Synthesis and anticancer activities of 1,4-phenylene-bis-N-acetyl- and N-phenylpyrazoline derivatives

Article abstract of DOI:10.1007/s11164-016-2697-2

Novel 1,4-phenylene-bis-N-acetyl- (3a–h) and bis-N-phenylpyrazoline derivatives (4a–h) were obtained by addition of hydrazine hydrate and phenylhydrazine to bis-chalcone derivatives (1a–h) in acetic acid and acetic acid/ethanol for 4 and 8?h in reflux con

Full text of DOI:10.1007/s11164-016-2697-2

Res Chem Intermed
DOI 10.1007/s11164-016-2697-2
Synthesis and anticancer activities of 1,4-phenylene-bis-
N-acetyl- and N-phenylpyrazoline derivatives
Meliha Burcu Gu¨rdere1 Onur Gu¨mu¨s¸1
•
•
Ayse Sahin Yaglioglu2 Yakup Budak1
•
•
Mustafa Ceylan1
Received: 20 January 2016 / Accepted: 9 August 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract Novel 1,4-phenylene-bis-N-acetyl- (3a–h) and bis-N-phenylpyrazoline
derivatives (4a–h) were obtained by addition of hydrazine hydrate and phenylhy-
drazine to bis-chalcone derivatives (1a–h) in acetic acid and acetic acid/ethanol for
4 and 8 h in reflux conditions, respectively. The structures of the obtained bis-N-
acetylpyrazoline and bis-N-phenylpyrazoline derivatives were characterized by
nuclear magnetic resonance (NMR) and infrared (IR) spectroscopic methods and
elemental analysis. Compounds 3a–h and 4a–h were investigated to evaluate their
anticancer activities against C6 (rat brain tumor cells) and HeLa (human uterus
carcinoma) in vitro using a dose-dependent assay from 5 to 100 lM with 5-fluo-
rouracil (5-FU) as standard anticancer drug. Compound 3a showed higher cell-
selective activity compared with 5-FU against HeLa cells. Compounds 3a–h (except
3d) were shown to have better activities than 5-FU against both cells, particularly at
high concentration. Compound 4c showed higher cell-selective activity compared
with 5-FU against C6 cells. Compound 3a may be particularly promising as an
anticancer drug against HeLa cells.
Keywords Anticancer activity Á Bis-N-acetylpyrazolines Á Bis-N-
biphenylpyrazolines Á C6 Á HeLa
Electronic supplementary material The online version of this article (doi:10.1007/s11164-016-2697-
2) contains supplementary material, which is available to authorized users.
&
Meliha Burcu Gu¨rdere
burcugurdere@gmail.com
1
2
Department of Chemistry, Faculty of Arts and Sciences, Gaziosmanpasa University,
60250 Tokat, Turkey
Department of Chemistry, Faculty of Sciences, Cankiri Karatekin University, 18100 Cankiri,
Turkey
123

M. B. Gu¨rdere et al.
Introduction
Cancer has become one of the major health problems ravaging the world today, and
one of the ways of treating cancer is known as chemotherapy [1–3]. Chemotherapy
causes intense side-effects, due to its cytotoxic effect on normal cells [4]. For this
reason, it is important that anticancer drugs show antiproliferative and cytotoxic
activity against tumor cells without affecting normal tissues. Although numerous
cytotoxic agents have been developed, there is a need to develop more potent and
safer chemotherapeutic agents [5].
In this area, pyrazolines are of great interest due to their synthetic feasibility and
biological importance [6–12]. Derivatives of pyrazolines are found to show good
anticancer and radioprotective activity [13]. Pyrazoline is a prominent structural
motif found in numerous antitumor agents [14]. Several 1,3,5-triaryl-4-alkylpyra-
zolines have been evaluated as breast cancer treatments with the goal of both
reducing toxicity and increasing the response rate as an antitumor agent [15]. In
addition, among anticancer pyrazolines, 1,3-diphenyl pyrazolines have been
reported to be highly potent and efficient cytotoxic agents [16–20]. Also, a series
of novel 3,5-diarylpyrazolines [21] and thiazolylpyrazoline derivatives were
recently reported as potent anticancer agents [22].
