Thiosemicarbazone-based selective proliferation inactivators inhibit gastric cancer cell growth, invasion, and migration

Article abstract of DOI:10.1039/c7md00353f

A series of novel thiosemicarbazone derivatives were synthesized and evaluated for their antiproliferative activity against several selected tumor cell lines of different origins using the MTT assay. The preliminary results indicated that the MGC-803 cell line was remarkably sensitive to all the synthesized compounds. Among this series, compound 5n showed the best inhibitory activity with an IC₅₀ value of 0.93 μM (about 10-fold more potent than 3-AP) against MGC-803. Further mechanism studies revealed that compound 5n could obviously inhibit the proliferation of MGC-803 cells by inducing apoptosis and arresting the cell cycle at the S phase. Compound 5n also showed marked inhibition of cell migration and invasion, without significant cytotoxicity against gastric epithelial immortalized GES-1 cells.

Full text of DOI:10.1039/c7md00353f

MedChemComm
View Article Online
View Journal
RESEARCH ARTICLE
Thiosemicarbazone-based selective proliferation
inactivators inhibit gastric cancer cell growth,
invasion, and migration†
Cite this: DOI: 10.1039/c7md00353f
Biao Hu,‡ Bo Wang,‡ Bing Zhao,‡ Qian Guo, Zhong-Hua Li, Xin-Hui Zhang,
Guang-Yao Liu, Ying Liu, Ying Tang, Fan Luo, Ya Du, Ya-Xin Chen,
*
*
Li-Ying Ma
and Hong-Min Liu
A series of novel thiosemicarbazone derivatives were synthesized and evaluated for their antiproliferative
activity against several selected tumor cell lines of different origins using the MTT assay. The preliminary re-
sults indicated that the MGC-803 cell line was remarkably sensitive to all the synthesized compounds.
Among this series, compound 5n showed the best inhibitory activity with an IC50 value of 0.93 μM (about
10-fold more potent than 3-AP) against MGC-803. Further mechanism studies revealed that compound 5n
could obviously inhibit the proliferation of MGC-803 cells by inducing apoptosis and arresting the cell cycle
at the S phase. Compound 5n also showed marked inhibition of cell migration and invasion, without signifi-
cant cytotoxicity against gastric epithelial immortalized GES-1 cells.
Received 14th July 2017,
Accepted 19th September 2017
DOI: 10.1039/c7md00353f
rsc.li/medchemcomm
novel thiosemicarbazone-based ligands with antiproliferative
Introduction
activity have been identified (Fig. 1), including 3-AP,15
Cancer, being one of the leading causes of death globally,
poses a major socioeconomic hazard to humanity at large. Al-
though there has been progress in the development of preven-
tion and treatment of cancer, successful treatment of cancer
remains a challenge. There is still an urgent need to search
for some newer and safer anticancer agents that have a
broader spectrum of cytotoxicity to cancer cells.
As reported, neoplastic cells have high requirements for
metal due to their generally higher rates of proliferation than
normal cells. Considering the crucial roles of these metals,
development of novel metal chelators has become a promis-
ing anticancer strategy.1 Many in vitro and in vivo studies and
clinical trials have demonstrated that metal (Fe, Cu, and Zn)
chelators are effective antiproliferative agents.2
Multi-functionalized thiosemicarbazones and their metal
complexes have been a focus of chemists and biologists be-
cause of their wide range of therapeutic uses with antiviral,
antiparasitic, antibacterial, anticonvulsant, and, most intrigu-
ingly, anticancer activity.3–14 Encouraged by the modest
results obtained with traditional metal chelators, a variety of
Dp44mT,16,17 Ap44mT,18–21 and DpC.22–24 In fact, 3-AP has
been investigated in over 20 phase I and phase II clinical tri-
als in a variety of advanced cancers.25–27 Unfortunately, 3-AP
has suffered multiple problems, including low efficacy in
some tumor-types and serious side effects, which limit its
clinical utility.28–30 Therefore, highly selective chelators with
strong toxicity toward cancer cells and less or no side effects
on normal cells remain to be identified.
