Zeolite-catalyzed synthesis of pyrazolo[1,2-a][1,2,4]triazole-1,3-dione derivatives as anti-breast cancer agents

Article abstract of DOI:10.1007/s13738-018-1310-6

A series of pyrazolo[1,2-a][1,2,4]triazole-1,3-dione derivatives were synthesized under the catalytic effect of Natrolite zeolite, and their cytotoxic effects on three human breast cancer cell lines viz. breast cancer (BT-474), triple negative breast cancer (MDA-MB 231), and breast carcinoma (MCF-7) were explored using MTT assay. Apoptotic effects of the compounds were investigated by western blot analysis on the caspase-3 protein. Moreover, we examined the level of reactive oxygen species in all three cell lines. The most promising compound, 7-Amino-1,3-dioxo-5-(4-nitrophenyl)-2-phenyl-1,2,3-trihydropyrazolo[1,2-a][1,2,4]triazole-6-carbonitrile (4c), showed high levels of cytotoxicity against all cell lines with IC₅₀ values of 2.3, 2.5 and 3?μg/ml, respectively.

Full text of DOI:10.1007/s13738-018-1310-6

Journal of the Iranian Chemical Society
https://doi.org/10.1007/s13738-018-1310-6
ORIGINAL PAPER
Zeolite‑catalyzed synthesis of pyrazolo[1,2‑a][1,2,4]triazole‑1,3‑dione
derivatives as anti‑breast cancer agents
Razieh Nejat1 · Mohammad Amin Mahjoub2 · Zahra Hekmatian1 · Mohammad Amin Javidi2 · Sadegh Babashah2
Received: 21 July 2017 / Accepted: 21 January 2018
© Iranian Chemical Society 2018
Abstract
A series of pyrazolo[1,2-a][1,2,4]triazole-1,3-dione derivatives were synthesized under the catalytic efect of Natrolite
zeolite, and their cytotoxic efects on three human breast cancer cell lines viz. breast cancer (BT-474), triple negative breast
cancer (MDA-MB 231), and breast carcinoma (MCF-7) were explored using MTT assay. Apoptotic efects of the com-
pounds were investigated by western blot analysis on the caspase-3 protein. Moreover, we examined the level of reactive
oxygen species in all three cell lines. The most promising compound, 7-Amino-1,3-dioxo-5-(4-nitrophenyl)-2-phenyl-1,2,3-
trihydropyrazolo[1,2-a][1,2,4]triazole-6-carbonitrile (4c), showed high levels of cytotoxicity against all cell lines with IC50
values of 2.3, 2.5 and 3 μg/ml, respectively.
Keywords Pyrazolo[1,2-a][1,2,4]triazole-1,3-dione · BT-474 · MDA-MB-231 · MCF-7
Introduction
activities [15–18]. These compounds have a wide range of
therapeutic properties and are used as drugs in modern med-
icine. They are useful for preventing breast cancer [19–21].
Therefore, triazoles, particularly 1,2,4-triazoles, are perspec-
tive scafolds for designing anticancer drugs [22]. Among
these heterocyclic compounds, 1,2,4-triazolidine-3,5-dione
containing heterocycles, so-called urazoles, have received
considerable interest in synthetic and industrial chemistry.
Also, these compounds have found immense technological
applications in the manufacture of herbicides, anti-bacterial
and antifungal compounds, production of plastics such as
automobile air bags and blowing agents [23, 24].
Cancer is a group of diseases characterized by uncontrolled
cell growth in the body, causing over 7 million deaths a
year [1]. Over the recent several decades, researchers have
been struggling to fnd efective clinical approaches for the
treatment of cancer and search for novel anticancer agents.
Breast cancer is the most prevalent cancer in women and
represents the second highest leading cause of cancer death
after lung cancer [2]. A wide range of naturally occurring
agents belongs to nitrogen-containing heterocyclic com-
pounds. Many of these heterocycles are of biological and
synthetic importance which acts as pharmaceuticals, agro-
chemicals, and polyfunctionalized fragments [3–14]. Azoles,
such as 1,2,3-triazole and 1,2,4-triazole, and its derivatives
are important heterocycles with diversifed pharmacological
Besides, pyrazolourazoles as their fused derivatives with
diverse structures have attracted much interest due to their
wide range of biological properties such as analgesic, anti-
bacterial, anti-infammatory, anti-diabetic and psychoana-
leptic activities [25–30].
