2-Pyridineformamide N(4)-ring incorporated thiosemicarbazones inhibit MCF-7 cells by inhibiting JNK pathway

Article abstract of DOI:10.1016/j.bmcl.2019.04.031

In an effort to develop a more potent anticancer therapeutic agent, a series of 2-pyridineformamide thiosemicarbazones (R = H, 4-CH₃, 5-F, 6-CH₃ and (Figure presented.)(Figure presented.))have been synthesized and evaluated for their

Full text of DOI:10.1016/j.bmcl.2019.04.031

Accepted Manuscript
2-Pyridineformamide N(4)-ring incorporated thiosemicarbazones inhibit
MCF-7 cells by inhibiting JNK pathway
Bhushan Shakya, Nerina Shahi, Faiz Ahmad, Paras Nath Yadav, Yub Raj
Pokharel
PII:
S0960-894X(19)30250-1
https://doi.org/10.1016/j.bmcl.2019.04.031
BMCL 26398
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
26 February 2019
8 April 2019
18 April 2019
Please cite this article as: Shakya, B., Shahi, N., Ahmad, F., Nath Yadav, P., Raj Pokharel, Y., 2-Pyridineformamide
N(4)-ring incorporated thiosemicarbazones inhibit MCF-7 cells by inhibiting JNK pathway, Bioorganic &
Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.04.031
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2-Pyridineformamide N(4)-ring incorporated
thiosemicarbazones inhibit MCF-7 cells by inhibiting JNK
pathway
Bhushan Shakyaa#, Nerina Shahib#, Faiz Ahmadb, Paras Nath Yadavc* and Yub Raj Pokharelb*
aAmrit Campus, Tribhuvan University, Thamel, Kathmandu, Nepal.
bFaculty of Life Science and Biotechnology, South Asian University, Akbar Bhawan, Chanakyapuri, New Delhi-
110021, India.
cCentral Department of Chemistry, Tribhuvan University, Kirtipur, Kathmandu, Nepal.
# Both authors have equal contribution
*Corresponding authors: Paras Nath Yadav, pnyadav219@gmail.com and Yuba Raj Pokharel,
yrp@sau.ac.in
ABSTRACT
In an effort to develop a more potent anticancer therapeutic agent, a series of 2-pyridineformamide
thiosemicarbazones (R = H, 4-CH3, 5-F, 6-CH3 and
N
N
N
O
N
=
,
,
,
N
N
S
N
,
,
) have been synthesized and evaluated for their anti-cancer
N
N
activities against the cancer cells MCF-7 (breast cancer cell line), A-431and A375 (epidermoid
carcinoma cell line), and HeLa (cervical cancer cell line) using MTT assay. All these 2-
pyridineformamide thiosemicarbazones exhibited anti-proliferative activities towards these cell lines.
5FAmPyrr possess most profound effects against MCF-7 cells with IC50 of 0.9 µM. In flow cytometry
using Propidium Iodide, 5FAmPyrr was found to induce cell death significantly in a dose dependent
manner (100 nM to 3 µM) and inhibited colony formation of MCF-7 cells. This compound induced pro-
apoptotic protein Bax and inhibited anti apoptotic protein Bcl-2 as well as both c-Jun and Jun N-terminal
kinase (abbreviated as JNK) in concentration dependent manner. Further pro-caspase 3 and PARP were
inhibited by 5FAmPyrr at concentration of 3 µM. The results suggest that 5FAmPyrr exhibit
anticancer potency and induced cell death by inhibiting MAPK signaling and inducing intrinsic
apoptotic pathway. All these indicate that 2-pyridineformamide thiosemicarbazones could be developed
as future therapeutics agents to treat cancer.
S
R
N
N
N
H
N
NH2
Key words: A431, A375, cell viability, HeLa, MCF-7, 2-pyridineformamide thiosemicarbazone.
