Synthesis and evaluation of new pyrazoline derivatives as potential anticancer agents

Article abstract of DOI:10.3390/molecules201019066

New pyrazoline derivatives were synthesized and evaluated for their cytotoxic effects on AsPC-1 human pancreatic adenocarcinoma, U87 and U251 human glioblastoma cell lines. 1-[((5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetyl]-3-(2-thienyl)-5-(4-chlor

Full text of DOI:10.3390/molecules201019066

Molecules 2015, 20, 19066-19084; doi:10.3390/molecules201019066
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molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Evaluation of New Pyrazoline Derivatives as
Potential Anticancer Agents
Muhammed Karabacak 1,†, Mehlika Dilek Altıntop 1,†, Halil İbrahim Çiftçi 2,†, Ryoko Koga 2,
Masami Otsuka 2, Mikako Fujita 3 and Ahmet Özdemir 1,*
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Anadolu University,
Eskişehir 26470, Turkey; E-Mails: rem1596@hotmail.com (M.K.);
mdaltintop@anadolu.edu.tr (M.D.A.)
2
Department of Bioorganic Medicinal Chemistry, School of Pharmacy, Kumamoto University,
Kumamoto 862-0973, Japan; E-Mails: 130y2011@st.kumamoto-u.ac.jp (H.İ.Ç.);
kk1205@kumamoto-u.ac.jp (R.K.); motsuka@gpo.kumamoto-u.ac.jp (M.O.)
3
Research Institute for Drug Discovery, School of Pharmacy, Kumamoto University,
Kumamoto 862-0973, Japan; E-Mail: mfujita@kumamoto-u.ac.jp
†
These authors contributed equally to this work.
* Author to whom correspondence should be addressed; E-Mail: ahmetefegunes@gmail.com;
Tel.: +90-222-3350580 (ext. 3753); Fax: +90-222-3350750.
Academic Editor: Jean Jacques Vanden Eynde
Received: 17 August 2015 / Accepted: 14 October 2015 / Published: 20 October 2015
Abstract: New pyrazoline derivatives were synthesized and evaluated for their cytotoxic effects
on AsPC-1 human pancreatic adenocarcinoma, U87 and U251 human glioblastoma cell lines.
1-[((5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)thio)acetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-
2-pyrazoline (11) was found to be the most effective anticancer agent against AsPC-1 and
U251 cell lines, with IC50 values of 16.8 µM and 11.9 µM, respectively. Tumor selectivity
of compound 11 was clearly seen between Jurkat human leukemic T-cell line and human
peripheral blood mononuclear cells (PBMC). Due to its promising anticancer activity,
compound 11 was chosen for apoptosis/necrosis evaluation and DNA-cleavage analysis in U251
cells. Compound 11-treated U251 cells exhibited apoptotic phenotype at low concentration
(1.5 µM). DNA-cleaving efficiency of this ligand was more significant than cisplatin and was
clearly enhanced by Fe(II)-H2O2-ascorbic acid systems. This result pointed out the relationship
between the DNA cleavage and the cell death.

Molecules 2015, 20
Keywords: pyrazoline; oxadiazole; anticancer activity; apoptosis; DNA cleavage
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1. Introduction
Cancer is not a single disease, but a large group of diseases characterized by uncontrolled, rapid, and
pathological proliferation of abnormally transformed cells. Despite recent advances in cancer therapy,
cancer is still the second leading cause of death after cardiovascular disorders throughout the world [1–4].
Resistance to chemotherapeutic agents remains a key challenge in the fight against cancer. Another
challenge for chemotherapy is lack of selectivity. Generally anticancer drugs destroy normal cells as
well as cancer cells and often cause serious adverse effects. Therefore, new antineoplastic agents are
continually under development to selectively destroy tumour cells or at least limit their proliferation [1–4].
Diversely substituted pyrazolines embedded with a variety of functional groups are found in many
important biologically-active compounds and considerable research on this class of agents has been carried
out. They exhibit a wide spectrum of biological activities such as antimicrobial, anti-inflammatory,
antidepressant, and anticancer effects. Among the reported activities, it is important to note that pyrazolines
are not only useful in treatment of various cancer types, including brain, bone, mouth, esophagus,
stomach, liver, bladder, pancreas, cervix, lung, breast, colon, rectum, and prostate cancers, but also some
of them act as cancer chemopreventive agents [5–23]. In many studies, pyrazoline derivatives were
reported as epidermal growth factor receptor tyrosine kinase (EGFR-TK) inhibitors [19], aurora kinase
inhibitors [20], COX-2/B-Raf inhibitors [21], telomerase inhibitors [22], tubulin assembling inhibitors [23].
Additionally, 1,3,4-oxadiazole has emerged as an important scaffold owing to its metabolic profile and ability
to engage in hydrogen bonding with receptor site. Recent studies have indicated that 1,3,4-oxadiazole
derivatives exhibit potent anticancer activity against different cancer cell lines through the inhibition of
different growth factors, enzymes and kinases including telomerase, histone deacetylase (HDAC),
methionine aminopeptidase (MetAP), thymidylate synthase (TS), glycogen synthase kinase-3 (GSK),
epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), and focal adhesion kinase
(FAK) [24–26]. Triazoles [27], tetrazoles [28], thiadiazoles [29], and pyrimidines [4] have also been reported
to show anticancer activity.
Prompted by the aforementioned findings and in the continuation of our ongoing research in the field
of design, synthesis, and biological evaluation of pyrazoline derivatives [30–35], herein we described
the synthesis and evaluation of a new series of heteroaryl substituted pyrazolines as potential anticancer
agents against AsPC-1 human pancreatic adenocarcinoma and two glioblastoma cell lines, U87 and U251
cell lines. Furthermore, tumor selectivity test on blood cells (PBMC and Jurkat cells) and the apoptotic,
necrotic, and DNA-cleavage analysis against U251 cells were carried out using the most effective compound.
2. Results and Discussion
The synthesis of new pyrazoline derivatives (1–12) was carried out according to the steps shown in
Scheme 1. In the initial step, 1-(2-thienyl)-3-(4-chlorophenyl)-2-propen-1-one was synthesized via the
base-catalyzed Claisen-Schmidt condensation of 2-acetylthiophene with 4-chlorobenzaldehyde. The
ring closure reaction of the chalcone with hydrazine hydrate afforded 5-(4-chlorophenyl)-3-(2-thienyl)-

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2-pyrazoline. 1-(Chloroacetyl)-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline was obtained by the reaction
of 5-(4-chlorophenyl)-3-(2-thienyl)-2-pyrazoline with chloroacetyl chloride in the presence of triethylamine.
The reaction of 1-(chloroacetyl)-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline with aryl thiols afforded
1-[(aryl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazolines (1–12). Thus, the synthetic procedure
was shown to be versatile, applicable to the preparation of many derivatives.
Scheme 1. The synthesis of compounds 1–12. Reagents and conditions: (i)
4-Chlorobenzaldehyde, 10% aqueous sodium hydroxide solution, ethanol, rt, 6–8 h; (ii) 80%
hydrazine hydrate, ethanol, reflux, 5 h; (iii) ClCOCH2Cl, TEA, toluene, rt, 1 h; (iv) Ar-SH,
acetone, rt, 8 h.
The structures of the compounds were elucidated by IR, 1H-NMR, 13C-NMR, mass spectral data, and
elemental analyses. In the IR spectra of compounds 1–12, all derivatives had a strong, characteristic
band in the region 1670–1641 cm−1 due to C=O stretching vibration. The asymmetric and symmetric
stretching bands for aliphatic C–H group occurred at 2983–2920 cm−1. The aromatic C–H stretching
vibrations gave rise to a band at 3134–3078 cm−1. The C=C, C=N and C–N stretching bands appeared
in the region of 1548–1408 and 1398–1012 cm−1, respectively.