On the other hand, reactive oxygen species (ROS), capa-
ble of causing damage to DNA, has been associated with
carcinogenesis, coronary heart disease, and many other
health problems related to advancing age [31]. Synthetic
antioxidants exert their efects by preventing the generation
of ROS [32] which can protect the formation of free radicals
and retard the progress of many diseases, such as cancer,
infammation and cardiovascular disorders [33]. Among
azoles, 1,2,4-triazole possesses urease inhibition and anti-
oxidant activities [34].
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s13738-018-1310-6) contains
supplementary material, which is available to authorized users.
* Razieh Nejat
organochem.nejat@yahoo.com
1
Department of Chemistry, Faculty of Science, Kosar
University of Bojnord, Bojnord, Iran
2
Department of Molecular Genetics, Faculty of Biological
Sciences, Tarbiat Modares University, Tehran, Iran
Vol.:(0123456789)
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Journal of the Iranian Chemical Society
In addition, one-pot multi-component reactions (MCRs)
have proven to be more advantageous over conventional
linear-type synthesis since the multi-step reactions are usu-
ally subjected to complex isolation procedures and produce
signifcant amounts of waste materials [35–45].
PL-STA 1500 apparatus with the rate of 10 °C min−1 in a
static atmosphere of argon (Supporting information).
General procedure for synthesis of pyrazolo[1,2‑a]
[1,2,4]triazole‑1,3‑diones (4)
Also, multi-component processes provide rapid and ef-
cient approach to organic transformations including diverse
synthesis of polyfunctionalized heterocycles as anticancer
drugs [46–49].
A mixture of malononitrile 1 (1 mmol), aldehyde 2
(1 mmol), 4-phenylurazole 3 (1 mmol), and zeolite (0.05 g)
was placed in a mortar. The reaction mixture was then heated
at 80 °C for 60 min. After completion of the reaction as
monitored by TLC analysis, the reaction mixture was cooled,
and the solid was dissolved in acetone (15 mL), fltered and
evaporated under vacuum to give the product, which was
crystallized from ethanol to aford pure compound 4.
In this paper, we report the synthesis of pyrazolo[1,2-
a][1,2,4]triazole-1,3-dione derivatives under the catalytic
efect of Natrolite zeolite as micro-porous crystalline alu-
minosilicates. Also, their cytotoxic and apoptotic activi-
ties were measured on three human breast cancer cell lines
including breast cancer (BT-474), breast carcinoma (MCF-
7,( and triple negative breast cancer (MDA-MB-231). The
reactive oxygen level (ROS) was also considered in the
treated cell lines. Most of the anticancer agents generate
ROS leading to oxidative damages in various molecules of
the cell. However, both processes including cytotoxic and
oxidative damage mainly induce apoptosis to kill cancer
cells.
MTT assay
Cell viability and proliferation were determined by using the
colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-
zolium bromide, according to the manufacture’s instruction.
Briefy, 6 h after treatment, 20 μl of MTT reagent (Sigma;
10 mg/ml) was added to 5 × 104 cells cultured in triplicate
in 96-well plates in a fnal volume of 200 μl. The cells were
incubated for 3 h at 37 °C and 5% CO2. Then 200 μl of dime-
thyl sulfoxide (DMSO) was introduced, as the solvent rea-
gent. The amount of formazan produced from MTT cleav-
age was quantitated with an ELISA plate reader (BioTech
Company, USA), at 490 nm wavelength. Means and standard
deviations generated from three independent experiments
were reported as the percentage of growth versus control at
72 h. To ensure a valid statistical analysis, only experimen-
tal data for which the linear correlation coefcient of the
median-efect plot was more than 0.9 were included.
Experimental
Chemistry
Chemicals used in this work were purchased from Fluka
and Merk chemical companies and used without purifca-
tion. FT-IR spectra were recorded on a Thermo Scientifc
NICOLET IR100 FT-IR Spectrometer from KBr pellets.
