N4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: Cytotoxicity against human tumor cells, structure-activity relationship studies and investigation on the mechanism of action

Article abstract of DOI:10.1016/j.bmc.2012.04.027

N⁴-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene)hydrazinecarbothioamide) and its N ⁴-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh, H2Ac4pFPh), N⁴-ortho-, -meta

Full text of DOI:10.1016/j.bmc.2012.04.027

Bioorganic & Medicinal Chemistry 20 (2012) 3396–3409
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N4-Phenyl-substituted 2-acetylpyridine thiosemicarbazones: Cytotoxicity
against human tumor cells, structure–activity relationship studies
and investigation on the mechanism of action
Marcella A. Soares a,b, Josane A. Lessa c, Isolda C. Mendes d, Jeferson G. Da Silva c, Raquel G. dos Santos b,e
Lívia B. Salum f, Hikmat Daghestani g, Adriano D. Andricopulo f, Billy W. Day h, Andreas Vogt i,
,
Jorge L. Pesquero a, Willian R. Rocha c, Heloisa Beraldo c,
⇑
a Departamento de Fisiologia e Biofísica, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
b Centro de Desenvolvimento de Tecnologia Nuclear, CDTN, 31270-901 Belo Horizonte, MG, Brazil
c Departamento de Química, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
d Escola de Belas Artes, Departamento de Artes Plásticas, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte, MG, Brazil
e Instituto Nacional de Ciência e Tecnologia de Medicina Molecular, Faculdade de Medicina, Universidade Federal de Minas Gerais, 30130-100 Belo Horizonte, MG, Brazil
f Centro de Biotecnologia Molecular Estrutural, Instituto de Física de São Carlos, Universidade de São Paulo, 13560-970 São Carlos, SP, Brazil
g Department of Structural Biology, University of Pittsburgh, Pittsburgh, PA 15260, USA
h Department of Pharmaceutical Sciences, University of Pittsburgh, Pittsburgh, PA 15261, USA
i University of Pittsburgh Drug Discovery Institute, University of Pittsburgh, Pittsburgh, PA 15261, USA
a r t i c l e i n f o
a b s t r a c t
N4-Phenyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-(1-(pyridin-2-yl)ethylidene)
hydrazinecarbothioamide) and its N4-ortho-, -meta- and -para-fluorophenyl (H2Ac4oFPh, H2Ac4mFPh,
H2Ac4pFPh), N4-ortho-, -meta- and -para-chlorophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh),
N4-ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh, H2Ac4pIPh) and N4-ortho-, -meta-
and -para-nitrophenyl (H2Ac4oNO2Ph, H2Ac4mNO2Ph, H2Ac4pNO2Ph) derivatives were assayed for their
cytotoxicity against human malignant breast (MCF-7) and glioma (T98G and U87) cells. The compounds
were highly cytotoxic against the three cell lineages (IC50: MCF-7, 52–0.16 nM; T98G, 140–1.0 nM; U87,
160–1.4 nM). All tested thiosemicarbazones were more cytotoxic than etoposide and did not present any
haemolytic activity at up to 10ꢀ5 M. The compounds were able to induce programmed cell death.
H2Ac4pClPh partially inhibited tubulin assembly at high concentrations and induced cellular microtubule
disorganization.
Article history:
Received 9 March 2012
Revised 5 April 2012
Accepted 10 April 2012
Available online 19 April 2012
Keywords:
2-Acetylpyridine thiosemicarbazones
Glioblastoma
Breast cancer
Cytotoxicity
Tubulin
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
most aggressive, grade IV, astrocytoma is referred to as glioblas-
toma multiforme.5 Traditionally, patients with glioblastoma multi-
In 2030 an estimated 12 million deaths from cancer are pro-
jected in the world.1 Breast cancer, one of the highest incident
types, affects more than one million women every year.2 Treat-
ment options for patients with advanced breast cancer are varied
and include endocrine therapies, chemotherapies and targeted
therapies. Nonetheless, the high mortality evoked by this pathol-
ogy is related to the resistance of breast tumor cells to conven-
tional therapy.3
Malignant gliomas are the most frequent and lethal cancers
originating in the central nervous system and the second most
common form of cancer in pediatric patients.4 Malignant gliomas
are classified as astrocytomas, oligodendrogliomas, and oligoastro-
cytomas and are histologically graded as II, III, or IV by WHO. The
forme have been managed with surgery followed by radiotherapy.
More recently, the oral alkylating agent temozolamide has become
the standard of care for the management of glioblastoma multi-
forme.6 Other alternative chemotherapy agents, such as etoposide,
carboplatin and nitrosurea-containing procarbazine, lomustine
and vincristine, have also been studied.7 These therapies produce,
however, only a short median survival time.8 Moreover, the low
tolerance of the central nervous system to conventional chemo-
therapeutic agents impairs the effectiveness of the treatment.9
Thus, it is important to search for novel drug candidates in order
to overcome these challenges.
Thiosemicarbazones constitute an interesting class of com-
pounds with a wide spectrum of pharmacological applications.10
a
(N)-Heterocyclic thiosemicarbazones have been extensively
investigated as potential anticancer agents.11 The search for an
effective anticancer agent led to the onset of clinical studies of
⇑
Corresponding author. Tel.: +55 31 34095740; fax: +55 31 34095700.
E-mail address: hberaldo@ufmg.br (H. Beraldo).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.027

M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
3397
Figure 1. Structural representation of 1 (H2Ac4Ph), 2 (H2Ac4oFPh), 3 (H2Ac4mFPh), 4 (H2Ac4pFPh), 5 (H2Ac4oClPh), 6 (H2Ac4mClPh), 7 (H2Ac4pClPh), 8 (H2Ac4oIPh), 9
(H2Ac4mIPh), 10 (H2Ac4pIPh), 11 (H2Ac4oNO2Ph), 12 (H2Ac4mNO2Ph), 13 (H2Ac4pNO2Ph).
3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP;
TriapineÒ, Vion Pharmaceuticals Inc., New Haven, CT).12,13 The
glioblastoma, and these results encouraged us to further investi-
gate their cytotoxic properties as well as those of novel
derivatives.25
antitumor activity of
a(N)-heterocyclic thiosemicarbazones has
been attributed to their ability to inhibit ribonucleoside diphos-
phate reductase (RDR), a rate-limiting enzyme in DNA syntheses
that catalyzes the conversion of ribonucleotides into deoxyribonu-
cleotides.11–14
The development of anticancer drugs requires the evaluation of
the possible mechanism of action involved in the process of cancer
cell death. During the past decade, advances in chemotherapy in
order to induce apoptosis in cancer cells were achieved. More
recently, the focus of the research has been on the non-apoptotic
types of programmed cell death, such as autophagy, which is
characterized by an increase in the number of autophagosomes,
vesicles that surround cellular organelles.26 Subsequently, auto-
phagosomes merge with lysosomes and digest the organelles, lead-
ing to cell death.27
The cytotoxic activity of thiosemicarbazones and their metal
complexes against a variety of human solid tumor cell lines as well
as leukemic cells has been reported by other authors15–19 and by
our group.20–24 Particularly, we have recently demonstrated that
1 (H2Ac4Ph) and its N4-ortho-, -meta- and -para-chlorophenyl
and N4-ortho-, -meta- and -para-tolyl derivatives showed cytotox-
icity at nanomolar concentrations against malignant glioma.25
and its chlorophenyl-derivatives were highly active against
1
Apoptosis and autophagy are predominantly distinct. However,
several studies have demonstrated cross-talk between them.26
A
Scheme 1. Synthesis of compounds 1-13.