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In the 1H-NMR spectra of the compounds, the CH2 protons of the pyrazoline ring resonated as a pair
of doublets at δ 3.16–3.24 ppm (C4-HA), 3.89–3.94 ppm (C4-HB). The CH proton appeared as a doublet
of doublets at δ 5.57–5.63 ppm (HX) due to the vicinal coupling with two magnetically non-equivalent
protons of the methylene group at position four of the pyrazoline ring (JAB = 17.60–18.00 Hz, JAX
=
4.00–4.80 Hz, JBX = 11.20–12.00 Hz). The CH2 protons of the acetyl group at position 1 of the pyrazoline
ring were observed at 4.32–4.83 ppm as a doublet (J = 15.20–16.40 Hz). This geminal coupling resulted
from the steric structure of the compound. These geminal protons were observed as a doublet due to two
different possible conformations since rigid protons occurred (Figure 1). All the other aromatic and
aliphatic protons were observed at expected regions.
HA
N
S
HB
HX
N
O
Cl
S
Ar
Figure 1. ABX system of the pyrazoline ring.
In the 13C-NMR spectra of the compounds, the signal due to the carbonyl carbon appeared at
163.92–166.37 ppm. The 13C-NMR chemical shift values of the carbon atoms at 43.34–43.42 ppm (C4),
60.04–60.24 ppm (C5) and 151.47–152.38 ppm (C3) corroborate 2-pyrazoline character deduced from
1
the H-NMR data. The signal due to the S-CH2 carbon was observed in the region 34.30–39.72 ppm.
The other aromatic and aliphatic carbons were observed at expected regions. The mass spectral data of
the synthesized compounds were found in full agreement with the proposed structures. All compounds
gave satisfactory elemental analysis.
The anticancer effects of new pyrazoline derivatives (1–12) and cisplatin (positive control) in the
range of 10–500 µM concentrations were tested. Since only limited choice of drugs are available for
pancreatic cancer and glioma, AsPC-1 human pancreatic adenocarcinoma, U87 and U251 glioblastoma
cell lines were used (Figure 2). Cisplatin was chosen as a control, considering its wide use in the treatment
of several types of human cancer.
Compounds 1–12 were evaluated for their cytotoxic effects on these cell lines by MTT assay (Figure 2
and Table 1), to determine their anticancer potential and selectivity. The activity of the tested compounds
was influenced considerably by the nature of the aryl group. Compounds 1, 10, 11, and 12 were found
to possess IC50 values lower than 500 μM against all three cell lines. Tetrazole-substituted compound 2 did
not show activity, whereas triazole-substituted compound 1 was active. Generally, oxadiazole-substituted
compounds 10, 11, and 12 exhibited good activity. The most effective cytotoxic agent against AsPC-1
and U251 cancer cell lines was found to be compound 11 with IC50 values of 16.8 µM, and 11.9 µM
respectively, followed by compound 12 with respective IC50 values of 62.1 µM, and 70.1 µM. On the
other hand, these compounds showed no significant cytotoxicity at the concentrations used for the U87
cell line.

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(a)
140
120
100
80
140
120
100
80
AsPC-1 Cell Line
AsPC-1 Cell Line
60
60
40
40
20
20
0
0
Concentration (10µM)
Concentration (50µM)
140
120
100
80
140
120
100
80
AsPC-1 Cell Line
AsPC-1 Cell Line
60
60
40
40
20
20
0
0
Concentration (100µM)
Concentration (200µM)
140
120
100
80
AsPC-1 Cell Line
60
40
20
0
Concentration (500µM)
(b)
140
120
100
80
140
120
100
80
U87 Cell Line
U87 Cell Line
60
60
40
40
20
20
0
0
Concentration (10µM)
Concentration (50µM)
140
120
100
80
140
120
100
80
U87 Cell Line
U87 Cell Line
60
60
40
40
20
20
0
0
Concentration (100µM)
Concentration (200µM)
140
120
100
80
U87 Cell Line
60
40
20
0
Concentration (500µM)
Figure 2. Cont.

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(c)
U251 Cell Line
140
120
100
80
140
120
100
80
U251 Cell Line
60
60
40
40
20
20
0
0
Concentration (10µM)
Concentration (50µM)
140
120
100
80
140
120
100
80
U251 Cell Line
U251 Cell Line
60
60
40
40
20
20
0
0
Concentration (100µM)
Concentration (200µM)
140
120
100
80
U251 Cell Line
60
40
20
0
Concentration (500µM)
Figure 2. Anticancer effects of compounds 1–12 and cisplatin at varying concentrations
(10 µM, 50 µM, 100 µM, 200 µM, and 500 µM) against AsPC-1 (a); U87 (b); and U251
(c) cells.
Table 1. The cytotoxic effects of the compounds 1–12 on the AsPC-1, U87, and U251
cell lines.
IC50 (μM)
Compound
AsPC1 Cell Line U87 Cell Line U251 Cell Line
1
166.7 ± 10.6
>500
112.2 ± 8.8
>500
126.9 ± 15.6
>500
2
3
>500
>500
>500
4
65.0 ± 5.4
215.7 ± 29.7
199.3 ± 32.8
236.2 ± 36.4
139.3 ± 26.8
108.1 ± 30.1
110.5±20.4
16.8 ± 2.1
62.1 ± 7.8
22.5 ± 2.0
>500
>500
5
>500
>500
6
>500
>500
7
>500
>500
8
>500
393.1 ± 60.4
>500
9
10
>500
250.6 ± 30.4
127.4 ± 12.8
159.2 ± 20.7
14.8 ± 1.4
166.6 ± 25.1
11.9 ± 1.1
70.1 ± 8.8
4.9 ± 1.0
11
12
Cisplatin
Among the tested compounds, compound 11 can be identified as the most promising anticancer agent.
Thus, detail of concentration dependency of this compound (0.1 µM–1000 µM) was examined against

