Synthesis of 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives and evaluation of their anticancer activity

Article abstract of DOI:10.3109/14756366.2012.724682

In the present study, 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives (1-6) were synthesized via the ring closure reaction of 1-(2-thienyl)-3-aryl-2-propen-1-ones with hydrazine hydrate in acetic acid. The chemical structures of the compounds were

Full text of DOI:10.3109/14756366.2012.724682

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; Early Online: 1–7
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2012.724682
ReseaRch aRtIcle
Synthesis of 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline
derivatives and evaluation of their anticancer activity
Ahmet Özdemir1, Mehlika Dilek Altıntop1, Zafer Asım Kaplancıklı1, Gülhan Turan-Zitouni1,
Gülşen Akalın Çiftçi2, and Şafak Ulusoylar Yıldırım3
1Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, Eskişehir, Turkey, 2Faculty of
Pharmacy, Department of Biochemistry, Anadolu University, Eskişehir, Turkey, and 3Faculty of Pharmacy,
Department of Pharmacology, Anadolu University, Eskişehir, Turkey
abstract
In the present study, 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives (1–6) were synthesized via the ring closure
reaction of 1-(2-thienyl)-3-aryl-2-propen-1-ones with hydrazine hydrate in acetic acid. The chemical structures of the
1
compounds were elucidated by IR, H-NMR, 13C-NMR and mass spectral data and elemental analyses. MTT assay,
analysis of DNA synthesis and caspase-3 activation assay were carried out to determine anticancer effects of the
compounds on A549 and C6 cancer cell lines. They exhibited dose-dependent anticancer activity against A549 and
C6 cancer cell lines. Anticancer activity screening results revealed that compounds 1, 2 and 4 were the most potent
derivatives among these compounds. But anticancer effects of these compounds may result from different death
mechanisms in A549 and C6 cell lines.
Keywords: Pyrazoline, anticancer activity, MTT assay, caspase-3 activation
Introduction
Over the past two decades, pyrazolines have attracted
Cancer, which is characterized by the uncontrolled
growth of abnormal cells in the body, has emerged as
the second leading cause of death throughout the world
after cardiovascular disorders. e incidence of cancer
has increased dramatically in the last decades. As a con-
sequence of this situation, the treatment of cancer has
a great deal of interest as privileged scaffolds owing to
their synthetic and biological importance in medicinal
chemistry. Pyrazoline derivatives have been reported to
exhibit a wide spectrum of biological effects including
anticancer activity6–16. Some compounds bearing pyr-
azole moiety exert their therapeutic action by inhibiting
different types of enzymes that play important roles in
gained great importance1–3
.
cell division16–18
.
Chemotherapy, which is used to destroy cancer cells
with drugs, is one of the most widely used treatment
options in cancer treatment. Although there are many
anticancer drugs currently available, their use in the
treatment is limited due to their adverse effects, and the
development of resistance. e side effects accompany-
ing the use of these drugs arise from the fact that antican-
Manna et al. synthesized 1-acetyl-3,5-diaryl-4,5-di-
hydro-(1H)-pyrazole derivatives and investigated their
anticancer activity and affinity binding to P-glycoprotein.
In the same study, some compounds were found to bind
to P-glycoprotein with greater affinity19.
Many researchers have also studied thiophenes
extensively due to the fact that new effective compounds
can be obtained by the bioisosteric replacement of
benzene ring with thiophene20,21. Some studies have
confirmed that compounds bearing thiophene moiety
cer agents act on both tumour cells and healthy cells3–6
.
From the above discussion, it is clear that the search
for new effective compounds which can selectively
inhibit the proliferation of abnormal cells only with least
or no affect on normal cells has gained great importance6.
possess antitumor activity22–24
.
Address for Correspondence: Ahmet Özdemir, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Anadolu University, 26470
Eskişehir, Turkey. Tel: +90-222-3350580/3774. Fax: +90-222-3350750. E-mail: ahmeto@anadolu.edu.tr
(Received 25 June 2012; revised 22 August 2012; accepted 23 August 2012)
1

2
A. Özdemir et al.
On the basis of these findings, we became interested
129.04 (2CH), 130.20 (CH), 133.22 (CH), 136.37 (CH),
137.17 (C), 138.44 (C), 139.61 (C), 150.57 (C), 167.23 (C).
