these observations, the colon cancer cells,
breast and U-251 glioma appeared to be similarly susceptible to TSCs. A particularly interest-
ing exception was the Hs683 cell line, which appeared to be relatively resistant to almost all of
the compounds with a ten-fold higher mean IC50. This phenomenon is important when con-
sidering the similar origin of the U-251 and Hs683 lines (from a malignant glioblastoma
tumor and from a glioma lesion of 75-year-old Caucasian male patient, respectively). The
explanation probably lies in a different level of the basal iron homeostasis, e.g. originating
from the different activity and concentration of the transferrin (Tf) receptors. It has previously
been reported that Hs683 is significantly more resistant than U-251 to Tf-toxins such as the
pokeweed antiviral protein, momordin or gelonin [69]. This suggests that in resistant cells, the
cellular metabolism of iron can be restored more easily.
To summarize, the novel compounds demonstrated the greatest promise as anti-cancer
agents with both a potent and selective anti-proliferative activity (Table 2). Accordingly, the
structure-activity relationship reveals that the combination of the piperazine ring with
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Piperazinyl fragment improves anticancer activity of Triapine
Triapine allows potent and selective anticancer chelators that warrant further in vivo examina-
tion to be identified. In particular, the compounds L9 and L10, which have fluorine atoms,
appeared to have the best activity against all of the cancer lines that were tested. For this rea-
son, we selected L9 for a more thorough investigation of the molecular mechanisms of its
activity.
Cell cycle analysis. The results presented in Fig 5 illustrate the effects of L9, which is one
of the most active TSCs of the current series, on the regulation of the cell cycle in the HCT116
p53+/+, U-251 and MCF-7 cell lines. In general, we observed a decrease in the percentage of
cells in the G0/G1 phase 24 h after treatment with L9 in all the cell lines. This effect was espe-
cially strong in the U-251 cells, in which L9 decreased the cell count in the G0/G1 phase to 50%
compared to the untreated cells (78%) (Fig 5B). Additionally, the L9 compound induced an
increase in the percentage of cells in the S phase in all of the tested cell lines. The strongest
Fig 5. Influence of L9 on the regulation of the cell cycle in the HCT116 p53+/+, U-251 and MCF-7 cells. The histograms show the percentage of cells in the G0/G1, S
and G2/M phases of the cell cycle for one of the experiments (A). The table shows the mean SD percentage of the cells in the G0/G1, S and G2/M phases of the cell cycle
from three independent experiments (B). Data were analyzed using one-way ANOVA with Bonferroni’s post-hoc test: Ãp<0.05, ÃÃp<0.01, ÃÃÃp<0.001 compared to the
control (C).
https://doi.org/10.1371/journal.pone.0188767.g005
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Piperazinyl fragment improves anticancer activity of Triapine
effect was observed for U-251 (41%). Moreover, 24 h after treatment with L9, we did not
observe any changes in the percentage of cells in the G2/M phase compared to the untreated
control. These results suggest that the tested TSCs may induce the arrest of the cell cycle in the
G1/S phase, thus contributing to the induction of cell death. This result is in good agreement
with literature data for TSC [70,71] as well as iron chelator in general [72–74]].
Analysis of programmed cell death–annexin V-FITC assay. The ability of L9 to induce
apoptosis in the HCT116, U-251 and MCF-7 cells was determined using Annexin V-FITC
staining. The results are presented in Fig 6. The largest percentage of apoptotic cells was
observed in the MCF-7 cell line. In this case, we detected a six-fold increase in the number of
dead cells (compared to the untreated cells). The total apoptotic value was 55% (Fig 6B). For
HCT116 p53+/+, we also noticed a four-fold increase in the number of dead cells. The situation
was different for the U-251 cell line, in which the percentage of total apoptotic cells was three
times higher in L9-treated cells than the control. All observed changes are statistically
significant.
Western blot analysis of the cell cycle and cell death proteins. Alterations in the iron
level are associated with the progression of the cell cycle. Due to their ability to bind with cellu-
lar iron, iron chelators may affect the expression of many proteins that are responsible for con-
trolling the cell cycle. The most important regulators of the cell cycle are cyclins, cyclin-
dependent kinases (cdks), p53 and cyclin-dependent kinase inhibitors such as p21 [73]. The
cdks are dependent on the cyclins to modulate their phosphorylation activity, and therefore
the activity of the cyclin-cdk complexes are affected by the cyclin-dependent kinase inhibitors.
The progression through the G1 phase and then transition to the S phase of the cell cycle are
controlled in part by the activation of the cyclin D1/cdk4 and cyclin E/cdk2 complexes [74].
Additionally, the activity of cyclin D1 is associated with the p21 protein, which plays a crucial
role in triggering various effects on cell cycle regulation. Thus, a decrease in the expression of
the p21CIP1/WAF1 protein may lead to the arrest of the cell cycle in the G1/S phase since this
protein can stabilize the cyclin D1-cdk complexes [75,76]. On the other hand, the effect of iron
chelation may up-regulate p21CIP1/WAF1, which may induce the signaling pathways, thus lead-
ing to apoptosis [74,77]. In turn, the cdc2 protein, which is the catalytic subunit that complexes
cyclin A, B, is responsible for the transition into the G2/M phase of the cell cycle [74,77]. With
this in mind, we evaluated the impact of L9, as well as reference 3-AP, on the expression of
cyclin E, p21 and the cdc2 proteins in HCT116 p53+/+, U-251 and MCF-7 cells. As is presented
in Fig 7, we observed various patterns of the expression of the cell cycle proteins in the investi-
gated cell lines. Treatment with L9 led to a slight increase in the expression of cyclin E in the
HCT116 cells. Reversely, we observed a slight down-regulation of this protein in the other cell
lines. A reference compound (3-AP) caused overexpression od cyclin E in all testes cell lines.