Thiosemicarbazones are important class of compounds with potential biological activities, such as
antibacterial, antiviral, antimalarial and antitumor activities.1-3 Anti-microbial activity of
thiosemicarbazone was first recognized by Domagk in 1946.4,5 Antiviral properties of
thiosemicarbazone was discovered in sixties and detailed structure activity relationship study of this
class of compound ultimately led to commercialization of methisazone, Marboran® for the treatment of
(1)

smallpox.6 Anticancer activity of heterocyclic thiosemicarbazone (HCT) was first reported by
Brockman et. al with pyridine-2-carboxaldehyde thiosemicarbazone which was found potent to prolong
the life span of mice bearing the L1210 leukemia.7 This observation encouraged the synthesis and
anticancer screening of different derivatives of thiosemicarbazones with modification in the
heterocyclic ring system, variations in the ring substituents and thiosemicarbazone side chain.8-10
Anticancer drugs exert their activity by inhibiting different biochemical process occurring in the cell
cycle resulting in cell death. The most common target of many currently used chemotherapy drugs is
inhibition of DNA or RNA synthesis and repair.11-13
Ribonucleotide reductase (RR) plays a central role for the generation of the cytosine, adenine, and
guanine deoxyribonucleotide 5'-triphosphate building blocks of DNA synthesis and repair.14 The α-(N)-
heterocyclic thiosemicarbazones are one of the most potent inhibitor of RR activity which ultimately
affect the DNA synthesis and prevents cancer cells from division.15 A strong inhibitor of RR would be
an effective anticancer agent. One of the member of thiosemicarbazone, 3-aminopyridine-2-
carboxaldehyde thiosemicarbazone (3-AP), Triapine®, has been found as a potent inhibitor of the RR
activity.16 Currently, Triapine® has reached phase II clinical trial on several cancer types; in patients
with recurrent or metastatic head and neck squamous cell carcinoma17 and a multicenter phase II trial
in combination with gemcitabine in advanced non-small-cell lung cancer18. Over expression of RR
activity correlates to an increase in cell invasion potential. Over expression of RR has also been reported
in human pancreatic adenocarcinoma, which is associated with the resistance to gemcitabine, a drug for
the treatment of advanced pancreatic cancer.19-20
We recently discovered that 2-pyridineformamide thiosemicarbazones retard the tolerance to nutrition
starvation of pancreatic cancer cell.21 Pancreatic cancer is known to be the most fatal form of cancer
having lowest 5 year survival rate of <5% known for all the cancers.22 The result of our previous study
encouraged us to explore further synthesis of 2-pyridineformamide thiosemicarbazone derivatives and
evaluate their preferential cytotoxic activity against other cancers also. Therefore, we prepared more
new 2-pyridineformamide thiosemicarbazone derivatives with variations in N(4)-substitution as
potential anti-cancer candidates. The synthetic route is illustrated in Scheme 1. The common
intermediate 4-methyl-4-phenyl-3-thiosemicarbazide (I) was first prepared according to the procedure
described by Scovill.23 Transamination of I with an amine gave the corresponding N- ring incorporated
thiosemicarbazide (II) which was converted to thiosemicarbazone (III). The structures and yields of the
23 compounds synthesized are presented in Table 1. The structures of all the synthesized compounds
were confirmed using NMR spectroscopic data and HRFABMS data.
S
NH2NH2.H2O (98%)
i. NaOH (1 equiv.)
ii. ClCH2COONa (1 equiv.)
iii. HCl (12 M)
S
S
+
NH2
OH
S
NH
N
S
N
N
O
CH3
CH3
CH3 H
(I)
R1
N
R1
N
R2
R3
R2
R3
S
(1 equiv.)
N
(1 equiv.)
HN
S
N
H2N
N
N
H
N
N
NH2
(III)
H
(II)
Scheme 1. Synthesis of N(4)-ring incorporated 2-pyridineformamide thiosemicarbazones
The synthesized compounds (Table 1) were screened for their anti-cancer potential in terms of viability
of following cancer cell lines- MCF-7 (breast cancer cell line), A-431and A375 (epidermoid carcinoma
cell line), HeLa (cervical cancer cell line) by MTT assay and percentage inhibition was calculated on
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the basis of percentage viability (see supplementary data). The IC50 values determined are shown in
Table 2.