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M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
Figure 2. Molecular plots of 2, 3, 4, 8, 10 and 11 showing the labelling scheme of the non-H atoms and their displacement ellipsoids at the 50% probability level.
variety of chemotherapeutic agents (alkylating agents and arsenic
trioxide), hormonal therapies (tamoxifen and vitamin D ana-
thiosemicarbazones 8–10 were characterized by means of their
infrared, NMR and HRMS spectra.
logues), natural compounds (resveratrol), cytokines (IFNc), gene
therapies (p53 and p27Kip1), microtubule disturbing agents and
radiotherapy have shown to trigger autophagic cell death in a pa-
nel of cancer cells.28
2.2. Characterization of the thiosemicarbazones
In the infrared spectra of 8–10 vibrations attributed to
at 3306–3264 and 3232–3208 cmꢀ1 were observed. Absorptions
at 1595–1581 and 786–782 cmꢀ1 were attributed to
(C@N) and
(C@S), respectively. Absorptions attributed to the in-plane defor-
mation mode of the pyridine ring were observed at 621–616 cmꢀ1
m(N–H)
In the present work we evaluated the cytotoxicities of N4-phe-
nyl 2-acetylpyridine thiosemicarbazone (H2Ac4Ph; N-(phenyl)-2-
(1-(pyridin-2-yl)ethylidene)hydrazinecarbothioamide) (1) and its
m
m
N4-ortho-,
-meta-
and
-para-fluorophenyl
(H2Ac4oFPh,
.
H2Ac4mFPh, H2Ac4pFPh) (2–4), N4-ortho-, -meta- and -para-chlo-
rophenyl (H2Ac4oClPh, H2Ac4mClPh, H2Ac4pClPh) (5–7), N4-
ortho-, -meta- and -para-iodophenyl (H2Ac4oIPh, H2Ac4mIPh,
H2Ac4pIPh) (8–10) and N4-ortho-, -meta- and -para-nitrophenyl
(H2Ac4oNO2Ph, H2Ac4mNO2Ph, H2Ac4pNO2Ph) (11–13) deriva-
tives (Fig. 1) against MCF-7 (breast adenocarcinoma), U87 (glio-
blastoma multiforme expressing wild-type p53 protein) and
T98G (glioblastoma multiforme expressing mutant p53) human
malignant tumor cells. A preliminary analysis of the compounds’
mechanism of action was carried out along with structure–activity
relationship (SAR) studies. The effect of H2Ac4pClPh on tubulin
assembly and its effects on cellular microtubule organization and
mitotic arrest were also investigated.
Duplicated signals were observed in the 1H NMR spectra
(DMSO-d6) of 8–10 suggesting a mixture of the E (95–87%) and Z
(13–5%) configurational isomers. This behavior has also been ob-
served for other 2-acetylpyridine-derived thiosemicarbazones.25
The N3–H chemical shifts at d 10.90–10.79 ppm, are characteristic
of the E configuration, in which N3–H is hydrogen bonded to the
solvent,20–22 while chemical shifts at d 14.69–14.66 ppm indicate
the presence of the Z configuration, in which N3–H is hydrogen
bonded to the pyridine nitrogen.20–22 Some hydrogen signals of
the Z isomer could not be observed separately since they appear
in the same region as those of the E isomer.
2.3. X-ray diffraction analysis
Upon slow evaporation in 9:1 acetone/DMSO crystals of 2–4, 8,
10 and 11 were formed and their crystal structures were deter-
mined by X-ray diffraction. The crystal structures of 130 and 5–
725 had been previously determined.
2. Results and discussion
2.1. Formation of the thiosemicarbazones
Figure 2 shows perspective views of 2–4, 8, 10 and 11. Table 1
reports the crystal data and refinement results for the determined
Preparation of all thiosemicarbazones was carried out by a
previously described method,29 as shown in Scheme 1. The novel

M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
3399
Table 1
Crystal data and refinement results for 2–4, 8, 10 and 11
Compound
2
3
4
8
10
11
Empirical formula
Formula weight
Crystal system
C
14H13FN4S
C14H13FN4S
288.34
C14H13FN4S
288.34
C14H13IN4S
396.24
Monoclinic
P 21/n
C14H13IN4S
396.24
Monoclinic
P 21/n
C14H13N5O2S
315.35
Monoclinic
P 21/c
288.34
Monoclinic
P 21/c
Triclinic
Triclinic
ꢀ
ꢀ
Space group
P 1
P 1
Unit cell dimensions
a, Å
b, Å
c, Å
5.4620(2)
10.5664(4)
24.1928(9)
90
94.868(3)
90
1391.22(9)
4/1.377
0.239
5.8660(4)
10.1714(6)
12.0075(7)
75.061(5)
85.873(5)
89.139(5)
690.42(7)
2/1.387
5.8396(4)
10.2292(6)
11.8164(7)
76.218(5)
87.761(5)
89.358(5)
685.00(7)
2/1.398
11.3298(3)
7.5376(2)
17.2904(5)
90
98.910(3)
90
1529.08(11)
4/1.721
2.226
10.4026(3)
5.6886(2)
25.9488(6)
90
92.499(2)
90
1534.09(8)
4/1.716
2.219
11.3298(3)
7.5376(2)
17.2904(5)
90
98.910(4)
90
1458.77(7)
4/1.436
0.237
a
, °
b, °
, °
c
Volume, Å3
Z/density calcd, Mg/m3
Absorption coefficient,
mmꢀ1
0.241
0.243
F(000)
600
300
300
776
776
656
Crystal size, mm
h range for data coll.,
Index range
0.20 ꢂ 0.12 ꢂ 0.06
3.19–26.37
ꢀ6 6 h 6 6
ꢀ9 6 k 6 13
ꢀ25 6 l 6 3
0.23 ꢂ 0.16 ꢂ 0.12
4.10–26.37
ꢀ7 6 h 6 6
ꢀ12 6 k 6 11
ꢀ14 6 l 6 11
0.25 ꢂ 0.12 ꢂ 0.08
3.02–26.37
ꢀ7 6 h 6 6
ꢀ12 6 k 6 12
ꢀ14 6 l 6 10
0.36 ꢂ 0.20ꢂ0.10
2.76–26.37
ꢀ15 6 h 6 14
ꢀ5 6 k 6 5
ꢀ34 6 l 6 34
0.39 ꢂ 0.20 ꢂ 0.07
3.00–26.37
ꢀ11 6 h 6 13
-6 6 k 6 7
0.23 ꢂ 0.17 ꢂ 0.08
2.77–26.37
ꢀ14 6 h 6 13
ꢀ9 6 k 6 9
ꢀ20 6 l 6 21
o
ꢀ31 6 l 6 32
Collected reflections/unique 10392/2834(0.0605) 4972/2809(0.0353) 5207/2804(0.0278) 9035/3120(0.0325)
13323/3120(0.0598) 11910/2987(0.0371)
(Rint
)
Completeness (%)
99.9 (to 26.37o)
0.9858 and 0.9538
2834/0/182
0.987
99.6 (to 26.37o)
0.9717 and 0.9467
2809/0/182
1.063
99.9 (to 26.37o)
0.9811 and 0.9660
2804/0/182
0.915
99.9 (to 26.37o)
1.00000 and 0.77529 0.8784 and 0.4782
3120/0/182
0.887
99.9 (to 26.37o)
99.9 (to 26.37o)
0.9813 and 0.9475
2966/0/200
1.061
Max and min transmission
Data/restraints/parameters
Goodness-of-fit on F2
3120/0/182
1.053
Final R indices [I > 2
r
(I)]
R1 = 0.0422
wR2 = 0.1155
R1 = 0.0601
wR2 = 0.1293
0.300/ꢀ0.326
R1 = 0.0480
wR2 = 0.1425
R1 = 0.0645
wR2 = 0.1495
0.598/ꢀ0.279
R1 = 0.0379
wR2 = 0.0846
R1 = 0.0660
wR2 = 0.0901
0.219/ꢀ0.178
R1 = 0.0323
wR2 = 0.0658
R1 = 0.0607
wR2 = 0.0706
0.607/ꢀ0.706
R1 = 0.0392
wR2 = 0.1096
R1 = 0.0511
wR2 = 0.1140
1.066/ꢀ1.246
R1 = 0.0369
wR2 = 0.0990
R1 = 0.0540
wR2 = 0.1039
0.161/ꢀ0.206
R indices (all data)
Largest peak & hole, e Åꢀ3
structures. Selected intra-molecular bond distances and angles for
130 and 5–7,25 2–4, 8, 10 and 11 are given in Tables 2 and 3,
respectively.
2.4. Cytotoxic activity against malignant breast and glioma cells
The results showed that all thiosemicarbazones were cytotoxic
in a dose-dependent way against all studied tumor cell lineages
(Supporting Material). The concentrations that inhibit 50% of cell
survival (IC50) (Table 5) were found in the 52–0.16, 140–1.0, and
160–1.4 nM ranges for MCF-7, T98G and U87 cells, respectively.
Breast tumor cells were significantly more sensitive to the thio-
semicarbazones’ cytotoxic effect than glioma cells. However, there
was no significant difference between their cytotoxic effect against
U87 and T98G cells, suggesting that the mutation extent in the p53
gene of T98G cells did not interfere in the thiosemicarbazones’
cytotoxic effect.
The bond distances are very similar in all structures. The S1–C8
bond varies from 1.654(3) to 1.677(2) Å. This bond is longer in 1
than in its derivatives. The C2–C7 bond varies from 1.472(4) to
1.497(2) Å. The N2–C7, N2–N3, N4–C8 and N4–C9 bonds are in
the 1.278(2)–1.290(3) Å, 1.361(3)–1.378(3) Å, 1.328(4)–
1.3514(19) Å and 1.3973(19)–1.430(3) Å ranges, respectively.
Compound 11 has the shortest N4–C9 bond, which can be attrib-
uted to the resonance withdrawing effect of the ortho nitro group.
In general the bond distances in the thiosemicarbazone skeleton of
11 were shorter than in the other derivatives (see Table 2).
As expected, in all thiosemicarbazones the bond angles are also
similar, but some differences were observed for the N4–C8–N3,
N4–C8–S1, C7–N2–N3 and C8–N4–C9 bond angles in 11 in com-
parison with the other thiosemicarbazones (see Table 3). These dif-
ferences might be attributed to a geometric constraint due to the
formation of an additional N4Hꢁ ꢁ ꢁO2 intra-molecular hydrogen
bond observed in the structure of 11 (see Table 4) which is absent
in thiosemicarbazones 2–4, 8 and 10.
All thiosemicarbazones 2–4, 8, 10 and 11 adopt the EE confor-
mation in relation to the C7–N2 and N3–C8 bonds. The presence
of the E conformation with respect to the N3–C8 bond, commonly
observed in the solid state, is associated with the formation of a
pair of N–Hꢁ ꢁ ꢁS hydrogen bonds that are interrelated across a cen-
ter of inversion generating dimmers.25 As can be seen in Table 4, all
the NHꢁ ꢁ ꢁS interactions are linked to a symmetry operation. A weak
N4–Hꢁ ꢁ ꢁN2 intra-molecular hydrogen bond (see Table 4) is present
in all structures, except in 8. This hydrogen bond probably hinders
rotation around the N3–C8 bond and might contribute to the sta-
bility of the EE conformation. Fig. 3 shows the packing of 3 and
11, with the inter-molecular N–Hꢁ ꢁ ꢁS hydrogen bonds.
Although in general the presence of substituents on the N4-phe-
nyl group does not lead to more cytotoxic compounds, in some
cases the substituted derivatives were as or more potent than 1.
Thus, in these cases the presence of different substituents was an
interesting strategy to improve the cytotoxicity of 1. Generally,
the ortho substituted thiosemicarbazones were more active against
the three cell lines than their meta and para position isomers.
All thiosemicarbazones presented higher cytotoxic activity than
the topoisomerase II inhibitor etoposide for which the IC50 values
were 480, 460 and 620 nM against MCF-7, T98G and U87 cells,
respectively. Moreover, the thiosemicarbazones’ antitumoral doses
resulted in no significant toxicity to red blood cells (IC50 >10ꢀ5 M),
indicating a good therapeutic index.
2.5. SAR studies
SAR studies were carried out to identify the stereo and electronic
properties determined by theoretical calculations that may be in-
volved in the mechanism of action of 1–13. Properties of interest
in this study were molecular surface area, theoretical octanol–water