1H and 13C NMR spectra were measured for samples in
DMSO-d6 using a 500 MHz Bruker instrument at 500.13
and 125.77 MHz, respectively, using Me4Si as internal
standard. Mass spectra were recorded with a spectrometer
FINNIGAN-MAT 8430 operating at an ionization poten-
tial of 70 eV. Melting points were measured on a SMPI
apparatus. The natural zeolite used in this study originated
from Zahedan city, Iran. Mineral identifcation using X-ray
diffraction showed that the zeolite consisted mainly of
Natrolite. The crystallinity of Natrolite zeolite was deter-
mined by on Holland Philips X-pert difractometer (Cu
Kα = 1.5418 Å). The XRF experiments were conducted on
a Phillips PW-1480 spectrometer, and the resulting data were
processed with a computer using X40 Phillips software.
The morphology of the sample was observed by scanning
electron microscopy (SEM) at an acceleration voltage of
15.0 kV by a Holland Philips XL30 microscope instrument.
The corresponding energy-dispersive X-ray (EDAX) spec-
trum of Natrolite zeolite was obtained on a Holland Philips
XL30 microscope instrument. Thermogravimetric–difer-
ential thermal analysis (TG–DTA) was performed using
Western blot analysis
Proteins were resolved in an SDS/PAGE gel and subjected
to western blot analysis using monoclonal antibodies against
caspase-3 and β-actin (Sigma–Aldrich). Antibodies were
used at 1 μg/ml of working concentration in PBS with 5%
dried milk. Detection by enzyme-linked chemiluminescence
was performed according to the manufacturer’s protocol
(ECL; Pierce Biotechnology Inc., Rockford, IL). Quanti-
fcation of protein bands was performed using the ImageJ
software.
Results and discussion
Chemistry
In continuation of our ongoing eforts to develop newer
and more benign approaches to the synthesis of various
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heterocyclic compounds [50], and also owing to wide range
of biological properties of nitrogen-containing heterocy-
clic compounds, we were encouraged to examine the use
of natural zeolite as efcient, low-cost and reusable catalyst
in the one-pot synthesis of pyrazolo[1,2-a][1,2,4]triazole-
1,3-diones 4a–i (Scheme 1) and evaluated for their cytotoxic,
apoptotic activities and also anti-oxidative efect.
interact with positive site of molecules that passes by these
structures. Accordingly, a possible mechanism to explain the
formation of pyrazolo[1,2-a][1,2,4]triazole-1,3-dione 4a–i
is depicted in Scheme 2.
As shown in this Scheme, the reaction is likely to be initi-
ated by a Knoevenagel-type condensation reaction between
malononitrile 1 and aromatic aldehyde 2 under the catalytic
efect of zeolite followed by dehydration to provide the
malononitrile derivative (A). This occurs through the activa-
tion of C–H bond in malononitrile by oxygen in the catalyst
from the one hand, and the activation of the carbonyl group
in aldehyde by the acidic part (cations) of the catalyst on
the other hand. Subsequently, a Michael-type addition of
intermediate (A) to 4-phenylurazole 3 under the catalytic
efect of natural zeolite occurs to yield a second interme-
diate (B) which successively undergoes cyclization to (C)
and natural zeolite promoted tautomerization to aford the
expected product 4.
The reaction between malononitrile 1, aldehydes 2a–i and
4-phenylurazole 3 for the synthesis of pyrazolo[1,2-a][1,2,4]
triazole-1,3-diones worked out best at 80 °C under solvent-
free conditions catalyzed with Natrolite zeolite (0.05 g). The
products 4a–i were characterized based on their physical
and spectral data which were in accord with those reported
in the literature [34].