Prompted by these results, we decided to investigate and report the synthesis and
anticancer activity of a series of novel 1,4-phenylene-bis-N-acetylpyrazoline (3a–
h) and 1,4-phenylene-bis-N-phenylpyrazoline derivatives (4a–h).
Results and discussion
Chemistry
The approach for synthesis of our target compounds is shown in Scheme 1. Initially,
the known key 1,4-phenylene-bis-chalcones intermediates 1a–h were resynthesized
according to published procedure [23–26]. Bis-N-acetylpyrazoline derivatives 3a–
h were obtained in good yields (75–93 %) by refluxing 4 equivalents of hydrazine
hydrate and the corresponding chalcones 1a–h in acetic acid for 4 h (Scheme 1;
Table 1). The compounds 3e–h are known [27]; the others are novel, and they were
synthesized by literature procedure [28]. The reaction involved nucleophilic attack
of amino group on polarized carbonyl group, followed by intramolecular
cyclization. Evidence for N-acetyl-4,5-dihydropyrazole ring formation was obtained
1
1
from H NMR spectra. The H NMR spectra of the compounds displayed a typical
ABX-type pattern of doublet of doublets due to three pyrazoline protons. Methine
proton of pyrazoline was found at around 5.50 ppm, as a doublet of doublets with
coupling constants of nearly 12 Hz and 4.0–4.8 Hz. Two methylene protons
displayed two signals: a doublet of doublets at around 3.80–3.65 ppm with coupling
constants of nearly 17 and 12 Hz, and a doublet of doublets at around
3.30–3.10 ppm. The 13C NMR spectra of compounds 3a–h confirmed their
proposed structures, since C4 and C5 of pyrazoline ring resonated at around 40–45
123

Synthesis and anticancer activities of 1,4-phenylene-bis-N…
Scheme 1 Synthesis of bis-N-acetyl- and bis-N-phenylpyrazoline derivatives 3 and 4
Table 1 IC50 and IC75 values
Compound
Ar
HeLa
IC50
a
C6
of 3a–h and 4a–h against C6
and HeLa cells (lM)
IC75
IC50
IC75
a
a
a
a
3a
3b
3c
1-Naphthyl
3-Thienyl
2-Thienyl
2-Furyl
5.18
9.79
43.90
55.52
79.09
73.60
59.49
34.33
64.11
82.10
78.74
91.24
72.34
75.30
86.19
81.30
12.58
41.88
51.24
24.72
41.84
49.74
3d
3e
7.65
Ph
29.43
a
55.24
a
3f
4-CH3Ph
4-CH3OPh
4-ClPh
28.93
a
a
a
a
a
a
3g
3h
4a
4b
4c
43.23
63.64
58.19
63.03
49.70
60.74
68.14
57.66
59.66
16.32
36.68
20.85
36.24
56.92
35.76
51.28
1-Naphthyl
3-Thienyl
2-Thienyl
2-Furyl
23.88
a
4d
4e
Ph
13.44
a
4f
4-CH3Ph
4-CH3OPh
4-ClPh
35.76
a
a
4g
4h
5-FU
77.80
b
53.56
5.8
78.82
b
a
b
\5 lM, no detection
and 55–60 ppm, respectively. Furthermore, in the 13C NMR spectra, signals
appearing at around 20–25 ppm indicated acetyl group connected to pyrazoline
ring.
123

M. B. Gu¨rdere et al.
Similarly, heating at reflux of 4 equivalents of phenylhydrazine and the chalcones
1a–h in acetic acid/ethanol mixture for 8 h afforded the corresponding bis-N-
phenylpyrazoline derivatives 4a–h in some good yields (76–93 %) (Scheme 1) [29].
Compound 4b is novel; the others are known [30–32]. The structures of compounds
4a–h were determined on the basis of spectral data. The spectral and microana-
lytical data of compounds 4a–h were consistent with their structures.