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M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
Table 2
Selected bond distances (Å) for 1 and its derivatives
Atoms
130
525,a
625
725
2
3
4
8
10
11
S1–C8
C2–C7
N2–C7
N2–N3
N3–C8
N4–C8
N4–C9
C–Rb
1.677(2)
1.486(2)
1.284(3)
1.376(2)
1.358(3)
1.346(3)
1.421(3)
—
1.654(3)
1.474(4)
1.281(3)
1.362(3)
1.359(3)
1.328(4)
1.413(3)
1.679(11)
1.800(14)
1.654(3)
1.472(4)
1.278(3)
1.361(3)
1.351(3)
1.336(3)
1.421(3)
1.724(3)
1.669(3)
1.487(4)
1.290(3)
1.378(3)
1.354(3)
1.349(3)
1.430(3)
1.741(3)
1.671(2)
1.484(2)
1.283(2)
1.374(2)
1.352(2)
1.341(2)
1.423(2)
1.7411(19)
1.6711(17)
1.488(2)
1.283(2)
1.3783(19)
1.346(2)
1.343(2)
1.412(3)
1.347(3)
1.671(2)
1.487(3)
1.287(3)
1.371(2)
1.359(3)
1.340(3)
1.421(3)
1.352(3)
1.672(2)
1.487(2)
1.280(2)
1.377(2)
1.349(2)
1.342(2)
1.428(2)
1.362(2)
1.665(3)
1.473(5)
1.285(4)
1.368(4)
1.361(4)
1.336(4)
1.419(4)
2.088(3)
1.670(3)
1.495(4)
1.285(4)
1.376(3)
1.352(4)
1.347(4)
1.417(4)
2.087(3)
1.6613(15)
1.497(2)
1.278(2)
1.3681(18)
1.361(2)
1.3514(19)
1.3973(19)
1.456(2)
a
There are two molecules in the asymmetric unit.
R = F, Cl, I or NO2.
b
Table 3
Selected angles for 1 and its derivatives
Atoms
130
525,a
625
725
2
3
4
8
10
11
N2N3C8
N3C8S1
N4C8N3
N4C8S1
C7N2N3
C8N4C9
118.9(2)
119.7(1)
114.8(2)
125.5(2)
118.8(2)
127.6(2)
118.2(2)
121.0(2)
114.7(2)
124.3(2)
119.1(2)
125.1(3)
118.9(2)
121.5(2)
114.4(2)
124.1(2)
118.7(2)
123.5(2)
119.0(2)
121.2(2)
114.4(2)
124.3(2)
118.7(2)
125.4(2)
118.66(15)
119.99(15)
114.71(17)
125.26(14)
118.86(15)
126.87(17)
118.14(14)
121.12(13)
114.99(15)
123.88(14)
118.79(14)
125.95(15)
118.67(17)
119.93(15)
114.43(17)
125.60(15)
118.37(18)
127.40(17)
119.18(14)
120.19(12)
114.58(15)
125.22(13)
118.91(13)
127.07(14)
119.1(3)
120.7(2)
114.9(3)
124.5(2)
118.9(3)
125.2(3)
118.7(2)
120.2(2)
114.9(2)
124.9(2)
118.1(2)
126.5(2)
118.23(13)
120.70(11)
112.28(13)
126.99(13)
121.28(14)
129.92(13)
a
There are two molecules in the asymmetric unit.
Table 4
Hydrogen bonds [Å and °] for 2–4, 8, 10 and 11
Compound
D—Hꢁ ꢁ ꢁA
d(D—H)
d(Hꢁ ꢁ ꢁA)
d(Dꢁ ꢁ ꢁA)
(D—Hꢁ ꢁ ꢁA)
Symmetry operation
2
N4—H4ꢁ ꢁ ꢁN2
N3—H3ꢁ ꢁ ꢁS1
N4—H4ꢁ ꢁ ꢁN2
N3—H3ꢁ ꢁ ꢁS1
N4—H4ꢁ ꢁ ꢁN2
N3—H3ꢁ ꢁ ꢁS1
N3—H3Aꢁ ꢁ ꢁS1
N4—H4ꢁ ꢁ ꢁN2
N3—H3Aꢁ ꢁ ꢁS1
N4—H4ꢁ ꢁ ꢁN2
N3—H3Aꢁ ꢁ ꢁS1
N4—H4ꢁ ꢁ ꢁO1
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
0.86
2.16
2.72
2.16
2.87
2.16
2.87
2.78
2.17
2.80
2.05
2.79
2.06
2.568(2)
3.5798(16)
2.580(2)
3.7215(18)
2.583(2)
3.7289(15)
3.635(3)
2.586(3)
3.652(3)
2.5208(19)
3.6383(14)
2.6137(17)
108.6
174.4
109.5
172.8
110.1
174.5
171.0
109.1
173.8
113.4
167.9
121.8
—
ꢀx+2,ꢀy+1,ꢀz+1
3
4
—
ꢀx,-y+1,ꢀz+2
—
ꢀx,-y+1,ꢀz+1
8
10
ꢀx+1,ꢀy+1,ꢀz
—
ꢀx+1,ꢀy+2,ꢀz+1
11
—
ꢀx+1,ꢀy+2,ꢀz
—
Figure 3. Molecular packing of 3 (left) and 11 (right), showing the N–Hꢁ ꢁ ꢁS hydrogen bond (dashed lines).