Moreover, the western blot analysis revealed the influence of L9, especially, by the significant
decrease of the expression of p21 protein in HCT116 cells. On the other hand, after treatment
with L9, we observed the considerable down-regulation of p21 in the MCF-7 cells. We
observed the similar pattern in changes in the expression of the cdc2 protein. Investigated
compound influenced on the slight down-regulation of the cdc2 in the HCT116, and MCF-7
cell lines. In the case of 3-AP this effect was much stronger (MCF-7). This suggests that the
TSCs have no influence on the entry of cells into the mitosis phase of the cell cycle. The
obtained results indicate that the novel analogs of Triapine induced the arrest of the cell cycle
in the G1/S phase and thus triggered apoptosis. Typically, this effect is connected with the acti-
vation of caspases–a family of endoproteases that participates in triggering the cellular
response to damage. Caspase-8 is the initiator that participates in the extrinsic apoptosis path-
way. Its activation comes via dimerization and leads to the initiation of the executioner cas-
pases (-3, -6, -7) or activates the intrinsic pathway of apoptosis. The second type of cell death is
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Piperazinyl fragment improves anticancer activity of Triapine
Fig 6. Evaluation of the induction of apoptosis in the HCT116 p53+/+, U-251 and MCF-7 cells 48 h after treatment with L9. The histograms show the percentage of
early and late apoptosis for one of three independent experiments (A). The table shows the mean SD percentage of live, early and late apoptotic cells from three
independent experiments (B). Data were analyzed using one-way ANOVA with Bonferroni’s post-hoc test: Ãp<0.05, ÃÃp<0.01, ÃÃÃp<0.001 compared to the control (C).
https://doi.org/10.1371/journal.pone.0188767.g006
also called mitochondrial apoptosis and is connected with the release of cytochrome c into the
cytosol [78,79]. Therefore, we examined the effect of a 24 h treatment with L9 on the regulation
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Piperazinyl fragment improves anticancer activity of Triapine
Fig 7. The effect of L9 on the expression of the proteins: GADPH, PARP, caspase-3, 8, 9, p53, cyclin E, cdc2, p21, and cytochrome c, in the HCT116 p53+/+, U-251
and MCF-7 cells. (A). Densitometric analyses of western blot images. Expression level signals are relative to GADPH expression. Data were analyzed using one-way
ANOVA with Bonferroni’s post-hoc test: Ãp<0.05, ÃÃp<0.01, ÃÃÃp<0.001 compared to the control (B).
https://doi.org/10.1371/journal.pone.0188767.g007
of cytochrome c, and caspase-3, 8, 9 proteins. Additionally we explored another proteins,
which are involved in the apoptosis pathway: p53, and PARP. Similar to previous results, we
observed some differences among the investigated cell lines, which may be associated with var-
ious lengths of their cell cycle. The obtained results confirm that treatment with L9 induces the
release of cytochrome c from the mitochondria into the cytosol in case of HCT116 cells. Addi-
tionally, we observed a slight down-regulation of cytochrome c in and MCF-7. For 3-AP we
detected release of cytochrome c in all tested cell lines. To further elucidate the mechanism, we
evaluated the influence of L9 on the extrinsic pathway of caspases activation.
In the case of caspase-3 we did not detect any significant changes in its expression for
HCT116, and U-251 cell lines. It is commonly known that MCF-7 cell line do not express cas-
pase-3 [80], and thus we investigate another caspase proteins. In general we observed the same
pattern for caspase-8. In the case of caspase-9 we detected slight down-regulation for HCT116,
and no influence on the MCF-7 cell line. A small up-regulation occurred for glioma cells.
Results of caspases expression may indicate another pathway of the apoptosis triggering.
Another investigated protein was p53, which is involved in many proliferating, and survival
signals in cell. We detected interesting dependence in glioma cells, for which the upregulation
of p53 was noticed. However it should be highlighted that U-251 cells are mutants with R273H
missense mutation of p53. This resulted in normally expressed protein, that is able to cross the
nucleus and interact with DNA [81,82]. Although overexpression of mutp53 in U-251 cells do
not correlate with apoptosis. With this in mind increased level of cytochrome c and caspase-9
may suggest p53-independent apoptosis pathway. In colon and breast cancer influence of p53
protein was unnoticeable. Very popular protein for detection of cell death is PARP. Analysis of
the lines on the gel indicated PARP cleavage for HCT116. This was also observed for glioma
cell, but just for 3-AP. Summarizing, our hypothesis suggests influence of the cytochrome c
(HCT116, MCF-7), and PARP (HCT116) on the apoptotic pathway.
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Piperazinyl fragment improves anticancer activity of Triapine
Conclusions
In the current study, a new class of TSCs, piperazinylogs of Triapine, was designed to fulfill the
di-substitution pattern at the TSCs N4 position, which is a crucial prerequisite for the high
activity of the previously obtained TSC compounds–DpC and Dp44mT. We tested the impor-
tant physicochemical characteristics of the novel compounds L1-L12. The studied ligands are
neutral at physiological pH, which allows them to permeate cell membranes and bind cellular
Fe pools more readily than less lipid-soluble ligands, e.g. DFO.
The selectivity and anti-cancer activity of the novel TSCs were examined in a variety of can-
cer cell types. In general, the novel compounds demonstrated the greatest promise as anti-can-
cer agents with both a potent and selective anti-proliferative activity (Table 2). Accordingly,
structure-activity relationship studies revealed that the combination of the piperazine ring
with Triapine allows potent and selective anticancer chelators that warrant further in vivo
examination to be identified. In particular, compounds L6 and L10 with a fluorine atom within
the piperazine fragment appeared to enhance the activity and selectivity of the new analogs.