Biological evaluation: anticancer activity
All compounds were tested for their potential growth inhibitory activity against rat
brain tumor cells (C6) and human uterus carcinoma (HeLa) cell line using a
proliferation BrdU enzyme-linked immunosorbent assay (ELISA). The tests were
performed at concentrations of 5, 10, 20, 30, 40, 50, 75, and 100 lM with
5-fluorouracil (5-FU) as standard. The antiproliferative activities of the compounds
increased with dose.
The activities of the N-acetylpyrazoline derivatives 3a–h against C6 are shown in
Fig. 1a and Table 1. All tested compounds 3a–h showed anticancer activity with
50 % inhibitory concentration (IC50) values ranging from\5 to 29.43 lM. In most
cases, compounds 3a–h were more active than 5-FU; in particular, compounds 3a,
b, f–h with IC50 values \5 lM exhibited significant activity at all concentrations.
The activities of the N-acetylpyrazoline derivatives 3a–h against HeLa are shown
in Fig. 1b and Table 1. As seen in Fig. 1b, the anticancer activities of all
compounds 3a–h (except 3d, e) were better than 5-FU at high concentrations (75
and 100 lM). Moreover, compound 3a exhibited very high activity with 95 %
inhibition value at 100 to 20 lM concentrations. According to the IC50 values, the
most active compounds were 3a, g with IC50 value \5 lM, followed by 3b
(9.79 lM), 3c (24.72 lM), and 3f (28.93 lM).
The results for the anticancer activities of the N-phenylpyrazoline derivatives 4a–
h against C6 cells are shown in Fig. 2a and Table 1. According to Fig. 2a, the
anticancer activity of 4c was better than 5-FU at high concentrations (75 and
100 lM), and it showed significant activity at all concentrations. Compound 4d
showed almost the same activity as 5-FU. Compounds 4b, e–h showed moderate
anticancer effects. However, 4a did not show significant activity compared with
5-FU. In addition, the compounds had very low IC50 values, except 4h; while the
IC50 values of 4a, b, d, f, g were \5 lM, the others were 23.88 lM for 4a,
13.44 lM for 4e, and 53.56 lM for 4h (Table 1).
The activities of 4a–h against HeLa are shown in Fig. 2b and Table 1. According
to these results, whereas compounds 4b–d, f showed moderate activity at 100 lM
concentration, the others were almost inactive compared with 5-FU.
According to these results, it was observed that compounds 3a–h showed higher
anticancer activities than compounds 4a–h against both cell lines. Moreover, while
compounds 3a–h exhibited significant activities against both cell lines, compounds
4a–h demonstrated significant activities against the C6 cell line, particularly at high
concentrations.
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Synthesis and anticancer activities of 1,4-phenylene-bis-N…
A
3a 3b 3c 3d 3e 3f 3g 3h 5-FU
against HeLa cell
B
95
75
55
35
15
-5
100
75
50
40
30
20
10
5
Concentration (µM)
Fig. 1 Antiproliferative activities of 3a–h against (a) C6 and (b) HeLa cells. *Each substance was tested
twice in triplicates against cell lines. Data show averages of two individual experiments (p \ 0.01)
Structure–activity relationship (SAR)
Comparing compounds 3a–h with 4a–h, it is obvious that the N-acetylpyrazolines
3a–h showed higher growth inhibitory effect than the N-phenylpyrazolines 4a–
h against both cell lines. The higher cytotoxicity of 3a–h is due to the acetyl
(CH3CO–) group attached to the pyrazoline ring, while in the case of compounds
4a–h, the Ph group lowered the cytotoxicity.
Considering the N-acetylpyrazolines 3a–h, all compounds showed high growth
inhibitory effects against the C6 cell line at almost all concentrations. Compounds
123

M. B. Gu¨rdere et al.
Fig. 2 Antiproliferative activities of 4a–h against (a) C6 and (b) HeLa cells. Asterisk each substance was
tested twice in triplicates against cell lines. Data show averages of two individual experiments (p \ 0.01)
3a and d with 1-naphthyl and furyl substituents, respectively, showed lower
activities, whereas the others exhibited activities higher than 5-FU against the C6
cell line at 100–50 lM concentrations.
