Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis via Activation of the Mitochondrial Pathway and Exhibit Antitumor Efficacy in Osteosarcoma Xenograft Nude Mice Model

Article abstract of DOI:10.1021/acschembio.6b01007

Mitochondria impart a crucial role in the regulation of programmed cell death and reactive oxygen species (ROS) generation, besides serving as a primary energy source. Mitochondria appeared as an important target for the therapy of cancer due to their significant contribution to cell survival and death. Here, we report the design and synthesis of a novel series of triazole-piperazine hybrids as potent anticancer agents. MCS-5 emerged as an excellent anticancer agent which showed better anticancer activity than the standard drug doxorubicin in in vitro and in vivo studies. MCS-5 displayed an IC₅₀ value of 1.92 μM and induced apoptosis in Cal72 (human osteosarcoma cell line) cells by targeting the mitochondrial pathway. This compound arrested the G2/M phase of the cell cycle and induced ROS production and mitochondrial potential collapse in Cal72 cells. MCS-5 displayed excellent anticancer activity in the Cal72 xenograft nude mice model, where it significantly reduced tumor progression, leading to enhanced life span in treated animals compared to control and doxorubicin treated animals without exerting noticeable toxicity. In addition, a 2DG optical probe guided study clearly evoked that MCS-5 remarkably reduced tumor metastasis in the Cal72 xenograft nude mice model. These results indicate that MCS-5 appeared as a novel chemical entity which is endowed with excellent in vitro as well as in vivo anticancer activity and may contribute significantly to the management of cancer in the future.

Full text of DOI:10.1021/acschembio.6b01007

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Article
Novel Triazole-Piperazine Hybrid Molecules Induce Apoptosis
via Activation of the Mitochondrial Pathway and Exhibit Anti-
tumor Efficacy in Osteosarcoma Xenograft Nude Mice Model
Chandra Bhushan Mishra, Raj Kumar Mongre, Shikha Kumari, Dong Kee Jeong, and Manisha Tiwari
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01007 • Publication Date (Web): 13 Jan 2017
Downloaded from http://pubs.acs.org on January 15, 2017
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Novel TriazoleꢀPiperazine Hybrid Molecules Induce Apoptosis via Activation of the
Mitochondrial Pathway and Exhibit Antitumor Efficacy in Osteosarcoma Xenograft Nude
Mice Model
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Chandra Bhushan Mishra‡1, Raj Kumar Mongre‡2, Shikha Kumari‡1, Dong Kee Jeong2*, Manisha Tiwari2*
1Bioꢀorganic Chemistry Laboratory, Dr. B.R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi
110007, India
2Laboratory of Animal Genetic Engineering and Stem Cell Biology, Department of Animal Biotechnology and
Advance Next Generation Convergence, Faculty of Biotechnology, Jeju National University, JejuꢀDo, Republic of
Korea
*Correspondence
Dr. Manisha Tiwari
Dr. B. R. Ambedkar Center for Biomedical
Research, University of Delhi, Delhi 110007, India
Eꢀmail: mtiwari07@gmail.com
Professor Dong Kee Jeong
Department of Animal Biotechnology and Advance Next Generation Convergence,
Faculty of Biotechnology, Jeju National University, Araꢀ1 Dong, Jejuꢀcity, JejuꢀDo 690ꢀ756,
Republic of Korea
Eꢀmail: dkjeong@jejunu.ac.kr
‡These authors have equal contribution.
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Abstract
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Mitochondria imparts crucial role in the regulation of programmed cell death, reactive oxygen
species (ROS) generation besides serving as a primary energy source. Mitochondria appeared as
an important target for therapy of cancer due to its significant contribution in cell survival and
death. Here, we report the design and synthesis of a novel series of triazoleꢀpiperazine hybrids as
potent anticancer agents. MCSꢀ5 emerged as an excellent anticancer agent which showed better
anticancer activity than standard drug Doxorubicin inꢀvitro and inꢀvivo studies. MCS5 displayed
an IC50 value of 1.92 ꢁM and induced apoptosis in Cal72 (human osteosarcoma cell line) cells by
targeting mitochondrial pathway. This compound arrested G2/M phase of cell cycle, induced
ROS production and mitochondrial potential collapse in Cal72 cells. MCSꢀ5 displayed excellent
anticancer activity in Cal72 Xenograft nude mice model where, it significantly reduced tumor
progression leading to enhanced life span in treated animals compared to control and
Doxorubicin treated animals without exerting noticeable toxicity. In addition, 2DG optical probe
guided study clearly evokes that MCSꢀ5 remarkably reduced tumor metastasis in Cal72
Xenograft nude mice model. These results indicate that MCSꢀ5 appeared as a novel chemical
entity which is endowed with excellent inꢀvitro as well as inꢀvivo anticancer activity and may
contribute significantly for the management of cancer in the future.
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Introduction
Cancer is a foremost reason of death worldwide and exerts a major health problem globally. 8.2
million deaths due to cancer in 2012 have been reported, which represents 13% of all deaths and
12.7 million new cases per year are being stated worldwide.1 Cancer is a result of uncontrolled
growth of abnormal cells and characterized by multiple structural, molecular and behavioral
features. Out of all cancers types, liver, lung, bone, stomach, colon and breast cancer are
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predominantly responsible for cancer mortality.2 Treatment of cancer relies heavily on
conventional therapies such as chemotherapy, radiotherapy, surgical removal as well as
immunotherapy.3ꢀ4Since long time, widespread efforts have been done to counteract
malignancies and in result various anticancer agents have been discovered.5ꢀ8 However, clinically
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running anticancer drugs exert dreadful side effects, including cytotoxicity against normal cells.9ꢀ
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Osteosarcomas, the utmost communal malignancy of bone which affects 900 people per year in
the United States.11 It represents 56% of malignant bone tumors in children and nearby 3.4% of
all childhood malignancies.12 Currently, most effective chemotherapeutic agents for the treatment
of osteosarcoma are Cisplatin, Doxorubicin and Methotrexate.13 In spite of this, numerous new
biological approaches to overcome osteosarcoma are being investigated which appear as fruitful
strategies to prevent osteosarcoma and its metastasis.14ꢀ16 Although several improvements in the
management of osteosarcoma to date, quiet seems as a challenge because the overall survival
rate of osteosarcoma patients has remained constant for over two decades therefore, new
treatment approaches are necessarily required.
The mitochondria is the most important organelle concerned with the cellular bioenergetic and
biosynthetic changes associated with cancer.17ꢀ18 Mitochondrial alterations contribute to the
invasive and metastatic properties in most of typical tumors. Therefore, the mitochondria is
considered as a valuable target to induce apoptosis in cancer cells and numerous anticancer drugs
follows the mitochondrial pathway to kill cancer cells.19ꢀ20 The molecular hybridization of
biologically active molecules is a powerful tool for drug discovery and appeared as a novel
strategy to identify potential drug molecules against various diseases, including cancer.21 Hybrid
drug molecules may serve as a combination therapy in single multifunctional agent, which would
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be more potent than conventional treatment approach.22 Therefore, by adopting a molecule
hybridization drug discovery approach, we aimed to design triazoleꢀpiperazine hybrid molecules
as potent anticancer agents.
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1,2,4ꢀtriazole is an important class of heterocycle which has been widely used to develop a large
variety of bioactive molecules such as antiꢀbacterial, antiꢀfungal, antiꢀdepressant, antiꢀoxidant,
antiꢀinflammatory, antiꢀHIV and antiꢀtubercular agents.23ꢀ26 1,2,4ꢀtriazoles have also a successful
history for the development of potent anticancer agent.27ꢀ29 Various derivatives of 1,2,4ꢀtriazole
derivatives were synthesized and tested for their anticancer potential in various inꢀvitro as well as
inꢀvivo models (Fig. 1). For example, 4ꢀaminoꢀ5ꢀ(5ꢀphenylthieneꢀ2ꢀyl)ꢀ2,4ꢀdihydroꢀ3Hꢀ1,2,4ꢀ
triazoleꢀ3ꢀthione 30 (1) displayed excellent cytotoxicity against thymocytes with an IC50 value of
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0.012 µM and 4ꢀaminoꢀ5ꢀmercaptoꢀ3ꢀ(2ꢀchlorophenyl)ꢀ1,2,4ꢀtriazole (2) displayed admirable
antiproliferative activity in Ehrlich Ascites Carcinoma mice. Romagnoli et al. also synthesized a
series of 1,5ꢀdisubstituted 1,2,4ꢀtriazole as potent anticancer agents.32 Compound 3 showed
excellent cytotoxicity against HeLa as well as Jurkat cells by inducing apoptosis.