A key feature of progressive tumors is having the potential
to invade neighboring tissue and to spread throughout the
body and form metastatic lesions at distant sites.31 In addi-
tion, there is increasing evidence that epithelial–mesenchy-
mal transition (EMT) is a key step for cancer cell migration,
invasion, and metastasis.32 During the process of EMT, cells
lose their epithelial characteristics such as epithelial mor-
phology, cell polarity, and cell–cell contact, and gain mesen-
chymal properties such as fibroblastic morphology and
increased migration and invasion, causing cancer cell
metastasis.33–36
We have previously reported a novel series of pyrimidine-
based hybrids with potent anticancer activity. One of the
compounds, 6-LM (Fig. 1), a thiosemicarbazone-containing
pyrimidine hybrid, showed marked inhibition of cell migra-
tion and invasion in vitro and in vivo.37 To explore the mecha-
nism of compound 6-LM regulated EMT more thoroughly, we
designed a series of small molecules and evaluated their
antitumor activity using thiosemicarbazone as a minimal
scaffold.
Collaborative Innovation Center of New Drug Research and Safety Evaluation,
Key Laboratory of Technology of Drug Preparation, Ministry of Education of
China, School of Pharmaceutical Sciences, Zhengzhou University, Zhengzhou
450001, Henan Province, PR China. E-mail: yingdejiqing1314@163.com,
liuhm@zzu.edu.cn
† Electronic supplementary information (ESI) available. See DOI: 10.1039/
c7md00353f
‡ These authors contributed equally to this work.
This journal is © The Royal Society of Chemistry 2017
Med. Chem. Commun.

View Article Online
Research Article
MedChemComm
Fig. 1 Representative examples of thiosemicarbazone derivatives.
Results and discussion
Chemistry
from the substitution with a methyl group in position R2:
compound 5p was found to exhibit a single-digit micromolar
inhibitory activity (IC50 = 3.06 μM). The same outcome was
observed for 5k (IC50 = 11.17 μM), an analog of compound 5a
(IC50 = 29.18 μM). Removing the hydroxyl group resulted in
decreased potency (5k vs. 5j), suggesting that the hydroxyl
group is a net contributor to the in vitro potency of the mole-
cule. In addition, it could be noticed that the 2-OH (5k) on
the phenyl group (R3) presented a more potent inhibitory ef-
fect than the 3-OH (5l) and 4-OH (5m) substitution. Also, the
substitution of the phenyl ring with a chloro group in posi-
tion 5 increased the potency (5p vs. 5k, 5b vs. 5a). These re-
sults revealed that the position of the substituent on the phe-
nyl group (R3) has a remarkable effect on the inhibitory
activity. In particular, compound 5n showed the best inhibi-
tory effect against MGC-803 with an IC50 value of 0.93 μM
(10-fold more potent than 3-AP).
Because compound 5n exhibits potent proliferative inhibi-
tory activity in vitro, we further characterized the selectivity of
this compound against GES-1 (normal human gastric epithe-
lial cell line). As can be seen in Table 2, we found that com-
pound 5n did not show a significant inhibitory effect on GES-
1, compared to the positive control, 3-AP. Our findings indi-
cate that compound 5n has good selectivity between cancer
and normal cells.
The general route for the synthesis of the target thio-
semicarbazones is depicted in Scheme 1. Commercially avail-
able 1a–c were reacted with CS2 and hydrazine hydrate in the
presence of triethylamine to form compounds 4a–c, which
were subjected to reaction with an appropriately substituted
aldehyde to afford the target compounds 5a–q with good
yields.
Evaluation of biological activity
Anti-proliferative activity. All the compounds synthesized
in this study were examined for their anti-proliferative activity
against three human cancer cell lines (commercially available
from Cell Bank, Shanghai Institutes for Biological Sciences,
Shanghai, China) such as MGC-803 (human gastric cancer
cell line), SMMC-7721 (human liver cancer cell line), and
MCF-7 (human breast cancer cell line). 3-AP was chosen as a
positive control. The results of preliminary evaluation are
summarized in Table 1.
During the SAR studies, we found that all synthesized
compounds showed a more potent inhibitory activity against
MGC-803 than SMMC-7721 and MCF-7. This observation sug-
gests that these compounds may act through targeting spe-
cific biological targets that are overexpressed in certain cells.