Significantly, this study proved the importance of the di-substitution pattern of the amine
N4 function, thus identifying new potent and selective anticancer chelators that warrant fur-
ther in vivo examination.
Experimental section
Chemistry
Microwave reactions were carried out in a Discover1 BenchMateTM (CEM) microwave
equipped with 10 mL vessels. Melting point measurements were determined in a Stanford
Research Systems OptiMelt (MPA 100). 1H and 13C NMR spectra were recorded on a Bruker
Ascend 500 MHz spectrometer at frequencies of 500 MHz and 126 MHz and a Bruker Avance
III 400 MHz FT-NMR spectrometer at frequencies of 400 MHz and 101 MHz using DMSO-d6
as the solvent and TMS as the internal standard. The NMR solvents were purchased from
ACROS Organics. The chemical shifts (δ) are given in ppm and the coupling constants (J) val-
ues are reported in hertz (Hz). The spin multiplicities are described as s (singlet), d (doublet),
dd (double of doublets), t (triplet), q (quartet) and m (multiplet). All evaporations were per-
formed on a rotary evaporator under diminished pressure at 60˚C. All reagents and solvents
were purchased from ACROS Organics, Asta-Tech, Maybridge, Santa Cruz Biotechnology and
Sigma-Aldrich and were used without further purification.
The purity of all compounds were tested using the HPLC/MS method. The HPLC–MS anal-
yses were performed on Varian model 920 liquid chromatograph equipped with the Varian
900-LC model autosampler, the gradient pump, the Varian Pro Star 510 model column oven,
the Varian 380-LC model evaporative light scattering detection (ELSD) detector. This was cou-
pled with Varian 500-MS IT. HRMS were determined with high resolution mass spectrometer
Waters LCT Premier XE with electrospray ionisation (ESI).
General procedure for the synthesis of thiosemicarbazides. The mixtures of (1,1’-thio-
carbonyl) bis-1H-imidazole (5 mmol) and a suitable derivative of piperazine (5 mmol) in
methylene chloride (25 mL) were stirred for 24 h at room temperature. The solutions were
extracted with distilled water three times and the organic phases were dried over anhydrous
magnesium sulfate, filtered and then evaporated on a rotary evaporator. The obtained deriva-
tives of thioketone were added to a solution of 5 mmol of hydrazine hydrate in 25 mL of etha-
nol at room temperature. The reaction mixture was refluxed for 2 h and cooled to obtain a
precipitate, which was collected via filtration. The final thiosemicarbazides were crystallized
from methanol.
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Piperazinyl fragment improves anticancer activity of Triapine
4-phenylpiperazine-1-carbothiohydrazide (K1)
White powder; yield 69%; mp: 173–174; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.15 (s,
4H, CH2), 3.87 (s, 4H, CH2), 6.80 (t, 1H; J = 7.1 Hz), 6.95 (d, 2H; J = 8.0 Hz), 7.22 (t, 2H; J = 7.7
Hz), 9.17 (s, 1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 48.3; 56.5; 116.0; 119.7; 129.4;
151.1; 183.0. MS (ESI+): m/z calculated for C11H16N4S: 236.11, found: 237.64 [M+H]+, 276.05
[M+K]+.
4-(4-fluorophenyl)piperazine-1-carbothiohydrazide (K2)
Light pink powder; yield 97%; mp: 180–181; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.09
(m, 4H, CH2), 3.87 (m, 4H, CH2), 4.77 (s, 2H, NH2), 6.97 (m, 2H, CH), 7.05 (m, 2H, CH), 9.19
(s, 1H, NH). 13C-NMR (101 MHz, d6-DMSO, ppm): δ 40.3; 49.1; 115.9; 117.9; 148.0; 157.8;
183.0. MS (ESI+): m/z calculated for C11H15FN4S: 254.10, found: 276.30 [M+Na]+.
4-(4-chlorophenyl)piperazine-1-carbothiohydrazide (K3)
White powder; yield 90%; mp: 195–196; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.16 (t, 4H,
J = 5.2 Hz), 3.86 (t, 4H, J = 5.2 Hz), 6.95 (m, 2H, CH), 7.24 (m, 2H, CH), 9.18 (s, 1H, NH). 13C-
NMR (126 MHz, d6-DMSO, ppm): δ 47.3; 48.0; 117.3; 123.0; 129.1; 149.8; 182.9. MS (ESI+): m/z
calculated for C11H15ClN4S: 270.07, found: 270.35 [M]+.
4-(4-methoxyphenyl)piperazine-1-carbothiohydrazide (K4)
White powder; yield 81%; mp: 194–195; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.00 (m,
4H, CH2), 3.68 (s, 3H, CH3), 3.86 (m, 4H, CH2), 4.76 (s, 2H, NH2), 6.83 (m, 2H, CH), 6.92 (m,
2H, CH), 9.17 (s, 1H, NH).13C-NMR (101 MHz, d6-DMSO, ppm): δ 19.0; 49.9; 56.5; 114.8;
118.2; 145.4; 153.7; 183.1. MS (ESI+): m/z calculated for C12H18N4OS: 266.12, found: 288.02
[M+Na]+.