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AsPC-1, U87 and U251 cell lines (Figure 3a). Swelling ratio curves of AsPC-1 and U251 cell lines suggested
biphasic mechanism of compound 11. Moreover, the activity of compound 11 against peripheral blood
mononuclear cells (PBMC) (IC50 = ~1000 μM) and Jurkat human leukemic T-cells (IC50 = 90 μM) indicated
significant tumor selectivity in blood cells (more than 10 times) as shown in Figure 3b. This outcome
pointed out the importance of 5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl moiety for anticancer activity.
Figure 3. Effects of compound 11 on cell viability of AsPC-1, U87, U251 cell lines (a) and
PBMC, Jurkat cell lines (b).
According to the MTT assay results, the most active anticancer compound 11 was chosen for the
evaluation of apoptosis and necrosis in U251 cells, which was carried out with annexin V/ethidium
homodimer III staining method. U251 cell lines were incubated with compound 11 or cisplatin at the
IC50 concentrations. U251 cells were stained and observed by fluorescence microscope (Figure 4). If the
cells are colored green with annexin V, and not stained red with ethidium homodimer III, the cells are
judged to be apoptotic. On the other hand, the completely opposite results indicate necrosis. The apoptotic
and necrotic effects of compound 11 were compared with cisplatin. The results indicate that cisplatin
has only apoptotic effects at 24 h (Figure 4b). In contrast, U251 cell lines treated with compound 11 at
24 h showed late stage apoptotic or necrotic effect (almost all cells were colored yellow, data not shown).
Therefore, the apoptotic/necrotic effects of compound 11 were tested against U251 cell line in earlier time
(3h) at IC50 (11.9 µM) and low (1.5 µM) concentrations. While compound 11 mainly induced necrosis at
high concentration (Figure 4d), it induced apoptosis at low concentration (Figure 4e).
Figure 4. Cont.

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Figure 4. Cellular and nuclear morphological changes of U251 cells following exposure to
IC50 concentrations of control (a); cisplatin (4.9 μM) (b); for 24 h and control (c); compound
11 (11.9 μM) (d); and (1.5 μM) (e) for 3 h.
The DNA cleavage activities of compound 11 and cisplatin at the IC50 concentrations in water and
Tris/boric acid buffer in the presence and absent of the iron complex, H2O2 and ascorbic acid as
an activator were studied using supercoiled pUC19 DNA (Figure 5). The reaction mixture was incubated
at 37 °C for 1.5 h and then agarose gel electrophoresis was performed at 100 V for 40 min. DNA was
visualized by photographing the fluorescence of intercalated ethidium bromide under a UV illuminator.
Control experiments using compound 11 with and without FeSO4, H2O2, and ascorbic acid showed that
the DNA cleavage efficiency was clearly enhanced in the case of iron (II) complex system (Figure 5b).
Thus, it is considered that DNA-cleavage was caused by oxygen activation. The DNA-cleaving

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efficiency of compound 11 was much greater than cisplatin. Compound 11 disintegrated pUC 19 DNA
and these results suggest the relationship between the DNA cleavage and the cell death.
Figure 5. The DNA cleaving capability of the Fe(II) complexes of compound 11 and cisplatin
in the presence of the iron (II) complex (a) and control experiments using compound 11 in the
presence and absence of FeSO4, H2O2, and ascorbic acid (b). It was studied by the relaxation
of the supercoiled pUC19 DNA and analyzed by agarose-gel electrophoresis.
3. Experimental Section
3.1. Chemistry
All reagents were purchased from commercial suppliers and were used without further purification.
Melting points were determined on an Electrothermal 9100 melting point apparatus (Weiss-Gallenkamp,
Loughborough, UK) and were uncorrected. IR spectra were recorded on a Shimadzu 8400 FT-IR
spectrophotometer (Shimadzu, Tokyo, Japan). 1H-NMR and 13C-NMR spectra were recorded on a Varian
Mercury-400 FT-NMR spectrometer (Agilent, Palo Alto, CA, USA). Mass spectra were recorded on an
Agilent LC-MSD-Trap-SL Mass spectrometer (Agilent Technologies). Elemental analyses were performed
on a Perkin Elmer EAL 240 elemental analyzer (Perkin-Elmer, Norwalk, CT, USA). Thin Layer
Chromatography (TLC) was performed on TLC silica gel 60 F254 aluminium sheets (Merck, Darmstadt,
Germany) using petroleum ether:ethyl acetate (3:1 v/v) as an eluent.
General Procedures for the Synthesis of Compounds
1-(2-Thienyl)-3-(4-chlorophenyl)-2-propen-1-one
A mixture of 2-acetylthiophene (0.04 mol), 4-chlorobenzaldehyde (0.04 mol) and 10% aqueous
sodium hydroxide solution (10 mL) in ethanol (30 mL) was stirred at room temperature for 6–8 h. The
progress of the reaction was checked by TLC. Upon completion, the reaction mixture was poured into
crushed ice. The precipitated solid was filtered, washed with water, and dried. The product was crystallized
from ethanol [36].