On the other hand the piperazine moiety has also been used constantly to develop various potent
anticancer agents and several piperazine containing molecules have shown admirable anticancer
activities in both preꢀclinical and clinical trials (Fig. 1).33ꢀ34 The successful anticancer drug
Imatinib 35 (4) also contains piperazine moiety and this drug is widely used to treat various types
of cancer including chronic myelogenous leukemia. Additionally, a novel piperazine derivative 5
also (LYGꢀ202) displayed potent antiꢀcancer activity in vitro and in vivo by inducing apoptosis.36
In continuation of our anticancer drug development agenda37ꢀ39 and enthused by diverse
anticancer properties of the triazole and piperazine moieties, we have designed a novel series of
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triazoleꢀpiperazine hybrid molecules to appraise their inꢀvitro and inꢀvivo anticancer activity.
This molecular hybridization might generate potential anticancer agent because both moieties
(triazole and piperazine) are well studied to produce anticancer activity. Synthesized derivatives
have been evaluated for their antiꢀproliferative activity against six types of cancer cell lines and
their structure activity relationship has been also studied. The most active novel derivative MCSꢀ
5 showed remarkable cytotoxicity against Cal72 (human osteosarcoma cell line) without
displaying cytotoxicity in normal cells. Therefore, MCSꢀ5 was selected for further studies
concerning its mechanism of action which governs apoptosis in Cal72 cells. Our studies have
shown that MCSꢀ5 activate the mitochondrial pathway to induce apoptosis process. MCSꢀ5 has
also exhibited noteworthy inꢀvivo antitumor activity in a xenograft mice model of human
osteosarcoma. Additionally, this novel compound successfully reduced tumor metastasis in 2DG
optical probe guided osteosarcomaꢀxenograft nude mice model. Our efforts have provided a
novel chemical entity (MCSꢀ5) with commendable anticancer activity which may help in the
management of osteosarcoma in the future.
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RESULTS AND DISCUSSION
Synthesis of triazoleꢀpiperazine hybrids. Designed novel hybrid molecules (5ꢀ16) have been
synthesized in four steps which are depicted in scheme 1 (Fig. 2). In short, commercially
available benzhydrazide 1 was reacted with carbon disulfide (CS2) under reflux condition in
dimethylformamide (DMF) to afford oxadiazole intermediate 2. After that, compound 2 was
reflux with hydrazine hydrate in 1,4ꢀdioxane to yield aminotriazole intermediate 3. Next, the
amino group of aminotriazole (3) was reacted with furanꢀ2ꢀ carboxyaldehyde in glacial acetic
acid under reflux conditions, which led to the formation of furoylꢀtriazole derivative 4 in high
yield. Finally, substituted piperazines were attached to triazole ring with a methylene linker by
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performing the Mannich reaction providing the Mannich bases 5ꢀ16. Synthesized compounds
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were fully characterized by 1HNMR, 13C NMR, Mass spectroscopy and elemental analysis.
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Compound
Number
RꢀGroup
Compound
Number
RꢀGroup
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14
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NO2
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Scheme 1: Reagents and conditions: A. CS2, DMF, reflux, 3ꢀ4 h; B. NH2NH2, 1,4 dioxane, reflux, 12ꢀ14 h; C.
Furanꢀ2ꢀ carboxyaldehyde, AcOH, reflux, 12ꢀ14 h; D. Substituted piperazine, formaldehyde, MeOH (reflux, 12ꢀ14
h)/ DMF(RT, 12ꢀ15 h).
Inꢀvitro cytotoxicity studies and structure activity relationship (SAR): Inꢀvitro cytotoxic
potential of synthesized hybrids 5ꢀ16 were assessed against six types of human cancer cell lines;
Cal72, HepGꢀ2, SaOSꢀ2, A549, MCFꢀ7 and Hela by using MTT assay. The results are presented
in terms of IC50 values as given in table 1. The structure activity relationship (SAR) has been
explicated on the basis of observed results in MTT assay. It was observed that phenyl
substitution (compound 5) of this core was completely ineffective against all six cell lines. In
electronegative element substituted phenyl derivatives, chloro substituted phenyl derivative
(compound 7) appeared more effective as compared to fluoro substituted derivative (compound
6). The chlorophenyl substituted derivative (compound 7) was effective in producing
cytotoxicity against Cal72 and MCF cell line even as fluorophenyl substituted derivative only
showed satisfactory cytotoxicity towards the Hela cell line as compared to Doxorubicin.
Ethanone group substitution on the phenyl ring (compound 8) also did not exert satisfactory
cytotoxicity against tested cell lines. However, this compound showed acceptable cytotoxicity
against SaOSꢀ2 cell line with an IC50 value of 15.76 µM which was almost similar to
Doxorubicin. The introduction of NO2 (strong electron withdrawing group) on the phenyl ring
(compound 9) displayed venerable cytotoxicity against the Cal72 cell line, while it did not show
promising cytotoxicity towards other cell lines as compared to Doxorubicin. Benzoyl group
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substitution (compound 10, MCSꢀ5) on this core made it a successful derivative and this
compound showed excellent cytotoxicity against all tested six cancer cell lines. This compound
displayed prominent cytotoxicity against Cal72, HepGꢀ2, SaoSꢀ2, MCFꢀ7 as well as Hela cell
line, where this compound showed much better IC50 value as compared to standard drug
Doxorubicin. Electron donor methyl substitution on the phenyl ring (compound 11) was unable
to produce significant cytotoxicity against all tested six cancer cell lines. However, methoxy
substituted phenyl derivative (compound 12) exhibited promising cytotoxicity towards MCFꢀ7
and Hela cell line as compared to Doxorubicin. Pyrimidine containing derivative (compound 13)
also did not produce a satisfactory cytotoxic effect against tested cancer cell lines. However,
flexible benzyl moiety bearing derivative 14 showed promising cytotoxic effect against Cal72,
HepGꢀ2, SaOSꢀ2 and Hela cell line as compared to Doxorubicin. The insertion of one more
phenyl ring to the benzyl derivative led to diphenyl derivative 15 which showed significant
cytotoxicity towards Cal72, SaoSꢀ2, MCFꢀ7 and Hela cell lines, where its efficacy was almost
similar to Doxorubicin. Fluoro substitution at pꢀposition of the diphenyl ring (compound 16) also
produced a remarkable cytotoxic effect against Cal72, HepGꢀ2, MCFꢀ7 and Hela cell line, where
it appeared more potent than Doxorubicin.
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Inꢀvitro cytotoxicity of synthesized derivatives revealed that most of the compounds displayed a
notable anticancer effect against tested cancer cell lines. Phase contrast microscopy images have
been taken to elucidate morphological changes associated with programmed cell death (Fig. 2a).
However, MCSꢀ5 emerged as a most effective derivative in whole series and it showed
admirable cytotoxicity against Cal72 with an IC50 value 1.97 µM in comparison with standard
drug Doxorubicin which possesses an IC50 value 16.43 µM (Fig. 2b). Moreover, MCSꢀ5 did not
show significant cytotoxicity against normal human bone marrow (hBM) cell line and it also
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demonstrated comparatively higher cell viability than Doxorubicin after 24 hours incubation
(Fig. 2c). Additionally, time course studies of MCSꢀ5 have been also performed against Cal72
cell line where, it gradually reduced cell viability up to 48 hours (Fig. 2d). Inspired with these
results, we have chosen MCSꢀ5 to proceed further for advance anticancer studies to confirm its
potency as effective anticancer agent.