Table 1. N(4)-Ring incorporated 2-Pyridineformamide thiosemicarbazones
R1
R2
4
3
S
5
2
N
7
6
R3
8
N
N
H
N
1
NH2
R3
N
Compound
R1
H
R2
Yield (%)
46
1'
2'
1
H
H
H
H
H
H
H
H
N
1'
2'
2
3
4
H
H
H
21
60
59
3'
N
N
N
1'
2'
O
1'
2'
S
2'
1'
3'
5
6
H
H
H
H
H
H
24
45
N
1'
2'
3"
4"
6"
2"
5"
N
N
N
1'
2'
7
8
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
24
29
41
32
N
1'
2'
3'
N
N
N
1'
2'
9
O
1'
2'
10
S
2'
1'
3'
11
12
CH3
CH3
H
H
H
H
45
63
N
1'
2'
3"
4"
6"
2"
5"
N
N
N
1'
2'
13
14
H
H
F
F
H
H
45
50
N
1'
2'
N
O
(3)

1'
2'
15
16
H
H
F
F
H
H
52
40
N
S
2'
1'
3'
N
1'
2'
3"
4"
6"
2"
5"
N
N
17
H
F
H
61
N
1'
2'
18
19
20
21
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
26
21
54
41
N
1'
2'
3'
N
N
N
1'
2'
O
1'
2'
S
2'
1'
3'
22
23
H
H
H
H
CH3
CH3
19
42
N
1'
2'
3"
4"
6"
2"
5"
N
N
N
Most of the synthesized compounds exhibited moderate to augmented effect against MCF-7 cell line
except 4MeAmMorp, 6MeAmTMorp and 6MeAmPypz with IC50 (0.9-74 µM). Likewise, all the
compounds have good anti-proliferative activity against epidermoid carcinoma cell lines A-431 and
A375 with IC50 (0.9-19.6 µM) and (0.6-14 µM) respectively. Synthesized compounds were also found
to be inhibitory against HeLa cell line with IC50 between 0.9-38.5 µM.
Table 2. IC50 values of compounds against different cancer cell lines
Compound
No.
Compound
Name
(IC50, µM)
MCF-7
3.6
A431
A375
0.6
HeLa
6.2
3.8
7.3
7.8
6
1
2
HAmPyrr
1.7
6.9
4.8
6.7
3
HAmPip
8.5
0.68
0.68
0.76
0.74
0.7
3
HAmMorp
HAmTmorp
HAmHexim
HAmPypz
4.7
4
8.8
5
2.6
6
13.7
1.2
4.3
0.9
18.9
6.8
7.2
8
8.6
1.9
7.7
38.5
15.5
7
7
4MeAmPyrr
4MeAmPip
4MeAmMorp
4MeAmTmorp
4MeAmHexim
4MeAmPypz
5FAmPyrr
0.72
1.9
8
18.8
>100
6.9
9
1.6
10
11
12
13
14
15
1.2
6.9
0.8
12.3
0.9
15.5
1
0.78
0.86
1.5
10.5
1.8
1.7
1.7
5FAmMorp
5FAmTmorp
22.1
21.4
4
1.2
0.9
(4)

16
17
18
19
20
21
22
23
5FAmHexim
5FAmPypz
21.1
22
1
0.96
1.4
2.9
1
0.9
2
13.4
4
6MeAmPyrr
6MeAmPip
61.8
74
2.7
2
12.4
4.3
6MeAmMorp
6MeAmTMorp
6MeAmHexim
6MeAmPypz
21.5
>100
54.7
>100
14
2.2
1.8
1
2.6
1.4
1.8
0.85
14.7
19.6
0.9
This primary screening result revealed that compound 5FAmPyrr was most active against all the cell
lines with IC50 values lying between 0.9-1.8 µM and among them exhibited profound anti-proliferative
activity against MCF-7 cell line with IC50 of 0.9 µM. This most potent candidate was further chosen for
detailed study, however lower concentrations (100 nM, 300 nM,1 µM and 3 µM) were chosen for
subsequent studies. To determine the cancer cell specific selectivity of 5FAmPyrr, crystal violet assay
was performed for cell viability with normal human cell lines, HEK 293 and PNT2 (normal prostate
epithelium cells), 3000 cells/well were incubated with 5FAmPyrr in 96 well plates (control, vehicle
control, 100nM, 300nM, 1µM and 3µM) for 72 hours. We observed that 5FAmPyrr showed partial
cytotoxic effects to non-cancerous cells but the percentage of cell death that was observed in
cancer cells was significantly higher as compared with non-cancerous cells (figure 1C). We
could conclude that 5FAmPyrr has potent anti-cancer effects and selectively target for cancer
cells. To explore the reason behind this anti-proliferative activity of 5FAmPyrr, cell death analysis
by flow cytometry using Propidium Iodide was done and it was found that 5FAmPyrr significantly
induced cell death in a dose dependent manner as shown in figure 1 A. Cell Viability assay was
performed in MCF-7 cells after the treatment of 5FAmPyrr in 100 nM, 300 nM, 1 µM and 3 µM
concentration for 72 hours. MTT assay results showed that 5FAmPyrr inhibit MCF-7 cells growth in
concentration dependent manner as shown in Figure 1B. Furthermore long term treatment of
5FAmPyrr significantly inhibits clonogenicity of MCF7 cells in dose dependent manner, as shown in
figure 2 A; 300 nM treated well showed the significant inhibition of both colony number and size
respectively.