MS-FAB+: m/z: 296 [M + 1]. For C16H13N3OS calculated:
65.06 % C, 4.44 % H, 14.23 % N; found: 64.88 % C, 4.31 %
H, 14.29 % N.
in biological evaluation of pyrazoline derivatives as
antitumor agents. Herein, we described the synthesis of
new pyrazoline derivatives bearing thiophene moiety
and focused on their potential anticancer effects against
A549 and C6 cancer cell lines using MTT assay. Among
these derivatives, the most effective compounds were
evaluated for their DNA synthesis inhibitory activity and
effects on caspase-3 activation.
1-Acetyl-3-(2-thienyl)-5-(4-isopropylphenyl)-2-pyrazoline (6)
IR [ʋ, cm−1, KBr]: 1648 (C=O), 1588 (C=N), 820 (1,4-disub-
stituted benzene C-H).
1H-NMR (500 MHz, DMSO-d6): δ 1.18 (6H, s, 2CH3),
2.26 (3H, s, COCH3), 2.87 (1H, m, CH(CH3)2), 3.17 (1H, dd
experimental
J
AM = 17.73 Hz, JAX = 4.18 Hz, C4-HA of pyrazoline ring), 3.84
Chemistry
(1H, dd JMA = 17.74 Hz, JMX = 11.80 Hz, C4–HM of pyrazoline
ring), 5.53 (1H, dd JMX = 11.61 Hz, JAX = 4.14 Hz, C5–HX of
pyrazoline ring), 7.09–7.45 (5H, m, aromatic protons),
7.75 (2H, d J = 5.03 Hz, phenyl protons).
All reagents were used as purchased from commer-
cial suppliers without further purification. Melting
points were determined by using an Electrothermal
9100 digital melting point apparatus and were uncor-
rected (Electrothermal, Essex, UK). e compounds
were checked for purity by TLC on silica gel 60 F254.
Spectroscopic data were recorded on the following
instruments: IR, Shimadzu 435 IR spectrophotometer
(Shimadzu, Tokyo, Japan); 1H-NMR, Bruker 500 MHz
NMR spectrometer (Bruker Bioscience, Billerica, MA,
USA) and 13C-NMR, Bruker Avance II 125 MHz NMR
spectrometer (Bruker Bioscience, Billerica, MA, USA)
in DMSO-d6 using TMS as internal standard; MS-FAB,
VG Quattro mass spectrometer (Fisons Instruments
Vertriebs GmbH, Mainz, Germany), Elemental analyses
were performed on a Perkin Elmer EAL 240 elemental
analyser (Perkin Elmer, Norwalk, CT, USA).
13C-NMR (125 MHz, DMSO-d6): δ 22.16 (CH3), 24.32
(2CH3), 34.22 (CH), 43.23 (CH2), 59.75 (CH), 125.81 (CH),
128.53 (CH), 129.67 (2CH), 130.81 (CH), 134.62 (CH),
136.86 (CH), 137.21 (C), 138.72 (C), 139.73 (C), 150.64
(C), 167.38 (C).
MS-FAB+: m/z: 313 [M + 1]. For C18H20N2OS calculated:
69.20 % C, 6.45 % H, 8.97 % N; found: 69.28 % C, 6.53 %
H, 8.95 % N.
Biochemistry
Cell culture and drug treatment
C6 glioma cells were incubated in Dulbecco’s modi-
fied Eagle’s medium (DMEM) (Sigma, Deisenhofen,
Germany) supplemented with 10% fetal calf serum
(Gibco, Paisley, Scotland). A549 cells were incubated in
90% RPMI supplemented with 10% fetal bovine serum
(Gibco, Paisley, Scotland). All media were supplemented
with 100 IU/mL penicillin-streptomycin (Gibco, Paisley,
Scotland) and cells were incubated at 37°C in a humidi-
fied atmosphere of 95% air and 5% CO2. Exponentially
growing cells were plated at 2 × 104 cells/mL into 96-well
microtiter tissue culture plates (Nunc, Denmark) and
incubated for 24 h before the addition of the drugs (the
optimum cell number for cytotoxicity assays was deter-
mined in preliminary experiments). Stock solutions
of compounds were prepared in dimethyl sulphoxide
(DMSO; Sigma Aldrich, Poole, UK) and further dilutions
were made with fresh culture medium (the concentra-
tion of DMSO in the final culture medium was <0.1%
which had no effect on the cell viability).