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5-(4-Chlorophenyl)-3-(2-thienyl)-2-pyrazoline
A mixture of 1-(2-thienyl)-3-(4-chlorophenyl)-2-propen-1-one (0.01 mol) and hydrazine hydrate (80%)
(0.02 mol) in ethanol (30 mL) was refluxed for 5 h. The reaction mixture was cooled and kept at 0 °C
overnight. The resulting solid was filtered and dried. The product was crystallized from ethanol [36].
1-(Chloroacetyl)-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline
5-(4-Chlorophenyl)-3-(2-thienyl)-2-pyrazoline (0.01 mol) and triethylamine (0.01 mol) were dissolved
in toluene (30 mL). The reaction mixture was cooled in an ice bath, and chloroacetyl chloride (0.01 mol)
was added dropwise with constant stirring. The mixture thus obtained was further agitated for 1 h at room
temperature. The solvent was evaporated to dryness under reduced pressure. The residue was washed with
water, and the product was recrystallized from ethanol [36].
1-[(Aryl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazolines (1–12)
A mixture of 1-(chloroacetyl)-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (0.005 mol) and aryl thiol
(0.005 mol) was treated in acetone (30 mL) at room temperature for 8 h. The solvent was evaporated, the
residue was washed with water and recrystallized from ethanol [37].
1-[(4-Methyl-4H-1,2,4-triazol-3-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (1). Yield:
83%; M.p. 165.1 °C. IR (KBr) νmax (cm−1): 3124 (aromatic C-H), 2922 (aliphatic C-H asymmetric), 1666
(C=O), 1490, 1458, 1417 (C=N and C=C), 1375, 1203, 1085, 1026 (C-N), 952, 817 (C-H out of plane
1
deformation). H-NMR (400 MHz, δ ppm, DMSO-d6): 3.16 (1H, dd, JAX = 4.40 Hz, JAB = 17.60 Hz,
pyrazoline C4-HA), 3.58 (3H, CH3), 3.89 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB),
4.33 (1H, d, J = 15.20 Hz, geminal proton, CO-CH), 4.46 (1H, d, J = 15.60 Hz, geminal proton, CO-CH),
5.57 (1H, dd, JAX = 4.80 Hz, JBX = 11.60 Hz, pyrazoline C5-HX), 7.16 (1H, m), 7.26 (2H, d, J = 8.80 Hz),
7.38 (2H, d, J = 8.80 Hz), 7.48 (1H, dd, J = 1.20 Hz, 4.00 Hz, thiophene C3-H), 7.78 (1H, dd, J = 1.20 Hz,
5.20 Hz, thiophene C5-H), 8.52 (1H, s, triazole). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 31.49 (CH3,
triazole), 39.72 (CH2, S-CH2), 43.36 (CH2, pyrazoline C4), 60.04 (CH, pyrazoline C5), 128.27 (2CH,
aromatic), 128.81 (CH, aromatic), 129.28 (2CH, aromatic), 130.83 (C, aromatic), 131.73 (CH, aromatic),
132.61 (CH, aromatic), 134.23 (C, aromatic), 141.05 (C, aromatic), 146.94 (CH, aromatic), 149.16 (C,
aromatic), 151.95 (C, pyrazoline C3), 164.96 (C, C=O). Anal. Calcd. for C18H16ClN5OS2: C, 51.73; H,
3.86; N, 16.76. Found: C, 51.66; H, 3.89; N, 16.81. MS (ESI) (m/z): 418 [M + H]+.
1-[(1-Methyl-1H-tetrazol-5-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (2). Yield: 86%;
M.p. 108.2 °C. IR (KBr) max (cm−1): 3107 (aromatic C-H), 2941 (aliphatic C-H asymmetric), 1658 (C=O),
1463, 1417 (C=N and C=C), 1396, 1278, 1219, 1089, 1026 (C-N), 968, 837, 819 (C-H out of plane
1
deformation). H-NMR (400 MHz, δ ppm, DMSO-d6): 3.22 (1H, dd, JAX = 4.80 Hz, JAB = 18.40 Hz,
pyrazoline C4-HA), 3.91 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB), 3.95 (3H, CH3), 4.57
(1H, d, J = 15.60 Hz, geminal proton, CO-CH), 4.71 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 5.59
(1H, dd, JAX = 4.80 Hz, JBX = 12.00 Hz, pyrazoline C5-HX), 7.17 (1H, m), 7.26 (2H, d, J = 8.40 Hz),
7.38 (2H, d, J = 8.80 Hz), 7.50 (1H, dd, J = 1.20 Hz, 3.60 Hz, thiophene C3-H), 7.79 (1H, dd, J = 0.80 Hz,
5.20 Hz, thiophene C5-H). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 34.32 (CH3, tetrazole), 37.15 (CH2,

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S-CH2), 43.40 (CH2, pyrazoline C4), 60.16 (CH, pyrazoline C5), 128.26 (2CH, aromatic), 128.84 (CH,
aromatic), 129.29 (2CH, aromatic), 130.96 (C, aromatic), 131.90 (CH, aromatic), 132.66 (CH, aromatic),
134.14 (C, aromatic), 140.96 (C, aromatic), 152.29 (C, pyrazoline C3), 153.97 (C, aromatic), 164.19 (C,
C=O). Anal. Calcd. for C17H15ClN6OS2: C, 48.74; H, 3.61; N, 20.06. Found: C, 48.65; H, 3.68; N, 19.95.
MS (ESI) (m/z): 419 [M + H]+, 420 [M + H]++.
1-[(1-Phenyl-1H-tetrazol-5-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (3). Yield: 84%;
M.p. 183.3 °C. IR (KBr) max (cm−1): 3107 (aromatic C-H), 2933 (aliphatic C-H asymmetric), 1656 (C=O),
1498, 1462, 1423 (C=N and C=C), 1388, 1220, 1089, 1012 (C-N), 831 (C-H out of plane deformation).
1H-NMR (400 MHz, δ ppm, DMSO-d6): 3.23 (1H, dd, JAX = 4.40 Hz, JAB = 18.00 Hz, pyrazoline C4-HA),
3.94 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB), 4.69 (1H, d, J = 16.00 Hz, geminal
proton, CO-CH), 4.83 (1H, d, J = 16.40 Hz, geminal proton, CO-CH), 5.60 (1H, dd, JAX = 4.00 Hz,
JBX = 11.20 Hz, pyrazoline C5-HX), 7.18 (1H, m), 7.27 (2H, d, J = 8.40 Hz), 7.38 (2H, d, J = 8.80 Hz),
7.51 (1H, dd, J=1.20 Hz, 3.60 Hz, thiophene C3-H), 7.64–7.68 (5H, m), 7.80 (1H, dd, J = 1.20 Hz, 4.80 Hz,
thiophene C5-H). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 37.35 (CH2, S-CH2), 43.41 (CH2, pyrazoline
C4), 60.21 (CH, pyrazoline C5), 125.10 (2CH, aromatic), 128.27 (2CH, aromatic), 128.87 (CH, aromatic),
129.29 (2CH, aromatic), 130.75 (2CH, aromatic), 131.01 (CH, aromatic), 131.34 (CH, aromatic), 131.98
(CH, aromatic), 132.65 (C, aromatic), 133.70 (C, aromatic), 134.13 (C, aromatic), 140.96 (C, aromatic),
152.38 (C, pyrazoline C3), 154.65 (C, aromatic), 163.92 (C, C=O). Anal. Calcd. for C22H17ClN6OS2: C,
54.94; H, 3.56; N, 17.47. Found: C, 54.99; H, 3.58; N, 17.51. MS (ESI) (m/z): 481 [M + H]+.
1-[(1-(4-Hydroxyphenyl)-1H-tetrazol-5-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (4).
Yield: 71%; M.p. 145.7 °C. IR (KBr) max (cm−1): 3601 (O-H), 3107 (aromatic C-H), 2932 (aliphatic
C-H asymmetric), 1641 (C=O), 1516, 1454, 1415 (C=N and C=C), 1278, 1228, 1087 (C-N), 837, 823
(C-H out of plane deformation). 1H-NMR (400 MHz, δ ppm, DMSO-d6): 3.22 (1H, dd, JAX = 4.80 Hz,
JAB = 18.00 Hz, pyrazoline C4-HA), 3.93 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB),
4.65 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 4.77 (1H, d, J = 16.00 Hz, geminal proton, CO-CH),
5.60 (1H, dd, JAX = 4.40 Hz, JBX = 11.60 Hz, pyrazoline C5-HX), 6.87 (2H, d, J = 8.80 Hz), 7.17 (1H, m),
7.26–7.33 (4H, m), 7.37 (2H, d, J = 8.40 Hz), 7.51 (1H, d, J = 3.60 Hz, thiophene C3-H), 7.79 (1H, d,
13
J = 5.20 Hz, thiophene C5-H), 9.35 (1H, s). C-NMR (100 MHz, DMSO-d6) δ (ppm): 37.04 (CH2,
S-CH2), 43.40 (CH2, pyrazoline C4), 60.20 (CH, pyrazoline C5), 117.35 (2CH, aromatic), 122.57 (C,
aromatic), 126.65 (2CH, aromatic), 128.27 (2CH, aromatic), 128.85 (CH, aromatic), 129.29 (2CH,
aromatic), 130.96 (CH, aromatic), 131.90 (CH, aromatic), 132.64 (CH, aromatic), 134.16 (C, aromatic),
140.98 (C, aromatic), 152.28 (C, pyrazoline C3), 154.65 (C, aromatic), 163.04 (C, aromatic), 164.05
(C, C=O). Anal. Calcd. for C22H17ClN6O2S2: C, 53.17; H, 3.45; N, 16.91. Found: C, 53.21; H, 3.54; N,
16.96. MS (ESI) (m/z): 497 [M + H]+, 498 [M + H]++.
1-[(1-(2-(Dimethylamino)ethyl)-1H-tetrazol-5-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline
(5). Yield: 76%; M.p. 112.1 °C. IR (KBr) max (cm−1): 3091 (aromatic C-H), 2943, 2827 (aliphatic C-H
asymmetric), 1654 (C=O), 1452, 1413 (C=N and C=C), 1394, 1220, 1093, 1029 (C-N), 831, 817 (C-H
out of plane deformation). 1H-NMR (400 MHz, δ ppm, DMSO-d6): 2.13 (6H, s), 2.64 (2H, t, J = 6.40 Hz,
12.00 Hz), 3.22 (1H, dd, JAX = 4.80 Hz, JAB = 18.00 Hz, pyrazoline C4-HA), 3.92 (1H, dd, JBX = 11.60 Hz,