M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
3401
Table 5
Cytotoxic effect of 1–13 and etoposide on malignant cells and their haemolytic activity
Compound
IC50 (nM)a
T98G
Haemolytic activity (nM)b
MCF-7
U87
1
2
3
4
5
6
7
8
0.18 0.06
0.39 0.20
9.00 2.00
1.20 0.73
0.16 0.14
24.00 1.50
8.50 0.40
4.10 1.90
45.00 37.00
6.60 3.20
5.70 0.19
38.00 32.00
52.00 21.00
480.00 70.00
6.80 0.83
3.10 2.20
12.00 5.40
17.00 1.90
1.10 0.96
1.00 0.40
5.90 0.76
8.10 4.20
61.00 25.00
64.00 36.00
6.60 3.30
140.00 110.00
140.00 11.00
460.00 25.00
1.40 0.39
5.00 0.47
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
18.00 1.70
10.00 10.00
2.60 2.10
31.00 9.60
1.70 0.82
6.10 2.10
9
10
11
12
13
Etoposide
84.00 10.00
59.00 37.00
4.30 0.82
69.00 7.10
160.00 56.00
620.00 150.00
a
IC50 determined by the MTT assay.
IC50 determined by direct haemolysis assay. Data were expressed as average standard deviation, n = 4.
b
partition coefficients (logP), dipole moment, highest occupied
molecular orbital (HOMO) and lowest unoccupied molecular orbital
(LUMO) energies, which were correlated to pIC50 (ꢀlogIC50). Molec-
ular surface area may offer information on stereo features for drug–
receptor interactions. LogP and dipole values may give some
insights on the degree of lipophilicity of these molecules. Addition-
ally, HOMO and LUMO energies are related to ionization potential
and electron affinity, respectively. These frontier orbitals are associ-
ated to the molecule’s reactivity. HOMO energy is closely related to
susceptibility to electrophilic attack while LUMO energy is closely
related to susceptibility to nucleophilic attack.
built simple correlation matrices (Supporting Material) for the E
and Z isomers.
SAR data for the E isomers indicate that there are similar corre-
lations between the chemical descriptors and cytotoxic activity
against MCF-7 and U87 cells. Different correlations were found be-
tween descriptors and cytotoxicity against T98G and U87 cells.
Although U87 and T98G are both glioma cells, the former is
wild-type while the latter is a p53-mutant cell. Thus, the mecha-
nisms of thiosemicarbazones’ cytotoxic action may be different in
these two cell lines. Reasonable correlations were observed be-
tween the cytotoxic activities against MCF-7 and U87 cells and
the molecular surface area (R = ꢀ0.71 and ꢀ0.60 for MCF-7 and
U87 cells, respectively), the HOMO energy (R = 0.69 and 0.67 for
MCF-7 and U87 cells, respectively) and the charge on sulfur
(R = 0.71 and 0.68 for MCF-7 and U87 cells, respectively). Thus,
Calculated stereo and electronic properties for 1–13 and exper-
imental pIC50 values against MCF-7, T98G and U87 cells are
shown in Table 6. Attempting to establish some correlation be-
tween stereo-electronic parameters and biological activity, we
Table 6
Experimental pIC50 (ꢀlog IC50) values for 1–13 against MCF-7, T98G and U87 cells and calculated stereo and electronic properties
Compound pIC50 MCF-7 pIC50 T98G pIC50 U87 Surface area (Å) HOMO (eV) LUMO (eV) LogP (ALOGPs) Dipole (D)
Charge (e. u.)a
Nazo
Npy
S
E Isomers
1
2
3
4
5
6
7
8
9.745
9.409
8.046
8.921
9.796
7.620
8.071
8.387
7.347
8.180
8.244
7.420
7.284
9.745
9.409
8.046
8.921
9.796
7.620
8.071
8.387
7.347
8.180
8.244
7.420
7.284
8.167 8.854 365.315
8.509 8.301 371.197
7.921 7.745 372.401
7.770 8.000 372.370
8.959 8.585 380.124
9.000 7.509 382.152
8.229 8.770 382.175
8.092 8.215 390.191
7.215 7.076 391.944
7.194 7.229 391.979
8.180 8.367 402.386
6.854 7.161 406.617
6.854 6.796 406.613
8.167 8.854 364.729
8.509 8.301 370.528
7.921 7.745 371.803
7.770 8.000 371.781
8.959 8.585 379.517
9.000 7.509 381.567
8.229 8.770 381.574
8.092 8.215 389.019
7.215 7.076 391.345
7.194 7.229 391.425
8.180 8.367 403.070
6.854 7.161 406.006
6.854 6.796 405.998
ꢀ8.171
ꢀ8.307
ꢀ8.400
ꢀ8.283
ꢀ8.346
ꢀ8.416
ꢀ8.304
ꢀ8.332
ꢀ8.407
ꢀ8.220
ꢀ8.370
ꢀ8.602
ꢀ8.688
ꢀ7.916
ꢀ8.063
ꢀ8.152
ꢀ8.030
ꢀ8.107
ꢀ8.169
ꢀ8.060
ꢀ8.119
ꢀ8.160
ꢀ7.998
ꢀ8.258
ꢀ8.390
ꢀ8.473
1.690
1.650
1.520
1.536
1.621
1.509
1.492
1.612
1.510
1.483
0.840
1.225
1.096
1.466
1.469
1.371
1.395
1.448
1.359
1.352
1.437
1.358
1.343
1.078
1.228
1.077
3.100
3.220
3.240
3.230
3.670
3.650
3.650
3.780
3.790
3.800
3.170
3.170
3.180
3.100
3.220
3.240
3.230
3.670
3.650
3.650
3.780
3.790
3.800
3.170
3.170
3.180
5.281
4.211
4.496
5.795
3.877
4.374
6.042
4.437
4.651
5.788
1.826
4.174
7.978
6.968
6.024
7.161
8.186
5.864
7.277
8.704
6.278
7.231
8.103
4.617
8.250
11.486
ꢀ0.484 ꢀ0.296 ꢀ0.200
ꢀ0.485 ꢀ0.296 ꢀ0.198
ꢀ0.484 ꢀ0.297 ꢀ0.193
ꢀ0.483 ꢀ0.297 ꢀ0.201
ꢀ0.485 ꢀ0.293 ꢀ0.196
ꢀ0.484 ꢀ0.298 ꢀ0.192
ꢀ0.483 ꢀ0.298 ꢀ0.195
ꢀ0.485 ꢀ0.290 ꢀ0.196
ꢀ0.484 ꢀ0.297 ꢀ0.193
ꢀ0.483 ꢀ0.297 ꢀ0.195
ꢀ0.489 ꢀ0.287 ꢀ0.192
ꢀ0.484 ꢀ0.300 ꢀ0.188
ꢀ0.483 ꢀ0.299 ꢀ0.178
ꢀ0.503 ꢀ0.290 ꢀ0.201
ꢀ0.503 ꢀ0.288 ꢀ0.201
ꢀ0.503 ꢀ0.291 ꢀ0.194
ꢀ0.503 ꢀ0.291 ꢀ0.203
ꢀ0.503 ꢀ0.286 ꢀ0.196
ꢀ0.503 ꢀ0.292 ꢀ0.194
ꢀ0.503 ꢀ0.292 ꢀ0.197
ꢀ0.503 ꢀ0.283 ꢀ0.194
ꢀ0.503 ꢀ0.291 ꢀ0.195
ꢀ0.503 ꢀ0.291 ꢀ0.196
ꢀ0.504 ꢀ0.279 ꢀ0.190
ꢀ0.504 ꢀ0.293 ꢀ0.190
ꢀ0.504 ꢀ0.292 ꢀ0.179
9
10
11
12
13
1
2
3
4
5
6
7
8
9
10
11
12
13
Z Isomers
a
e. u.: electrostatic unit.