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Table 1: Inꢀvitro cytotoxic effect of novel synthesized compounds (5ꢀ16) a
IC50 (µM)b
Test
Cal72
HepGꢀ2
SaOSꢀ2
A549
MCFꢀ7
Hela
compound
5
44.73 ± 38.64
± 32.31±
5.908
27.623±
2.109
23.20±
1.225
41.07±
3.645
1.902
0.816
17.20±
0.130
19.95±
0.022
39.32±
0.725
22.36±
1.556
42.13±
1.902
13.25±
0.380
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7
16.04±
0.227
56.23±
0.492
51.87±
0.537
32.15±
0.529
19.25±
0.596
32.78±
0.759
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9
18.75±
0.183
52.25±
0.435
15.76±
0.115
25.468±
0.602
34.28±
0.378
38.25±
0.642
3.93±
0.169
19.95±
0.663
16.65±
0.733
30.29±
0.943
35.39±
0.482
22.64±
0.466
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10 (MCSꢀ5)
1.97 ±
0.090
7.64±
0.509
9.99±
0.681
13.58±
6.34±
0.465
10.24±
0.442
0.779
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9
23.56±
0.345
19.02±
0.350
16.52±
0.328
26.81±
0.623
27.51±
0.104
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27.31±
0.160
18.14±
0.252
81.86±
36.234
19.91±
0.405
13.42±
0.170
10.21±
0.520
11.26±
1.234
22.52±
0.240
75.80±
0.600
39.94±
0.723
40.25±
1.418
52.36±
0.378
29.37±
0.352
16.18±
0.334
9.784±
0.285
8.678±
0.372
12.54±
0.755
16.25±
0.111
12.52±
0.200
16.39±
0.352
19.65±
0.226
15.07±
0.755
21.45±
1.211
14.27±
1.139
15.67±
0.236
4.702±
0.432
10.75±
0.837
24.78±
0.959
18.39±
0.459
12.73±
1.100
9.43±
0.429
Doxorubicinc 16.43±
17.31±
0.136
15.23±
0.220
8.27±
0.164
21.90±
0.432
14.52±
0.654
0.352
aThe data denoted the mean of three experiments in triplicate and were value represented as mean.
b The IC50 denotes the concentration at which 50% of cells survived.
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C Used as positive control
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MCSꢀ5 induces cell apoptosis. The generation of apoptosis has been regarded as a praiseworthy
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strategy for therapy of cancer and nearly all anticancer drugs kill tumor cells by inducing
apoptotic processes.40ꢀ41 Therefore, apoptotic effect of MCSꢀ5 was assessed by Hoechst 33342
staining and propidium iodide (PI)/AnnexinꢀV FITC double staining method in Cal72 cells.
Phase contrast microscopy images have been taken to elucidate morphological alterations in the
apoptotic cells by both staining methods as shown in Fig 3a. In Hoechst 33342 staining, MCSꢀ5
treated cells showed distinctive apoptotic characteristics, including chromatin condensation,
apoptotic body and nuclear fragmentation as compared to control and Doxorubicin (10 µM).
Whereas, untreated cells displayed uniformly dispersed chromatin and stained meager
homogeneous blue. Further, apoptosis induced by MCSꢀ5 was also examined by microscopic
study and flow cytometer using PI/AnnexinꢀV FITC double staining method. Phase contrast
microscopy of PI/AnnexinꢀV FITC double staining MCSꢀ5 treated cells displayed significant
early and late apoptosis as compared to untreated cells (Fig. 3b). Flow cytometry studies
markedly indicate that MCSꢀ5 showed superior apoptosis against Cal72 cells as compared to
Doxorubicin (Fig. 3c). The result of this study indicates that MCSꢀ5 showed significant (P<0.05)
apoptosis against Cal72 cells at both concentrations (5 µM and 10 µM). MCSꢀ5 showed 14.82%
and 31.40% apoptosis at 5 µM and 10 µM concentration, respectively, while Doxorubicin
displayed only 7.00% apoptosis (Fig. 3c, 3d).
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Effect of MCSꢀ5 on Cell cycle arresting. Spontaneous cell divisions of cancer cells occur due
to dysregulation of cell cycle, therefore inhibition of the cell cycle plays an important role in
treating proliferative disease like cancer.42 Most of the anticancer drugs disturbs the proliferation
cycle of cancer cells by blocking cell cycle events which activate apoptosis.43 Cell cycle arresting
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potential of MCSꢀ5 was assessed in Cal72 cells by a flow cytometric analysis. Results showed
that MCSꢀ5 effectively arrested the cell cycle in G2/M phase (Fig. 2e, 2f) and it has significantly
raised Cal72 cell population in G2/M phase as compared to untreated cells. Upon MCSꢀ5
treatment, the percentage of cells in G2/M phase were 25.47% and 21.65% at 5 and 10 µM,
respectively, while, untreated cells displayed 10.79% cells in G2/M phase.
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MCSꢀ5 activates intrinsic apoptotic pathway: In order to explore the detailed mechanism of
action of MCSꢀ5 which directs apoptosis in Cal72, various associated proꢀapoptotic and antiꢀ
apoptotic protein expression levels were checked by immunocytochemistry (ICC), mRNA
quantification as well as immunoblotting assay. Bclꢀ2 act as a suppressor of apoptosis by
blocking migration of cytochrome c, while proꢀapoptotic members such as Bax work as
promoters of apoptosis.44 Our results revealed that MCSꢀ5 treatment significantly (P<0.05)
reduced antiꢀapoptotic protein Bclꢀ2 and increased proꢀapoptotic Bax protein at transcriptional as
well as translational level (Fig. 4aꢀg). In qualitative ICC analysis, down regulation of Bclꢀ2 and
upꢀregulation of Bax was also noticed (Fig.4c). Next, we intended to analyze migration of
cytochrome c from mitochondria to cytosol, which is a hallmark marker of apoptosis mediated
through intrinsic pathway. qꢀPCR, Immunofluorescence, and Immunoblot studies revealed that
treatment of MCSꢀ5 markedly decreases mitochondrial cytochrome c that led to increased levels
of cytochrome c in cytosol. (Fig. 4hꢀk). Cytochrome c binds with apoptotic activator factorꢀ
1(APAFꢀ1) and thereby, forming apoptosome which activates caspaseꢀ9 followed by activation
of caspaseꢀ3 leading to precede apoptosis through intrinsic pathway.45 Therefore, we made an
attempt to explicate the effect of MCSꢀ5 on APAFꢀ1, caspaseꢀ3 and caspaseꢀ9 regulation by ICC,
qPCR and western blot analysis. Interestingly, our findings clearly demonstrated that treatment
of MCSꢀ5 significantly (P<0.05) up regulate APAFꢀ1, caspaseꢀ9 and capaseꢀ3 at both
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transcriptional and translational level, which further confirmed their involvement in the intrinsic
pathway to induce apoptosis by formation of apoptosome complex (Fig. 5aꢀ5i). These proteins
up regulation have been also visualized in ICC studies where strong fluorescence intensity was
seen in MCSꢀ5 treated cells as compared to untreated cells (Fig.5c, 5g). After that, we have
inspected impact of MCSꢀ5 on p53 activation; p53 is the most extensively studied tumor
suppressor which mediates apoptosis primarily through the intrinsic apoptotic pathway and may
also transꢀactivate numerous components of the apoptotic effectors machinery.46 Consequently,
our results showed that after 24 hours treatment MCSꢀ5 remarkably increased p53 expression in
Cal72 cells as compared to untreated cells, which was quantified by qPCR analysis and also
captured in immunofluorescence staining (Fig. 6a, 6b). Over expression of p53 was also
appeared in western blot analysis as compared to untreated cells and Doxorubicin treated cells
(Fig. 6c, 6d). p53 activation is escorted by down regulation of cꢀMYC expression and both are
considered as a decisive factor of cell to undergo apoptosis.47 Thus, activation of p53 by MCSꢀ5
stimulated apoptotic effectors machinery, which led to induce apoptosis via intrinsic pathway.
Overall, all these studies evidently supports, triggering of the intrinsic pathway of apoptosis by
MCSꢀ5 which directs apoptosis in Cal72 cells.
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MCSꢀ5 down regulates NFꢀκB and ILꢀ6 level. Suppression of NFꢀκB activity plays important
role in stimulation of apoptotic process and it is a key decisive factor in the selection of drugs for
therapy of cancer.48 Moreover, interleukinꢀ6 (ILꢀ6) is an attractive target for cancer therapy and
its inhibition encourages apoptosis progression in cancer cells. ILꢀ6 is regulated by the
transcription factor NFꢀκB and built a significant link between inflammation initiation and
progression of angiogenesis.49 ICC studies visibly pointed out the down regulation of NFꢀκB and
ILꢀ6 after MCSꢀ5 treatment (Fig. 6j, 6k). Addition to this, MCSꢀ5 treatment significantly
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reduced mꢀRNA level of NFꢀκB and ILꢀ6 as compared to untreated and Doxorubicin treated cells
(Fig. 6e, 6f). Furthermore, western blot analysis also certified reduction of NFꢀκB and ILꢀ6 at the
proteomic level in MCSꢀ5 treated cells as compared to untreated cells (Fig. 6gꢀi). Thus, our
results indicate that MCSꢀ5 impressively down regulates NFꢀκB and ILꢀ6 resulting to collapse
mitochondrial integrity and initiate apoptosis in Cal72 cells.
