Synthesis, characterization, antioxidant, and anticancer studies of 6-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-3-[(2-naphthyloxy) methyl][1,2,4] triazolo[3,4-b][1,3,4]thiadiazole in HepG2 cell lines

Article abstract of DOI:10.1007/s00044-010-9436-9

Triazolo- thiadiazoles exhibit a variety of pharmacological properties, due to their cytotoxicity. In continuation of a previous study on triazolo-thiadiazoles, the authors have synthesized a new thiadiazole, 6-[3-(4-chlorophenyl)- 1-H-pyrazol-4-yl]-3-[(2

Full text of DOI:10.1007/s00044-010-9436-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2011) 20:1074–1080
DOI 10.1007/s00044-010-9436-9
ORIGINAL RESEARCH
Synthesis, characterization, antioxidant, and anticancer studies
of 6-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-3-[(2-naphthyloxy)
methyl][1,2,4]triazolo[3,4-b][1,3,4]thiadiazole in HepG2 cell lines
•
Dhanya Sunil Arun M. Isloor Prakash Shetty
•
•
•
K. Satyamoorthy A. S. Bharath Prasad
Received: 2 April 2010 / Accepted: 13 September 2010 / Published online: 6 October 2010
Ó Springer Science+Business Media, LLC 2010
Abstract Triazolo- thiadiazoles exhibit a variety of phar-
macological properties, due to their cytotoxicity. In contin-
uation of a previous study on triazolo-thiadiazoles, the
authors have synthesized a new thiadiazole, 6-[3-(4-chlo-
rophenyl)-1-H-pyrazol-4-yl]-3-[(2-naphthyloxy)methyl]
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (CPNT), which was
further characterized by advanced spectral techniques and
elemental analysis. The compound exhibited a dose-depen-
dent cytotoxic effect on hepatocellular carcinoma cell line,
HepG2 with very low IC50 value of 0.8 lg/ml in 24 h when
compared with standard drug, doxorubicin. Incorporation of
[3H] thymidine in conjunction with cell cycle analysis sug-
gested that CPNT inhibited the growth of HepG2 cells. Flow
cytometric studies revealed more percentage of cells in
subG1 phase, indicating apoptosis, which was further con-
firmed through chromatin condensation studies by Hoechst
staining. In vitro antioxidant activity of CPNT was deter-
mined by DPPH and ABTS free radical scavenging assays
which revealed increasing scavenging activity with
increasing concentration of the compound when compared
with reference ascorbic acid.
Keywords CPNT ꢀ HepG2 cell lines ꢀ Cytotoxicity ꢀ
Antioxidant
Introduction
Hepatocellular carcinoma is one of the most common
cancers in the world and accounts for very high percentage
of deaths annually (Courtney et al., 2008). The majority of
patients with liver cancer die within a year after diagnosis.
Presently practiced treatment for the disease mainly
includes surgery and chemotherapy, but the curative effects
of the existing chemotherapeutic drugs are not good
enough as they have several side effects. Therefore,
searching for highly efficient antitumor drug remains a hot
research area. It has been proposed recently that apoptosis
might be an important and ubiquitous mode of cell death
for cells treated with chemotherapeutic drugs.