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M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
the smaller the molecular surface area, the higher the cytotoxic
activity against MCF-7 and U87 cells. A direct correlation was
found between the HOMO energies and the activities against
MCF-7 and U87 cells, which is an interesting result since the
HOMO energy is closely related to the molecules’ reactivity. Be-
sides, the direct correlation between cytotoxicity and the charge
on sulfur indicates that the more negatively charged the sulfur
atom, the higher anti-proliferative effect of the thiosemicarbazones
against MCF-7 and U87 cells. It’s noteworthy that HOMO energy
and negative charge on sulfur are correlated (R = 0.90) and both
properties are correlated to the activities against MCF-7 and U87
cells. Thus, the sulfur atom may play an important role in the thio-
semicarbazones’ reactivity and therefore, in their cytotoxic
activity.
stained with 40,6-diamidine-2-phenylindole dihydrochloride
(DAPI) (Fig. 6). These results suggest that the reduction of cell sur-
vival after treatment with thiosemicarbazones occurred, at least in
part, due to apoptosis induction.
Although DAPI is a commonly used assay for detection of apop-
tosis, this method cannot rule out other kinds of cell death and it is
very important to analyze cell death using other fluorescent DNA
binding dyes as acridine orange/ethidium bromide.31
Figures. 5 and 6 show, as examples, breast and glioma cells trea-
ted with compound 7.
2.7. Acridine orange–ethidium bromide (AO/EB) double staining
assay
Comparison of HOMO density plots for all E isomers (Fig. 4)
shows distinct electronic delocalization among the studied thio-
semicarbazones, which may partially account for the difference
in biological activity of these compounds, although other parame-
ters may not be excluded.
Concerning correlation between the properties of E isomers and
activity against T98G cells, we only found an inverse correlation
between cytotoxicity and molecular surface area (R = ꢀ0.60).
The same correlations observed for E isomers were also verified
for the Z isomers. In addition, for the Z isomers the cytotoxic activ-
ity against T98G cells is correlated to the LUMO energy (R = 0.63)
and the dipole (R = 0.62).
Acridine orange/ethidium bromide (AO/EB) staining can be used
to differentiate live, apoptotic and necrotic cells. Moreover, acri-
dine staining is very useful to show cytoplasmic acidic vacuoles
characteristics of autophagy. Under AO/EB staining untreated con-
trol breast and glioma cells showed cytoplasm and nucleus with
homogeneous green with minimal orange fluorescence indicative
of healthy cells. On the other hand, cells treated with the thiosemi-
carbazones presented chromatin condensation, visible as bright
green fragments, and absence of EB fluorescence, indicating pre-
served membrane. All of these characteristics are typical of early
apoptosis. Moreover, treated cells presented large acidic compart-
ments in the cytoplasm, and visible red fluorescence suggesting
the presence of autophagolysosomes, considered a characteristic
feature of cells engaged in autophagy.32 However, more specific
tests, like the quantification of mammalian autophagy protein
(LC3),33 are necessary to confirm this hypothesis. Further studies
should be performed in order to determine the exact mechanism
of cell death triggered by these compounds.
2.6. Morphological analysis of tumor cells
Treatment with compounds 1–13 induced visible membrane
and nuclear alterations characteristics of programmed cell death
on breast and glioma cells. The main morphological changes ob-
served were: irregularities in cellular shape, cell shrinkage and
membrane blebbing (Fig. 5). Moreover, chromatin condensation
and DNA fragmentation were observed in all treated cells when
Chemotherapeutics like cisplatin and etoposide can induce
apoptosis and autophagy in tumor cells.34,35 Taken together, our
results suggest that, like cisplatin and etoposide, the studied
Figure 4. HOMO density distributions of: 1 (A), 2 (B), 3 (C), 4 (D), 5 (E), 6 (F), 7 (G), 8 (H), 9 (I), 10 (J), 11 (K), 12 (L) and 13 (M). Contour value = 0.018.

M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
3403
Figure 5. Compound 7 induces morphological changes on MCF-7 breast and T98G and U87 glioma tumor cell lines. Tumor cells were treated with 7 (1
l
M) for 48 h. Cell
shrinkage and irregularity in cellular shape were seen in cells treated with 7. Photomicrographs from phase-contrast microscopy. Control cells are represented above and
treated cells bellow. Magnification: 400ꢂ.
Figure 6. Compound 7 induces nuclear changes on MCF-7 breast and T98G and U87 glioma tumor cell lines. Tumor cells were treated with 7 (1 lM) for 48 h. After treatment
cells were stained with DAPI. Nuclear condensation and formation of apoptotic bodies were seen in cells treated with 7. Photomicrographs from fluorescence microscopy.
Control cells are represented above and treated cells bellow. Magnification: 400ꢂ.
thiosemicarbazones were able to induce these two types of pro-
grammed cell death. Figure 7 shows, as example, breast and glioma
cells treated with compound 7 and stained with acridine orange/
ethidium bromide.
even at concentrations close to the limit of solubility of the
compound.
2.9. High content analysis of mitotic arrest
2.8. Effects on tubulin assembly
Microtubule interacting agents characteristically provoke cell
cycle arrest and cellular microtubule perturbations.37 The fact that
7 apparently inhibited in vitro polymerization of purified tubulin
motivated the investigation on the effects of this compound on
cellular microtubule perturbation and mitotic arrest. The cellular
phenotypic changes associated with mitotic arrest caused by 7
were investigated as previously described.37,38 Asynchronously
growing HeLa cells were treated with the assayed compounds
and incubated with primary antibodies for tubulin and the mitotic
marker protein phosphohistone H3, followed by fluorescein-
isothiocyanate (FITC) and Cy3-conjugated secondary antibodies,
respectively. Cells were detected by nuclear counterstaining with
Hoechst 33342, which also provided information about chromatin
condensation and cell density as markers of cell death.
Interference with either assembly or disassembly of microtu-
bules in rapidly dividing cells results in cell cycle arrest, triggering
signals that induce apoptosis.36 In light of the finding that cyto-
toxic thiosemicarbazones induce apoptosis, we investigated
whether the mechanisms of either cytotoxicity or apoptosis were
related to inhibition of tubulin assembly or microtubule stabiliza-
tion. Four twofold concentrations of 7 were screened for their
effects on in vitro tubulin polymerization (Fig. 8). As it can be seen,
at high concentrations 7 seemed to cause a partial concentration-
dependent inhibition of tubulin assembly. Although 2.5 lM of
colchicine completely abolished tubulin polymerization, total inhi-
bition of tubulin assembly in the presence of 7 was not observed