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Figure 1: Effect of 5FAmPyrr on cell survival in MCF-7 cells. A &B. MCF-7 cells (30,000cells/well)
were incubated at 37˚C with 5FAmPyrr (control, vehicle control, 100nM, 300nM, 1µM and 3µM) for
48h. Following incubation, cells were harvested and stained with propidium iodide and analyzed by
flow cytometry. Bar diagram showing % of cell death after treating with increasing concentrations
(control, vehicle control, 100nM, 300nM, 1µM and 3µM). C. HEK 293 and PNT2 (normal prostate
epithelium cells) (3000 cells/well) were incubated with 5FAmPyrr in 96 well plates (control, vehicle
control, 100nM, 300nM, 1µM and 3µM) for 72 hours and crystal violet assay (CVs) was performed to
determine the cytotoxic effect.
Next to unravel the molecular machinery, participation of which led to significant cell death in MCF-7
cells, expression of Bcl-2 family members like pro-apoptotic Bax and anti-apoptotic Bcl-2 were
analyzed by immunoblotting. It was found that pro-apoptotic Bax was equally upregulated in all
concentration as shown in figure 2B, whereas anti-apoptotic Bcl-2 was equally down-regulated at all
concentration of 5FAmPyrr. This modulation of Bax and Bcl-2 following treatment of MCF-7 cells
with 5FAmPyrr committed cells to undergo cell death, as the ratio of Bax to Bcl-2 is a critical aspect
for a cell to undergo apoptosis and then cell death.24
Figure 2: Effect of 5FAmPyrr on expression of apoptotic proteins and mechanism of action.
A. MCF-7 cells (1200/well) were treated with increasing concentration of 5FAmPyrr (Control, 100nM,
300nM, 1µM and 3µM) for 21 days with every 72 hours treatment and after the colonies developed,
they were fixed, stained with crystal violet solution. Colony formation assay showed that 5FAmPyrr
effectively inhibit colony formation in a concentration dependent manner.
B & C. Expression of anti- and pro-apoptotic proteins following treatment with 5FAmPyrr. Cell
lysate was prepared after treating MCF-7 cells with 5FAmPyrr (Control, 100nM, 300nM, 1µM and
3µM for 48 h). Protein lysate was resolved on SDS-PAGE and immunoblotting was performed using
appropriate primary and secondary antibodies. Protein expression was studied for BAX, BCL2, Pro-
Caspase-3, PARP, JNK, and C-Jun respectively and β-actin was used as loading control in each
experiment.
We further checked the status of caspase 3 an executioner caspase, and Poly(ADP-ribose) polymerase
(PARP) one of the substrate of caspase 3 and whose cleavage induces apoptosis in cancer cells.25 Verily
we found inhibition in pro casapase-3 and PARP-1 as shown in figure 2C, which suggest an intrinsic
nature of cell death induction by 5FAmPyrr against breast cancer MCF-7 cell lines. This activation of
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cell death led us to check the expression of Jun N-terminal kinase (JNK), a critical and one of major
member of MAPK super family whose involvement has been reported both in cell survival and
apoptosis26 and its hyperactivation is a common anomaly in a number of pathological conditions
including cancer and a growing body of evidence suggests that impairing JNK signaling could be an
important cancer therapeutic strategy.27 Compound 5FAmPyrr was found to be inhibiting JNK at 1 and
3 µM as shown in figure 2 which supports the apoptotic cell death mechanism followed by treatment
of cells. Moreover c-Jun an important downstream target of JNK and multi-faceted protein possess both
proliferative and apoptosis inducing activities28 was also found to be inhibited in a dose dependent
manner which further strengthen the finding that 5FAmPyrr induces cell death hampering JNK/c-Jun
proliferative signals.