In addition, a slight difference in inhibition of growth of
MGC-803 cells was observed for compounds with different
substitution patterns. A significant boost in potency came
Among those compounds, we chose compound 5n,
exhibiting the most potent activity against MGC-803 with an
IC50 value of 0.93 0.02 μmol L−1, to further investigate its
inhibitory effect on MGC-803. As shown in Fig. 2A, the
Scheme 1 Reagents and conditions: a: (i) methanol, triethylamine, rt, 1 h; (ii) hydrazine hydrate 80%, reflux, 3 h; b: methanol, appropriate
aldehyde, reflux, 4 h.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2017

View Article Online
MedChemComm
Research Article
Table 1 Inhibitory results of the novel thiosemicarbazone derivatives against three cancer cell lines
IC50a (μM)
Comp
R1
R2
H
R3
MGC-803
SMMC-7721
37.28 1.57
MCF-7
5a
29.18 1.46
>64
5b
5c
H
H
16.31 1.21
20.38 1.46
35.32 1.54
32.62 1.51
>64
>64
5d
5e
H
H
56.05 1.74
38.26 1.58
>64
>64
>64
>64
5f
H
H
32.59 1.51
>64
>64
>64
5g
>64
44.88 1.65
5h
5i
H
H
10.17 1.00
2.42 0.38
27.34 1.43
34.06 1.53
41.92 1.62
12.38 1.09
5j
CH3
CH3
CH3
32.61 1.10
11.17 0.85
>64
>64
>64
>64
44.64 1.65
49.23 1.69
>64
5k
5l
5m
5n
CH3
CH3
>64
>64
>64
0.93 0.02
19.18 1.28
9.99 1.00
5o
5p
5q
CH3
CH3
CH3
—
>64
>64
>64
3.06 0.48
58.37 1.76
9.68 0.93
>64
29.15 1.46
>64
>64
3-AP
—
—
42.81 1.63
18.85 1.27
a
Inhibitory activity was assayed by exposure to substances for 72 h and expressed as the concentration required to inhibit tumor cell
proliferation by 50% (IC50). Data are presented as mean SD of three independent experiments.
antiproliferative activity of compound 5n against MGC-803 is
presented in a dose-dependent and time-dependent manner
with IC50 values of >64, 9.22 0.96, and 0.93 0.02 μmol L−1
at 24 h, 48 h, and 72 h, respectively. The colony formation
assay shown in Fig. 2B also demonstrated that compound 5n
could inhibit the proliferation of MGC-803.
This journal is © The Royal Society of Chemistry 2017
Med. Chem. Commun.

View Article Online
Research Article
MedChemComm
Table 2 Inhibitory results of compound 5n and 3-AP against the normal
gastric epithelial cell line GES-1 and the gastric cancer cell line MGC-803
this transition. As shown in Fig. 3, wound healing, transwell
and matrigel-coated transwell assays all demonstrated that
compound 5n could inhibit the migration and invasion abil-
ity of MGC-803 in a dose-dependent manner.
IC50a (μM)
Comp
GES-1
MGC-803
We also examined the expression of some typical proteins
of the EMT process. The western blotting assay showed that
compound 5n could up-regulate the expression of the epithe-
lial cell biomarker, E-cadherin, while the mesenchymal cell
biomarker, N-cadherin, is down-regulated. These data indi-
cated that compound 5n may impair the migration and inva-
sion ability of MGC-803 cells through inhibiting the EMT
process.
5n
3-AP
27.93 1.254
5.40 0.73
0.93 0.02
9.68 0.93
a
Inhibitory activity was assayed by exposure for 72 h to substances
and expressed as the concentration required to inhibit normal and
tumor cell proliferation by 50% (IC50). Data are presented as mean
SD of three independent experiments.
To better understand the anti-proliferative activity of
compound 5n against MGC-803, cell-cycle analysis was
performed. After treatment with compound 5n for 48 h, it
was observed that the percentage of cells in the S phase was
28.4%, 32.82%, 46.07%, and 51.85% at different concentra-
tions of 0, 0.1, 0.25, and 0.5 μmol L−1, respectively, which
demonstrated that compound 5n caused an obvious S-phase
arrest pattern against MGC-803 in a dose-dependent manner.