4-(4-cyanophenyl)piperazine-1-carbothiohydrazide (K5)
White powder; yield 86%; mp: 179–180; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.42 (m,
4H, CH2), 3.88 (m, 4H, CH2), 4.76 (s, 2H, NH2), 6.99 (m, 2H, CH), 7.59 (m, 2H, CH), 9.15 (s,
1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 40.6; 48.8; 98.6; 114.3; 120.5; 133.8; 153.1;
182.9. MS (ESI+): m/z calculated for C12H15N5S: 261.10, found: 284.75 [M+Na]+.
4-[2-nitro-4-(trifluoromethyl)phenyl]piperazine-1-carbothiohydrazide (K6)
Light orange crystals; yield 75%; mp: 167–168; 1H-NMR (500 MHz, d6-DMSO, ppm): δ
3.26 (m, 4H, CH2), 3.88 (m, 4H, CH2), 4.76 (s, 2H, NH2), 7.44 (d, 1H; J = 8.8 Hz), 7.86 (dd, 1H;
J1 = 9.0 Hz, J2 = 2.3 Hz), 8.17 (dd, 1H; J1 = 2.3 Hz, J2 = 0.9 Hz), 9.15 (s, 1H, NH). 13C-NMR
(126 MHz, d6-DMSO, ppm): δ 46.9; 49.4; 119.7; 121.2; 123.0; 124.4; 130.5; 139.1; 147.5; 183.0.
MS (ESI+): m/z calculated for C12H14F3N5O2S: 349.08, found: 349.05 [M]+.
tert-butyl 4-(hydrazinylcarbonothioyl)piperazine-1-carboxylate (K7)
White powder; yield 70%; mp: 165–167; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 1.41 (s,
9H, CH3), 3.32 (m, 4H, CH2), 3.71 (m, 4H, CH2), 4.72 (s, 2H, NH2), 9.11 (s, 1H, NH). 13C-
NMR (126 MHz, d6-DMSO, ppm): δ 19.0; 28.5; 47.4; 49.0; 79.6; 154.3; 183.1. MS (ESI+): m/z
calculated for C10H20N4O2S: 260.13, found: 283.77 [M+Na]+.
4-(pyridin-2-yl)piperazine-1-carbothiohydrazide (K8)
White powder; yield 98%; mp: 172–173; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.18 (s,
4H, CH2), 3.85 (m, 4H, CH2), 4.77 (s, 2H, NH2), 6.83 (m, 1H, CH), 7.56 (m, 1H, CH), 8.13 (m,
1H, CH), 9.13 (s, 1H, NH). 13C-NMR (101 MHz, d6-DMSO, ppm): δ 39.6; 51.0; 107.6; 113.7;
138.0; 148.0; 159.1; 183.0. MS (ESI+): m/z calculated for C10H15N5S: 237.10, found: 237.65
[M]+.
4-[5-(trifluoromethyl)pyridin-2-yl]piperazine-1-carbothiohydrazide (K9)
White powder; yield 70%; mp: 206–207; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.70 (s,
4H, CH2), 3.87 (s, 4H, CH2), 4.76 (s, 2H, NH2), 6.93 (d, 1H, J = 9.1 Hz), 7.82 (s, 1H, CH), 8.42
(s, 1H, CH), 9.14 (s, 1H, NH). 13C-NMR (101 MHz, d6-DMSO, ppm): δ 43.8; 46.9; 49.1; 106.7;
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Piperazinyl fragment improves anticancer activity of Triapine
134.9; 145.6; 145.7; 160.3; 183.0. MS (ESI+): m/z calculated for C11H14F3N5S: 305.09, found:
305.25 [M]+.
4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]piperazine-1-carbothiohydrazide (K10)
White powder; yield 85%; mp: 191–192; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.51 (m,
4H, CH2), 3.88 (m, 4H, CH2), 4.83 (s, 2H, NH2), 8.20 (d, 1H; J = 2.1 Hz), 8.56 (s, 1H, CH), 9.18 (s,
1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 47.3; 48.1; 120.2; 122.8; 125.0; 136.8; 143.5;
159.7; 183.2. MS (ESI+): m/z calculated for C11H13ClF3N5S: 339.05, found: 340.28 [M+H]+.
4-(pyrimidin-2-yl)piperazine-1-carbothiohydrazide (K11)
White powder; yield 92%; mp: 208–209; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.75 (m,
4H, CH2), 3.83 (m, 4H, CH2), 4.76 (s, 2H, NH2), 6.66 (t, 1H, J = 4.7 Hz), 8.38 (d, 2H, J = 4.7
Hz), 9.13 (s, 1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 43.2; 47.3; 110.9; 158.4; 161.5;
183.0. MS (ESI+): m/z calculated for C9H14N6S: 238.10, found: 238.35 [M]+.
4-(pyrazin-2-yl)piperazine-1-carbothiohydrazide (K12)
Light pink crystals; yield 88%; mp: 177–178; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.61
(m, 4H, CH2), 3.87 (m, 4H, CH2), 4.77 (s, 2H, NH2), 7.86 (d, 1H; J = 2.7 Hz), 8.09 (dd, 1H; J1 =
2.7 Hz, J2 = 1.5 Hz), 8.31 (d, 1H, J = 1.5 Hz), 9.16 (s, 1H, NH). 13C-NMR (126 MHz, d6-
DMSO, ppm): δ 43.7; 47.0; 131.7; 133.0; 141.9; 154.7; 182.9. MS (ESI+): m/z calculated for
C9H14N6S: 238.10, found: 238.57 [M+H]+.
General procedure for synthesis of thiosemicarbazones. We added two drops of glacial
acetic acid as a catalyst to the mixtures of thiosemicarbazides (0.5 mmol) and 3-aminopyri-
dine-2-carboxaldehyde (0.5 mmol) in ethanol (5 mL). The glass tubes were sealed and placed
into a microwave reactor at 83 ºC for 20 minutes (the reactor power did not exceed 50 W).
The obtained thiosemicarbazones were crystallized from methanol.