The activities of all the compounds (except 3d containing furyl ring) were better
than 5-FU against HeLa cell lines at high concentrations. In addition, the activity of
3e was low compared with 5-FU. However, compounds 3a and g with 2-naphthyl
and 4-methoxyphenyl substitution, respectively, exhibited activities higher than
5-FU against HeLa cell lines at 100 to 20 lM concentrations. This high cytotoxicity
123

Synthesis and anticancer activities of 1,4-phenylene-bis-N…
of both compounds 3a and g is attributed to the presence of naphthyl moieties and
methoxide group, respectively.
Considering the N-phenylpyrazolines 4a–h, all compounds 4a–h showed selec-
tive growth inhibitory effects against the C6 cell line. All compounds (except 4a
containing naphthyl ring) exhibited remarkable activities, particularly at high
concentrations. Among these compounds, the most active was 4c (containing
2-thienyl ring), which showed significant activity at all concentrations. The
remarkable activity of 4c is due to the presence of the thiophene moiety.
All compounds showed very low activities except at 100 lM concentration
against the HeLa cell line. The most active compound was 4d, which is attributed to
the presence of furan ring.
Conclusions
A series of 1,4-phenylene-bis-N-acetylpyrazoline (3a–h) and 1,4-phenylene-bis-N-
phenylpyrazoline derivatives (4a–h) were synthesized in good yields by addition of
hydrazine hydrate and phenyl hydrazine to 1,4-phenylene-bis-chalcones and
evaluated for their anticancer activities against C6 (rat brain tumor) and HeLa
(human uterus carcinoma) cell lines. Compounds 3a–h showed potent anticancer
activities against both cell lines. Most of them were higher in activity than 5-FU
against the C6 and HeLa cell lines at high concentration. Compound 3a exhibited
very high activity against HeLa cell line cell selectively at 100 to 20 lM
concentrations. Also, compounds 4a–h (except 4a) demonstrated significant
activities against the C6 cell line, when compared with 5-FU at high concentrations.
Moreover, only one compound (4d) showed remarkable activity against HeLa cell
lines at 100 and 75 lM concentrations.
Experimental
The major chemicals were purchased from Sigma-Aldrich and Fluka. IR spectra
1
(KCl disc) were recorded on a Jasco FT/IR-430 spectrometer. H and 13C NMR
spectra were recorded on a Bruker Avance DPX-400 instrument. Tetramethylsilane
1
(TMS) and CDCl3 served as internal standard for H and 13C NMR spectroscopy
with d values of 0.00 and 77.0 ppm, respectively. J values are given in Hz. Signal
multiplicities in the H NMR spectra are abbreviated as s (singlet), d (doublet), t
1
(triplet), q (quartet), m (multiplet), br (broad), and combinations thereof. Melting
points were measured on Electrothermal 9100 apparatus. Elemental analyses were
obtained using a LECO CHNS 932 elemental analyzer.
General method for synthesis of 1,4-phenylene-bis-N-acetylpyrazoline (3a–
h) and 1,4-phenylene-bis-N-phenylpyrazoline derivatives (4a–h)
Bis-chalcone (1a–h) (1 mmol) and hydrazine hydrate (4 mmol) in acetic acid and/or
phenylhydrazine (4 mmol) in acetic acid/ethanol mixture were refluxed for 4 and
123

M. B. Gu¨rdere et al.
8 h, respectively. The reaction mixture was poured into ice bath at the end of the
reaction, and was checked with pH paper to ensure neutralization of the medium by
addition of ammonia. It was observed that the collapse occurred when the mixture
was allowed to stand overnight, and the resulting precipitate was filtered off and
allowed to dry. The resulting material was obtained as pure.