General procedure for the synthesis of compounds
1-(2-Thienyl)-3-aryl-2-propen-1-ones (Chalcones)
A mixture of 2-acetylthiophene (0.04 mol), aromatic
aldehyde (0.04 mol) and 10% aqueous sodium hydroxide
(10 mL) in ethanol (50 mL) was stirred at room tempera-
ture for 4 h. e resulting solid was washed, dried and
crystallized from ethanol26.
1-Acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives (1–6)
To a solution of chalcone derivative (1 mmol) in acetic
acid (3 mL), hydrazine hydrate (80%) (0.3 mL, 6 mmol)
was added. e mixture was refluxed under stirring for
5 h, and then poured onto crushed ice. e precipitate
was filtered off, washed with cold water, and crystallized
from methanol to give pyrazolines9.
1-Acetyl-3-(2-thienyl)-5-(4-cyanophenyl)-2-pyrazoline (5)
IR [ʋ, cm−1, KBr]: 1648 (C=O), 1597 (C=N), 827 (1,4-disub-
stituted benzene C-H).
1H-NMR (500 MHz, DMSO-d6): δ 2.27 (3H, s, COCH3),
3.16 (1H, dd JAM = 17.90 Hz, JAX = 4.89 Hz, C4-HA of pyr-
azoline ring), 3.92 (1H, dd JMA = 17.95 Hz, JMX = 11.96 Hz,
C4–HM of pyrazoline ring), 5.66 (1H, dd JMX = 11.88 Hz,
MTT assay for cytotoxicity of compounds
e level of cellular 3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyltetrazolium bromide (MTT) (Sigma) reduction
was quantified as previously described in the literature
with small modifications27,28. After 24 h of preincuba-
tion, the tested compounds were added to give final
concentration in the range 3.9–500 µg/mL and the
cells were incubated for 24 h. At the end of this period,
MTT was added to a final concentration of 0.5 mg/mL
and the cells were incubated for 4 h at 37oC. After the
medium was removed, the formazan crystals formed by
J
AX = 4.87 Hz, C5-HX of pyrazoline ring), 6.78–7.76 (7H, m,
aromatic protons).
13C-NMR (125 MHz, DMSO-d6): δ 22.04 (CH3), 43.36
(CH2), 59.76 (CH), 124.71 (C), 125.89 (CH), 127.14 (CH),
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives
3
MTT metabolism were solubilized by addition of 200 µL
DMSO to each well and absorbance was read at 540 nm
with a microtitre plate spectrophotometer (Bio-Tek plate
reader). Every concentration was repeated in three wells
and IC50 values were defined as the drug concentrations
that reduced absorbance to 50% of control values.
medium was replaced with fresh medium containing dif-
ferent concentrations of compounds 1, 2, 4 and mitoxan-
trone. e cells, without treatment, were used as negative
controls. Cells then were incubated for 24 h in humidified
atmosphere at 37°C in 5% CO2. e medium was removed
and cells were washed three times with PBS. en cells
were fixed with 70% ethanol and incubated for 5 min in
RT. Cells were washed with PBS. en working Solution,
ethidium bromide/acridin orange (a mixture of acri-
dine orange and ethidium bromide 1:1, 100 µg/mL) was
added and incubated for 5 min. After cells were washed
with PBS, cover slip, containing cells was removed and
covered on the object glass. en assessment can be car-
ried out under fluorescence microscope (Olympus)31.
Acridine Orange/Ethidium Bromide combination was
used to visualize cells with aberrant chromatin organiza-
tion. Acridine Orange was used to visualize the number
of cells which has undergone apoptosis. e differential
uptake of these two dyes allows the identification of
viable and non-viable cells32.
Analysis of DNA synthesis
Analysis of DNA synthesis was measured by Roche Cell
Proliferation ELISA, BrdU (colorimetric) kit. is immu-
nostaining procedure is based on measuring the incor-
poration of bromodeoxyuridine (BrdU) into nuclear
DNA in place of thymidine during the S-phase of the cell
cycle using specific anti-BrdU antibodies29. Hence, such
method provides a colorimetric measurement for DNA
synthesis inhibition ratio of the carcinogenic cells. Firstly
cells were seeded into 96-well flat-bottomed microtiter
plates at a density of 2 × 103. e tumor cells were cul-
tured in the presence of various doses of compounds 1,
2, 4 and mitoxantrone. Microtiter plates were incubated
at 37°C in a 5% CO2/95% air humidified atmosphere for
24 h. Cells were labeled with 10 µL BrdU solution for 2h
and then fixed. Anti-BrdU-POD (100 µL) was added and
incubated for 90 min. Finally, wells were washed with
BPS and cells were incubated with substrate. Absorbance
of the samples was measured with an ELX808-IU Bio-
Tek apparatus at 492 nm. All experiments were repeated
two times. For each compound dose, duplicate wells
were used.