N’-[(3-aminopyridin-2-yl)methylidene]-4-phenylpiperazine-1-carbothiohydrazide (L1)
Light yellow crystals; yield 83%; mp: 189–190; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.26
(s, 4H, CH2), 4.09 (s, 4H, CH2), 6.82 (t, 1H; J = 7.3 Hz), 6.99 (d, 2H; J = 8.2 Hz), 7.10 (m, 2H,
CH), 7.18 (s, 2H, NH2), 7.25 (t, 2H; J = 7.8 Hz), 7.84 (dd, 1H; J1 = 4.0 Hz, J2 = 1.6 Hz), 8.53 (s,
1H, CH), 11.44 (s, 1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 48.3; 48.6; 116.0; 119.6;
122.4; 124.5; 129.5; 134.2; 137.2; 144.2; 149.5; 150.9; 180.0. HRMS (ESI): m/z calculated for
C17H21N6S: 341.1548, found: 341.1547 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(4-fluorophenyl)piperazine-1-carbothiohydrazide
(L2)
Yellow crystals; yield 70%; mp: 210–211; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.18 (s,
4H, CH2), 4.08 (s, 4H, CH2), 6.96–7.03 (m, 2H, CH), 7.04–7.13 (m, 4H, CH), 7.17 (s, 2H,
NH2), 7.84 (d, 1H; J = 3.9 Hz), 8.53 (s, 1H, CH), 11.44 (s, 1H, NH). 13C-NMR (126 MHz, d6-
DMSO, ppm): δ 48.6; 49.3; 115.8; 117.9; 122.4; 124.5; 134.2; 137.2; 144.2; 147.8; 149.5; 155.8;
157.6; 180.1. HRMS (ESI): m/z calculated for C17H20FN6S: 359.1454, found: 359.1466 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(4-chlorophenyl)piperazine-1-carbothiohydrazide
(L3)
Yellow powder; yield 83%; mp: 206–207; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.27 (t,
4H, J = 5.2 Hz), 4.08 (t, 4H, J = 5.3 Hz), 6.99 (m, 2H, CH), 7.06–7.08 (dd, 1H; J1 = 8.3 Hz, J2 =
4.2 Hz), 7.09–7.12 (dd, 1H; J1 = 8.4 Hz, J2 = 1.6 Hz), 7.14–7.22 (s, 2H, NH2), 7.25–7.28 (m, 2H,
CH), 7.84 (dd, 1H; J1 = 4.2 Hz, J2 = 1.6 Hz), 8.53 (s, 1H, CH), 11.45 (s, 1H, NH). 13C-NMR
(126 MHz, d6-DMSO, ppm): δ 48.0; 48.4; 117.3; 122.5; 123.1; 124.5; 129.1; 134.2; 137.2; 144.2;
149.5; 149.7; 180.1. HRMS (ESI): m/z calculated for C17H20ClN6S: 375.1159, found: 375.1155
[M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(4-methoxyphenyl)piperazine-1-carbothiohydrazide
(L4)
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Piperazinyl fragment improves anticancer activity of Triapine
Yellow powder; yield 59%; mp: 195–196; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.11 (s,
4H, CH2), 3.70 (s, 3H, CH3), 4.07 (s, 4H, CH2), 6.85 (m, 2H, CH), 6.96 (m, 2H, CH), 7.09 (m,
2H, CH), 7.17 (s, 2H, NH2), 7.84 (dd, 1H; J1 = 4.1 Hz, J2 = 1.7 Hz), 8.52 (s, 1H, CH), 11.43 (s,
1H, NH). 13C-NMR (101 MHz, d6-DMSO, ppm): δ 48.8; 50.0; 55.7; 114.8; 118.3; 122.4; 124.5;
134.2; 137.1; 144.2; 145.3; 149.4; 153.7; 180.1. HRMS (ESI): m/z calculated for C18H23N6OS:
371.1654, found: 371.1645 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(4-cyanophenyl)piperazine-1-carbothiohydrazide
(L5)
Light orange powder; yield 74%; mp: 209–210; 1H-NMR (400 MHz, d6-DMSO, ppm): δ
3.53 (s, 4H, CH2), 4.10 (s, 4H, CH2), 7.02 (d, 2H; J = 8.8 Hz), 7.09 (m, 2H, CH), 7.17 (s, 2H,
NH2), 7.62 (d, 2H; J = 8.7 Hz), 7.84 (dd, 1H; J1 = 4.1 Hz, J2 = 1.6 Hz), 8.54 (s, 1H, CH), 11.42 (s,
1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 45.8; 47.9; 98.5; 114.0; 120.6; 122.5; 124.5;
133.8; 134.2; 137.2; 144.2; 149.6; 152.9; 179.9. HRMS (ESI): m/z calculated for C18H20N7S:
366.1501, found: 366.1503 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-[2-nitro-4-(trifluoromethyl)phenyl]piperazine-
1-carbothiohydrazide (L6)
Yellow powder; yield 75%; mp: 179–180; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.38 (m,
4H, CH2), 4.09 (m, 4H, CH2), 7.07 (dd, 1H; J1 = 8.3 Hz, J2 = 4.2 Hz), 7.11 (dd, 1H; J1 = 8.4 Hz,
J2 = 1.6 Hz), 7.13–7.21 (s, 2H, NH2), 7.47 (d, 1H; J = 8.9 Hz), 7.83–7.91 (m, 2H, CH), 8.20 (d,
1H, J = 2.7 Hz), 8.53 (s, 1H, CH), 11.42 (s, 1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ
19.0; 47.9; 49.3; 56.5; 121.1; 122.5; 123.0; 124.4; 130.5; 134.2; 137.2; 138.9; 144.3; 147.5; 149.7;