D. Sunil
Department of Chemistry, Manipal Institute of Technology,
Manipal University, Manipal, Udupi 576 104, India
1,2,4-Triazole derivatives represent an interesting class
of heterocyclic compounds with the advantage of broad
spectrum activity, high oral availability, and toxicity
(Shu-Sheng et al., 2007). Triazoles and thiadiazoles have
been reported to possess analgesic (Mathew et al., 2007),
anti-inflammatory (Mathew et al., 2007, Kamotra et al.,
2007), antiviral (Tomita et al., 2002), antimicrobia
(Demirbas et al., 2004, Swamy et al., 2006), antifungal
(Tsukuda et al., 1998, Hirpara et al., 2003, Isloor et al.,
2009), antibacterial (Wang et al., 1996), antitubercular
(Udupi et al., 1999), and antitumor (Shivarama et al., 2002)
activities. Reports of compounds that contain two active
groups, triazole and thiadiazole, in a single molecule have
rarely been studied for its anticancer activity (Shu-Sheng
A. M. Isloor (&)
Organic Chemistry Division, Department of Chemistry,
National Institute of Technology-Karnataka, Surathkal,
Mangalore 575 025, India
e-mail: isloor@yahoo.com
P. Shetty
Department of Printing and Media Engineering,
Manipal Institute of Technology, Manipal University,
Manipal, Udupi 576 104, India
K. Satyamoorthy ꢀ A. S. Bharath Prasad
Department of Biotechnology, Manipal Life Sciences Centre,
Manipal University, Manipal, Udupi 576 104, India
123

Med Chem Res (2011) 20:1074–1080
1075
Cl
et al., 2007). Also, in vitro and in vivo data suggest that
certain antioxidants selectively inhibit the growth of tumor
cells, may induce cellular differentiation, and may alter the
intracellular redox state, thereby enhancing the effects of
cytotoxic therapy (Lamson and Brignall, 1999, Conklin
2000, 2002). In continuation of an earlier study (Dhanya
et al., 2009) on the synthesis of triazolo-thiadiazoles, the
authors report in this article the synthesis of 6-[3-(4-chloro-
phenyl)-1-H-pyrazol-4-yl]-3-[(2-naphthyloxy)methyl][1,2,4]
triazolo[3,4-b][1,3,4] thiadiazole (CPNT) and its dose-
dependant anti-cell proliferative efficiency leading to
apoptosis in HepG2 cell lines. The high antioxidant
capacity of CPNT was confirmed by 1,1-diphenyl-2-picryl
hydrazide (DPPH) and 2,2-azino bis 3-ethyl benzo-thia-
zoline-6-sulphonic acid (ABTS) assays.
Cl
HOOC
OHC
KMnO4
N
Pyridine
N
N
H
N
H
2
1
N
N
O
Reflux18h
POCl3
SH
3
N
NH2
N
N
O
Cl
Results and discussion
S
N
N
Synthesis
N
N
H
3-Methyl-b-naphthyloxy-4-amino-5-mercapto-1,2,4-triazole
3 was synthesized as per the method in the literature (Isloor
et al., 2000). 3-(4-Chloro phenyl)pyrazole aldehyde 1 was
prepared as reported in the literature (Kalluraya et al., 2001,
2004, Singh et al., 2005). The aldehyde obtained was con-
verted into pyrazole acid 2 by oxidation using potassium
permanganate in pyridine medium (Bratenko et al., 2001).
4
Scheme 1 Synthetic route for triazolothiadiazole
Anticancer studies
Unless otherwise mentioned, all the chemicals used in this
study were from Sigma-Aldrich, USA. The hepatocellular
carcinoma cell line, HepG2, was used for all the analyses in
this study. HepG2 cell lines were purchased from National
Center for Cell Science, Pune, India. The cell line was
cultured in DMEM containing 10% FBS at 37°C in an
atmosphere containing 5% CO2.
Subsequently,
3-methyl-b-naphthyloxy-4-amino-5-mer-
capto-1,2,4-triazole 3 and 3-(4-chloro phenyl)pyrazole acid
2 were refluxed for 18 h in phosphorous oxychloride
medium which resulted in cyclized title compound 4. The
reaction sequence is outlined in Scheme 1.
Characterization
MTT assay
The characterization of CPNT was done using advanced
spectroscopic studies and elemental analysis. Melting point
was determined by open capillary method. Thin layer
chromatography was conducted on 0.25 9 10-3 m silica
gel plates to monitor the progress of the reaction and to
check the purity of the compound. A 1:1 mixture of ethyl
acetate and petroleum ether solution was used as the eluent.
Visualization was made by using iodine vapors. The IR
spectrum in KBr pellets was recorded on a Schimadzu
FTIR 8400S spectrophotometer. 1H-NMR spectrum was
recorded in deuterated dimethyl sulphoxide on an AV500
NMR spectrometer using TMS as an internal standard. The
mass spectrum was recorded on a Schimadzu GCMS-
QP5050 mass spectrometer. The elemental analysis was
done using Flash thermo 1112 series CHN analyzer.
