Discovery of 2-pyridineformamide thiosemicarbazones as potent antiausterity agents

Article abstract of DOI:10.1016/j.bmcl.2013.12.044

Series of 2-pyridineformamide thiosemicarbazones were synthesized. Their preferential cytotoxicity in nutrient deprived medium (NDM) was evaluated using PANC-1 human pancreatic cancer cells by employing an antiausterity strategy. 2-Pyridineformamide thios

Full text of DOI:10.1016/j.bmcl.2013.12.044

Bioorganic & Medicinal Chemistry Letters 24 (2014) 458–461
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Discovery of 2-pyridineformamide thiosemicarbazones as potent
antiausterity agents
Bhushan Shakya a, Paras Nath Yadav a, Jun-ya Ueda b, Suresh Awale b,
⇑
a Central Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal
b Frontier Research Core for Life Sciences, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
Series of 2-pyridineformamide thiosemicarbazones were synthesized. Their preferential cytotoxicity in
nutrient deprived medium (NDM) was evaluated using PANC-1 human pancreatic cancer cells by
employing an antiausterity strategy. 2-Pyridineformamide thiosemicarbazones induced apoptosis and
exhibited preferential cytotoxic activity toward PANC-1 cells in NDM, with potencies in the
submicromolar range. These compounds are potential candidates for the development of therapeutics
against pancreatic cancer.
Received 18 November 2013
Revised 9 December 2013
Accepted 11 December 2013
Available online 18 December 2013
Keywords:
Antiausterity agents
Pancreatic cancer
Ó 2013 Elsevier Ltd. All rights reserved.
Nutrition starvation
2-Pyridineformamide thiosemicarbazones
Apoptosis
Pancreatic cancer is one of the most deadly forms of cancer. It is
associated with the lowest 5-year survival rate known for human
cancers (<5%).1 Almost all patients with pancreatic cancer rapidly
develop metastases and die within a short period after diagnosis.2
It is resistant to conventional chemotherapeutic agents, including
paclitaxel, doxorubicin, cisplatin, and camptothecin, and there
are currently no reliable chemotherapeutic agents available to
treat this disease. Therefore, there is an urgent need for the discov-
ery of novel agents to treat this disease.3 Pancreatic cancers are
hypovascular in nature, which causes an inadequate supply of
nutrition and oxygen to aggressively proliferating tumor cells.4
However, these tumor cells show an extraordinary tolerance to
nutrient starvation for a prolonged period of time, enabling them
to survive in the hypovascular (austere) tumor microenviron-
ment.5 Development of drugs that specifically target the resistance
of tumor cells to nutrient starvation has been termed the antiaus-
terity therapeutic strategy.6–11
Thiosemicarbazones are an important class of compounds that
have long attracted interest among medicinal chemists owing to
their incredible biological activities, which include antibacterial,
antiviral, antimalarial, and anti-tumor activities.12–14 MarboranÒ
(methisazone), which was marketed for the treatment of smallpox,
is a notable example of a successful commercial thiosemicarba-
zone drug.15 A more recent development was the discovery of
TriapineÒ (3-aminopyridine-2-carboxaldehyde thiosemicarbazone,
3-AP, Fig. 1), which has undergone both phase II clinical trial in pa-
tients with metastatic squamous cell carcinoma of the head and
neck16 and phase II clinical trial, in combination with gemcitabine,
in patients with advanced non-small cell lung cancer.17 TriapineÒ,
a potent antiproliferative that is effective against many cancer
types, presents a marked selectivity for tumor cells.18 It obstructs
tumor growth by inhibiting ribonucleotide reductase (RR), a key
enzyme involved in the conversion of ribonucleotides into
deoxyribonucleotides, the building blocks of DNA synthesis.19
Overexpression of RR, which has been reported in human pancre-
atic adenocarcinoma, is associated with resistance to gemcita-
bine,19,20 a drug that has been prescribed most frequently for the
management of advanced pancreatic cancer.
In our antiausterity strategy-based anticancer drug discovery
program, we found that almost all of the conventional chemother-
apeutic agents, including gemcitabine, are virtually ineffective
against pancreatic cancer cells in the tumor mimicking austere
environment of nutrient starvation.21 In contrast, thiosemicarba-
zones have been reported to show improved activity against gem-
citabine resistant human cancers.22 Therefore, we speculated that
thiosemicarbazones could be the new antiausterity agents and
may possess the ability to diminish cancer cells’ tolerance to nutri-
ent starvation. To test this hypothesis, we synthesized series of 2-
pyridineformamide thiosemicarbazone derivatives with variations
in their ring and N-4 substitution. The synthetic route is illustrated
in Scheme 1. The common intermediate 4-methyl-4-phenyl-3-thi-
osemicarbazide (I) was first prepared according to the procedure
described by Scovill.21 Transamination of I with an amine gave
⇑
Corresponding author.
E-mail address: suresh@inm.u-toyama.ac.jp (S. Awale).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.12.044