180.1. HRMS (ESI): m/z calculated for C18H19F3N7O2S: 454.1293, found: 474.1269 [M+H]+.
tert-butyl 4-({2-[(3-aminopyridin-2-yl)methylidene]hydrazinyl}carbonothioyl)piperazine-
1-carboxylate (L7)
Yellow powder; yield 20%; mp: 193–194; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 1.43 (s,
9H, CH3), 3.44 (m, 4H, CH2), 3.93 (m, 4H, CH2), 7.05–7.12 (m, 2H, CH), 7.13–7.19 (s, 2H,
NH2), 7.84 (dd, 1H; J1 = 4.2 Hz, J2 = 1.6 Hz), 8.51 (s, 1H, CH), 11.39 (s, 1H, NH). 13C-NMR
(126 MHz, d6-DMSO, ppm): δ 28.5; 48.4; 79.7; 122.8; 124.5; 133.9; 136.9; 144.3; 149.2; 154.3;
180.2. HRMS (ESI): m/z calculated for C16H25N6O2S: 365.1760, found: 365.1769 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(pyridin-2-yl)piperazine-1-carbothiohydrazide (L8)
Yellow crystals; yield 38%; mp: 210–211; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.64 (m,
4H, CH2), 4.07 (m, 4H, CH2), 6.68 (m, 1H, CH), 6.86 (d, 1H; J = 8.6 Hz), 7.09 (m, 2H, CH),
7.18 (s, 2H, NH2), 7.58 (m, 1H, CH), 7.84 (dd, 1H; J1 = 4.1 Hz, J2 = 1.6 Hz), 8.15 (d, 1H, J = 4.9
Hz), 8.54 (s, 1H, CH), 11.41 (s, 1H, NH). 13C-NMR (101 MHz, d6-DMSO, ppm): δ 44.4; 48.3;
107.5; 113.7; 122.5; 124.5; 134.2; 137.1; 138.1; 144.2; 148.0; 149.5; 159.0; 180.1. HRMS (ESI): m/
z calculated for C16H20N7S: 342.1501, found: 342.1514 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-[5-(trifluoromethyl)pyridin-2-yl]piperazine-1-car-
bothiohydrazide (L9)
Yellow crystals; yield 47%; mp: 232–233; 1H-NMR (400 MHz, d6-DMSO, ppm): δ 3.80 (s,
4H, CH2), 4.09 (s, 4H, CH2), 6.97 (d, 1H; J = 9.1 Hz), 7.08 (m, 2H, CH), 7.18 (s, 2H, NH2), 7.85
(m, 2H, CH), 8.46 (d, 1H, J = 2.5 Hz), 8.54 (s, 1H, CH), 11.42 (s, 1H, NH). 13C-NMR (101
MHz, d6-DMSO, ppm): δ 43.8; 47.9; 106.7; 113.8; 122.5; 124.5; 126.4; 134.2; 135.0; 137.1; 144.2;
145.7; 149.6; 160.3; 180.0. HRMS (ESI): m/z calculated for C17H19F3N7S: 410.1374, found:
410.1365 [M+H]+.
4.1.2.10. N’-[(3-aminopyridin-2-yl)methylidene]-4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]
piperazine-1-carbothiohydrazide (L10)
Dark yellow powder; yield 64%; mp: 199–200; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.63
(m, 4H, CH2), 4.10 (m, 4H, CH2), 7.05–7.09 (dd, 1H; J1 = 8.3 Hz, J2 = 4.2 Hz), 7.09–7.12 (dd,
1H; J1 = 8.3 Hz, J2 = 1.6 Hz), 7.14–7.21 (s, 2H, NH2), 7.84 (dd, 1H; J1 = 4.1 Hz, J2 = 1.6 Hz),
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Piperazinyl fragment improves anticancer activity of Triapine
8.23 (d, 1H, J = 2.2 Hz), 8.53 (s, 1H, CH), 8.59 (dd, 1H; J1 = 2.3 Hz, J2 = 1.1 Hz), 11.44 (s, 1H,
NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 48.1; 48.3; 119.1; 120.0; 122.5; 124.5; 125.0;
134.2; 136.9; 137.2; 143.6; 144.2; 149.6; 159.5; 180.3. HRMS (ESI): m/z calculated for
C17H18ClF3N7S: 444.0985, found: 464.0988 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(pyrimidin-2-yl)piperazine-1-carbothiohydrazide
(L11)
Light yellow powder; yield 39%; mp: 198–199; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.86
(m, 4H, CH2), 4.05 (m, 4H, CH2), 6.69 (t, 1H, J = 4.7 Hz), 7.07 (dd, 1H; J1 = 8.3 Hz, J2 = 4.2 Hz),
7.11 (dd, 1H; J1 = 8.3 Hz, J2 = 1.6 Hz), 7.13–7.22 (s, 2H, NH2), 7.84 (dd, 1H; J1 = 4.2 Hz, J2 = 1.6
Hz), 8.41 (d, 2H, J = 4.7 Hz), 8.53 (s, 1H, CH), 11.43 (s, 1H, NH). 13C-NMR (126 MHz, d6-
DMSO, ppm): δ 43.2; 48.4; 111.0; 122.5; 124.5; 134.2; 137.1; 144.2; 149.4; 158.5; 161.5; 180.1.
HRMS (ESI): m/z calculated for C15H19N8S: 343.1453, found: 343.1464 [M+H]+.