Cytotoxic effect of CPNT was assessed using 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide
(MTT) assay (Mosmann 1983, Kishore et al., 2008). Cells
were seeded in duplicates in 96-well plates at 1 9 104
cells/well. After 24 h, CPNT was added at a concentration
of 0.1, 1, 10, and 100 lg/ml and incubated for another
24 h. 5 mmol of MTT reagent was added and incubated for
additional 4 h. The purple formazan crystals were dis-
solved in 0.1 ml of hydrochloric acid (0.4 N): isopropanol
(1:24). Cells grown in culture media alone and with
appropriate concentration of DMSO were used as control
and vehicle control, respectively. Doxorubicin was used as
the standard drug. The optical density of each well was
measured at 570 nm in an ELISA plate reader. The results
were reported as percentage survival of the cells in
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Med Chem Res (2011) 20:1074–1080
Fig. 1 Cytotoxic analysis of CPNT in HepG2 cell lines using MTT
assay. DMSO acts as vehicle control. In case of control, neither CPNT
nor DMSO was added. Cells were treated with 0.1, 1, 10, and 100 lg/ml
of CPNT. The percentage viability was calculated considering control
wells as 100% and plotted with representation of error bars
Fig. 2 [3H] thymidine incorporation assay to determine effect of
CPNT on cell proliferation. The data presented are the results of
duplicates, and error bars are indicated
centrifuged at 3000 rpm for 10 min, and alcohol was dis-
carded. The cells were washed in 2 ml PBS, and to the
pellet, 0.015 ml of RNase was added, followed by 0.01 ml
of propidium iodide. The cells were incubated for 1 h and
subjected to flow cytometry. The results were analyzed
using cell quest pro software using excitation at 488 nm
laser and emission at 560/670 nm. A minimum of 10,000
cells were acquired and histograms analyzed. The total
number of cells included in analysis was taken as 100%.
The histogram of DMSO-treated cells showed a standard
cell cycle pattern, which includes G1 and G2/M peaks
separated by S phase peak. The subG1 peak showed very
less percentage of dead cells. Interestingly after addition of
CPNT, a concentration-dependent change was observed in
the percentage of cells in each phase of the cell cycle.
There was a remarkable dose-dependent increase in the
percentage of sub-ploid cells in the subG1 phase. We could
also observe more cells in the G1 phase when compared to
S and G2phases indicating a cell cycle arrest probably in
the G1 phase of the cell cycle allowing fewer cells to enter
into the S phase confirming the results obtained in the [3H]
thymidine incorporation assay. The bar diagram quantify-
ing the percentage of cells in the different phases of the cell
cycle in control and treated with different concentrations of
CPNT is shown in Fig. 3. The flow cytometry data are
represented in Table 1.
comparison to that of the untreated control cells stan-
dard deviation (Fig. 1). The IC50 of CPNT was found to be
0.8 lg/ml which was significantly low when compared to
that of the standard drug, doxorubicin.
[3H] Thymidine incorporation assay
Since the above MTT assay suggested that CPNT affects the
cell viability, we were interested in knowing whether it could
inhibit cell division. DNA synthesis was monitored by
labeling cells using [3H] thymidine (Kavitha et al., 2009).
Around 1 9 106 cells/ml were seeded in duplicates, CPNT
was added at a concentration of 1, 10, and 100 lg/ml and
incubated for 3 h. 18.5 9 103 Bq of [3H] thymidine was
added and incubated for additional 2 h. The cells were col-
lected by Millipore filtration, washed with cold 10% tri-
chloro acetic acid (TCA), methanol, and water. Filters were
removed into liquid scintillation vials, dried, cocktail added.
and radioactivity was measured using a liquid scintillation
beta counter. Radioactivity was expressedas cpm (counts per
minute), which was proportional to the amount of [3H] thy-
midine incorporated into the DNA of cultured cells stan-
dard deviation. CPNT treatment showed a dose-dependant
reduced incorporation of [3H] thymidine drastically, sug-
gesting that it affects the cell viability by inhibiting cell
division probably by interfering with DNA replication
(Fig. 2). However, it is also possible that in addition to its
effect on cell division, CPNT could induce apoptosis.
Chromatin condensation studies (Hoechst staining)
To confirm the action of CPNT through growth inhibition
mediated by a DNA replication defect followed by apop-
tosis, chromatin condensation studies were conducted.
Condensation of chromatin is usually the late event in
apoptosis. HepG2 cells were grown in 96-well plates in
duplicates and subjected to the treatment of CPNT in two
different concentrations of 10 and 100 lg/ml and incubated
for 24 h. About 0.05 ml of the medium was aspirated, the
Cell cycle analysis (Kavitha et al., 2009,
Ormerod et al., 1994)
Hep G2 cells were cultured and treated with two concen-
trations, 0.06 and 0.08 lg/ml of CPNT, in duplicates for
24 h to study concentration-dependent effect on the cell
cycle arrest. Cells were fixed in 2 ml of 70% alcohol,
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Med Chem Res (2011) 20:1074–1080
1077
same amount of diluted Hoechst dye was added and
incubated for 5 min. 0.05 ml of the medium was removed
and viewed under fluorescence microscope. Hoechst dye
binds at the adenine–thymine-rich regions of DNA and
emits blue fluorescence when excited by UV light about
350 nm. DMSO-treated wells were used as control
(Fig. 4a). The presence of dose-dependent increase in
condensed apoptotic chromatin confirms the apoptotic
action of CPNT (Fig. 4b, c).