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M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
Figure 7. Detection of thiosemicarbazone-induced acidic autophagossomes and apoptosis. MCF-7 breast and T98G and U87 glioma tumor cell lines were treated with 7
(1 M) for 48 h. After treatment cells were stained with acridine orange/ethidium bromide. Photomicrographs from fluorescence microscopy. Control cells are represented
l
above and treated cells bellow. Magnification: 400ꢂ.
Fluorescence micrographs of nuclei (blue), tubulin (green) and
phosphohistone H3 (red) staining demonstrated that vehicle-trea-
ted cells have highly organized microtubules and a low percentage
of mitotic cells, while nanomolar concentrations of colchicine in-
duced cellular microtubule disorganization and mitotic arrest (
Fig. 9A and B). Cells treated with low micromolar cytotoxic concen-
trations of 7 had a phenotype of generally disorganized microtu-
bules with seemingly lower overall tubulin content. Moreover,
the general cellular structure was lost (Fig. 9C). Hoechst 33342
staining revealed the presence of condensed and fragmented nuclei
characteristic of apoptosis (Fig. 9B and C). However, in contrast to
colchicine, which caused an increased number of phosphohistone
H3-positive cells, the same phenotype was not observed with 7,
suggesting that 7 did not arrest cells in mitosis.
The multi-parametric mitotic arrest assay also showed that both
colchicine and 7 caused cell loss, either through detachment or ly-
sis. Although we had previously demonstrated that 7 inhibited cell
growth of different cancer cell lines at nanomolar doses, higher
doses (in the micromolar range) were required to inhibit attach-
ment and survival of HeLa cells (Table 7).25 The EC50 values were
obtained from the dose-response cell density curves. The form of
the chromatin condensation, microtubule density and mitotic index
graphs precluded IC50 determinations. Accordingly, we determined
Figure 8. In vitro inhibition of tubulin assembly. Electrophoretically homogeneous
bovine brain tubulin (final concentration 10 l
M; 1 mg mLꢀ1) was pre-incubated
with the studied compounds dissolved in DMSO (1% v/v final concentration) and
monosodium glutamate (0.8 M final concentration) at 30 °C in 96-well plates. The
reaction mixtures were cooled to 0 °C for 10 min and guanosine-50-triphosphate
(GTP, 0.4 mM final concentration) was added. The absorbance at 350 nm was
immediately followed in a spectrophotometer. Baselines were established and
temperature was quickly raised to 37 °C. At high concentrations compound 7
partially inhibits tubulin polymerization in an apparent concentration-dependent
manner.
Figure 9. Immunofluorescence staining for markers of mitotic arrest in HeLa cells. HeLa cells were treated with (a) vehicle (DMSO 0.1%), (b) colchicine (62 nM) or (c) 7
(12.5 M), followed by simultaneous immunostaining of -tubulin (green), phosphohistone H3 (red), and Hoechst 33342 (blue). Vehicle-treated cells have highly organized
l
a
microtubules and a low percentage of mitotic cells. Colchicine and 7 caused a heterogeneous response of tubulin disorganization, chromatin condensation, and nuclear
fragmentation. Typical apoptotic morphology is observed for 7. Images are representative fields from a single experiment repeated three times with similar results.
Magnification: 200ꢂ.

M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
3405
Table 7
Results of high-content analysis of mitotic arrest in HeLa cellsa
Compound
EC50
(lM) cell density
MDEC (l
M)b
Nuclear condensation
Tubulin intensity
Mitotic índexc
Colchicine
7
0.03
9.2 0.8
0.01
5.8 2.0
0.01
3.6 0.1
0.02
> 50
a
b
c
Average SD of four independent experiments, performed in quadruplicate.
Minimum detectable effective concentration.
Percentage of phosphohistone H3-positive cells.
Figure 10. High-content analysis of mitotic arrest profiles in cells treated with the compounds being investigated. Cells were treated in 384-well plates with 10 twofold
dilutions of either colchicine and 7 and analyzed by high-content analysis for (A) cell density, (B) chromatin condensation, (C) microtubule density and (D) mitotic index. All
agents caused cell loss, enhanced nuclear condensation and induced cellular microtubule perturbations. Colchicine increased mitotic index, but this effect was not observed
for 7.
the minimum detectable effective concentration (MDEC) as an
alternative measurement of 7 and colchicine activities.37
T98G glioma cells, with good therapeutic index, as shown by their
reduced haemolytic activity. In general, among the N4-phenyl
substituted derivatives, the ortho derivatives proved to be more
cytotoxic than their meta- and para congeners.
Our results suggest that the studied thiosemicarbazones were
able to induce programmed cell death in tumor cells. The re-
sults described thus far suggest that these compounds induce
both apoptosis and autophagy in the studied malignant tumor
cells.
Despite their abilities to partially inhibit tubulin assembly at
high concentrations and to provoke cellular microtubule disorgani-
zation, the thiosemicarbazones did not behave as mitotic arresters.
As a consequence, direct interaction with tubulin is probably not
primarily responsible for their cytotoxic and pro-apoptotic effects.
The high cytotoxic activity of the studied compounds, together
with their good therapeutic index, indicates that they are promis-
ing as antitumor drug candidates for the treatment of breast
tumors and malignant gliomas.
Cells treated with colchicine and 7 showed chromatin conden-
sation and microtubule alterations (Fig. 10B, C and Table 7). Colchi-
cine also provoked a concentration-dependent increase in the
percentage of cells with elevated phosphohistone H3 levels,
whereas 7 did not (Fig. 10D and Table 7). Therefore, despite its abil-
ity to partially inhibit tubulin assembly at high concentrations and
to provoke cellular microtubule disorganization, 7 does not appear
to cause mitotic arrest. As a consequence, direct interaction with
tubulin is probably not responsible for the cytotoxic and pro-apop-
totic effects of this compound.
3. Conclusion
Compound 1 and its N4-ortho -meta- and -para-phenyl substi-
tuted derivatives presented cytotoxicity in nanomolar doses
against malignant MCF-7 breast tumor cells and against U87 and