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MCSꢀ5 induce ROS level enhancement in Cal72 cells: Reactive oxygen species (ROS) are
generated as byꢀproducts of respiration and their levels are very high during cell apoptosis.50
Mitochondria is the major creator of ROS in mammalian cells, and its dysfunction increases high
ROS levels, which led to induce cell apoptosis process.51 Hence, we assessed intercellular ROS
levels using DCFDA dye by FACS and phase contrast microscopy. FACS analysis showed that
MCSꢀ5 increases ROS levels in a concentration dependent manner where, it showed 38 %, 58 %
and 62 % ROS induced cells at 5, 10 and 15 µM concentration, respectively (Fig. 7c, 7d).
Enhanced fluorescence intensity after treatment of MCSꢀ5 as compared to untreated cells in
phase contrast microscopy further validate ROS inducing capability of MCSꢀ5 (Fig. 7e). Both
studies noticeably indicate that MCSꢀ5 induced ROS production in Cal72 cells, which impart
progression of cell apoptosis through the mitochondria.
MCSꢀ5 induces depolarization and loss of mitochondrial transmembrane potential (ꢁΨm).
A decrease of mitochondrial transmembrane potential (ꢁΨm) is well acknowledged during an
early event of apoptosis and considered as a hallmark of cells, which proceeding apoptosis
through mitochondrial insult.52 We have measured loss of ꢁΨm in MCSꢀ5 treated Cal72 cells
using JCꢀ1 staining by flow cytometer and ꢁΨm was also pictured by phase contrast microscopy.
Microscopic results showed that MCSꢀ5 induced strong green fluorescence and reduced red
fluorescence as compared to untreated cells and Doxorubicin treated cells in Cal72 cells, which
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suggests potential deduction of ꢁΨm by this compound at both concentrations (5 µM and 10 µM)
(Fig. 7b). Consequently, flow cytometry result also showed a loss of mitochondrial potential in
MCSꢀ5 treated cell as compared to untreated cell. At 5 µM and 10 µM concentrations it showed
18.17 % and 34.67 % cell population, which emit green fluorescence, respectively, while
untreated cells displayed 1.97 % green fluorescence emitting cells (Fig 7a). In this study
Doxorubicin at 5 µM and 10 µM have been shown 14.83 % and 12.56 % cell population,
respectively which produce green fluorescence. Hence, MCSꢀ5 strongly induced mitochondrial
potential deficit in Cal72 cells as compared to Doxorubicin (Fig. 7a, b). These studies suggest
that MCSꢀ5 significantly induced mitochondrial potential collapse in Cal72 cells, which lead to
activate cell apoptosis.
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MCSꢀ5 exhibits tumoricidal effects in Cal72 xenograft nude mice model. The inꢀvivo
anticancer action of MCSꢀ5 was judged in osteosarcoma xenograft animal model using
immunodeficient nude mouse at the dose of 5 and 10 mg/kg body weight (bwt). A successfully
developed model was treated with both doses for 45 days and Doxorubicin (10 mg/kg, bwt) was
used as a positive control. Firstly, routine investigation of MCSꢀ5 efficacy on survivability of
mice has been analyzed by Logꢀrank test. The KaplanꢀMeier curves demonstrate that orthotopic
tumorꢀbearing mice treated with MCSꢀ5 at 10 mg/kg/day exhibited significantly enhanced
survival rate than 5 mg/kg/day MCSꢀ5 or 10 mg/kg/day Doxorubicin or vehicleꢀtreated controls
(Fig. 8a, b). The results point out that MCSꢀ5 increased almost double life span (survival rate=
63 %) of animals as compared to untreated animals (survival rate= 32 %). Additionally, MCSꢀ5
at the dose 10 mg/kg significantly increased life span of tumor bearing mice 32 to 62 %
compared to Doxorubicin (32 to 52 %). Furthermore, MCSꢀ5 (10 mg/kg/day) significantly
attenuated tumor size as compared to control animals (Fig. 8c). To validate our studies, tumors
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were removed and weighed and it was witnessed that MCSꢀ5 (10 mg/kg) significantly (P<0.01)
reduced tumor weight as compared to Doxorubicin (Fig. 8d). Also, MCSꢀ5 (10 mg/kg)
significantly reduced tumor volume over time as compared to untreated and Doxorubicin
(10mg/kg) treated animals (Fig. 8e). These results clearly evoke the efficacy of MCSꢀ5 to
conquer Cal72 induced osteosarcoma nude mice model.
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MCSꢀ5 inhibits tumor metastasis in Cal72 xenograft nude mice model. To evaluate whether
the robust antiꢀneoplastic efficacy of MCSꢀ5 observed inꢀvivo live tumor resection and
dissemination, we established optical probe guided tumor imaging model. Infraꢀred (IR) dyeꢀ
800CWꢀ2ꢀdeoxyꢀDꢀglucose (2DG) wavelengthꢀbased whole body images were captured to
visualize the effect of MCSꢀ5 to slow down tumor metastases in MCSꢀ5 treated animals (Fig. 9aꢀ
c). 2DG is extensively used tracer to mark out tumor progression and metastases because the rate
of glycolysis in tumor cells and metastasized body organs is significantly enhance.53 2DG optical
probe guided fluorescence imaging studies displayed that MCSꢀ5 at the dose of 10 mg/kg/day
successfully stopped tumor metastasis in tumor bearing animals. Tumor metastasis was clearly
visible in untreated tumor bearing animals while MCSꢀ5 treated animals did not show any
metastasized tumor. Thus, we can conclude that MCSꢀ5 rewardingly abridged tumor and its
metastasis in the osteosarcoma nude mice model.
Toxicity evaluation. Toxicity and sideꢀeffects of the MCSꢀ5 were assessed by monitoring the
relative body weight, behavioral appearance, intake of water and food of animals on a daily
basis. Analysis showed that there was no statistically significant difference seen in body weight
(assessed as a percentage of the initial body weight) between the MCSꢀ5 treatment group and the
control (normal mice) observed which suggests that MCSꢀ5 treatment was not overtly toxic for
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the mice. Moreover, no behavioral changes were observed, including any alteration in food and
water intake during whole experimental period. Although, a significantly reduction the body
weight was observed in Doxorubicin treated group (Fig. 9d).
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Conclusion
In conclusion, synthesized novel triazoleꢀpiperazine hybrid molecules displayed moderate to
excellent cytotoxicity against six cell lines. MCSꢀ5 appeared as a most active derivative and it
displayed better cytotoxicity as compared to Doxorubicin in Cal72 cells. This compound showed
magnificent apoptotic potential in Cal72 cells and arrested cells in the G2/M phase of the cell
cycle. Mechanistic studies revealed that MCSꢀ5 activate the mitochondrial pathway to induce
apoptosis in Cal72 cells where it up regulates/down regulates a number of proteins which are
involved in mitochondrial associated intrinsic pathway of apoptosis. Further, MCSꢀ5 excellently
induced ROS generation and loss of ꢁΨm in Cal72 cells. Furthermore, this derivative displayed
remarkable tumoricidal effect in Cal72ꢀxenograft nude mice model without producing significant
toxicity in animals. In addition to this MCSꢀ5 significantly stop tumor metastasis in the
osteosarcoma nude mice model. Thus, investigation provided a potent anticancer agent MCSꢀ5
which showed the excellent anticancer effect inꢀvitro and inꢀvivo models by triggering
mitochondrial pathway of apoptosis.