Molecules 2015, 20
19077
JBA = 17.60 Hz, pyrazoline C4-HB), 4.38 (2H, t, J = 6.00 Hz, 12.00 Hz), 4.57 (1H, d, J = 15.60 Hz,
geminal proton, CO-CH), 4.71 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 5.59 (1H, dd, JAX = 4.80 Hz,
JBX = 12.00 Hz, pyrazoline C5-HX), 7.17 (1H, m), 7.26 (2H, d, J = 8.80 Hz), 7.38 (2H, d, J = 8.80 Hz),
7.49 (1H, dd, J = 0.80, 3.60 Hz, thiophene C3-H), 7.79 (1H, dd, J = 5.20, 0.80 Hz, thiophene C5-H).
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 37.46 (CH2, S-CH2), 43.38 (CH2, pyrazoline C4), 45.65 (2CH3,
H3C-N-CH3), 45.99 (CH2, CH2-tetrazole), 57.84 (CH2, CH2-N(CH3)2), 60.15 (CH, pyrazoline C5),
128.25 (2CH, aromatic), 128.84 (CH, aromatic), 129.29 (2CH, aromatic), 130.97 (CH, aromatic), 131.91
(CH, aromatic), 132.64 (C, aromatic), 134.15 (C, aromatic), 140.99 (C, aromatic), 152.25 (C, pyrazoline
C3), 154.19 (C, aromatic), 164.23 (C, C=O). Anal. Calcd. for C20H22ClN7OS2: C, 50.46; H, 4.66; N, 20.60.
Found: C, 50.42; H, 4.69; N, 20.57. MS (ESI) (m/z): 476 [M], 477 [M + H]+, 478 [M + H]++.
1-[(5-Methyl-1,3,4-thiadiazol-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (6). Yield:
82%; M.p. 140.9 °C. IR (KBr) max (cm−1): 3103 (aromatic C-H), 2920 (aliphatic C-H asymmetric), 1645
(C=O), 1456, 1417 (C=N and C=C), 1398, 1325, 1219, 1072, 1029 (C-N), 840, 819 (C-H out of
plane deformation). 1H-NMR (400 MHz, δ ppm, DMSO-d6): 2.66 (3H, s), 3.22 (1H, dd, JAX = 4.80 Hz,
J
AB = 18.00 Hz, pyrazoline C4-HA), 3.92 (1H, dd, JBX = 11.60 Hz, JBA = 17.60 Hz, pyrazoline C4-HB),
4.54 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 4.67 (1H, d, J = 16.00 Hz, geminal proton,
CO-CH), 5.60 (1H, dd, JAX = 4.40 Hz, JBX = 11.60 Hz, pyrazoline C5-HX), 7.17 (1H, m), 7.27 (2H, d,
J = 8.40 Hz), 7.38 (2H, d, J = 8.80 Hz), 7.50 (1H, dd, J = 0.80 Hz, 3.60 Hz, thiophene C3-H), 7.79 (1H,
13
dd, J = 1.20 Hz, 5.20 Hz, thiophene C5-H). C-NMR (100 MHz, DMSO-d6) δ (ppm): 15.85 (CH3,
thiadiazole), 37.33 (CH2, S-CH2), 43.36 (CH2, pyrazoline C4), 60.13 (CH, pyrazoline C5), 128.29 (2CH,
aromatic), 128.83 (CH, aromatic), 129.29 (2CH, aromatic), 130.93 (CH, aromatic), 131.83 (CH, aromatic),
132.63 (C, aromatic), 134.20 (C, aromatic), 141.05 (C, aromatic), 152.08 (C, pyrazoline C3), 164.40 (C,
aromatic), 164.98 (C, aromatic), 166.37 (C, C=O). Anal. Calcd. for C18H15ClN4OS3: C, 49.70; H, 3.48;
N, 12.88. Found: C, 49.72; H, 3.49; N, 12.83. MS (ESI) (m/z): 434 [M], 435 [M + H]+, 436 [M + H]++,
437 [M + H]+++
.
1-[(Pyrimidin-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (7). Yield: 78%; M.p.
126.6 °C. IR (KBr) max (cm−1): 3109 (aromatic C-H), 2929 (aliphatic C-H asymmetric), 1653 (C=O),
1548, 1448, 1409 (C=N and C=C), 1379, 1201, 1176, 1089, 1016 (C-N), 821 (C-H out of plane
1
deformation). H-NMR (400 MHz, δ ppm, DMSO-d6): 3.19 (1H, dd, JAX = 4.40 Hz, JAB = 18.00 Hz,
pyrazoline C4-HA), 3.92 (1H, dd, JBX = 12.00 Hz, JBA = 17.60 Hz, pyrazoline C4-HB), 4.32 (1H, d,
J = 16.40 Hz, geminal proton, CO-CH), 4.60 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 5.61 (1H,
dd, JAX = 4.80 Hz, JBX = 11.60 Hz, pyrazoline C5-HX), 7.15–7.18 (1H, m), 7.22 (1H, t, J = 4.80 Hz,
5.20 Hz, pyrimidine), 7.28 (2H, d, J = 8.00 Hz), 7.40 (2H, d, J = 8.00 Hz), 7.47–7.49 (1H, m, thiophene
C3-H), 7.77 (1H, dd, J = 0.80 Hz, 4.40 Hz, thiophene C5-H), 8.63 (2H, d, J = 4.80 Hz, pyrimidine).
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 34.30 (CH2, S-CH2), 43.34 (CH2, pyrazoline C4), 60.09 (CH,
pyrazoline C5), 118.01 (CH, aromatic), 128.13 (2CH, aromatic), 128.81 (CH, aromatic), 129.27 (2CH,
aromatic), 130.68 (CH, aromatic), 131.58 (CH, aromatic), 132.54 (C, aromatic), 134.43 (C, aromatic),
141.33 (C, aromatic), 151.47 (C, pyrazoline C3), 158.42 (2CH, aromatic), 165.28 (C, C=O), 170.99 (C,
aromatic). Anal. Calcd. for C19H15ClN4OS2: C, 55.00; H, 3.64; N, 13.50. Found: C, 55.11; H, 3.51; N,
13.43. MS (ESI) (m/z): 414 [M], 415 [M + H]+, 416 [M + H]++.