Zeolites are the most important group of porous alu-
minum silicates with large tunnels and cages. They are
inorganic materials with tetrahedrally connected framework
structures based on corner-sharing silicate (SiO4) and alu-
minate (AlO4) tetrahedrals that are linked to each other by
shared oxygen atoms. For each Si4+ ion replaced by an Al3+,
the charge must be balanced by having other positive ions
such as Na+, K+, and Ca2+ ones. As a result, the frame work
is negatively charged. Thus, the oxygen atoms are sites to
The str uctural for mula of zeolite is
Mx/n[(AlO2)x(SiO2)y]∙wH2O, where M is an alkali or alkaline
earth cation, n is the valence of the cation, w is the number
of water molecules per unit cell, x and y are the total number
of tetrahedral atoms per unit cell, and the y/x ratio (Si/Al
Scheme 1 Synthesis of
pyrazolo[1,2-a][1,2,4]triazole-
1,3-diones 4a–i
Scheme 2 Possible mechanism
to explain the formation of
pyrazolo[1,2-a][1,2,4]triazole-
1,3-dione 4a–i
(B)
(A)
(C)
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Journal of the Iranian Chemical Society
ratio) usually ranges from 1 to 5 or 10 to 100 for high-silica
zeolites. There are several types of natural zeolites distrib-
uted in various parts of the world. Generally, these materials
are found in rocks near active or extinct volcanoes. Among
the natural zeolites, Natrolite zeolite, Na2[Al2Si3O10]∙2H2O,
as natural catalyst is widely used in the synthesis of specialty
and fne chemicals. Natural Natrolite zeolite as a heterogene-
ous catalyst used in the present reactions was characterized
by powder XRD, XRF, SEM, EDAX, TG–DTA analysis and
FT-IR spectra as explained below.
and 1637 cm−1 attributed to the O–H stretching vibration
due to H2O interporous structure and H–O–H bending
vibration, respectively. Also, the vibrational frequencies
appearing at 415–622 and 978–1066 cm−1 are likely allo-
cated to the pseudo-lattice, and the Si–O–Si and Si–O–Al
vibrations, respectively. In addition, the band appearing at
622 cm−1 can be assigned to Al–O vibrational frequency,
and the bands shown at 415–502 cm−1 are assigned to the
Si–O–Si bending vibration.
The advantages of SEM analysis include high-resolu-
tion imaging, and greater magnifcation which provides
textural information and much greater depth of the feld.
The morphology of zeolite demonstrates a surface with a
cubic shape, which is a typical structure for zeolite. It is
noted that the shape of Natrolite zeolite grains is heteroge-
neous in nature. The SEM micrograph of Natrolite zeolite
presented in Fig. 3 shows that the size of surface particles
is in the range 1–10 µm. As noticed, zeolites tend to form
bulkier crystals.
The X-ray difraction (XRD) spectrum of Natrolite zeo-
lite is shown in Fig. 1. The sharp intense peaks reveal the
good crystallinity of the zeolite material. The comparison
of the X-ray difractogram pattern of Natrolite zeolite with
the correspondence values for the standard sample (Interna-
tional Zeolite Association) show which pattern completely
matched with that of the Natrolite zeolite. The actual phases
for the natural Iranian Natrolite zeolite were silicon oxide
cristobalite-SiO2 (cubic) and aluminum silicate zeolite.
Chemical analysis of Natrolite zeolite based on the XRF
technique indicated that the natrolite content of zeolite min-
eral was 99.98% (w/w) (see Table 1). The LOI (loss on igni-
tion) method has been used to estimate the water and organic
matter contents of Natrolite zeolite. The SiO2/Al2O3 weight
ratio of the material is found to be 1.64 corresponding to
Natrolite zeolite used in this study.
Energy dispersion X-ray spectroscopy (EDS) was
recorded and is shown in Fig. 4. In the EDS spectrum of
the catalyst, all the peaks related to Si, Al, Na, and O were
observed (sample was coated with Au).
Thermal methods such as thermogravimetric analy-
sis (TGA) provide qualitative or quantitative analytical
information by the efect of heat on a sample. In these
techniques, the temperature in the specifc atmosphere is
The FT-IR spectrum recorded from zeolite is shown in
Fig. 2. As seen in this spectrum, the bands at 3330–3544
Fig. 1 XRD difraction pattern
of natural Natrolite zeolite
Table 1 Chemical composition of Natrolite zeolite used in this study; all data are expressed for oxides as (%, w/w)
SiO2
Al2O3
Na2O
Sr
CaO
SO3
LOIa
44.74
27.31
12.64
0.064
1.31
0.035
13.89
aLoss on ignition (this method has been used to estimate the amounts of water and organic matter)
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Journal of the Iranian Chemical Society
Fig. 2 FT-IR spectrum of the
natural Natrolite zeolite
Fig. 3 Scanning electron microscopy (SEM) analysis of the natural Natrolite zeolite
Fig. 4 EDS analysis of the
natural Natrolite zeolite
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Journal of the Iranian Chemical Society
programmed while the property of this sample is moni-
tored against time or temperature.