METHODS
Chemistry. All the commercial starting material and organic solvents were procured from TCI
Chemicals (Tokyo, Japan), Sigma Aldrich (St. Louis, MO, USA), Alfa Aesar (Massachusetts,
U.S States), and Merck (Darmstadt, Germany). The solvents which were used for reaction
medium were dried by standard methods. 1H and 13C nuclear magnetic resonance (NMR) spectra
were acquired by high resolution Jeol NMR spectrophotometer (USA) at 400 and 100 MHz,
respectively. Chemical shifts are reported in parts per million (ppm) relative to the solvent peak
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or tetramethylsilane (TMS). The coupling constants (J) are reported in hertz (Hz) and the
splitting patterns are described by using the following abbreviations: s, (singlet); d, (doublet); t,
(triplet); m, (multiplet); brs, (broad singlet); dd, (double doublet). LC/MS data were recorded on
Agilent 6310 Ion trap LC/MS system. Melting points were determined in open capillaries using
model KSPII, KRUSS, (Germany) and elemental analysis (C, H and N) was carried on
Elementar analysensysteme. Purity of the target compounds was determined by on a Shimadzu
HPLC system (Kyoto, Japan) coupled with a photodiode array detector (PDA) and Cꢀ18 column.
All final compounds reported here showed more than 95% HPLC purity
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Procedure for the synthesis of 5ꢀphenylꢀ1,3,4ꢀoxadiazoleꢀ2(3H)ꢀthione (2)
Benzoic hydrazide (1 Molar) and carbon disulphide (3 Molar) were reacted in dried DMF at
room temperature (RT) for 20 minutes. Then after, the reaction mixture was heated at a reflux
temperature for 3ꢀ4 hours. Upon completion, the reaction mixture was poured into iceꢀcold water
to obtain white colored precipitate. The obtained product was washed twice with water and
petroleum ether to yield pure compound 2.
5ꢀphenylꢀ1,3,4ꢀoxadiazoleꢀ2(3H)ꢀthione (2)
1
Yield: 95%; White solid; mp: 207ꢀ209˚C; H NMR (DMSOꢀd6, 400 MHz): δ 7.58ꢀ7.56 (m, 3H),
7.85ꢀ7.84 (m, 2H, Ar), 14.81 (brs, 1H, SH); LCꢀMS: m/z, 179 (M+1).
Procedure for the synthesis of 4ꢀaminoꢀ3ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (3)
A suspension of intermediate 2 (1 Molar) and hydrazine hydrate (50 Molar), in 1,4ꢀdioxane was
refluxed for 12ꢀ14 hours. The color of the reaction mixture was changed into green, with the
evolution of hydrogen sulfide gas. After completion, the reaction mixture was cooled on ice and
acidified with conc. hydrochloric acid. The product obtained was washed twice with tap water and
then with petroleum ether to yield pure compound 3.
4ꢀaminoꢀ3ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (3)
1
Yield: 88%; White solid; mp: 206ꢀ208˚C; H NMR (DMSOꢀd6, 400 MHz): δ 5.74 (s, 2H, NH2),
7.49ꢀ7.47 (m, 3H, Ar), 7.96ꢀ7.94 (m, 2H, Ar), 13.89 (brs, 1H, SH); LCꢀMS: m/z, 193 (M+1).
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Procedure for the synthesis of 4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ
5(4H)ꢀthione (4)
2ꢀFuraldehyde and intermediate 3 were taken in equimolar ratio and refluxed for 12ꢀ14 hours.
Upon reaction completion, the reaction mixture was poured into iceꢀcold water to crude product.
The obtained product was filtered and washed with water thrice. Then after, it was crystallized
from chloroform/methanol mixture to afford pure compound 4.
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4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (4)
Yield: 74%; Creamish solid; mp: 182ꢀ184˚C; 1H NMR (DMSOꢀd6, 400 MHz): δ 6.78ꢀ6.76 (m, 1H,
Ar), 7.37 (d, 1H, Ar, J=4.2 Hz), 7.52ꢀ7.50 (m, 3H, Ar), 7.86ꢀ7.81 (m, 2H, Ar), 8.06 (m, 1H, Ar),
9.54 (s, 1H, CH), 14.19 (s, 1H, SH); LCꢀMS: m/z, 271 (M+1).
General Procedure for the synthesis of 4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1ꢀ
(substitutedꢀpiperazinꢀ1ꢀyl)methyl)ꢀ1Hꢀ1,2,4ꢀtriazoleꢀ5(4H)ꢀthione
The equimolar molar ratio of substituted piperazine derivative and intermediate 4 was reacted
either in methanol (anhydrous) under reflux condition for 12ꢀ14 hours or DMF at room
temperature for 12ꢀ15 hours in the presence of formaldehyde (equimolar ratio). The reaction
mixture was diluted with water to give precipitation of the desired compound. The crude products
were filtered and purified by column chromatography using chloroform/methanol (97:03) as
eluent.
4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1ꢀ((4ꢀphenylpiperazinꢀ1ꢀyl)methyl)ꢀ1Hꢀ1,2,4ꢀ
triazoleꢀ5(4H)ꢀthione (5)
1
Yield: 69%; Creamish solid; mp: 68ꢀ70˚C; H NMR (DMSOꢀd6, 400 MHz): δ 2.89ꢀ2.93 (m, 4H,
piperazine), 3.10ꢀ3.13 (m, 4H, piperazine), 5.24 (s, 2H, CH2), 6.72ꢀ6.79 (m, 2H, Ar), 6.88 (d,
2H, Ar, J=8.2 Hz), 7.16 (t, 2H, Ar, J=8.2Hz), 7.39 (d, 1H, Ar, J=3.6Hz), 7.50ꢀ7.56 (m, 3H, Ar),
7.84ꢀ7.88 (m, 2H, Ar), 8.07 (s, 1H, Ar), 9.48 (s, 1H, CH); LCꢀMS: m/z, 444 (M+); Anal. Calcd
for C24H24N6OS: C, 64.84; H, 5.44; N, 18.90; Found: C, 64.59; H, 5.64; N, 19.11.
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1ꢀ((4ꢀ(4ꢀfluorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1Hꢀ
1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (6)
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Yield:73%; White solid; mp: 75ꢀ77˚C; 1H NMR (DMSOꢀd6, 400 MHz): δ 2.92 (s, 4H, piperazine),
3.07 (s, 4H, piperazine), 5.23 (s, 2H, CH2), 6.77ꢀ6.78 (m,1H, Ar), 6.90ꢀ6.92 (m, 2H, Ar), 7.00
(m, 2H, Ar), 7.38 (d, 1H, Ar, J=3.0 Hz), 7.75ꢀ7.54 (m, 3H, Ar), 7.86ꢀ7.88 (m, 2H, Ar), 8.06 (s,
1H, Ar), 9.51 (s, 1H, CH); 13C NMR (CDCl3, 100 MHz): δ 50.3, 50.4, 69.2, 112.5, 115.3, 115.5,
118.1, 118.9, 125.1, 128.5, 128.7, 130.7, 147.0, 147.7, 147.9, 148.1, 151.8, 156.0, 158.3, 163.6;
LCꢀMS: m/z, 463 (M+1); Anal. Calcd for C24H23FN6OS: C, 62.32; H, 5.01; N, 18.17; Found: C,
62.63; H, 5.28; N, 17.95.
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1ꢀ((4ꢀ(4ꢀchlorophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1Hꢀ
1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (7)
Yield:88%; White solid; mp: 85ꢀ87˚C; 1H NMR (DMSOꢀd6, 400 MHz): δ 2.90 (s, 4H, piperazine),
3.13 (s, 4H, piperazine), 5.24 (s, 2H, CH2), 6.78ꢀ6.80 (m, 1H, Ar), 6.91 (d, 2H, Ar, J=9.1
Hz),7.19 (d, 2H, Ar, J=8.5 Hz), 7.39 (d, 1H, Ar, J=3.6 Hz), 7.54ꢀ7.56 (m, 3H, Ar), 7.85ꢀ7.87 (m,
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2H, Ar), 8.08 (s, 1H, Ar), 9.48 (s, 1H, CH); C NMR (DMSOꢀd6, 100 MHz): δ 48.1, 49.7, 68.9,
113.1, 117.0, 121.0, 122.3, 124.9, 125.0, 128.4, 128.6, 128.7, 130.9, 147.0, 147.4, 148.4, 149.8,
155.8, 163.1; LCꢀMS: m/z, 480 (M+1); Anal. Calcd for C24H23ClN6OS: C, 60.18; H, 4.84; N,
17.55; Found: C, 60.37; H, 4.61; N, 17.82.