Anti-migration and anti-invasion assays. As described in
the introduction, the migration and invasion ability of tumor
cells is an intractable problem in cancer therapy, while EMT
is tightly associated with the migration and invasion of tu-
mor cells. Excitingly, compound 5n could potently inhibit
Cell apoptosis analysis. Inducing the apoptosis of tumor
cells is considered a major way to defeat cancer. Due to its
potent activity against MGC-803 cells, compound 5n was
chosen to evaluate its influence on the apoptosis of MGC-
803 cells. We observed the morphology of MGC-803 under
the treatment of compound 5n at different concentrations
(0, 0.1, 0.25, and 0.5 μmol L−1) for 48 h, and the changes in
the morphology were observed by fluorescence microscopy
using Hoechst 33258 staining. As shown in Fig. 4A, with
increasing compound concentration, the typical apoptotic
markers, including cell rounding, chromatin shrinkage and
apoptotic bodies, become more and more frequently com-
pared with the control. Meanwhile, the data obtained using
Fig. 2 Effects of compound 5n on the proliferation of the human gastric cancer cell line MGC-803. (A) MGC-803 were treated with compound 5n
at different concentrations for 24, 48, and 72 h, respectively, then the MTT assay was used to evaluate its effect on cell viability; (B) the colony for-
mation after treatment with compound 5n at different concentrations for 7 days; (C) effect of compound 5n on the cell cycle distribution of MGC-
803. Cells were treated with different concentrations of compound 5n for 48 h. Then, the cells were fixed and stained with PI to analyze their DNA
content by flow cytometry. The experiments were performed three times, and a representative experiment is shown.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2017

View Article Online
MedChemComm
Research Article
Fig. 3 Compound 5n blocked the migration and invasion of MGC-803. (A) Anti-migration analysis performed using the wound healing assay after
treatment of MGC-803 cells with compound 5n for 48 h; (B) anti-migration and anti-invasion analyses performed using transwell and matrigel-
coated transwell assays, respectively, after treatment of MGC-803 cells with compound 5n for 48 h; (C) expression of E-cadherin and N-cadherin
in MGC-803 cells after treatment with compound 5n for 48 h. **P < 0.01 was considered statistically highly significant. Data are presented as
mean SD. All experiments were carried out at least three times.
Annexin V-FITC/PI double staining implied that after 48 h
of treatment with compound 5n, the total percentage of
early apoptosis and late apoptosis increased up to 10.9%,
16.4%, and 32.3% compared to the control (3.7%), which
was presented in
(Fig. 4B).
To further clarify the mechanism of the apoptosis induced
by compound 5n, we investigated the key proteins in the
a
concentration-dependent manner
This journal is © The Royal Society of Chemistry 2017
Med. Chem. Commun.

View Article Online
Research Article
MedChemComm
Fig. 4 Compound 5n induced apoptosis in MGC-803. (A) Apoptosis analysis using Hoechst-33258 staining after treatment of MGC-803 cells with
compound 5n for 48 h; (B) quantitative analysis of apoptotic cells using Annexin V-FITC/PI double staining and flow-cytometry calculation; quad-
rant Q2 represents live cells, quadrant Q3 is for early/primary apoptotic cells, and quadrant Q4 is for late/secondary apoptotic cells, while quadrant
Q1 represents cells damaged during the procedure; (C) expression of Bax and Bcl-2 in MGC-803 cells after treatment with compound 5n for 48 h.
**P < 0.01 was considered statistically highly significant. Data are presented as mean SD. All experiments were carried out at least three times.
mitochondria-related apoptotic pathway. As shown in Fig. 4C,
the pro-apoptotic protein, Bax, was up-regulated after the
treatment with compound 5n, while the anti-apoptotic pro-
tein, Bcl-2, was down-regulated, which were all presented in a
concentration-dependent manner. These findings indicated
that compound 5n may be involved in mitochondria-related
apoptosis.
study showed that compound 5n had the best inhibitory
activity with an IC50 value of 0.93 μM (about 10-fold more
potent than 3-AP) against MGC-803 without significant cyto-
toxicity against gastric epithelial immortalized GES-1 cells.
Further mechanism studies revealed that compound 5n
regulates the proliferation activity of MGC-803 cells by induc-
ing apoptosis, arresting the cell cycle at the S phase, and
inhibiting the EMT. Further SAR and mechanistic studies on
the selectivity toward MGC-803 cells are ongoing in our labo-
ratories and will be reported in due course.
Conclusions
In summary, we have synthesized and identified a series of
thiosemicarbazone-based analogues as potent and selective
antiproliferative agents to modulate cancer cell growth,
migration, and invasion. The structure–activity relationship
Conflicts of interest
The authors declare no competing interests.
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2017

View Article Online
MedChemComm
Research Article
Acknowledgements
11 S. I. Pascu, P. A. Waghorn, T. D. Conry, B. Lin, H. M. Betts,
J. R. Dilworth, R. B. Sim, G. C. Churchill, F. I. Aigbirhio and
J. E. Warren, Cellular confocal fluorescence studies and
cytotoxic activity of new Zn(II) bisIJthiosemicarbazonato)
complexes, Dalton Trans., 2008, 2107–2110.