B. Shakya et al. / Bioorg. Med. Chem. Lett. 24 (2014) 458–461
459
Table 1
H2N
S
2-Pyridineformamide thiosemicarbazones
N
R3
H2N
N
H
N
4
5
6
3
2
S
R1
N
Figure 1. Structure of TriapineÒ
.
7
8
N
R2
N
H
N
1
NH2
the corresponding N-disubstituted thiosemicarbazides (II) which
was converted into thiosemicarbazone (III) (see Supplementary
data S2–S4).23 For this study, we prepared twelve 2-pyridinefor-
mamide thiosemicarbazones with varied substituents.24,25 Their
structures and yields are presented in Table 1. The structures of
all synthesized compounds were established using NMR spectro-
scopic data and HRFABMS data (See Supplementary data S4–S7).
All of these synthesized compounds were tested for their pref-
erential cytotoxicity against PANC-1 human pancreatic cancer cell
line under both nutrient-rich and nutrient-deprived conditions,
utilizing an antiausterity strategy26 (Fig. 2). The concentration at
which 50% of the cells were preferentially killed in the nutrient
deprived medium (PC50) are also presented in Fig. 2. Arctigenin,
an antiausterity strategy-based anticancer agent, was used as a
positive control. It showed preferential cytotoxicity with a PC50
Compd
–NR1R2
R3
H
Yield of III (%)
1'
2'
1
46
21
60
59
1'
1'
1'
2'
2'
2'
2
3
4
H
H
H
3'
2'
1'
1'
3'
5
6
H
H
24
45
3"
2'
4"
value of 0.85 lM. Of interest, all thiosemicarbazones were highly
5"
active against PANC-1 human pancreatic cancer cell lines. More-
over, presence or absence of methyl substituent at C-4 position
did not found to alter the activity of thiosemicarbazones. 7 showed
the most potent preferential cytotoxicity among the tested com-
2"
6"
7
8
4-CH3
4-CH3
4-CH3
4-CH3
24
29
41
32
pounds with a PC50 of 0.37
lM, approximately two fold as potent
as the positive control arctigenin (PC50, 0.86
l
M). Among others,
1, 6, 7, and 12 also displayed more potent activity than the positive
control. Paclitaxel, a well known anti cancer agent, was virtually
inactive. Similarly, gemcitabine, a clinically used anticancer drug
for the treatment of pancreatic cancer, was virtually inactive after
24 h in both NDM and Dulbecco’s modified Eagle medium (DMEM)
9
10
11
12
4-CH3
4-CH3
45
63
at the maximum tested dose, with PC50 >200 lM (Fig. 2). This
evidence suggests that thiosemicarbazones are powerful lead
candidates for antipancreatic cancer drug development and
demand urgent further investigation.
Among the synthesized compounds, 6 was selected for further
study because it was synthesized in larger quantities. PANC-1 cells
were treated with 1 lM 6 for 24 h in NDM, stained with ethidium
AO is a cell permeable dye that emits bright green fluorescence
in viable cells. EB is impermeable and does not stain viable cells.
In late apoptotic and necrotic cells, the integrity of the plasma
and nuclear membranes decreases, allowing EB to pass through
the membranes, intercalate into DNA and other nucleic acids,
and emits red fluorescence. As shown in Figure 3a, control cells
showed intact cell morphology with bright green AO fluorescence,
bromide/acridine orange (EB/AO) reagent, and then visualized
under fluorescent and phase contrast modes of the microscope.27
S
1. NaOH (1 equiv.)
CS2
+
OH
2. ClCH2COONa (1 equiv.)
3. HCl (12 M)
NH
suggesting the cells are viable. However, cells treated with a 1 lM
N
S
6 showed exclusively red fluorescence due to EB, indicative of
nonviable cells (Fig. 3b). Phase contrast microscopic observation
of the treated cells showed a dramatic alteration in the PANC-1 cell
morphology (Fig. 3c), including swelling, rupture of cell
membranes, and disruption of cellular organelles.
CH3
CH3
O
S
S
R1
NH2
NH2
HNR1R2 (1 equiv.)
N
N
H
NH2NH2, H2O (98%)
N
N
H
We further performed western blot analysis28 to check whether
6 modulated the key proteins involved in cell survival mechanisms
(Fig. 4). A number of antiausterity agents have been found to inhi-
bit Akt activation, leading to preferential cell death under nutrient-
deprived conditions.6 However, in present study, 6 was not found
to inhibit p-Akt (S437) or p-Akt (T308), suggesting that Akt signal-
ing is unlikely to be a target of 6. However, 6 was found to activate
apoptosis, resulting in the cleavage of caspase-3 even at a very
short exposure time of 4 h in a concentration dependent manner
(Fig. 4). Therefore, much stronger effect at lower concentration
could be expected with increase in the exposure time period. The
evidence for apoptosis was further con-firmed using an annexin
R2
CH3
(I)
(II)
R3
N
R3
S
(1 equiv.)
N
R1
N
N
N
N
H
R2
NH2
(III)
Scheme 1. Synthesis of 2-pyridineformamide thiosemicarbazones.