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M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
(ppm, E isomer (87%)) 177.01 (C8), 154.32 (C2), 148.39 (C6),
4. Material and methods
4.1. Chemicals
140.43 (C7), 136.28 (C4), 121.21 (C3), 149.54 (C9), 124.08 (C5),
12.55 (C15); HRMS m/z [MꢀH]ꢀ calcd for C14H12N4SI: 394.9833,
found: 394.9835.
2-Acetylpyridine, hydrazine dihydrate, o-fluorophenyl-, m-fluo-
rophenyl-, p-fluorophenyl-, o-chlorophenyl-, m-chlorophenyl-,
p-chlorophenyl-, o-iodophenyl-, m-iodophenyl-, p-iodophenyl-,
o-nitrophenyl-, m-nitrophenyl- and p-nitrophenyl-isothiocyanate
were purchased from Aldrich or Alfa Aesar and were used as re-
ceived. 1 and its fluoro (2–4), chloro (5–7) and nitro (11–13) deriv-
atives were prepared as previously described.29
4.3.3. 2-Acetylpyridine N4-para-iodo-phenyl thiosemicarbazone
(10)
Light yellow powder. Yield: 89%; Melting point: 205.0–206.1 °C;
IR (KBr, cmꢀ1): 3294, 3232 (
m(NH)), 1581 (m(C@N)), 783 (m(C@S)),
621 ((py)); 1H NMR (200 MHz, DMSO-d6): d (ppm, E isomer
(94%)) = 10.79 (s, 1H, N3-H), 10.19 (s, 1H, N4-H), 8.60 (d,
J = 4.39 Hz, 1H, H6), 8.52 (d, 1H, J = 8.04 Hz, H3), 7.82 (t, 1H,
J = 7.83 Hz, H4), 7.50-7.53 (m, 1H, H5), 2.47 (s, 3H, H15); d (ppm,
Z isomer (6%)) = 14.66 (s, 1H, N3-H), 10.33 (s, 1H, N4-H), 8.79 (d,
J = 4.12 Hz, 1H, H6), 8.10 (t, J = 7.79 Hz, 1H, H4); 13C NMR
(200 MHz, DMSO-d6): d (E isomer (94%)) 176.98 (C8), 154.34
(C2), 148.41 (C6), 149.51 (C7), 136.70 (C4), 121.19 (C3), 138.93
(C9), 124.07 (C5), 12.54 (C15); HRMS m/z [MꢀH]ꢀ calcd for
C14H12N4SI: 394.9833, found: 394.9837.
4.2. Physical measurements
Infrared spectra were recorded on a Perkin Elmer FT-IR Spec-
trum GX spectrometer using KBr plates (4000–400 cmꢀ1). NMR
spectra were obtained at room temperature with a Brucker DRX-
400 Avance (400 MHz) spectrometer using DMSO-d6 as the solvent
and tetramethylsilane (TMS) as internal reference. Melting Points
were determined on a Mettler FP 90 equipment. Compound’s puri-
ties were established by HPLC (HPLC Agilent Technologies 1200)
with an UV detector at 314 nm, using Supelcosil C18 column
4.4. Crystal structures determination
The crystal structures of 2–4, 8, 10 and 11 were determined by
using single-crystal X-ray diffractometry. The measurements were
carried out on an Oxford-Diffraction GEMINI-Ultra diffractometer
(5
l
m particle size, 25 cm ꢂ 4.6 mm) and eluting with methanol.
HPLC data indicated purity >98% for all compounds except for
compounds 6 and 7 (purities = 97 and 94%, respectively). High
resolution MS measurements were performed on a Shimadzu
LCMS-IT-TOF mass spectrometer.
using graphite-Enhance Source Mo K radiation (k = 0.71073 Å) at
a
293(2) K. The data collection, cell refinements, and data reduction
were performed using the CRYSALISPRO software.39 Semi-empiri-
cal from equivalents absorption correction method was applied.39
The structures were solved by direct methods using SHELXS-
97.40 Full-matrix least-squares refinement procedure on F2 with
anisotropic thermal parameters was carried on using SHELXL-
97.40 Positional and anisotropic atomic displacement parameters
were refined for all non-hydrogen atoms. Hydrogen atoms were
placed geometrically and the positional parameters were refined
using a riding model. Hydrogen atoms were placed geometrically
and the positional parameters were refined using a riding model.
Molecular graphics and packing figures were plotted using
ORTEP41 and PLATON,42 respectively.
4.3. Synthesis of 2-acetylpyridine N4-ortho-(8), N4-meta- (9) and
N4-para-iodophenyl (10) thiosemicarbazones
Hidrazine dihydrate (39 mmol, 30% of excess) was added to 2-
acetylpyridine (30 mmol) in methanol under stirring at room tem-
perature. After 24 h, the product 2-acetylpyridine hydrazone was
filtered off. The thiosemicarbazones were prepared by stirring 2-
acetylpyridine hydrazone with o-, m- or p-iodophenyl isothiocya-
nate in 1:1 molar ratio under reflux in ethanol for 6 h. The solids
were filtered off, washed with ethanol followed by diethyl ether,
and dried in vacuo.
4.5. Cell lines and culture conditions
4.3.1. 2-Acetylpyridine N4-ortho-iodo-phenyl
thiosemicarbazone (8)
Malignant human tumor cell lines MCF-7, U87 and T98G were
obtained from the American Type Culture Collection (ATCC, USA)
and maintained in Dulbecco´s Modified Eagle’s Medium (DMEM,
Gibco), supplemented with 10% foetal bovine serum (Cultilab)
White powder. Yield: 70%; Melting point: 173.0–173.9 °C; IR
(KBr,cmꢀ1): 3306, 3208 (
m
(NH)), 1582 (
m(C@N)), 782 (m(C@S)),
621 (
q
(py)); 1H NMR (200 MHz, DMSO-d6): d (ppm, E isomer
(95%)) = 10.90 (s, 1H, N3-H), 10.14 (s, 1H, N4-H), 8.61 (d,
J = 6.06 Hz, 1H, H6), 8.57 (d, 1H, J = 8.35 Hz, H3), 7.82 (t, 1H,
J = 7.99 Hz, H4), 7.55–7.35 (m, 1H, H5), 2.49 (s, 3H, H15); d (ppm,
Z isomer (5%)) = 14.66 (s, 1H, N3-H), 10.21 (s, 1H, N4-H), 8.81 (d,
J = 5.59 Hz, 1H, H6), 8.11 (t, J = 8.00 Hz, 1H, H4); 13C NMR
and antibiotics (50 U mLꢀ1 penicillin/50
lM streptomycin), in a
humidified atmosphere air/CO2 (5%/95%) at 37 °C. For all experi-
ments, cells were seeded in 96-well plates, at a density of 1–
2 ꢂ 103 cells/well.
(200 MHz, DMSO-d6):
d (ppm, E isomer (95%)) 177.43 (C8),
4.6. Cytotoxic activity
154.42 (C2), 148.39 (C6), 141.07 (C7), 136.33 (C4), 121.07 (C3),
148.87 (C9), 124.01 (C5), 12.44 (C15); HRMS m/z [MꢀH]ꢀ calcd
for C14H12N4SI: 394.9833, found: 394.9837.
Cytotoxicity was evaluated by the 3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide (MTT) assay which measures
the cellular metabolic viability.43 The cells were cultured in 96-well
plates and, 12 h after incubation, they were treated with different
concentrations of test compounds (1 ꢂ 10ꢀ12 Mꢀ1 ꢂ 10ꢀ5 M).
Another group of cells was treated with the same concentrations
of etoposide (positive control), an antineoplasic agent that inhibits
the enzyme topoisomerase II. Compounds were previously
dissolved in DMSO and the final concentrations were adjusted,
through an 8-fold serial dilution, in DMEM in such manner that
the final DMSO concentration was lower than 0.5%.
4.3.2. 2-Acetylpyridine N4-meta-iodo-phenyl
thiosemicarbazone (9)
Yellow powder. Yield: 94%; Melting point: 175.9–177.9 °C; IR
(KBr, cmꢀ1): 3264 (
m
(NH)), 1595 (
m
(C@N)), 786 (
m
(C@S)), 616
((py)); 1H NMR (200 MHz, DMSO-d6):
d
(ppm,
E
isomer
(87%)) = 10.83 (s, 1H, N3-H), 10.22 (s, 1H, N4-H), 8.62 (d,
J = 4.68 Hz, 1H, H6), 8.55 (d, 1H, J = 8.04 Hz, H3), 7.84 (t, 1H,
J = 7.64 Hz, H4), 7.42 (t, 1H, J = 5.32 Hz, H5), 2.49 (s, 3H, H15); d
(ppm, Z isomer (13%)) = 14.69 (s, 1H, N3-H), 10.37 (s, 1H, N4-H),
8.80 (d, J = 4.24 Hz, 1H, H6); 13C NMR (200 MHz, DMSO-d6): d
After 48 h-treatment the cells were incubated with MTT
(0.5 mg mLꢀ1), and formazan crystals were solubilised in DMSO.