1ꢀ(4ꢀ(4ꢀ((4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ5ꢀthioxoꢀ4,5ꢀdihydroꢀ1Hꢀ1,2,4ꢀtriazolꢀ1ꢀ
yl)methyl)piperazinꢀ1ꢀyl)phenyl)ethanone (8)
1
Yield:59%; White solid; mp: 105ꢀ107˚C; H NMR (DMSOꢀd6, 400 MHz): δ 2.41 (s, 3H, CH3),
2.90 (s, 4H, piperazine), 3.35 (s, 4H, piperazine), 5.25 (s, 2H, CH2), 6.77ꢀ6.79 (m, 1H, Ar), 6.94
(d, 2H, Ar, J=8.5 Hz), 7.37 (d, 1H, Ar, J=4.8 Hz), 7.51ꢀ7.56 (m, 3H, Ar), 7.76 (d, 2H, Ar, J=9.1
Hz), 7.84ꢀ7.87 (m, 2H, Ar), 8.07 (s, 1H, Ar), 9.47 (s, 1H, CH); 13C NMR (DMSOꢀd6, 100 MHz):
δ 26.0, 46.6, 49.6, 68.9, 113.1, 121.1, 125.1, 126.7, 128.4, 128.8, 130.0, 130.9, 148.4, 153.6,
155.8, 163.3, 195.6; LCꢀMS: m/z, 487 (M+1); Anal. Calcd for C26H26N6O2S: C, 64.18; H, 5.39;
N, 17.27; Found: C, 63.84; H, 5.23; N, 17.07.
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4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ1ꢀ((4ꢀ(4ꢀnitrophenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylꢀ1Hꢀ
1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (9)
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8
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Yield:73%; Pale yellow solid; mp: 151ꢀ153˚C; H NMR (DMSOꢀd6, 400 MHz): δ 2.86 (t, 4H,
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piperazine, J=5.4 Hz), 3.43 (t, 4H, piperazine, J=5.4Hz), 5.21 (s, 2H, CH2), 6.75ꢀ6.72 (m, 1H,
Ar), 6.94 (d, 2H, Ar, J=9.1Hz), 7.32 (d, 1H, Ar, J=4.2Hz), 7.52ꢀ7.45 (m, 3H, Ar), 7.81 (d, 2H,
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Ar, J=7.9Hz), 7.96 (d, 2H, Ar, J=9.2Hz), 8.02 (s, 1H, Ar), 9.44 (s, 1H, CH); C NMR (DMSOꢀ
d6, 100 MHz): δ 46.3, 49.5, 68.7, 112.6, 113.1, 120.8, 124.9, 125.7, 128.4, 128.7, 131.0, 136.8,
147.0, 147.3, 148.4, 154.6, 155.8, 163.1; LCꢀMS: m/z, 490 (M+1); Anal. Calcd for C24H23N7O3S:
C, 58.88; H, 4.74; N, 20.03; Found: C, 59.16, H, 4.90; N, 19.71.
(4ꢀ((4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ5ꢀthioxoꢀ4,5ꢀdihydroꢀ1Hꢀ1,2,4ꢀtriazolꢀ1ꢀ
yl)methyl)piperazinꢀ1ꢀyl)(phenyl)methanone (10)
1
Yield:78%; White solid; mp: 72ꢀ74˚C; H NMR (CDCl3, 400 MHz): δ 2.80ꢀ3.03 (m, 4H,
piperazine), 3.34 (s, 2H, piperazine), 3.81 (s, 2H, piperazine), 5.28 (s, 2H, CH2), 6.59ꢀ6.61 (m,
1H, Ar), 7.10 (d, 1H, Ar, J=3.8 Hz),7.34ꢀ7.39 (m, 5H, Ar), 7.45ꢀ7.49 (m, 3H, Ar), 7.68 (s, 1H,
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Ar), 7.95ꢀ8.01 (m, 2H, Ar), 9.99 (s, 1H, CH); C NMR (CDCl3, 100 MHz): δ 42.0, 47.6, 50.6,
69.2, 112.6, 119.0, 125.0, 127.0, 128.3, 128.5, 128.6, 129.6, 130.8, 135.4, 147.0, 147.7, 148.2,
151.9, 163.6, 170.3; LCꢀMS: m/z, 473 (M+1); Anal. Calcd for C25H24N6O2S: C, 63.54; H, 5.12; N,
17.78; Found: C, 63.27; H, 4.93; N; 18.04.
4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1ꢀ((4ꢀ(pꢀtolyl)piperazinꢀ1ꢀyl)methyl)ꢀ1Hꢀ1,2,4ꢀ
triazoleꢀ5(4H)ꢀthione (11)
Yield:83%; White solid; mp: 152ꢀ154˚C; 1H NMR (CDCl3, 400 MHz): δ 2.25 (s, 3H, CH3), 3.04ꢀ
3.06ꢀ3.15 (m, 8H, piperazine), 5.32 (s, 2H, CH2), 6.59ꢀ6.60 (m, 1H, Ar), 6.82 (d, 2H, Ar, J=8.4
Hz), 7.04ꢀ7.10 (m, 3H, Ar), 7.45ꢀ7.50 (m, 3H, Ar), 7.67 (s, 1H, Ar), 7.96ꢀ7.98 (m, 2H, Ar), 10.01
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(s, 1H, CH); C NMR (CDCl3, 100 MHz): δ 20.3, 49.9, 50.4, 69.3, 112.5, 116.7, 118.9, 125.2,
128.5, 128.7, 129.4, 129.5, 130.7, 147.0, 147.8, 148.1, 149.1, 151.8, 163.6; LCꢀMS: m/z, 459
(M+1); Anal. Calcd for C25H26N6OS: C, 65.48; H, 5.71; N, 18.33; Found: C, 65.22; H, 5.88; N,
18.64.
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4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ1ꢀ((4ꢀ(4ꢀmethoxyphenyl)piperazinꢀ1ꢀyl)methyl)ꢀ3ꢀphenylꢀ
1Hꢀ1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (12)
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Yield:69%; Cream solid; mp: 160ꢀ162˚C; H NMR (CDCl3, 400 MHz): δ 3.08ꢀ3.09 (m, 8H,
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piperazine), 3.75 (s, 3H, CH3), 5.31 (s, 2H, CH2), 6.58ꢀ6.61(m, 1H, Ar), 6.81ꢀ6.89 (m, 4H, Ar),
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7.09 (d, 1H, Ar, J=7.9Hz), 7.48ꢀ7.49 (m, 3H, Ar), 7.67 (m, 1H, Ar), 7.96ꢀ8.00 (m, 2H, Ar), 10.01
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(s, 1H, CH); C NMR (CDCl3, 100 MHz): δ 29.6, 59.3, 62.4, 112.6, 119.2, 124.5, 124.7, 125.1,
128.0, 128.2, 128.5, 130.8, 146.9, 147.1, 147.6, 151.1, 152.0, 163.3; LCꢀMS: m/z, 475 (M+1);
Anal. Calcd for C25H26N6O2S: C, 63.27; H, 5.52; N, 17.71; Found: C, 63.54; H, 5.75; N, 17.47.
4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1ꢀ((4ꢀ(pyrimidinꢀ2ꢀyl)piperazinꢀ1ꢀyl)methyl)ꢀ1Hꢀ
1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (13)
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Yield:83%; Creamish solid; mp: 128ꢀ130˚C; H NMR (CDCl3, 400 MHz): δ 2.96 (t, 4H,
piperazine, J= 4.8 Hz), 3.87 (t, 4H, piperazine, J=5.1Hz), 5.31 (s, 2H, CH2), 6.44 (t, 1H, Ar,
J=4.5Hz), 6.60 (t, 1H, Ar, J= 1.8Hz), 7.07 (d, 1H, Ar, J=3.6Hz), 7.48ꢀ7.44 (m, 3H, Ar), 7.67 (s,
1H, Ar), 7.96ꢀ7.94 (m, 2H, Ar), 8.25 (d, 2H, Ar, J=4.8Hz), 10.0 (s, 1H, CH); 13C NMR (CDCl3,
100 MHz): δ 43.5, 50.4, 69.5, 109.7, 112.5, 118.9, 125.1, 128.5, 128.7, 130.7, 147.0, 147.8,
148.1, 151.7, 157.6, 161.4, 163.6; LCꢀMS: m/z, 447 (M+1); Anal. Calcd for C22H22N8OS: C,
59.18; H, 4.97; N, 25.09; Found: C, 59.52; H, 5.12; N, 24.79.