Statistical analyses
Statistical Package for the Social Science (SPSS) for
Windows 15.0 was used for statistical analysis. Data was
expressed as Mean SD. Comparisons were performed
by one way ANOVA test for normally distributed continu-
ous variables and post hoc analyses of group differences
were expressed by the Tukey test.
Results and discussion
Spectrofluorometric analysis of Caspase-3 activation
Caspase-3activationwasanalysedbySpectrofluorometric
Caspase-3 Assay kit (BD Pharmingen). is kit is designed
to measure caspase-3 or DEVD-cleaving activity, an
early marker of cells undergoing apoptosis30. Firstly cells
(1–106 cells/mL) were washed with PBS and resuspended
in cold cell lysis buffer and incubated for 30 min on ice.
en cell lysates were prepared after 24 h incubation with
compounds 1, 2, 4 and mitoxantrone. For each reaction, 5
µL reconstituted Ac-DEVD-AMC (synthetic tetrapeptide
fluorogenic substrate for Caspase-3) was added to a well
containing 0.2 mL of 1 X HEPES buffer. 20 µL cell lysate
was added to each well/reaction. Reaction mixtures were
incubated for 1 h at 37°C. e amount of AMC liberated
from Ac-DEVD-AMC was measured using plate reader
(Perkin Elmer/Victor/X3) with an excitation wavelenght
of 380 nm and an emmision wavelength of 460nm.
Apoptotic cell lysates containing active Caspase-3 yield
a considerable emission as compared to controls. Non-
apoptotic control cell lysates AMC emission was accepted
as 100% and other cell lysates emissions were measured
according to control cells emissions. All experiments
were repeated two times. For each compound dose,
duplicate wells were used.
e synthesis of pyrazoline derivatives (1–6) was carried
out according to the steps shown in Scheme 1. In the
initial step, 1-(2-thienyl)-3-aryl-2-propen-1-ones were
synthesized via the base-catalyzed Claisen-Schmidt
condensation of 2-acetylthiophene with appropriate
aldehydes. e ring closure reaction of chalcones with
hydrazine hydrate in acetic acid afforded 1-acetyl-3-(2-
thienyl)-5-aryl-2-pyrazolines (1–6). Some properties of
the compounds were given in Supplementary Table S1.
e structures of all compounds were confirmed by
IR, 1H-NMR, 13C-NMR, mass spectral data and elemental
analyses. e IR data were very informative and provided
evidence for the formation of the expected structures.
C=O, C=N, and 1,4-disubstituted benzene C-H func-
tions absorbed strongly in the expected regions: C=O at
1640–1655 cm−1, C=N at 1614-1402 cm−1 and 1,4-disub-
stituted benzene C-H at 820–830 cm−1, respectively. e
1H-NMR spectral data were also consistent with the
1
assigned structures. In the H-NMR spectra of all com-
pounds, the CH2 protons of the pyrazoline ring resonated
as a pair of doublets of doublets at δ = 3.10–3.17, and
3.81–3.92 ppm. e CH proton appeared as doublet of
doublets at δ = 5.45–5.66 ppm due to vicinal coupling
with two magnetically non-equivalent protons of the
methylene group at position 4 of the pyrazoline ring
(JAM = 17.71–17.95 Hz, JAX = 4.12–4.89 Hz, JMX = 11.61–11.96
Hz). Moreover, the methyl protons of the acetyl group
Acridine orange/ethidium bromide staining methods
C6 glioma cells were cultured on cover slips at 1 × 105
cells/well onto 6 wells plate until 50% confluent. e
© 2012 Informa UK, Ltd.

4
A. Özdemir et al.
All compounds gave satisfactory elemental analysis. e
mass spectra (MS-FAB+) of all compounds showed [M +
1] peaks, in agreement with their molecular formula. e
spectral data of compounds 1, 2, 3 and 4 were given in
Supplementary Material.
Anticancer activity screening of the compounds
against A549 and C6 cell lines was carried out at four steps.