N’-[(3-aminopyridin-2-yl)methylidene]-4-(pyrazin-2-yl)piperazine-1-carbothiohydrazide
(L12)
Yellow crystals; yield 70%; mp: 217–218; 1H-NMR (500 MHz, d6-DMSO, ppm): δ 3.72 (m,
4H, CH2), 4.09 (m, 4H, CH2), 7.05–7.12 (m, 2H, CH), 7.14–7.22 (s, 2H, NH2), 7.84 (dd, 1H; J1 =
4.2 Hz, J2 = 1.6 Hz), 7.88 (d, 1H; J = 2.6 Hz), 8.12 (dd, 1H; J1 = 2.7 Hz, J2 = 1.5 Hz), 8.35 (d, 1H,
J = 1.6 Hz), 8.54 (s, 1H, CH), 11.44 (s, 1H, NH). 13C-NMR (126 MHz, d6-DMSO, ppm): δ 43.8;
48.0; 122.5; 124.5; 131.7; 133.1; 134.2; 137.2; 141.9; 144.2; 149.6; 154.7; 180.0. HRMS (ESI): m/z
calculated for C15H19N8S: 343.1453, found: 343.1454 [M+H]+.
Materials and physico-chemical measurements
Starting Materials and Solvents. The solution studies were carried out in bidistilled water. All
the chemicals were commercial products of reagent grade and were used without further
purification.
The Iron(III) stock solution was prepared from FeCl3Á6H2O (Aldrich) in 1.01 × 10−2
M
HCl (Chempur 38%) immediately before use. The Copper(II) solution was prepared from
CuCl2 Á2H2O (Aldrich). All metal stock solutions were standardized using ICP-AES. The HCl
solution was titrated using standardized NaOH (0.1 M Fluka standard solution).
pH-dependent UV-visible titrations. To determine the acid-base properties of the stud-
ied ligands, UV-visible spectrophotometric experiments as a function of p[H] were carried out
in the pH range 2–11.
The isosbestic curves were used to determine the ability of a binding event of the ligand
with Fe(III) and Cu(II). A series of six samples were prepared for each ligand. The ligand was
dissolved in 0.1 M KCl in MeOH/H2O (80/20 w/w) ionic strength and mixed with a freshly
prepared FeCl3Á6H2O or CuCl2Á2H2O solution at various ratio concentrations of the metal
ions. The complex that was formed for each reaction mixture was allowed to stand for 30 min
before the analysis. Absorption spectra were recorded in a Hellma quartz optical cell (1 cm) on
a Jasco V630 spectrophotometer.
All the titrations were carried out on 3.2 mL samples. The metal-ligand system titrations
were performed on solutions of ligand concentrations of 5x10-5 M and metal-to-ligand molar
ratios of 1:1, 1:2, 1:3, 1:4 and 1:5.
UV-Visible titrations were also carried out. The absorption spectra (200–800 nm) were
recorded using a Jasco V630 spectrophotometer. The initial pH of 3.2 mL ligand samples was
adjusted to be acidic or basic and the titration of the solution was then carried out by adding
known volumes of NaOH or HCl, respectively. The spectrophotometric data were fitted using
the HypSpec [83] program. Distribution diagrams of the species were calculated using Hyper-
quad Simulation and Speciation (HySS) software [84].
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Piperazinyl fragment improves anticancer activity of Triapine
Antitumor activity
Cell lines. The human colon carcinoma cell line HCT116 wild type (p53+/+), human
breast carcinoma cell line MCF-7 and glioblastoma cell line Hs683 were obtained from ATCC.
The glioma cell line U-251 was purchased from Sigma Aldrich and the normal human fibro-
blast cell lines NHDF from PromoCell. The human colon cancer cell line HCT116 with a p53
deletion (p53-/-) was kindly provided by Prof. M. Rusin from the Maria Sklodowska-Curie
Memorial Cancer Centre and Institute of Oncology in Gliwice, Poland. Cells were grown as
monolayer cultures in Dulbecco’s modified Eagle’s medium with an antibiotic gentamicin
(200 μL/100 mL medium) in 75 cm2 flasks (Nunc). DMEM for HCT116, MCF-7, U-251,
Hs683 were supplemented with 12% heat-inactivated fetal bovine serum (Sigma) and for
NHDF with 15% non-inactivated fetal bovine serum (Sigma). All the cell lines were cultured
under standard conditions at 37˚C in a humidified atmosphere at 5% CO2.
Cytotoxicity studies. The cells were seeded in 96-well plates (Nunc) at a density of 5,000
cells/well (HCT116, MCF-7, U-251, Hs683) and 4,000 cells/well (NHDF) and incubated at
37˚C for 24 h. The assay was performed following a 72 h incubation with varying concentra-
tions of the compounds that were tested. Then, 20 μL of The CellTiter 961AQueous One
Solution-MTS (Promega) solution was added to each well (with 100 μL DMEM without phe-
nol red) and incubated for 1 h at 37˚C. The optical densities of the samples were analyzed at
490 nm using a multi-plate reader (Synergy 4, Bio Tek). Results were expressed as a percentage
of the control and calculated as the inhibitory concentration (IC50) values using GraphPad
Prism 5. The IC50 parameter was defined as the compound concentration that was necessary
to reduce the proliferation of cells to 50% of the untreated control. Each individual compound
was tested in triplicate in a single experiment with each experiment being repeated four to five
times.