1ꢀ((4ꢀbenzylpiperazinꢀ1ꢀyl)methyl)ꢀ4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1Hꢀ1,2,4ꢀ
triazoleꢀ5(4H)ꢀthione (14)
Yield:71%; Cream solid; mp: 60ꢀ62˚C; 1H NMR (DMSOꢀd6,400 MHz): δ 2.36 (s, 4H, piperazine),
2.77 (s, 4H, piperazine), 3.41 (s, 2H, CH2), 5.16 (s, 2H, CH2), 6.78ꢀ6.79 (m, 1H, Ar), 7.19ꢀ7.26 (m,
5H, Ar), 7.38 (d, 1H, Ar, J=3.6Hz), 7.51ꢀ7.54 (m, 3H, Ar), 7.84ꢀ7.89 (m, 2H, Ar), 8.08 (s, 1H, Ar),
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9.49 (s, 1H, CH); C NMR (DMSOꢀd6, 100 MHz): δ 49.8, 52.3, 62.0, 70.9, 113.0, 120.3, 125.0,
126.9, 128.0, 128.4, 128.7, 130.9, 148.4, 155.4, 163.0; LCꢀMS: m/z, 459 (M+1); Anal. Calcd for
C25H26N6OS: C, 65.48; H, 5.71; N, 18.33; Found: C, 65.71; H, 5.50; N, 18.06.
1ꢀ((4ꢀbenzhydrylpiperazinꢀ1ꢀyl)methyl)ꢀ4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀphenylꢀ1Hꢀ1,2,4ꢀ
triazoleꢀ5(4H)ꢀthione (15)
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Yield:89%; White solid; mp: 122ꢀ124˚C; H NMR (DMSOꢀd6, 400 MHz): δ 2.30 (s, 4H,
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piperazine), 2.80 (s, 4H, piperazine), 4.23 (s, 1H, CH), 5.15 (s, 2H, CH2), 6.78ꢀ6.80 (m, 1H, Ar),
7.14 (t, 2H, Ar, J=8.8 Hz), 7.23 (t, 4H, Ar, J=7.6 Hz), 7.34ꢀ7.40 (m, 5H, Ar), 7.52ꢀ7.58 (m, 3H,
Ar), 7.87ꢀ7.93 (m, 2H, Ar), 8.08 (s, 1H, Ar), 9.49 (s, 1H, CH); 13C NMR (DMSOꢀd6, 100 MHz):
δ 49.9, 51.4, 69.0, 75.1, 113.1, 120.9, 125.0, 126.7, 127.4, 128.4, 128.7, 130.8, 142.8, 147.0,
147.2, 148.4, 155.7, 163.1; LCꢀMS: m/z, 535 (M+1); Anal. Calcd for C31H30N6OS: C, 69.64; H,
5.66; N, 15.72; Found: C, 69.49; H, 5.48; N, 16.05.
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1ꢀ((4ꢀ(bis(4ꢀfluorophenyl)methyl)piperazinꢀ1ꢀyl)methyl)ꢀ4ꢀ((furanꢀ2ꢀylmethylene)amino)ꢀ3ꢀ
phenylꢀ1Hꢀ1,2,4ꢀtriazoleꢀ5(4H)ꢀthione (16)
1
Yield:76%; Creamish solid; mp: 125ꢀ127˚C; H NMR (CDCl3, 400 MHz): δ 2.39 (s, 4H,
piperazine), 2.90 (t, 4H, piperazine, J=4.5 Hz), 4.19 (s, 1H, CH), 5.24 (s, 2H, CH2), 6.60ꢀ6.61
(m, 1H, Ar), 6.91ꢀ6.95 (m, 4H, Ar), 7.10 (d, 1H, Ar, J=3.0 Hz), 7.28ꢀ7.31 (m, 4H, Ar), 7.48ꢀ7.50
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(m, 3H, Ar), 7.68 (d, 1H, Ar, J=1.8 Hz), 8.00ꢀ8.08 (m, 2H, Ar), 10.0 (s, 1H, CH); C NMR
(DMSOꢀd6, 100 MHz): δ 50.0, 51.3, 69.0, 73.2, 113.2, 115.4, 121.1, 125.1, 128.6, 128.9, 129.4,
131.2, 138.8, 147.1, 147.4, 148.6, 156.0, 159.0, 163.3; LCꢀMS: m/z, 571 (M+1); Anal. Calcd for
C, 65.25; H, 4.95; N, 14.73; Found: C, 65.58; H, 5.08; N, 14.44.
Cell lines and culture conditions. HepGꢀ2 (liver), A549 (lung), MCFꢀ7 (breast) and Hela
(cervical) cancer cell lines were procured from ATCC (Manassas, VA, US). The HepGꢀ2 cell
line was cultured in media formulated by ATCC (Eagle's Minimum Essential Medium, Catalog
No. 30ꢀ2003). A549, MCFꢀ7 and Hela cells were grown in DMEM {Dulbecco’s modified
Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS), 2 mmol/ liter glutamine,
and 100 units/ml penicillin and streptomycin}. Rest Cal72 and SaOSꢀ2 cell lines were obtained
from Korean Cell Bank (Seoul, Korea) and was propagated in DMEM added with FBS (10%)
and antibioticꢀantimycotic (1%) solution. All cell lines were kept at 37°C in a humidified 95%
air, 5% CO2 atmosphere incubator designated as culture at a steadyꢀstate condition.37ꢀ39
Cytotoxicity assay. Briefly, a 100 ꢁl cell suspension at a density of 4,000 cells each well was
seeded in 96ꢀwell plate (Nunc™, Wiesbaden, Germany). After 24 hours recovery period, the
cells were incubated in standard humid conditions and treated with synthesized compounds 5ꢀ16.
The cells were treated with varying concentrations of compounds 5ꢀ16 and Doxorubicin (10 µM)
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separately. Tested compounds 5ꢀ16 were dissolved in molecular grade DMSO, reaching a final
DMSO concentration of 0.5%. After 24 hours, incubation with compounds 5ꢀ16 and
Doxorubicin, 25 µl of PBS containing 2.5 mg/ml of MTT was added to each well. After 4 hours,
the medium was discarded and 100 µl DMSO was added to dissolve the formazan crystals and
then cells were incubated for 4 hours (37˚C and 5% CO2). The light absorbance was measured at
590 nm by the Model 680 microplateꢀreader (BioꢀRad, Berkeley, CA, USA).37ꢀ39
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Hoechst 33342 staining: The Cal72 cells were visualized for the apoptotic morphological
changes in their nuclear chromatin by Hoechst 33342 staining by following our earlier reported
methods.37,39 The standard drug Doxorubicin was tested at 10 µM while, MCSꢀ5 was tested by
using 5 and 10 ꢁM concentration.
Apoptosis assay by flow cytometry: Cal72 harvested cells were trypsinized (0.2 % EDTAꢀ
Trypsin) and then suspended in 4 ml fresh complete media and propagated in T25 flask and
incubated overnight. Then, MCSꢀ5 at 5 and 10 ꢁM final concentrations was added and incubated
for 24 hours. After 24 hours, PI/AnnexinꢀV FITC double staining was carried out according to
our standardized protocol.39
Early and Late apoptosis by PI/AnnexinꢀVꢀFITC double staining. On the basis of
morphological changes and pre and late apoptotic cells induced by MCSꢀ5 in Cal72 cells were
analyzed using PI/AnnexinꢀVꢀFITC double staining assay. Contrast, Cal72 cells were seeded in
four chambered culture slide (SPL, Life Sciences, Korea) followed by treatment with MCSꢀ5 (5
ꢁM/mL and 10 ꢁM/ml) for 24 hours. After the incubation time, extractꢀuntreated and treated
Cal72 cells were harvested and washed with cold 1X PBS. Afterwards, 2 µl AnnexinꢀVꢀFITC
and 2 µl PI (50 µg/ml) were added to the suspension. The mixture was incubated for 30 min in
the dark at room temperature. Stained cells were examined on slides via fluorescence microscopy
(Olympus) using an excitation wavelength of 488 nm and a 515 nm filter in the detection
pathway.