In the first step, dose-dependent cytotoxic effects of the
compounds were tested on A549 and C6 cancer cell lines
using MTT assay. en, DNA synthesis inhibition assay
appeared as a singlet peak at δ = 2.23–2.27 ppm. All the
other aromatic and aliphatic protons were observed at
expected regions. e signals obtained from 13C-NMR
spectra further confirmed the proposed structures; the
C4 and C5 carbons of the pyrazoline ring resonated at
43.23–43.56 and 59.49–59.96 ppm, respectively. All com-
pounds showed a signal at 150.57–150.68 ppm, which
was assignable to azomethine carbon of pyrazoline ring.
In the 13C-NMR spectra of the compounds, the acetyl
carbon was observed in the region 167.16–167.57 ppm.
Scheme 1. e synthesis of the compounds (1–6).
Table 1. Cytotoxicity of compounds 1–6 against A549 cell line.
Concentration (µg/mL)
Compound
3.9
7.8
15.6
31.2
62.5
125
250
500
IC50 (µg/mL)a
86.7 2.9
53.3 5.8
>500
1
2
3
4
5
6
Mito
87.4 6.0
88.9 9.2
111.5 5.0
96.0 8.1
111.4 11.4
98.8 5.3
81.1 5.3
83.1 3.2
81.0 3.4*
105.7 7.6
90.8 1.8
87.1 8.8
80.5 7.4*
77.5 6.7*
85.8 8.5
82.2 4.0*
99.6 7.3
89.2 16.7
81.8 10.9
73.6 7.7*
81.8 4.9
67.3 23.5* 34.3 8.9*
33.8 1.5*
28.3 2.6*
86.4 3.3
46.8 1.9*
59.4 3.9
55.7 2.2*
30.9 5.4*
25.5 2.9*
24.6 1.5*
84.6 6.7
79.9 2.1* 41.35 5.5*
29.6 2.7*
87.3 2.6
57.4 2.8*
60.2 3.4
59.2 5.9*
36.5 5.1*
97.4 6.8
82.1 5.8
74.9 8.3
64.5 9.1*
94.1 6.1
73.6 4.8*
69.6 8.6
55.9 0.8*
39.9 4.4*
31.0 3.1* 193.3 40.4
57.3 4.7 >500
47.5 4.1* 435.0 68.7
24.8 1.9* 24.3 2.1
58.8 17.1* 37.8 3.6*
aResults are expressed as the mean % of MTT absorbance. Data points represent means of three independent experiments SD.
*significantly differences from control p < 0.05.
Table 2. Cytotoxicity of compounds 1–6 against C6 cell line.
Concentration (µg/mL)
Compound
3.9
7.8
15.6
31.2
62.5
125
250
500
IC50 (µg/mL)a
50.7 3.8
45.0 8.8
>500
1
2
3
4
5
6
Mito
112.9 11.9 123.7 20.7 121.8 17.7 109.8 8.6
36.4 5.1*
40.7 3.4*
82.7 8.7
91.6 8.8
31.4 4.8*
17.7 0.8*
64.9 7.8*
12.7 0.7*
14.7 3.4*
59.2 8.9*
12.3 1.4*
13.2 1.6*
62.7 10.6*
113.2 13.4 118.9 21.0 111.9 7.5
70.7 15.1
91.2 1.3
102.8 5.1
108.2 12.1
97.9 6.6
98.5 11.2
89.5 14.5 104.5 16.3 104.9 11.5
66.5 10.6* 48.4 3.2*
42.1 4.7* 216.0 29.5
41.7 7.0 * 415 73.7
36.5 5.8* 256.7 40.4
16.5 2.1* 11.0 1.0
116.2 22.2 120.4 8.6
111.9 16.5 127.9 13.4 102.5 5.1
90.2 8.8
83.1 12.1
34.7 4.7*
71.5 9.9
50.9 5.0*
25.1 4.1*
106.6 3.9
74.9 8.4*
98.7 12.2 111.8 12.6 121.4 23.6 107.6 7.5
57.6 3.3* 45.8 8.3* 35.9 3.9* 37.4 7.0*
aResults are expressed as the mean % of MTT absorbance. Data points represent means of three independent experiments SD.