M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
3407
Absorbance was measured in a microplate reader at 570 nm. Tests
incubated with serum-free medium, containing 1
orange (Sigma) and 1 l
l
g mLꢀ1 acridine
using DMSO (0.5% in DMEM) as negative control were carried out
in parallel. IC50 values were calculated as the concentration of com-
pound that induced 50% of cytotoxicity. Data were presented as
average standard deviation. All experiments were carried out in
quadruplicates and repeated in at least three independent experi-
ments with full agreement between the results. The statistical sig-
nificance was assessed using Student’s t-test (p <0.05).
g mLꢀ1 ethidium bromide (Sigma), for
15 min. The medium was removed and fluorescent micrographs
were obtained using an inverted fluorescence microscope (Nikon
– 530–650 nm). Viable cells presented uniform bright green nuclei;
early apoptotic cells (which still have intact membranes and did
not stain with ethidium bromide) have green nuclei, but chromatin
condensation is visible as bright green fragments; late apoptotic
cells have orange to red nuclei with condensed or fragmented
chromatin and necrotic cells have uniformly orange to red nuclei.
4.7. SAR studies
Conformational analysis of thiosemicarbazones was performed
using the Merck Molecular Force Field (MMFF)44 implemented in
the Tinker Molecular Modeling Program.45 For all compounds the
E and Z isomers were analyzed. The minimum energy structures
of the isomers obtained in the conformational analysis step were
further optimized at the Density Functional Theory46 level,
employing the hybrid B3LYP47,48 exchange-correlation functional
and using the cc-pVDZ49 all electron basis set50,51 for all atoms.
In order to have better properties and energetic results, single
point energy calculations at the second order Møller-Plesset per-
turbation theory level52,53 were then performed on the optimized
B3LYP/cc-pVDZ structures, using the same basis set (MP2/cc-
pVDZ//B3LYP/cc-pVDZ). The inner shell electrons of iodine were
treated by the effective core potential LANL2DZ54 and its associ-
ated double-f basis set was used for the valence electrons
(5s,5p). The charge distribution on the most stable conformers
was computed using the Natural Bonding Orbital (NBO) formal-
ism.55,56 All quantum mechanical calculations were performed
using the Gaussian program.57 HOMO and LUMO energies, dipole,
NBO charges of sulfur and nitrogen from pyridine (Npyr) and azo-
methine group (Nazo) were obtained after full optimization and
were used as descriptors for further SAR studies. Superficial molec-
ular area and logP were also used as descriptors for SAR studies.
The three-dimensional structures obtained from optimization of
thiosemicarbazones were used as input in the Marvin software58
to calculate the superficial molecular area. Calculations of logP
for the thiosemicarbazones were performed using ALOGPS 2.1 soft-
ware, accessed via the Virtual Computational Chemistry Labora-
tory interface.59
4.10. Direct haemolysis assay
The haemolytic activity of thiossemicarbazones was evaluated
according to Fisher et al.61 Human blood, collected in tubes con-
taining EDTA, was centrifuged at 700xg for 10 min. The pellet
was washed tree times with cold PBS pH 7.4 by centrifugation,
and re-suspended in the same buffer. Thiossemicarbazones’ solu-
tions of different concentrations were added to the erythrocytes
and were incubated for 1 h at 37 °C in a shaker water bath. The re-
lease of haemoglobin was determined after centrifugation by pho-
tometric analyses at 540 nm. Complete haemolysis was achieved
using 5% Triton X-100 yielding the 100% control value.
4.11. Inhibition of tubulin assembly
Since all studied thiosemicarbazones induced cellular pheno-
typic changes characteristics of apoptosis in a similar way, the
inhibition of tubulin assembly and microtubule stabilization assays
were only performed for H2Ac4pClPh (7), one of the most cytotoxic
compounds.
Electrophoretically homogeneous bovine brain tubulin (final
concentration 10 l
M; 1 mg mLꢀ1) was pre-incubated with test
agents (colchicine and compound 7) dissolved in DMSO (1% v/v fi-
nal concentration) and monosodium glutamate (0.8 M final con-
centration) at 30 °C in 96-well plates. The reaction mixtures were
cooled to 0 °C for 10 min and GTP (0.4 mM final concentration)
was added. The absorbance at 350 nm was immediately followed
in a spectrophotometer. Baselines were established and tempera-
ture was quickly raised to 37 °C. The turbidity value after 20 min
at 30 °C for DMSO 1% was assigned as 100% assembly, and for col-
4.8. Morphological analysis of tumor cells
chicine 2.5 lM as 0% assembly. To observe concentration-depen-
dent effects of the tested compounds, four twofold dilutions of
Cells were plated in 96-well plates and treated with test com-
them were evaluated.
pounds 1–13 (1 lM). Morphological changes were analyzed 48 h
after the treatment by contrast-phase microscopy (Nikon).
4.12. Automated high-content cellular analyses
DNA alterations were detected by DAPI staining. After 48 h-
treatment with 1
phosphate buffered saline (PBS) and fixed with 70% methanol at
l
M of test compounds, cells were washed with
The effects of colchicine and 7 on mitotic arrest, nuclear mor-
phology and cellular microtubules were studied as previously de-
scribed.37,38 HeLa human cervical carcinoma cells (8000/well)
were plated in collagen-coated 384-well microplates and treated
in quadruplicate with vehicle (DMSO 0.1% final concentration) or
10 twofold dilutions of test compounds within 4–6 h of seeding.
Cells were incubated for 18 h at 37 °C and CO2 5%, fixed with form-
room temperature for 30 min. Cells were incubated with
0.4 l
g mLꢀ1 DAPI (Sigma) for 1 h in the dark after washing with
PBS. DNA alterations such as condensation and fragmentation were
observed by fluorescence microscopy (Nikon – 385–410 nm).
4.9. Acridine orange-ethidium bromide (AO/EB) doubling
staining assay
aldehyde, and labeled with 10 l
g mLꢀ1 Hoechst 33342 in Hank’s
balanced salt solution (HBSS). Cells were permeabilized with 0.5%
(w/w) Triton-X100 for 5 min at room temperature and incubated
with a primary antibody cocktail consisting of an HBSS solution con-
taining rabbit polyclonal antiphosphohistone H3 (Ser10, 1:500, Up-
Acridine orange, a cationic dye, has been the subject of exten-
sive studies in recent years because of its metachromatic staining
properties and can be used in conjunction with ethidium bromide
to differentiate among alive, apoptotic, necrotic and autophagic
cells.60
Acridine orange/ethidium bromide doubling staining was per-
formed according to Baskic et al.34 with modifications. MCF-7,
U87 and T98G cells were seeded in 96-well plates and treated with
state, Charlottesville, VA, USA), and mouse monoclonal anti-a-
tubulin (1:3000, Sigma, St. Louis, MO, USA), followed by a mixture
of FITC-labeled donkey anti-mouse IgG (1:500) and Cy3-labeled
donkey anti-rabbit IgG (1:500) as secondary cocktail. Cells were
rinsed once with HBSS and stored at 4 °C in HBSS until analysis.
Microplates were analyzed with an ArrayScanII instrument (Cello-
mics, Pittsburgh, PA, USA) using the Target Activation Bioapplication
test compounds 1–13 (1 lM) for 48 h. After treatment, cells were

3408
M. A. Soares et al. / Bioorg. Med. Chem. 20 (2012) 3396–3409
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This work was supported by CNPq (Conselho Nacional de
Desenvolvimento Científico e Tecnológico), CNEN (Comissão Nac-
ional de Energia Nuclear), FAPEMIG (Fundação de Amparo à Pesqu-
isa do Estado de Minas Gerais), INCT-INOFAR (Instituto Nacional de
Ciência e Tecnologia de Fármacos e Medicamentos, Proc. CNPq
573.364/2008-6), INCT-MM (Instituto Nacional de Ciência e Tecno-
logia de Medicina Molecular, Proc. FAPEMIG: CBB-APQ-00075-09/
CNPq 573646/2008-2), INCT-Catálise (Instituto Nacional de Ciência
e Tecnologia de Catálise em Sistemas Moleculares e Nanoestrutur-
ados) and FAPESP (Fundação de Amparo à Pesquisa do Estado de
São Paulo). The authors express sincere thanks to LabCRI (UFMG),
particularly to Professor Nilvado L. Speziali for the X-ray facilities
and measurements.
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the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.027.
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