12 C. R. Kowol, R. Trondl, V. B. Arion, M. A. Jakupec, I.
Lichtscheidl and B. K. Keppler, Fluorescence properties and
cellular distribution of the investigational anticancer drug
triapine (3-aminopyridine-2-carboxaldehyde thiosemicarbazone)
and its zinc(II) complex, Dalton Trans., 2010, 39, 704–706.
13 A. E. Stacy, D. Palanimuthu, P. V. Bernhardt, D. S.
Kalinowski, P. J. Jansson and D. R. Richardson, ZincIJII)-
Thiosemicarbazone Complexes Are Localized to the
Lysosomal Compartment Where They Transmetallate with
Copper Ions to Induce Cytotoxicity, J. Med. Chem., 2016, 59,
4965–4984.
This work was supported by the National Natural Science
Foundation of China (Project No. 81430085 and No. 21372206
for H.-M. L. and No. 81703328 for L.-Y. M.); the National Key
Research Program of Proteins (No. 2016YFA0501800); the Key
Research Program of Henan Province (No. 1611003110100 for
H.-M. L.).
Notes and references
1 Y. Yu, J. Wong, D. B. Lovejoy, D. S. Kalinowski and D. R.
Richardson,
Chelators
at
the
cancer
coalface:
desferrioxamine to Triapine and beyond, Clin. Cancer Res.,
2006, 12, 6876–6883.
2 J. L. Buss, F. M. Torti and S. V. Torti, The role of iron
chelation in cancer therapy, Curr. Med. Chem., 2003, 10,
1021–1034.
3 T. B. Chaston, D. B. Lovejoy, R. N. Watts and D. R.
Richardson, Examination of the antiproliferative activity of
iron chelators: multiple cellular targets and the different
mechanism of action of triapine compared with
desferrioxamine and the potent pyridoxal isonicotinoyl
hydrazone analogue 311, Clin. Cancer Res., 2003, 9,
402–414.
14 Y. Yu, D. S. Kalinowski, Z. Kovacevic, A. R. Siafakas, P. J.
Jansson, C. Stefani, D. B. Lovejoy, P. C. Sharpe, P. V.
Bernhardt and D. R. Richardson, Thiosemicarbazones from
the old to new: iron chelators that are more than just
ribonucleotide reductase inhibitors, J. Med. Chem., 2009, 52,
5271–5294.
15 R. A. Finch, M. Liu, S. P. Grill, W. C. Rose, R. Loomis, K. M.
Vasquez, Y. Cheng and A. C. Sartorelli, Triapine
(3-aminopyridine-2-carboxaldehyde-thiosemicarbazone):
A
4 Z. Kovacevic, D. S. Kalinowski, D. B. Lovejoy, Y. Yu, Y. Suryo
Rahmanto, P. C. Sharpe, P. V. Bernhardt and D. R.
Richardson, The medicinal chemistry of novel iron chelators
for the treatment of cancer, Curr. Top. Med. Chem., 2011, 11,
483–499.
5 Y. Yu, E. Gutierrez, Z. Kovacevic, F. Saletta, P. Obeidy, Y.
Suryo Rahmanto and D. R. Richardson, Iron chelators for
the treatment of cancer, Curr. Med. Chem., 2012, 19,
2689–2702.
6 F. Tisato, C. Marzano, M. Porchia, M. Pellei and C. Santini,
Copper in diseases and treatments, and copper-based anti-
cancer strategies, Med. Res. Rev., 2010, 30, 708–749.
7 D. B. Lovejoy, P. J. Jansson, U. T. Brunk, J. Wong, P. Ponka
and D. R. Richardson, Antitumor activity of metal-chelating
compound Dp44mT is mediated by formation of a redox-
active copper complex that accumulates in lysosomes, Can-
cer Res., 2011, 71, 5871–5880.
potent inhibitor of ribonucleotide reductase activity with
broad spectrum antitumor activity, Biochem. Pharmacol.,
2000, 59, 983–991.
16 J. Yuan, D. B. Lovejoy and D. R. Richardson, Novel di-2-
pyridyl-derived iron chelators with marked and selective
antitumor activity: in vitro and in vivo assessment, Blood,
2004, 104, 1450–1458.