1,10-(5,50-(1,4-Phenylene)bis(3-(naphthalen-1-yl)-4,5-dihydro-1H-pyrazole-5,1-diyl))
diethanone (3a) Yield: 86 %. M.p. 250–252 °C. IR (KCl) v/cm-1: 3046, 1648,
1508, 1419, 1394, 1268, 1143, 1014, 958, 804, 773, 557. H NMR (400 MHz,
1
DMSO, ppm): d 9.24 (d, J = 8.4 Hz, 2H), 8.03–7.98 (m, 4H), 7.75 (d, J = 6.8 Hz,
2H), 7.68 (t, J = 7.2 Hz, 2H), 7.60 (t, J = 7.2 Hz, 2H), 7.55–7.52 (m, 2H), 7.24
(brs, 4H), 5.55–5.51 (dd, J = 11.4, 3.2 Hz, 2H), 4.10–4.03 (dd, J = 17.6, 12.4 Hz,
2H), 3.33–3.28 (m, 2H), 2.41 (s, 6H). 13C NMR (100 MHz, DMSO, ppm): d 168.4
(2C), 155.5 (2C), 141.6 (2C), 134.1 (2C), 131.5 (2C), 130.1 (2C), 129.5 (2C), 129.2
(4C), 128.3 (2C), 127.4 (2C), 126.8 (2C), 126.6 (2C), 126.3 (2C), 125.7 (2C), 58.5
(2C), 45.0 (2C), 22.2 (2C). Anal. Calcd. for C36H30N4O2: C, 78.52; H, 5.49; N,
10.17. Found: C, 78.48; H, 5.32; N, 10.11.
1,10-(5,50-(1,4-Phenylene)bis(3-(thiophen-3-yl)-4,5-dihydro-1H-pyrazole-5,1-diyl))
diethanone (3b) Yield: 77 %. M.p. 223–225 °C. IR (KCl) v/cm-1: 3023, 2924,
1644, 1438, 1409, 1338, 1102, 1037, 1018, 982, 813, 701, 635, 548. H NMR
1
(400 MHz, DMSO, ppm): d 7.55–7.54 (m, 2H), 7.47–7.46 (m, 2H), 7.42–7.38 (m,
2H), 7.18–7.16 (m, 4H), 5.57–5.51 (dd, J = 11.6, 4.8 Hz, 2H), 3.73–3.65 (dd,
J = 17.6, 11.6 Hz, 2H), 3.14–3.07 (dt, J = 17.6, 4.8 Hz, 2H), 2.43 (s, 6H). 13C
NMR (100 MHz, CDCl3, ppm): d 168.8 (2C), 150.2 (2C), 141.0 (2C), 133.9 (2C),
126.9 (2C), 126.4 (4C), 126.1 (2C), 125.5 (2C), 59.5 (2C), 42.8 (2C), 21.9 (2C).
Anal. Calcd. for C24H22N4O2S2: C, 62.31; H, 4.79; N, 12.11. Found: C, 62.28; H,
4.67; N, 12.06.
1,10-(5,50-(1,4-Phenylene)bis(3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazole-5,1-diyl))
diethanone (3c) Yield: 75 %. M.p. 306–308 °C. IR (KCl) v/cm-1: 3033, 2929,
1644, 1440, 1413, 1326, 1141, 1037, 1018, 952, 831, 761, 630, 555. H NMR
1
(400 MHz, DMSO, ppm): d 7.45–7.41 (dd, J = 5.2, 0.8 Hz, 2H), 7.21–7.20 (m,
6H), 7.09–7.07 (dd, J = 5.0, 3.6 Hz, 2H), 5.58–5.55 (dd, J = 11.6, 4.4 Hz, 2H),
3.77–3.69 (dd, J = 17.4, 11.6 Hz, 2H), 3.19–3.13 (dd, J = 17.2, 4.4 Hz, 2H), 2.40
(s, 6H). 13C NMR (100 MHz, CDCl3, ppm): d 168.7 (2C), 149.6 (2C), 140.9 (2C),
134.8 (2C), 128.8 (2C), 128.7 (2C), 127.6 (2C), 126.1 (4C), 59.7 (2C), 42.8 (2C),
21.9 (2C). Anal. Calcd. for C24H22N4O2S2: C, 62.31; H, 4.79; N, 12.11. Found: C,
62.25; H, 4.72; N, 12.09.