Cell cycle analysis. Cell cycle analysis was accomplished by using FACS according to our
previously described process.37 Cal72 cells were treated with MCSꢀ5 at 5 ꢁM and 10 ꢁM
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concentration, whereas 10 ꢁM concentration of Doxorubicin was taken separately to compare the
results.
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Immunofluorescence staining: Cal72 cells were seeded in 4ꢀwell chambered slide with glass
coverslips (SPL Life Sciences Co., Ltd., Korea). The cells were incubated with MCSꢀ5 (5 and 10
µM) and Doxorubicin (10 µM) and nonꢀtreated cells incubated with DMEM high glucose
medium for 24 hours. After the incubation time period, cells were washed carefully with 1X cold
PBS and fixed with 4% paraformaldehyde for 15 minutes Then after, cells were permeabilized
with 0.2% Triton Xꢀ100 for 10 min followed by washing with PBS and blocked with 1% bovine
serum albumin (BSA) for 30 minutes Further, the cells were incubated with the primary
antibodies (detailed in western blot analysis) and were detected using 1:200 rabbit IgG and
mouse IgG. Afterward, the cells were incubated with Alexa Fluor 488, Alexa Fluor 647 and GFP
conjugated (donkey antiꢀrabbit/antiꢀmouse) secondary antibodies for 1 hour at 25˚C in the dark.
All of the antibodies except cytochromeꢀc and Apafꢀ1 (Cell Signaling) used were obtained from
Santa Cruz Biotechnology, Inc. CA. Cells were then washed with cold PBS and were
counterstained with 4',6ꢀdiamidinoꢀ2ꢀphenylindole (DAPI, Invitrogen) for 5 minutes Once again
cells were washed with cold 1X PBS and 1 ml mounting media was added and cells were
observed under a fluorescence microscope (Olympus, Milan, Italy).38
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Quantitative realꢀtime polymerase chain reaction (qPCR): The relative cDNA synthesis was
performed according to our formerly described laboratory protocol.37ꢀ38 The cDNA was
employed to qPCR for the quantification of the relative transcript levels of NFꢀκB (p65), Bclꢀ2,
Bax, p53, cytochromeꢀc, Apafꢀ1, inflammatory cytokine ILꢀ6 caspꢀ3, and caspꢀ9, using the
specific primers (Table S1; supplementary information). βꢀactin opted as endogenous control.
Western blot analysis. Western blot analysis of protein of interest was carried out using
procedure as described earlier.37ꢀ38 Altogether, the antibodies excluding cytochromeꢀc/Apafꢀ1
(Cell Signaling) used were bought from Santa Cruz Biotechnology, USA. The PVDF membrane
was incubated with the primary antibodies βꢀactin (1:1,000), antiꢀNFꢀκB (1:200), caspꢀ3, caspꢀ9,
antiꢀhumanꢀILꢀ6, Bclꢀ2, Bax, antiꢀp53, cytochromeꢀc, Apafꢀ1 (mouse monoclonal IgG) were
detected using 1:200 dilution. The proteins of interest were recognized by a greater
chemiluminescence detection kit using LAS4000 machine (GE Healthcare, USA).
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Determination of mitochondrial membrane potential (ꢁΨm). Cal72 cells were originally
seeded onto T25 flask and allowed to grow to 70 percent confluence. The cells were then exposed
for 24 hours with the addition of MCSꢀ5 (5 and 10 µM) and Doxorubicin (10 µM) to determine
the state of mitochondrial membrane potential. The JCꢀ1 dye was acquired from Sigma Aldrich
and prepared in DMSO as a 5 mg/ml stock solution. Next day, to stain the cells, monolayers
were rinsed with DMEM without phenol red (SigmaꢀAldrich, USA). Cell monolayers were
incubated with DMEM complete media and JCꢀ1 5 (µg/ml) at 37˚C for 30 minutes. Further the
cells were rinsed two times with DMEM and imaged using confocal fluorescence microscope set
to excitation at 488 nm and detection at 510 nm to 525 nm (green) and 590 nm (red) channels
using a dual bandꢀpass filter. On the other hand, to determine ꢁΨm, the cells were harvested and
washed twice in cold PBS and then Centrifuge cells at 1200 rpm for 5 minutes at RT. The
supernatent was removed and reꢀsuspend cells in the 500 µl EDTAꢀ1X PBS. Pipette and vortex
to disrupt cellꢀtoꢀcell clumping and the samples were analyzed by flow cytometry within one
hour
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Generation of intracellular ROS estimation by FACS. The Cal72 cells were seeded in T25
flask and incubated at 37ºC with 5% CO2 until at least 70% confluent. Next day, cells were
treated with varying concentrations, i.e. 5, 10 and 15 µM of MCSꢀ5 and Doxorubicin,
respectively. After 24 hours of treatment exposure, the cells were then washed with cold HEPES
buffered salt solution to remove traces of the original medium. The cells were trypsinized,
harvested and reꢀsuspended in 2 ꢁM (final concentration) 2′7′ꢀdichlorofluorescein diacetate
fluorescent dye (DCFꢀDA, Sigma Aldrich) and then incubated for 30 minutes in dark at room
temperature. Obtained fluorescence intensity was quantified with the help of FACS (BD
Biosciences, India) and fluorescence microscope. The FL1ꢀH channel was used to calculate the
mean DCF fluorescence intensity based on measurements of 10,000 cells. The M1 range was
deliberated as each subꢀpopulation percentage of cells displaying enhanced DCFꢀDA
fluorescence.
Cal72ꢀinduced xenograft nude mice model. Immunodeficient nude mice (SPF/VAF; 5 weeks
old) were procured from KS HIꢀTEC, Inc Korea. The experimental processes were prior
approved by the institutional review board of the Department of Animal Biotechnology, Jeju
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National University, Jeju Special SelfꢀGoverning Province, Korea. The animals were housed by
providing appropriate environmental and nutritional conditions. The mice (10, 11, and 12 weekꢀ
age) were sacrificed by following the standard protocols of the Jeju National University. In order
to design tumorigenesis and metastasis induction experiment, 24 nude mice were distributed into
four groups. Group one as positive control (n = 6, treated with Doxorubicin 10 mg/kg/day). A
second group used as test group low dose (n = 6, treated with MCSꢀ5, 5 mg/kg/day). Another
third group opted as a test group for high dose (n = 6, treated with MCSꢀ5 10 mg/kg/day). The
fourth group was inoculated nonꢀtreated tumor group as negative control (n = 6). To induce
Cal72 mediated tumorgenesis, 1x106 Cal72 cells/ml concentrations of cells into lower right/left
flanks of nude mouse by microꢀneedle syringe (29 gauge x 1ꢂ2'' 12.7 mm needle, UltraꢀThin
Plus™ Korea). After generation of successful tumor model MCSꢀ5 (5 and 10 mg/kg) and
Doxorubicin (10 mg/kg) treatment were provided up to 45 days. Then the animals sacrificed and
the tumors were removed from all animals and weight.
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Optical probe guided molecular imaging to study tumor metastasis. Inꢀvivo tumor
localization was performed to study preꢀ and intraoperative tumor localization in the realꢀtime
resection by using 2ꢀDG optical probe, according to our established method.38 The metastatic
potential of Cal72 was studied by injecting subcutaneously Cal72 cells in nude mice. To study
the potential of MCSꢀ5 (5 and 10 mg/kg) against metastasis was analyzed as compared to
Doxorubicin (10 mg/kg). Spontaneous metastasis induced by Cal72 cells in nude mice and effect
of MCSꢀ5 to vanish/slow down metastasis were carefully analyzed.
Acknowledgements
The authors CB Mishra is thankful to the University Grants Commission (UGC) for the award of
Dr. D.S. Kothari postdoctoral fellowship, S Kumari is thankful to UGC for financial support, M
Tiwari is thankful to the University of Delhi for financial assistance. RJ Mongre and DK Jeong
are thankful to Next Generation Bio Green 21 Program organization for providing financial
support; grant number PJ01117401, Rural Development Administration, Republic of Korea.
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University Science Instrumentation (USIC), University of Delhi is acknowledged for providing
NMR and Mass spectral characterization of the synthesized compounds.
Financial Interests
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The authors declare no conflict of interest.
Supporting Information
Supporting Information Available: This material is available free of charge via the Internet.
NMR spectra, HPLC chromatogram and primer table
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