*significantly differences from control p < 0.05.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives
5
Figure 3. Effect of Ac-DEVD-amc on the activity of caspase-3
induced by compounds 1, 2, 4 and mitoxantrone. A546 cells were
maintained in cultures for 24 h and then exposed to Ac-DEVD-amc
(1.0 mM) 30 min before exposure to 1a 31.2 µg/mL; 1b 86.7 µg/mL;
2a 31,2 µg/mL; 2b 53.3 µg/mL; 4a 62.5 µg/mL; 4b 193.3 µg/mL;
Mito a 7.8 µg/mL; Mito b 24.3 µg/mL). Values represent mean
S.D. from duplicate samples for each experiment. ***significantly
different from respective control cells (p < 0.001).
Figure 1. DNA Synthesis inhibitory activity of compounds 1, 2,
4 and mitoxantrone on A549 cells. Mean percent absorbance
of untreated control cells were assumed 0%. ree different
concentrations (1a 31.2 µg/mL; 1b 62.5 µg/mL; 1c 125µg/mL; 2a
31.2 µg/mL; 2b 53.3 µg/mL; 2c 125 µg/mL; 4a 62.5 µg/mL; 4b 125
µg/mL; 4c 250 µg/mL; Mito a 7.8 µg/mL; Mito b 24.3 µg/mL; Mito c
31.2 µg/mL) of test compounds and mitoxantrone were given. Data
points represent means for two independent experiments SD of
four independent wells. p < 0.05.
Figure 4. Effect of Ac-DEVD-amc on the activity of caspase-3
induced by compounds 1, 2, 4 and mitoxantrone. C6 cells were
maintained in cultures for 24 h and then exposed to Ac-DEVD-amc
(1.0 mM) 30 min before exposure to (1a 31.2 µg/mL; 1b 50.7 µg/mL;
2a 31.2 µg/mL; 2b 45 µg/mL; 4a 125 µg/mL; 4b 216 µg/mL; Mito a
3.9 µg/mL; Mito b 11 µg/mL). Values represent mean S.D. from
dublicate samples for each experiment.*: significantly different
from respective control cells (p < 0.05).**significantly different from
respective control cells (p < 0.01).***significantly different from
respective control cells (p < 0.001).
Figure 2. DNA Synthesis inhibitory activity of compounds 1,
2, 4 and mitoxantrone on C6 cells. Mean percent absorbance
of untreated control cells were assumed 0%. ree different
concentrations (1a 31.2 µg/mL; 1b 50 µg/mL; 1c 125 µg/mL;
2a 31.2 µg/mL; 2b 45µg/mL; 2c 125 µg/mL; 4a 125 µg/mL; 4b 216
µg/mL; 4c 250 µg/mL; Mito a 3.9 µg/mL; Mito b 11 µg/mL; Mito c
15.6 µg/mL) of test compounds and mitoxantrone were given. Data
points represent means for two independent experiments SD of
four independent wells. p < 0.05.
of the cancer cell lines with IC50 values of 24.3 2.1 µg/mL
and 11.0 1.0 µg/mL, respectively. Compounds 1 and 4
exhibited cytotoxic activity against A549 cell line with IC50
values of 86.7 2.9 and 193.3 40.4 µg/mL, respectively.
IC50 values of compounds 1 and 4 against C6 cell line were
also determined as 50.7 3.8 and 216.0 29.5 µg/mL,
respectively. Cytotoxicity of compound 3 was lower than
compound 6. Cytotoxicity of compound 5 was also simi-
lar to compound 3. ese results suggest that 4-methyl,
4-bromo and 3,4-methylenedioxy moieties contribute
to the increase in cytotoxicity. On the other hand, low
cytotoxicity of compounds 3, 5 and 6 can be attributed to
4-hydroxy, 4-cyano and 4-isopropyl moieties.
was carried out on A549 and C6 cell lines for compounds
1, 2 and 4 which exhibited significant cytotoxic activity
in MTT assay. Afterwards, compounds 1, 2 and 4 were
also subjected to caspase-3 activation assay to determine
the possible mechanism of action. Finally, cellular and
nuclear morphological changes of C6 cells following
exposure to various concentrations of compounds 1, 2, 4
and mitoxantrone were also observed.
In MTT assay, cytotoxicity of compounds 1, 2 and
4 was closer to that of mitoxantrone than other com-
pounds and the most effective cytotoxic agent against two
cancer cell lines was found as compound 2 followed by
compounds 1 and 4 (Tables 1 and 2). Compound 2 exhib-
ited anticancer activity against A549 and C6 cell lines with
IC50 values of 53.3 5.8 and 45.0 8.8 µg/mL, whereas
mitoxantrone exhibited anticancer activity against both
DNA synthesis inhibition activity of compounds 1,
2 and 4 was evaluated for 24 h. A549 and C6 cell lines
were incubated with three different concentrations of
compounds that were determined according to their IC50
© 2012 Informa UK, Ltd.