Molecules 2015, 20
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1-[(5-Phenyl-1,3,4-oxadiazol-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (8). Yield:
74%; M.p. 162.3 °C. IR (KBr) max (cm−1): 3107 (aromatic C-H), 2983 (aliphatic C-H asymmetric), 1670
(C=O), 1465, 1450, 1408 (C=N and C=C), 1238, 1197, 1068, 1012 (C-N), 827 (C-H out of plane
1
deformation). H-NMR (400 MHz, δ ppm, DMSO-d6): 3.23 (1H, dd, JAX = 4.40 Hz, JAB = 18.00 Hz,
pyrazoline C4-HA), 3.93 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB), 4.63 (1H, d,
J = 16.00 Hz, geminal proton, CO-CH), 4.77 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 5.62 (1H,
dd, JAX = 4.80 Hz, JBX = 11.60 Hz, pyrazoline C5-HX), 7.16–7.19 (1H, m), 7.26 (2H, d, J = 8.40 Hz),
7.34 (2H, d, J = 8.80 Hz), 7.51–7.64 (4H, m), 7.78–7.80 (1H, m, thiophene C5-H), 7.92–7.95 (2H, m).
13C-NMR (100 MHz, DMSO-d6) δ (ppm): 36.11 (CH2, S-CH2), 43.40 (CH2, pyrazoline C4), 60.19 (CH,
pyrazoline C5), 123.66 (C, aromatic), 127.03 (2CH, aromatic), 128.24 (2CH, aromatic), 128.85 (CH,
aromatic), 129.29 (2CH, aromatic), 130.07 (2CH, aromatic), 131.00 (CH, aromatic), 131.95 (CH,
aromatic), 132.66 (C, aromatic), 132.69 (CH, aromatic), 134.12 (C, aromatic), 140.99 (C, aromatic),
152.35 (C, pyrazoline C3), 164.02 (C, aromatic), 164.13 (C, aromatic), 165.77 (C, C=O). Anal. Calcd.
for C23H17ClN4O2S2: C, 57.43; H, 3.56; N, 11.65. Found: C, 57.47; H, 3.55; N, 11.61. MS (ESI) (m/z):
481 [M + H]+.
1-[(5-(Pyridin-3-yl)-1,3,4-oxadiazol-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline (9).
Yield: 70%; M.p. 156.9 °C. IR (KBr) max (cm−1): 3080 (aromatic C-H), 2981 (aliphatic C-H asymmetric),
1668 (C=O), 1456, 1417 (C=N and C=C), 1199, 1076, 1016 (C-N), 833 (C-H out of plane deformation).
1H-NMR (400 MHz, δ ppm, DMSO-d6): 3.24 (1H, dd, JAX = 4.80 Hz, JAB = 18.00 Hz, pyrazoline
C4-HA), 3.94 (1H, dd, JBX = 11.60 Hz, JBA = 18.40 Hz, pyrazoline C4-HB), 4.65 (1H, d, J = 16.00 Hz,
geminal proton, CO-CH), 4.79 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 5.63 (1H, dd, JAX = 4.80 Hz,
JBX = 12.00 Hz, pyrazoline C5-HX), 7.16–7.18 (1H, m), 7.28 (2H, d, J = 8.40 Hz), 7.35 (2H, d, J = 8.40 Hz),
7.50 (1H, dd, J = 1.20 Hz, 4.00 Hz, thiophene C3-H), 7.58–7.62 (1H, m, pyridine C5-H), 7.78 (1H, dd,
J = 0.80 Hz, 4.80 Hz, thiophene C5-H), 8.29-8.32 (1H, m, pyridine), 8.79 (1H, dd, J = 1.60 Hz, 4.80 Hz,
pyridine), 9.12 (1H, d, J = 1.60 Hz, pyridine). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 36.28 (CH2,
S-CH2), 43.42 (CH2, pyrazoline C4), 60.23 (CH, pyrazoline C5), 120.31 (CH, aromatic), 124.98 (C,
aromatic), 128.27 (2CH, aromatic), 128.85 (CH, aromatic), 129.31 (2CH, aromatic), 130.99 (CH,
aromatic), 131.93 (CH, aromatic), 132.69 (CH, aromatic), 134.12 (C, aromatic), 134.64 (C, aromatic),
140.99 (C, aromatic), 147.74 (CH, aromatic), 152.37 (C, pyrazoline C3), 153.16 (CH, aromatic), 164.04
(C, aromatic), 164.08 (C, aromatic), 164.76 (C, C=O). Anal. Calcd. for C22H16ClN5O2S2: C, 54.82; H, 3.35;
N, 14.53. Found: C, 54.83; H, 3.33; N, 14.60. MS (ESI) (m/z): 481 [M], 482 [M + H]+, 483 [M + H]++.
1-[(5-(4-Chlorophenyl)-1,3,4-oxadiazol-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline
(10). Yield: 77%; M.p. 176.5 °C. IR (KBr) max (cm−1): 3099 (aromatic C-H), 2981 (aliphatic C-H
asymmetric), 1643 (C=O), 1463, 1417 (C=N and C=C), 1392, 1188, 1174, 1082, 1008 (C-N), 952, 840,
819 (C-H out of plane deformation). 1H-NMR (400 MHz, δ ppm, DMSO-d6): 3.23 (1H, dd, JAX = 4.80 Hz,
JAB = 18.40 Hz, pyrazoline C4-HA), 3.93 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB),
4.62 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 4.75 (1H, d, J = 16.00 Hz, geminal proton,
CO-CH), 5.62 (1H, dd, JAX = 4.80 Hz, JBX = 11.20 Hz, pyrazoline C5-HX), 7.17 (1H, dd, J = 3.60 Hz,
5.20 Hz thiophene C4-H), 7.27 (2H, d, J = 8.40 Hz), 7.35 (2H, d, J = 8.40 Hz), 7.50 (1H, dd, J = 0.80 Hz,
3.60 Hz, thiophene C3-H), 7.62 (2H, d, J = 8.80 Hz), 7.77 (1H, dd, J = 0.80 Hz, 5.20 Hz, thiophene