1,10-(5,50-(1,4-Phenylene)bis(3-(furan-2-yl)-4,5-dihydro-1H-pyrazole-5,1-diyl))di-
ethanone (3d) Yield: 93 %. M.p. 251–253 °C. IR (KCl) v/cm-1: 3124, 1643,
1421, 1388, 1010, 983, 827, 769, 563. 1H NMR (400 MHz, DMSO, ppm): d 7.57 (s,
2H), 7.18 (s, 4H), 6.73 (d, J = 2.8 Hz, 2H), 6.54–6.52 (m, 2H), 5.58–5.54 (dd,
J = 12.0, 4.8 Hz, 2H), 3.71–3.63 (dd, J = 17.6, 12.0 Hz, 2H), 3.12–3.06 (dt,
J = 17.6, 3.6 Hz, 2H), 2.42 (s, 6H). 13C NMR (100 MHz, CDCl3, ppm): d 168.8
(2C), 146.7 (2C), 145.7 (2C), 144.8 (2C), 140.8 (2C), 126.1 (4C), 112.8 (2C), 111.9
123

Synthesis and anticancer activities of 1,4-phenylene-bis-N…
(2C), 59.1 (2C), 41.9 (2C), 21.9 (2C). Anal. Calcd. for C24H22N4O4: C, 66.97; H,
5.15; N, 13.02. Found: C, 66.89; H, 5.09; N, 12.98.
1,10-(5,50-(1,4-Phenylene)bis(3-phenyl-4,5-dihydro-1H-pyrazole-5,1-diyl))diethanone
(3e) Yield: 78 %. M.p. 296–298 °C. IR (KCl) v/cm-1: 3060, 1644, 1454, 1409,
1118, 958, 829, 771, 555. 1H NMR (400 MHz, DMSO, ppm): d 7.78 (d,
J = 4.0 Hz, 4H), 7.46 (s, 6H), 7.14 (s, 4H), 5.54–5.51 (dd, J = 11.8, 4.0 Hz, 2H),
3.87–3.80 (dd, J = 18.0, 12.0 Hz, 2H), 3.15–3.10 (dd, J = 18.0, 4.0 Hz, 2H), 2.43
(s, 6H). 13C NMR (100 MHz, CDCl3, ppm): d 167.8 (2C), 154.6 (2C), 141.8 (2C),
131.5 (2C), 130.7 (2C), 129.2 (4C), 127.4 (2C), 127.0 (4C), 126.2 (2C), 59.5 (2C),
42.4 (2C), 22.1 (2C). Anal. Calcd. for C28H26N4O2: C, 74.65; H, 5.82; N, 12.44.
Found: C, 74.59; H, 5.79; N, 12.38.
1,10-(5,50-(1,4-Phenylene)bis(3-(p-tolyl)-4,5-dihydro-1H-pyrazole-5,1-diyl))diethanone
(3f) Yield: 78 %. M.p. 268–270 °C. IR (KCl) v/cm-1: 3027, 2921, 1662, 1417,
1
1326, 1143, 1027, 1010, 960, 867, 819, 543. H NMR (400 MHz, CDCl3, ppm): d
7.63 (d, J = 7.2 Hz, 4H), 7.27 (d, J = 7.6 Hz, 4H), 7.18 (s, 4H), 5.59–5.53 (dd,
J = 12.0, 4.8 Hz, 2H), 3.73–3.66 (dd, J = 17.4, 12.0 Hz, 2H), 3.19–3.12 (dt,
J = 17.4, 4.4 Hz, 2H), 2.45 (s, 6H), 2.42 (s, 6H). 13C NMR (100 MHz, CDCl3,
ppm): d 168.8 (2C), 154.2 (2C), 141.1 (2C), 140.7 (2C), 129.4 (4C), 128.4 (2C),
126.5 (4C), 126.1 (4C), 59.5 (2C), 42.2 (2C), 21.9 (2C), 21.5 (2C). Anal. Calcd. for
C30H30N4O2: C, 75.29; H, 6.32; N, 11.71. Found: C, 75.21; H, 6.27; N, 11.63.