6
A. Özdemir et al.
Figure 5. Cellular and nuclear morphological changes of C6 cells following exposure to various concentrations of compound 1, 2, 4 and
mitoxantrone for 24 h. (1b 50 µg/mL; 2b 45 µg/mL 4b 216 µg/mL; Mito b 11 µg/mL). Cells were distinguished according to the fluorescence
emission and the morphological aspect of chromatin condensation in the stained nuclei. Viable cells have uniform bright green nuclei with
organized structure. Early apoptotic cells have green nuclei, but perinuclear chromatin condensation is visible as bright green patches or
fragments. White arrows indicate apoptotic cells. (See colour version of this figure online at www.informahealthcare.com/enz)
values. Mitoxantrone was used as positive control. e
tested compounds showed inhibitory activity in dose-
dependent manner.
increases in caspase-3 activation on C6 cells when com-
pared with controls (Figure 4). Compound 2 showed the
highest activity in caspase-3 activation assay.
DNA synthesis inhibitory activity of the com-
pounds on A549 and C6 cell lines is presented in
Figures 1 and 2. ese results show that compounds tested
in this assay cause DNA synthesis inhibition. Among these
compounds, the most potent inhibitor of DNA synthesis
against A549 and C6 cell lines was found as compound 2.
Caspase-3 activation, which plays a pivotal role in
initiation of cellular events during the early apoptotic
process, can be used in cellular assays as a hallmark of
apoptosis25. A549 and C6 cell lines were incubated with
the compounds at two different concentrations. IC50 and
lower concentrations were preferred for this purpose. As
seen in Figure 3, A549 cells treated with compounds 1,
2 and 4 had no significant differences in caspase-3 acti-
vation when compared with controls. is result may be
explained by different death mechanism in A549 cells
caused by compounds other than apoptosis. On the other
hand, these compounds showed significantly different
Morphological evaluation of early apoptosis in C6
cells was carried out with acridine orange/ethidium bro-
mide staining method due to the fact that only C6 cells
displayed early apoptotic response to these compounds.
C6 cell lines were incubated with compounds 1, 2, 4 and
mitoxantrone at IC50 concentrations. When incubation
period (24h) was over, C6 cells were stained with acri-
dine orange/ethidium bromide and cellular and nuclear
morphological changes were monitored in fluorescence
microscope (Figure 5). Viable cells had uniform bright
green nuclei with organized structure. But early apop-
totic cells caused by compounds 1, 2 and 4 had green
nuclei and perinuclear chromatin condensation.
conclusion
In the present paper, we synthesized a series of pyrazoline
derivatives and evaluated their anticancer activity against
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis of 1-acetyl-3-(2-thienyl)-5-aryl-2-pyrazoline derivatives
7
containing pyrazoline moiety and evaluation of their anticancer
activity. Eur J Med Chem 2009;44:1396–1404.
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A549 and C6 cancer cell lines by means of MTT, DNA syn-
thesis inhibition and caspase-3 activation assays. Among
these derivatives, compounds 1, 2 and 4 exhibited dose-
dependent anticancer activity against A549 and C6 can-
cer cell lines. Although these three compounds showed
substantial cytotoxicity and caspase-3 activation in C6
cells, they did not show any early apoptotic effects on
A549 cells. Hence, anticancer effects of these compounds
may result from different death mechanism in A549 and
C6 cell lines.
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3-(1H-benzimidazol-2-yl)-5-isoquinolin-4-ylpyrazolo[1,2-b]
pyridine, a potent cyclin dependent kinase 1 (CDK1) inhibitor.
Bioorg Med Chem Lett 2007;17:1243–1245.
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
18. Zhu GD, Gong J, Gandhi VB, Woods K, Luo Y, Liu X et al. Design
and synthesis of pyridine-pyrazolopyridine-based inhibitors of
protein kinase B/Akt. Bioorg Med Chem 2007;15:2441–2452.
19. Manna F, Chimenti F, Fioravanti R, Bolasco A, Secci D, Chimenti
P et al. Synthesis of some pyrazole derivatives and preliminary
investigation of their affinity binding to P-glycoprotein. Bioorg
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