460
B. Shakya et al. / Bioorg. Med. Chem. Lett. 24 (2014) 458–461
Figure 2. Preferential cytotoxic activity of synthesized 2-pyridineformamide thiosemicarbazones against the PANC-1 human pancreatic cancer cell line in nutrient deprived
medium (NDM) and Dulbecco’s modified Eagle medium (DMEM).
Figure 4. Western blot of the effect of 6 for 4 h against Akt, pAkt, pro-caspase 3, and
Figure 3. Fluorscent [ethidim bromide (EB)/acridine orange (AO)] and phase
cleaved-caspase 3.
contrast images of PANC-1 cells at 24 h. (a) Control, live cells stained only with
AO emitted bright green fluorescence, (b) treatment with 6 (1
of PANC-1 cells within 24 h; cells stained with EB emitted red fluorescence, (c)
phase contrast image of PANC-1 cells treated with 6 (1 M) showing morphological
alteration. (d) Merged image of phase contrast and red fluorescence image of PANC-
1 cells treated with 6 (1 M).
lM) led to total death
l
the key reasons for the failure of chemotherapy, and lead to
aggressive growth and metastasis. In the present study, apoptosis
induced by 6 indicated that 2-pyridineformamide thiosemicarba-
zones are potential candidates for antipancreatic cancer drug
development.
In conclusion, 2-pyridineformamide thiosemicarbazones are
novel powerful antiausterity agents for the development of thera-
peutics against deadly pancreatic cancers. Detailed studies of both
their mechanism of action and their in vivo activity are under
consideration.
l
v (AV) and propidium iodide (PI) staining assay. During apoptosis,
alterations in the plasma membrane lead to the translocation of
phosphatidylserine (PS), which is exposed at the external surface
of the cell. AV specifically binds to the exposed PS, which is
detected by green fluorscence (Supplementary data S8). Pancreatic
cancer cells are known to be resistant to apoptosis, which is one of

B. Shakya et al. / Bioorg. Med. Chem. Lett. 24 (2014) 458–461
461
17. Traynor, A. M.; Lee, J.-W.; Bayer, G. K.; Tate, J. M.; Thomas, S. P.; Mazurczak, M.;
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Acknowledgments
This work was supported by a Grant from Toyama Support
Center for Young Principal Investigators in Advanced Life Sciences,
Japan, and a Grant in Aid for Scientific Research (No. 24510314)
from the Japan Society for the Promotion of Science (JSPS) to S.A.,
and the University Grants Commission of the Government of Nepal
to B.S. We thank Dr. Rajendra Kumar Shakya, Wayne State
University for the NMR data acquisition.
22. Di Marco, M.; Di Cicilia, R.; Macchini, M.; Nobili, E.; Vecchiarelli, S.; Brandi, G.;
Biasco, G. Oncol. Rep. 2010, 23, 1183.
23. Scovill, J. P. Phosphorus Sulfur Silicon 1991, 60, 15.
24. Compounds 2 was previously reported by Ketcham, K. A.; Swearingen, J. K.;
Castiñeiras, A.; Garcia, I.; Bermejo, E.; West, D. X. Polyhedron 2001, 20, 3265.
25. Compound 5 was previously reported by Santos, I. G.; Hagenbach, A.; Abram, U.
Dalton Trans. 2004, 21, 677.
26. PANC-1 cell line, culture and in vitro preferential cytotoxicity: The PANC-1 human
pancreatic cancer cell lines were purchased from Riken BRC cell bank. These
cell lines were maintained in standard D-MEM 10% FBS supplement, 0.1%
NaHCO3, and 1% antibiotic antimycotic solution. For preferential cytotoxicity
experiment, PANC-1 cells were seeded in 96-well plates (1.5 Â 104/well) and
incubated in fresh DMEM at 37 °C under humidified 5% CO2 and 95% air for
24 h. After the cells were washed with D-PBS, the medium was changed to
serially diluted test samples in DMEM or NDM, with the control and blank in
each plate. After 24 h incubation, cells were washed twice with D-PBS and
Supplementary data
Supplementary data (general experimental procedures, annex-
in-V/PI staining assay, synthetic protocol, and NMR spectral data
for compounds 1–12) associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
12.044.
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