Molecules 2015, 20
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C5-H), 7.93 (2H, d, J = 8.40 Hz). 13C-NMR (100 MHz, DMSO-d6) δ (ppm): 36.16 (CH2, S-CH2), 43.41
(CH2, pyrazoline C4), 60.23 (CH, pyrazoline C5), 122.55 (C, aromatic), 128.25 (2CH, aromatic), 128.81
(3CH, aromatic), 129.29 (2CH, aromatic), 130.21 (2CH, aromatic), 130.93 (CH, aromatic), 131.82 (CH,
aromatic), 132.71 (C, aromatic), 134.15 (C, aromatic), 137.42 (C, aromatic), 140.96 (C, aromatic), 152.32
(C, pyrazoline C3), 164.12 (C, aromatic), 164.30 (C, aromatic), 165.05 (C, C=O). Anal. Calcd. for
C23H16Cl2N4O2S2: C, 53.59; H, 3.13; N, 10.87. Found: C, 53.48; H, 3.17; N, 10.75. MS (ESI) (m/z): 515
[M], 516 [M + H]+, 517 [M + H]++.
1-[(5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline
(11). Yield: 79%; M.p. 194.0 °C. IR (KBr) max (cm−1): 3078 (aromatic C-H), 2926 (aliphatic C-H
asymmetric), 1668 (C=O), 1469, 1450, 1409 (C=N and C=C), 1197, 1072 (C-N), 829 (C-H out of plane
deformation). 1H-NMR (400 MHz, δ ppm, DMSO-d6): 2.38 (3H, s, CH3), 3.22 (1H, dd, JAX = 4.80 Hz,
J
AB = 18.00 Hz, pyrazoline C4-HA), 3.93 (1H, dd, JBX = 11.60 Hz, JBA = 18.00 Hz, pyrazoline C4-HB),
4.60 (1H, d, J = 15.60 Hz, geminal proton, CO-CH), 4.73 (1H, d, J = 15.60 Hz, geminal proton, CO-CH),
5.61 (1H, dd, JAX = 4.80 Hz, JBX = 11.20 Hz, pyrazoline C5-HX), 7.15–7.18 (1H, m, thiophene C4-H),
7.26 (2H, d, J = 8.80 Hz), 7.33 (2H, d, J = 8.80 Hz), 7.36 (2H, d, J = 8.40 Hz), 7.49 (1H, m, thiophene
13
C3-H), 7.77 (1H, d, J = 4.80 Hz, thiophene C5-H), 7.81 (2H, d, J = 8.00 Hz). C-NMR (100 MHz,
DMSO-d6) δ (ppm): 21.80 (CH3), 36.09 (CH2, S-CH2), 43.41 (CH2, pyrazoline C4), 60.22 (CH, pyrazoline
C5), 120.97 (C, aromatic), 127.00 (2CH, aromatic), 128.24 (2CH, aromatic), 128.81 (CH, aromatic),
129.29 (2CH, aromatic), 130.59 (2CH, aromatic), 130.93 (CH, aromatic), 131.82 (CH, aromatic), 132.69
(C, aromatic), 134.16 (C, aromatic), 140.98 (C, aromatic), 142.84 (C, aromatic), 152.29 (C, pyrazoline
C3), 163.60 (C, aromatic), 164.17 (C, aromatic), 165.91 (C, C=O). Anal. Calcd. for C24H19ClN4O2S2: C,
58.23; H, 3.87; N, 11.32. Found: C, 58.19; H, 3.92; N, 11.25. MS (ESI) (m/z): 495 [M], 496 [M + H]+,
497 [M + H]++.
1-[(5-(4-Methoxyphenyl)-1,3,4-oxadiazol-2-yl)thioacetyl]-3-(2-thienyl)-5-(4-chlorophenyl)-2-pyrazoline
(12). Yield: 72%; M.p. 146.8 °C. IR (KBr) max (cm−1): 3082 (aromatic C-H), 2933 (aliphatic C-H
asymmetric), 1670 (C=O), 1504, 1477, 1452, 1413 (C=N and C=C), 1251, 1178, 1026 (C-N), 840 (C-H
1
out of plane deformation). H-NMR (400 MHz, δ ppm, DMSO-d6): 3.21 (1H, dd, JAX = 4.80 Hz,
JAB = 18.00 Hz, pyrazoline C4-HA), 3.84 (3H, s, OCH3), 3.92 (1H, dd, JBX = 11.60 Hz, JBA = 17.60 Hz,
pyrazoline C4-HB), 4.57 (1H, d, J = 16.00 Hz, geminal proton, CO-CH), 4.71 (1H, d, J = 15.60 Hz,
geminal proton, CO-CH), 5.61 (1H, dd, JAX = 4.80 Hz, JBX = 11.60 Hz, pyrazoline C5-HX), 7.08 (2H, d,
J = 9.20 Hz), 7.16 (1H, J = 3.60 Hz, 4.80 Hz, thiophene C4-H), 7.25 (2H, d, J = 8.80 Hz), 7.34 (2H, d,
J = 8.80 Hz), 7.50 (1H, dd, J = 1.20 Hz, 4.00 Hz, thiophene C3-H), 7.77 (1H, dd, J = 1.20 Hz, 4.80 Hz,
13
thiophene C5-H), 7.86 (2H, d, J = 8.80 Hz). C-NMR (100 MHz, DMSO-d6) δ (ppm): 36.03 (CH2,
S-CH2), 43.42 (CH2, pyrazoline C4), 56.23 (CH3, methoxy), 60.24 (CH, pyrazoline C5), 115.54 (2CH,
aromatic), 116.10 (C, aromatic), 128.24 (2CH, aromatic), 128.80 (CH, aromatic), 128.92 (2CH,
aromatic), 129.29 (2CH, aromatic), 130.89 (CH, aromatic), 131.74 (CH, aromatic), 132.70 (C, aromatic),
134.19 (C, aromatic), 140.98 (C, aromatic), 152.25 (C, pyrazoline C3), 162.77 (C, aromatic), 163.11 (C,
aromatic), 164.26 (C, aromatic), 165.80 (C, C=O). Anal. Calcd. for C24H19ClN4O3S2: C, 56.41; H, 3.75;
N, 10.96. Found: C, 56.39; H, 3.73; N, 10.98. MS (ESI) (m/z): 511 [M], 512 [M + H]+, 513 [M + H]++.