and 68 mg/ml, respectively (Table 2). Compound 4c inhib-
ited the growth of BT-474 and MCF-7 cells compared to
that of MDA-MB-231 cells. Moreover, compounds 4b, 4d
and 4h inhibited the growth of BT-474 and MCF-7 cells
compared to MDA-MB-231 cells. As shown in Table 2,
compound 4c exhibited between 10 and 30 times greater
cytotoxic activity against all used breast cancer cell line,
whereas it showed 5–10 times less toxicity compared with
that of the standard anticancer agent doxorubicin. Of note,
these concentrations had no signifcant efects on fbroblast
cell viability (Supplementary data).
TGA is a technique by which the sample weight can be
followed during specifc period of time while its temperature
is being raised gradually. Many materials produce specifc
curves when they are heated over the range of temperatures.
This change can be due to the particular thermal event such
as loss of water of crystallization. TGA experiments were
carried out by heating samples under air up to 800 °C in
order to check the thermal stability of natural Natrolite zeo-
lite. As seen in Fig. 5, two weight loss stages were observed
in fowing air. About 0.68 wt% weight loss was observed in
the frst stage at 80–240 °C corresponding to desorption of
surface water. In the second stage at 240–400 °C, weight loss
is about 8.98 wt%, which can be attributed to desorption of
lattice water.
Further analysis clearly revealed that the potency of com-
pounds was extremely dependent on the electronic proper-
ties of the substituent(s) on the phenyl moiety. Introduc-
tion of electron with electron-withdrawing groups (EWGs)
at the para-position of the phenyl ring (Ar) was favorable
for improved cytotoxic activity, whereas the presence of
electron-donating groups (EDGs), (4i), showed a decreased
activity compared to unsubstituted phenyl ring (4a). It can be
concluded that EWGs on the phenyl ring are more efective
than EDGs. These results could be ascribed to the degree of
electron density on the phenyl ring as well as the specifcity
of cancer cells. In addition, the nature of the halogen atoms
on the phenyl ring plays an important role on anticancer
activity. Compound 4b, with a chlorine atom at the para-
position, promoted the anticancer potency toward all cancer
cell lines as compared to 4e and 4f, which contain a fuorine
and a bromine, respectively, at the same region, suggesting
that chlorine is better tolerated than fuorine and bromine at
the para-position.
Biological activity
MTT assay in vitro
The cytotoxic activities of compounds 4a–i were evaluated
against human breast cancer lines (BT-474, MDA-MB-231,
and MCF-7) and fbroblast cells (as normal control) by the
standard MTT assay in vitro. Table 2 lists the IC50 values
obtained from these compounds. Most compounds showed
moderate antiproliferative efects and displayed IC50 values
in the micromolar concentration range (2.3 < IC50 < 78 μg/
ml) with three human breast cancer cell lines. Compound 4c
inhibited cancer cell growth in a dose-dependent manner,
and lower concentrations compared to all other compounds
after 48 h of treatment.
Western blot analysis
Among these compounds, 4c one showed the most potent
activity against BT-474, MDA-MB-231 and MCF-7 cells
with IC50 values of 2.3, 2.5 and 3 mg/ml, respectively; mean-
while, 4i agents showed the lowest activity against BT-474,
MDA-MB-231 and MCF-7 cells with IC50 values of 72, 78
To further demonstrate the apoptosis-inducing efect of
pyrazolo[1,2-a][1,2,4]triazole-1,3-dione derivatives, the
activation of protease caspase-3, which is activated in the
classic apoptosis pathway and used as a marker of apoptosis
Fig. 5 Thermogravimetric
analysis (TGA) of natural
Natrolite zeolite
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Table 2 Cytotoxicity of pyrazolo[1,2-a][1,2,4]triazole-1,3-diones 4a–
i against human breast cancer lines
increased the intensity of the band, particularly for com-
pound 4b.