Immunoblotting. The HCT116 (p53+/+), U-251 and MCF-7 cells were seeded in 3 cm
Petri dishes (Nunc) at a density of 0.5Á106 cells/well and incubated overnight. The next day, a
solution of L9 at a four-fold IC50 concentration for each cell line was added and incubated for
24 h. Cells were harvested by trypsinization, washed with cold PBS and cell pellets were
obtained by centrifugation at 2,000 rpm. Total cell lysates were obtained by dissolving the cell
pellets in 150 μL of a RIPA buffer (Thermo Scientific) containing a Halt Protease Inhibitor
Cocktail (Thermo Scientific) or Halt Phosphatase Inhibitor Cocktail (Thermo Scientific) along
with 0.5 M EDTA and lysed for 20 min on ice on a rocking plate. The lysates were then soni-
cated, centrifuged at 10,000 rpm for 10 min at 4˚C and the supernatants were collected for fur-
ther analysis. The protein concentration was determined using a Micro BCA™ Protein Assay
Kit (Thermo Scientific) according to the manufacturer’s instructions. Equal amounts of pro-
teins (15 μg) were electrophoresed on SDS-Page gels and transferred onto a nitrocellulose
membranes. The membranes were blocked in 5% non-fat milk prepared in PBS containing
0.1% Tween-20 (TPBS) for 1 h. After blocking, the membranes were incubated with specific
primary antibodies: PARP, p53, p21Waf1/Cip1, cdc2, cyclin E, cytochrome c, caspase-3,8,9 and
GAPPH overnight at 4˚C, then washed and incubated with horseradish peroxidase (HRP)-
conjugated secondary antibodies for 1 h at room temperature. All the antibodies were pur-
chased from Cell Signaling and were diluted 1:1000 in 5% milk in TPBS. Finally, the mem-
branes were washed and incubated with a SuperSignal™ West Pico Chemiluminescent
Substrate (Thermo Scientific). The chemiluminescence signals were captured using a Chemi-
Doc™ XRS+ System (BioRad). The experiments were performed at least three times.
Cell cycle assay. The HCT116 (p53+/+), U-251 and MCF-7 cells were seeded in 3 cm Petri
dishes (Nunc) at a density of 0.25Á106 cells/well and incubated at 37˚C for 24 h. Then, the
medium was removed and a freshly prepared solution of the tested compound–L9 at a 0.5 μM
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Piperazinyl fragment improves anticancer activity of Triapine
concentration was added. After 48 h of treatment, assays were performed using a Muse Cell-
Cycle Kit (Millipore) according to the manufacturer’s instructions. Briefly, the cells were col-
lected, washed with cold PBS and were centrifuged at 300 g. Then, the cells were fixed in ice
cold 70% ethanol and stored at -20˚C overnight. Afterwards, the cells were centrifuged and
resuspended in 200 μL of Muse™ Cell Cycle Reagent and incubated for 30 min at room temper-
ature in the dark. After staining, the cells were processed for cell cycle analysis using a Muse
Cell Analyzer (Millipore). The experiments were performed at least three times.
Annexin V binding assay. The HCT116 (p53+/+), U-251 and MCF-7 cells were seeded in
3 cm Petri dishes (Nunc) at a density of 0.25Á106 cells/dish and incubated at 37˚C for 24 h.
After treatment with 0.5 μM of L9 for 48 h, the assays were performed using an Annexin V &
Dead Cell Kit (Millipore) according to the manufacturer’s instructions. Briefly, detached and
adherent cells were collected and centrifuged at 500 g for 5 min. Next, the resuspended cells
were incubated with 100 μL of Muse™ Annexin V & Dead Cell Reagent for 20 min at room
temperature in the dark. After staining, the events for live, early and late apoptotic cells were
counted using a Muse Cell Analyzer (Millipore). The experiments were performed at least
three times.
Statistical analysis. All of data were expressed as the mean standard deviation (SD) of
the results obtained from at least three independent experiments. Statistical differences was
performed using one-way ANOVA with a Bonferroni post-hoc test (comparison to control). A
p-value of 0.05 or less was considered to be statistically significant. GraphPad Prism v.5.0 soft-
ware (GraphPad Software, USA) was used for analysis.
Supporting information
S1 Fig. The spectra and distribution forms of the ligands. (a) Absorption spectrophotomet-
ric titration vs. pH of the free L1, L8, L11 and L12 ligands; (b) electronic spectra of the proton-
ated species; (c) concentration distribution curves for chosen ligands species. I = 0.1 M (KCl)
in 80% (w/w) MeOH/H2O; T = 25.0˚C; [L] = 5x10-5 M; pH 1.90–11.5.
(DOCX)
S2 Fig. The effect of L9 on the expression of the proteins–full view from Western blot anal-
ysis. (A) GADPH, (B) caspase-8, (C) caspase-9, (D) GADPH, (E) p53, (F) PARP, (G) cyclin E,
(H) cdc2, (I) caspase-3, (J) p21, (K) cytochrome c.
(DOCX)
S1 Table. Changes in absorption spectra and isosbestic points for chosen ligands.
(DOCX)
S2 Table. Therapeutic indexes of the novel Triapine analogs describing their selectivity
against normal cells. TI values I > 25, TI 2.5–25, TI < 2.5 were marked with red, yellow and
grey color, respectively.
(DOCX)
Acknowledgments
The financial support of the National Center of Science grants 2014/13/D/NZ7/00322 (AMW),
2013/09/B/NZ7/00423 (RM) and NCBiR Warsaw ORGANOMET No: PBS2/A5/40/2014 (JP)
is greatly appreciated.
Author Contributions
Conceptualization: Robert Musiol, Jaroslaw Polanski.
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Piperazinyl fragment improves anticancer activity of Triapine
Formal analysis: Kamila Gajcy, Robert Musiol, Jaroslaw Polanski.
Investigation: Marta Rejmund, Anna Mrozek-Wilczkiewicz, Katarzyna Malarz, Monika Pyr-
kosz-Bulska, Mieczyslaw Sajewicz.
Methodology: Anna Mrozek-Wilczkiewicz.
Supervision: Jaroslaw Polanski.
Writing – original draft: Kamila Gajcy, Robert Musiol, Jaroslaw Polanski.
Writing – review & editing: Kamila Gajcy, Robert Musiol, Jaroslaw Polanski.
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