Molecules 2015, 20
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3.2. Biochemistry
3.2.1. Cell Culture and Drug Treatment
U251 and U87 human glioblastoma cells were incubated in Dulbecco’s-modified Eagle’s medium
(DMEM) (Wako Pure Chemical Industries, Osaka, Japan) supplemented with 10% fetal bovine serum
(FBS) (Equitech-Bio, Kerrville, TX, USA). AsPC-1 human pancreas adenocarcinoma and Jurkat human
leukemic T-cells were incubated RPMI 1640 (Wako Pure Chemical Industries), supplemented with 10%
FBS. Peripheral blood mononuclear cells (PBMC) (Precision Bioservices, Frederick, MD, USA) were
incubated RPMI 1640 and supplemented with 10% human serum AB (HS) (Gemini, Woodland, CA,
USA). All media were supplemented with 89 µg/mL streptomycin (Meiji Seika Pharma, Tokyo, Japan)
and cells were incubated at 37 °C in a humidified atmosphere of 95% air and 5% CO2. Growing cells were
plated at 1 × 105 cells/mL into 24-well microtiter tissue culture plates (Iwaki brand Asahi Glass Co., Chiba,
Japan) and incubated for 24 h before the addition of the drugs (the optimal cell number for cytotoxicity
assays was determined in preliminary experiments). Stock solutions (1 mM, 5 mM, 10 mM, 20 mM, and
50 mM) of compounds and cisplatin (Sigma-Aldrich, St. Louis, MO, USA) were prepared in dimethyl
sulfoxide (DMSO; Wako Pure Chemical Industries) and in dimethylformamide (DMF; Wako Pure
Chemical Industries) respectively, then were added to fresh culture medium. The concentration of DMSO
and DMF in the final culture medium was 1%.
3.2.2. MTT Assay for Cytotoxicity of Compounds
The level of cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
(Dojindo Molecular Technologies, Kumamoto, Japan) was quantified as previously described in the
literature with small modifications [38]. The tested compounds were incubated with cells to give a final
concentration in the range 50–500 µM for 24 h. At the end of this period, MTT was added to cells in
culture to give a final concentration of 0.275 mg/mL and incubated further for 4 h at 37 °C. The medium
was removed and the formazan crystals were solubilized by addition of 100 µL DMSO to each well. After
the solubilized crystals were diluted (1:10) with DMSO, 100 µL was transferred to wells of 96-well
microtiter plates (Iwaki brand Asahi Glass Co.) and the absorbance at 550 nm was measured using a
microplate spectrophotometer Infinitive M1000 (Tecan, Groding, Austria). Every concentration was
repeated in three wells and IC50 values were defined as the drug concentrations which reduced absorbance
to 50 % of control values.
3.2.3. Detection of Apoptotic and Necrotic Cells
U251 cells were incubated with compound 11 at IC50 concentration for 3 h and 24 h. Then,
apoptotic/necrotic/healthy cells detection kit protocol was applied according to the manufacturer’s
instruction manual (PromoKine, Heidelberg, Germany) [39,40]. After the cells were washed twice with
1 × binding buffer, a staining solution containing 50 µL of 1 × binding buffer, 2 µL of FTIC-Annexin V
solution, 2 µL of ethidium homodimer III solution and 2 µL of Hoechst 33342 solution was added and
the cells were incubated for 15 min at RT, protected from light. Cells were washed with 1 × binding

Molecules 2015, 20
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buffer and analyzed by all-in-one fluorescence microscope Biorevo Fluorescence BZ-9000 (Keyence,
Osaka, Japan).
3.2.4. DNA Cleavage Assay
The DNA cleavage activities of the compounds were studied using supercoiled pUC19 DNA and
analyzed by agarose (Takara, Kyoto, Japan) gel electrophoresis Mupid-2x (Mupid, Tokyo, Japan). pUC19
DNA (2 µg) was treated with compounds in water and Tris/boric acid (Nacalai Tesque, Kyoto, Japan)
buffer (10 mM, pH 8.5) in the presence and absence of iron (II) sulfate heptahydrate (FeSO4·7H2O; 30 µM)
(Wako Pure Chemical Industries), hydrogen peroxide (H2O2; 30 µM) (Tokyo Chemical Industry Co.,
Tokyo, Japan) and ascorbic acid (30 µM) (Tokyo Chemical Industry Co.) as an activator. The reaction
mixture was incubated at 37 °C for 1.5 h before the addition of EDTA (Dojindo Molecular Technologies)
and loading buffer (Takara, Kyoto, Japan). Agarose gel electrophoresis of pUC 19 DNA was performed
at 100 V for 40 min in 1% slab gels containing ethidium bromide (Wako Pure Chemical Industries) in
Tris/Boronic acid/EDTA buffer. DNA was visualized by photographing the fluorescence of intercalated
ethidium bromide under a UV illuminator (Nippon Genetics Co., Tokyo, Japan).
4. Conclusions
In the present paper, new pyrazoline derivatives were synthesized and investigated for their
antiproliferative effects on AsPC-1 human pancreatic adenocarcinoma, U87, and U251 human glioblastoma
cell lines. Detailed investigation of compound 11 against AsPC-1, U87, U251 cell lines and tumor
selectivity of this compound on blood cells (PBMC and Jurkat cells) were also carried out. Among these
compounds, compound 11 was the most effective anticancer agent against AsPC-1 and U251 cancer cell
lines and exhibited significant tumor selectivity. Therefore, compound 11 was chosen for apoptosis/necrosis
evaluation and DNA-cleavage analysis in U251 cells. Compound 11-treated U251 cells showed apoptotic
activity at low concentration (1.5 µM). Interestingly, DNA-cleaving efficiency of this ligand was more
significant than cisplatin. This outcome pointed out the relationship between the DNA cleavage and the
cell death.
In the view of this study, further research can be carried out on the development of new effective
anticancer agents by the modification of compound 11.
Acknowledgments
This study was supported by Anadolu University Scientific Research Projects Commission under the
grant no: 1505S371.
Author Contributions
A.Ö., M.D.A. and M.K. designed the research; M.D.A. and M.K. performed the synthetic work, M.O.,
M.F. and H.İ.Ç. were responsible for the direction of the biological research, H.İ.Ç. and R.K. performed
the anticancer activity. A.Ö. was also responsible for the correspondence of the manuscript, whereas
M.D.A. and H.İ.Ç. mainly wrote the manuscript. All authors discussed, edited and approved the
final version.

Molecules 2015, 20
19082
Conflicts of Interest
The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 1–12 are available from the authors.
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