17 D. R. Richardson, P. C. Sharpe, D. B. Lovejoy, D. Senaratne,
D. S. Kalinowski, M. Islam and P. V. Bernhardt, Dipyridyl
thiosemicarbazone chelators with potent and selective
antitumor activity form iron complexes with redox activity,
J. Med. Chem., 2006, 49, 6510–6521.
18 C. R. Kowol, R. Berger, R. Eichinger, A. Roller, M. A.
Jakupec, P. P. Schmidt, V. B. Arion and B. K. Keppler,
GalliumIJIII) and iron(III) complexes of alpha-N-heterocyclic
thiosemicarbazones: synthesis, characterization, cytotoxicity,
and interaction with ribonucleotide reductase, J. Med.
Chem., 2007, 50, 1254–1265.
19 C. R. Kowol, R. Eichinger, M. A. Jakupec, M. Galanski, V. B.
Arion and B. K. Keppler, Effect of metal ion complexation
and chalcogen donor identity on the antiproliferative activity
of 2-acetylpyridine N,N-dimethylIJchalcogen)semicarbazones,
J. Inorg. Biochem., 2007, 101, 1946–1957.
8 B. M. Zeglis, V. Divilov and J. S. Lewis, Role of metalation in
the topoisomerase II alpha inhibition and antiproliferation
activity of
a series of alpha-heterocyclic-N4-substituted
thiosemicarbazones and their Cu(II) complexes, J. Med.
Chem., 2011, 54, 2391–2398.
9 D. Palanimuthu, S. V. Shinde, K. Somasundaram and A. G.
Samuelson, In vitro and in vivo anticancer activity of copper
bisIJthiosemicarbazone) complexes, J. Med. Chem., 2013, 56,
722–734.
10 A. R. Cowley, J. Davis, J. R. Dilworth, P. S. Donnelly, R.
Dobson, A. Nightingale, J. M. Peach, B. Shore, D. Kerr and L.
Seymour, Fluorescence studies of the intra-cellular distribu-
tion of zinc bisIJthiosemicarbazone) complexes in human
cancer cells, Chem. Commun., 2005, 845–847.
20 D. R. Richardson, D. S. Kalinowski, V. Richardson, P. C.
Sharpe, D. B. Lovejoy, M. Islam and P. V. Bernhardt,
2-Acetylpyridine thiosemicarbazones are potent iron
chelators and antiproliferative agents: redox activity, iron
complexation and characterization of their antitumor
activity, J. Med. Chem., 2009, 52, 1459–1470.
21 D. B. Lovejoy, D. M. Sharp, N. Seebacher, P. Obeidy, T.
Prichard, C. Stefani, M. T. Basha, P. C. Sharpe, P. J. Jansson,
This journal is © The Royal Society of Chemistry 2017
Med. Chem. Commun.

View Article Online
Research Article
D. S. Kalinowski, P. V. Bernhardt and D. R. Richardson,
MedChemComm
combination with gemcitabine for patients with advanced
cancer, Cancer Chemother. Pharmacol., 2004, 54, 331–342.
29 J. J. Knox, S. J. Hotte, C. Kollmannsberger, E. Winquist, B.
Fisher and E. A. Eisenhauer, Phase II study of Triapine in
patients with metastatic renal cell carcinoma: a trial of
the National Cancer Institute of Canada Clinical Trials
Group (NCIC IND.161), Invest. New Drugs, 2007, 25,
471–477.
30 A. M. Traynor, J. W. Lee, G. K. Bayer, J. M. Tate, S. P.
Thomas, M. Mazurczak, D. L. Graham, J. M. Kolesar and
J. H. Schiller, A phase II trial of triapine (NSC# 663249) and
gemcitabine as second line treatment of advanced non-small
cell lung cancer: Eastern Cooperative Oncology Group Study
1503, Invest. New Drugs, 2010, 28, 91–97.
31 S. C. Chu, C. C. Yu, L. S. Hsu, K. S. Chen, M. Y. Su and P. N.
Chen, Berberine reverses epithelial-to-mesenchymal transi-
tion and inhibits metastasis and tumor-induced angiogene-
sis in human cervical cancer cells, Mol. Pharmacol., 2014, 86,
609–623.
32 S. Lamouille, J. Xu and R. Derynck, Molecular mechanisms
of epithelial-mesenchymal transition, Nat. Rev. Mol. Cell
Biol., 2014, 15, 178–196.