Ar
Product (4)
IC50 (µg/ml)a
Apoptotic assay by Acridine orange staining
BT-474 MDA-MB-231 MCF-7
C6H5
70
75
72
Fluorescence microscopy analysis was used to examine the
apoptotic feature in BT-474, MDA-MB-231 and MCF-7
cells induced by IC50 concentration of each compound. In
order to investigate whether the inhibition of cancer cell
proliferation promoted by IC50 concentration was associ-
ated with apoptosis, three cell populations, including viable,
early apoptotic, and late apoptotic cells or dead cells, were
studied. Untreated cells were green after staining with AO,
showing their viability, while treated cells indicated stages
of apoptosis with AO staining. Figure 7 shows the stage of
apoptosis in three breast cells with nine compounds in their
IC50 concentrations.
4-ClC6H4
4-NO2C6H4
4-CNC6H4
4-FC6H4
25
27.5
25
2.3
28
45
52
2.5
30
48
50
3
23
55
41
Intracellular ROS measurement
As shown in Fig. 8a, treatment of BT-474 cells with lower
concentrations of pyrazolo[1,2-a][1,2,4]triazole-1,3-diones
4a–i decreased ROS production compared to higher con-
centrations, except 4d. Treatment with high doses of all
the agents increased ROS levels. However, all of the agents
could show an antioxidant activity in vitro but in higher
doses induced ROS production because of toxic activity.
The pattern of ROS level changes after treatment of MCF-7
and MDA-MB-231 cells with compounds 4a–i was shown
in Fig. 8b, c.
4-BrC6H4
Pyridin-3-yl
2,4-Cl2C6H4
40
23
43
32
18
15
Conclusions
The present study reports an efcient and convenient method
for synthesis of pyrazolo[1,2-a][1,2,4]triazole-1,3-diones
using the natural Natrolite zeolite as a non-toxic catalyst, as
well as their biological evaluation as cytotoxic and apopto-
sis-inducing agents. Operational simplicity, low cost, easy
availability and recyclability of the catalyst, also easy work
up are the attractive features of this methodology. Using the
three-component condensation reaction, compounds were
prepared and their cytotoxicity against three human breast
cancer cell lines, e.g., breast cancer (BT-474), breast car-
cinoma (MCF ( and triple negative breast cancer (MDA-
MB-231), were evaluated. Compound 4c containing nitro
substituent showed the highest cytotoxic activity against all
cancer cell lines. Based on ROS measurement of all deri-
vations, our results showed that all tested compounds in
this study can be potentially used as an antioxidant agent
and reduce ROS level in lower doses of IC50. But in IC50
doses, our derivation had an apoptotic role during treatment
of three breast cancer cell lines. Most of the compounds
4-MeC6H4
72
78
68
Determined by MTT assay (48 h drug exposure)
aCompounds tested in triplicate, data expressed as mean values of
three independent experiments
induction, was studied. The results showed that treatment
with compounds 4b, 4c, 4d and 4h for 24 h increased the
activation of caspase-3 in IC50 concentration. As shown in
Fig. 6, the western blot analysis results revealed one band
at 45 kDa, which was the band for caspase-3. The caspase-3
bands of the 4a, 4e, 4f, 4g and 4i were extremely faint.
Treatment with compounds 4b, 4c, 4d and 4h for 24 h
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Journal of the Iranian Chemical Society
Fig. 6 Activation of protease
caspase-3 as a marker of apop-
tosis induction by pyrazolo[1,2-
a][1,2,4]triazole-1,3-dione
derivatives in human breast
cancer cells
Fig. 7 Comparison of a 96-well-based and the AO method for
BT-474, MCF-7 and MDA-MB-231 adherent cells. Cells were treated
with IC50 concentration of each compound for induction of apopto-
sis. Green cells show live cells, while orange cells indicate apoptotic
cells, and red cells indicate necrotic cells
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Journal of the Iranian Chemical Society
Fig. 8 Intracellular ROS evaluation in a MCF-7, b MDA-MB-231, c BT474 cell lines after treatment with compounds 4a–i
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Fig. 8 (continued)
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showed signifcant apoptotic activity against all cell lines,
whereas compound 4c showed greater potent activity against
three cell lines. Moreover, we found in IC50 concentration of
each compound, intracellular ROS level elevation may cause
apoptosis and cytotoxic induction. This pathway could play
a role in cell death in IC50 concentration.
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