Antioxidant studies
DPPH radical scavenging assay (Sreejayan and Rao,
1996, John and Steven, 1984)
The DPPH antioxidant assay is based on the ability of
DPPH, a stable, free radical, to decolorize in the presence
of antioxidants. DPPH radical scavenging is mediated by
transfer of hydrogen atom. The DPPH radical contains an
odd electron, which is responsible for the absorbance at
517 nm and also for visible deep purple color. When DPPH
accepts an electron donated by an antioxidant compound,
the DPPH is decolorized which can be quantitatively
measured from the changes in absorbance. 100 ll of vari-
ous concentrations of CPNT was added to respective wells
of a 96-well micro-plate. Equal amount of DPPH (1,1-
diphenyl-2-picryl hydrazide) was also added to each well
to make up a final volume of 200 ll. After 20-min incu-
bation in the dark, the ability of CPNT to scavenge the free
radical DPPH was measured by recording the absorbance at
517 nm using an ELISA plate recorder. Experiment was
Fig. 3 Bar diagram showing quantification of cells in each phase of
the cell cycle in flow cytometry studies
Table 1 % cells in different
stages of the cell cycle—flow
cytometric analysis
Concentration
M1 (%subG1 cells)
M2 (%G1 cells)
M3 (%S cells)
M4 (%G2/M cells)
Control (DMSO)
0.06 lg/ml
3.57 0.03
4.00 0.06
12.33 0.09
50.68 1.42
56.59 1.96
52.65 1.37
32.84 1.06
28.82 0.98
23.68 0.67
12.92 0.27
10.58 0.51
11.33 0.24
0.08 lg/ml
Fig. 4 Chromatin condensation
studies (Hoechst test). Dose-
dependent increase in
condensed apoptotic chromatin
is visible on treatment with
CPNT when compared to
control
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Med Chem Res (2011) 20:1074–1080
performed in triplicates, and average values were consid-
ered. An equal amount of each of methanol and DPPH was
added to the control. Ascorbic acid was used as reference
standard. Comparison between the antioxidant activity of
CPNT and ascorbic acid is shown in Fig. 5.
%Scavenging of DPPH
Absorbanceof controlꢁAbsorbanceof testsample
¼
ꢂ100
Absorbance of control
The IC50 values for CPNT and ascorbic acid for scavenging
DPPH were found to be 1.023 lg/ml and 1.46 lg/ml,
respectively. CPNT was found to be more potent than
ascorbic acid.
Fig. 6 ABTS radical scavenging activity of CPNT measured at
734 nm as compared to standard ascorbic acid. Values are the average
of triplicate experiments
ABTS radical scavenging assay (Vaijanathappa et al.,
2008)
CPNT was found to be more potent when compared with
ascorbic acid in ABTS assay.
ABTS is chemically 2,2-azino bis 3-ethyl benzo-thiazoline-
6-sulphonic acid. The reduction of this radical by CPNT is
measured at 690 nm. The electron transfer capability of
CPNT was studied using ABTS radical scavenging assay.
In a 96-welled microtiter plate, 40 ll of the CPNT/ascorbic
acid, 200 ll of methanol, and 30 ul of ABTS solution were
added. The plate was then incubated at 37°C for 20 min
after which the absorbance was measured at 690 nm using
an ELISA plate reader. Sample blank and control were also
taken. The experiment was performed in triplicates, and
average values were considered. The comparative values of
the antioxidant activity of CPNT and ascorbic acid is
depicted in Fig. 6.
Conclusion
In this study, a new triazolo thiadiazole CPNT was synthe-
sized, which showed a dose-dependent cytotoxic effect, the
IC50 being 0.8 lg/ml which is found to be significantly low
when compared to standard drug, doxorubicin with IC50
19 lg/ml. Treatment of HepG2 cells with CPNT showed a
dose-dependent decreased cell division in [3H] thymidine
incorporation assay and increase in the subG1 population in
flow cytometric analysis. The chromatin condensation
studies confirmed the apoptotic action of CPNT. The sig-
nificant antioxidant activity of CPNT with low IC50 values
when compared with standard ascorbic acid is clearly evi-
dent from DPPH and ABTS free radical scavenging assays.