33 J. P. Thiery and J. P. Sleeman, Complex networks orchestrate
epithelial-mesenchymal transitions, Nat. Rev. Mol. Cell Biol.,
2006, 7, 131–142.
34 J. P. Thiery, H. Acloque, R. Y. Huang and M. A. Nieto,
Epithelial-mesenchymal transitions in development and
disease, Cell, 2009, 139, 871–890.
35 G. Berx, E. Raspe, G. Christofori, J. P. Thiery and J. P.
Sleeman, Pre-EMTing metastasis? Recapitulation of
morphogenetic processes in cancer, Clin. Exp. Metastasis,
2007, 24, 587–597.
36 M. Jechlinger, S. Grunert, I. H. Tamir, E. Janda, S.
Ludemann, T. Waerner, P. Seither, A. Weith, H. Beug and N.
Kraut, Expression profiling of epithelial plasticity in tumor
progression, Oncogene, 2003, 22, 7155–7169.
Novel second-generation di-2-pyridylketone thiosemicarbazones
show synergism with standard chemotherapeutics and dem-
onstrate potent activity against lung cancer xenografts after
oral and intravenous administration in vivo, J. Med. Chem.,
2012, 55, 7230–7244.
22 D. B. Lovejoy and D. R. Richardson, Novel “hybrid” iron
chelators derived from aroylhydrazones and thiosemicarbazones
demonstrate selective antiproliferative activity against tumor
cells, Blood, 2002, 100, 666–676.
23 Z. Kovacevic, S. Chikhani, D. B. Lovejoy and D. R.
Richardson, Novel thiosemicarbazone iron chelators induce
up-regulation and phosphorylation of the metastasis sup-
pressor N-myc down-stream regulated gene 1: a new strategy
for the treatment of pancreatic cancer, Mol. Pharmacol.,
2011, 80, 598–609.
24 Y. Yu, Y. Suryo Rahmanto and D. R. Richardson, Bp44mT:
an orally active iron chelator of the thiosemicarbazone class
with potent anti-tumour efficacy, Br. J. Pharmacol.,
2012, 165, 148–166.
25 M. J. Mackenzie, D. Saltman, H. Hirte, J. Low, C. Johnson, G.
Pond and M. J. Moore, A Phase II study of 3-aminopyridine-
2-carboxaldehyde thiosemicarbazone (3-AP) and gemcitabine
in advanced pancreatic carcinoma, A trial of the Princess
Margaret hospital Phase II consortium, Invest. New Drugs,
2007, 25, 553–558.
26 J. E. Karp, F. J. Giles, I. Gojo, L. Morris, J. Greer, B. Johnson,
M. Thein, M. Sznol and J. Low, A phase I study of the novel
ribonucleotide reductase inhibitor 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (3-AP, Triapine) in combi-
nation with the nucleoside analog fludarabine for patients
with refractory acute leukemias and aggressive myeloprolif-
erative disorders, Leuk. Res., 2008, 32, 71–77.
27 B. Ma, B. C. Goh, E. H. Tan, K. C. Lam, R. Soo, S. S.
Leong, L. Z. Wang, F. Mo, A. T. Chan, B. Zee and T.
Mok, A multicenter phase II trial of 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (3-AP, Triapine) and
gemcitabine in advanced non-small-cell lung cancer with
pharmacokinetic evaluation using peripheral blood mononu-
clear cells, Invest. New Drugs, 2008, 26, 169–173.
37 L. Y. Ma, Y. C. Zheng, S. Q. Wang, B. Wang, Z. R. Wang,
L. P. Pang, M. Zhang, J. W. Wang, L. Ding, J. Li, C. Wang, B.
Hu, Y. Liu, X. D. Zhang, J. J. Wang, Z. J. Wang, W. Zhao and
H. M. Liu, Design, synthesis, and structure-activity relation-
ship of novel LSD1 inhibitors based on pyrimidine-thiourea
hybrids as potent, orally active antitumor agents, J. Med.
Chem., 2015, 58, 1705–1716.
28 Y. Yen, K. Margolin, J. Doroshow, M. Fishman, B. Johnson,
C. Clairmont, D. Sullivan and M. Sznol, A phase I trial of
3-aminopyridine-2-carboxaldehyde thiosemicarbazone in
Med. Chem. Commun.
This journal is © The Royal Society of Chemistry 2017