Acknowledgments
This work was supported by a Grant from University Grants Commission of Government of Nepal to
B.S. We thank Dr. Rajendra Shakya, Wayne State University, USA for IR and NMR data acquisition
and Dr. Suresh Awale, University of Toyama, Japan for HR-FAB MS analysis. South Asian University
for providing support to performed biological assays.
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23. Scovill, J. P. Phosphorus Sulfur Silicon 1991, 60, 15.
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29. Cell culture and treatment: MCF7, A431, A375 and HeLa cells were cultured in DMEM medium
supplemented with 10% fetal bovine serum and penicillin-streptomycin at 37 ⁰C in 5% CO2
incubator. Cells were treated with varying concentrations of a series of 23 synthetic 2-
pyridineformamide thiosemicarbazones (1, 3, 10, 100 µM) in DMSO for 72 h for screening and
followed by 5FAmPyrr experimentation with (0.1, 0.3, 1, 3µM) concentration respectively.0.1%
DMSO was used as vehicle control.
30. MTT Assay (cell viability assay): The colorimetric MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide) assay was used to measure cell viability. MCF7, A431, A375 and
HeLa cells as well as HEK 293 and PNT2 (normal prostate epithelial cells) were seeded in a 96 well
plate at a density of 3000 cells/100µL/well. Following attachment of cells left overnight, cells were
treated with varying concentrations (1-100 µM) of the series of 23 compounds as well as (100 nM-
3 µM) 5FAmPyrrin another 100 µL of media and were kept in incubation for 72 h. Afterwards, 20
µL of MTT (5 mg/mL in PBS) was added to each well and cells were incubated for another 3 h at
37 C. The supernatant was removed carefully and 150 µL DMSO was added to each well to
dissolve MTT formazan crystals. The plate was shaken on a rocker for 15-20 min and absorbance
value of each well was determined using a multi-well plate reader (Biotek, Winooski, USA) at 570
nm. The wells treated with DMSO were taken as vehicle control respectively. Data were taken from
the number of replicates and percentage viability was determined with respect to DMSO treated
cells.
31. Apoptosis and cell death assays: For flow cytometry study MCF7 cells were cultured in a six-well
culture plates, incubated with concentrations 100 nM - 3 µM of 5FAmPyrr for 72 h. Cells were
harvested, washed with PBS and incubated with Propidium Iodide (5µg/mL) for 20 min at 4 C.
Samples were acquired using FACS verse (BD Biosciences) for cell death using flow cytometry.
Cells with DMSO treatment were taken as control.
32. Western Blotting: 1.25105 MCF7 cells were seeded into each well of six well plates and exposed
to different concentration of 5FAmPyrr (0.1, 0.3, 1, 3, and 10 µM) for 72 h. Following incubation,
cells were washed with cold PBS and lysed using 2XSDS lysis buffer (0.5 MTris-HCl, pH 6.8,
glycerol, 10% (w/v) SDS). Cell lysates were sonicated once for 10 sec. at 30% amplitude. Protein
estimation was done by BCA method and 20-25 µg protein samples were resolved on 12%
polyacrylamide SDS gel and electrophoretically transferred to PVDF membranes. Membranes were
then blocked with 5% BSA in TBST buffer for 1 h at room temperature, incubated with primary
antibodies (1:500 and 1:1000) for overnight, and subsequently with the conjugated secondary
antibody (1:5000) for 1 h at room temperature. Proteins were detected by enhanced
chemiluminescence (Bio-Rad).
33. Colony Formation Assay: MCF7 cells were seeded in six well plates at a density of 500 cells per
well. Following adherence of cells left for overnight and next day cells were treated with
5FAmPyrr. After every 72 h media was changed and cells were treated with 5FAmPyrr and the
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experiment lasts for 21 days. After the colony were fully grown, cells were washed with PBS, fixed
with 3.7% formaldehyde for 20 min, washed with PBS and then stained using 0.4% crystal violet
followed by rinsing the wells with PBS 4 to 5 times. Colonies were counted using Image J software.
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