As ABTS scavenging occurred at a lower concentration than
DPPH scavenging, it may be presumed that the antioxidant
action of CPNT is primarily through the mechanism of
electron transfer rather than hydrogen transfer.
% Scavenging of ABTS
Absorbanceof controlꢁAbsorbanceof testsample
¼
ꢂ 100
Absorbance of control
The ability to cross the biomembranes and bioavail-
ability of CPNT is dependent on the lipophilic moiety
attached. The title compound has naphthyloxy methyl and
pyrazole substituents. The chlorine atoms also might have
added to the potency of the compound as a chlorine
substituent produces simultaneously an increase in lipo-
philicity, an electron-attracting effect, and a metabolic
obstruction. In CPNT, since the bulky chlorine atom is
present at the para position, it may provide less steric
hindrance and could increase the binding affinity of the
molecule. As this study reveals CPNT as a potent cytotoxic
compound with low IC50 values compared to the standard
drug doxorubicin, leading to apoptotic pathway along with
significant antioxidant activity, there is ample scope of
further study in this area.
Fig. 5 DPPH radical scavenging activity of CPNT added to methanol
solution of DPPH. Radical scavenging activity was measured at
517 nm as compared to standard ascorbic acid. Values are the average
of triplicate experiments
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Med Chem Res (2011) 20:1074–1080
1079
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Experimental
Preparation of 6-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-
3-[(2-naphthyloxy)methyl][1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazole (4)
To a mixture of 2.72 g, 10 mmol of 3-methyl-b-naphthy-
loxy-4-amino-5-mercapto-1,2,4-triazole
3
and 2.22 g,
Dhanya S, Isloor AM, Prakash S (2009) Synthesis, characterization
and anticancer activity of 1,2,4-triazolo[3,4-b]-1,3,4-thiadiazoles
on HepG2 cell lines. Der Pharma Chemica 1(2):19–26
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Microwave assisted synthesis and biological activities of some
10 mmol of 3-(4-chloro phenyl) pyrazole 4-carboxylic acid
2, and 20 ml of phosphorous oxychloride were added, and
the contents were heated under reflux for 18 h. Excess
phosphorous oxychloride was distilled off, and the residue
was poured onto crushed ice with vigorous stirring. The
resulting solid 4 was washed with cold water, 20% sodium
hydrogen carbonate solution, and recrystallized from a
mixture of ethanol and dioxane (1:1 mixture).
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Characterization data of 6-[3-(4-chlorophenyl)-
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[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole (4)
John A, Steven DA (1984) Microsomal lipid peroxidation. Methods
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Yield: 78%, m.p.; 150-152°C, IR (KBr) [cm-1]; 1620 (C=N str.),
3090(Ar–H str.), 1495, 1380 (Ar.C=C str.), 820 (Ar.C–H
def.), 750, 820, 840(naphthalene C–H), 1050, 1280 (Ar.C–
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1
O–C str.), H NMR (deuterated DMSO) [ppm]; 5.5 (2H,
O–CH2), 6.9 (1H, pyrazole C–H), 7.1 (1H, pyrazole N–H),
7.5-8 (11Ar–H), 13C–NMR : 174.1, 167.1, 162.4, 157.2,
145.2, 136.1, 131.1, 129.4, 110.0, 105.9, MS(m/z); 458(M?),
460 (M?2), 315 (M? of 6- [3-(4-chlorophenyl)-1 H-pyrazol-
4-yl]-3-methyl[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole), 317
(M?2 of 6-[3-(4-chlorophenyl)-1H-pyrazol-4-yl]-3-methyl
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazole), 144 (M? of 2-naphthol),
280 (M? of 3-methyl-6-(3-phenyl-1Hpyrazol-4-yl)[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazole), elemental analysis calcd.
(%) for C23H15N6OClS: C, 60.19; H, 3.27; N, 18.32. Found
(%): C, 60.06; H, 3.23; N, 18.34.
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Acknowledgments The authors are thankful to the Head-SIF,
Indian Institute of Science, Bangalore for providing NMR and mass
spectral data. AMI is grateful to the Director, NITK, Surathkal, India
for providing research facilities. The authors express their gratitude to
the Chairman, RGCB, Thiruvananthapuram, for permission to carry
out the chromatin condensation studies. One of the authors (D.S) is
thankful to the Director and Head-Department of Chemistry, Manipal
Institute of Technology, Manipal University for providing us with the
necessary laboratory facilities.
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