1,10-(5,50-(1,4-Phenylene)bis(3-(4-methoxyphenyl)-4,5-dihydro-1H-pyrazole-5,1-diyl))
diethanone (3g) Yield: 79 %. M.p. 248–250 °C. IR (KCl) v/cm-1: 3002, 2933,
2836, 1656, 1606, 1517, 1423, 1255, 1174, 1035, 960, 831, 547. 1H NMR
(400 MHz, CDCl3, ppm): d 7.71–7.66 (dd, J = 8.8, 2.8 Hz, 4H), 7.18 (s, 4H),
6.97–6.93 (dd, J = 8.8, 2.4 Hz, 4H), 5.58–5.54 (dd, J = 12.0, 4.8 Hz, 2H), 3.87 (s,
6H), 3.72–3.64 (dd, J = 17.4, 12.0 Hz, 2H), 3.17–3.12 (dt, J = 17.4, 4.4 Hz, 2H),
2.42 (s, 6H). 13C NMR (100 MHz, CDCl3, ppm): d 168.7 (2C), 161.3 (2C), 153.9
(2C), 141.1 (2C), 128.2 (4C), 126.2 (2C), 126.1 (2C), 123.9 (2C), 114.5 (4C), 59.5
(2C), 55.4 (2C), 42.2 (2C), 21.9 (2C). Anal. Calcd. for C30H30N4O4: C, 70.57; H,
5.92; N, 10.97. Found: C, 70.52; H, 5.83; N, 10.84.
1,10-(5,50-(1,4-Phenylene)bis(3-(4-chlorophenyl)-4,5-dihydro-1H-pyrazole-5,1-diyl))
diethanone (3h) Yield: 81 %. M.p. 253–255 °C. IR (KCl) v/cm-1: 1666, 1592,
1417, 1398, 1322, 1145, 1091, 1010, 956, 827, 565, 538. H NMR (400 MHz,
1
CDCl3 ppm): d 7.70–7.65 (dd, J = 8.8, 2.8 Hz, 4H), 7.42–7.39 (dd, J = 8.6,
2.4 Hz, 4H), 7.19 (s, 4H), 5.61–5.55 (m, 2H), 3.73–3.62 (m, 2H), 3.17–3.10 (dt,
J = 17.6, 4.4 Hz, 2H), 2.42 (s, 6H). 13C NMR (100 MHz, CDCl3, ppm): d 169.0
(2C), 153.0 (2C), 140.9 (2C), 136.3 (2C), 129.7 (2C), 129.0 (4C), 127.8 (4C), 126.1
(4C), 59.7 (2C), 42.0 (2C), 21.9 (2C). Anal. Calcd. for C28H24Cl2N4O2: C, 64.74; H,
4.66; N, 10.79. Found: C, 64.66; H, 4.59; N, 10.72.
1,4-Bis(3-(naphthalen-1-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzene
(4a)
Yield: 79 %. M.p. 271–273 °C. IR (KCl) v/cm-1: 3446, 3322, 1598, 1540, 1498,
1
1247, 1133, 997, 802, 779, 690. H NMR (400 MHz, DMSO, ppm): d 9.58 (d,
123

M. B. Gu¨rdere et al.
J = 8.4 Hz, 2H), 8.09–7.99 (m, 4H), 7.72 (t, J = 7.2 Hz, 2H), 7.68 (t, J = 7.2 Hz,
2H), 7.46–7.41 (m, 2H), 7.25–7.21 (m, 4H), 6.99–6.97 (m, 4H), 6.90–6.87 (m, 4H),
6.06–6.02 (m, 4H), 5.33–5.23 (m, 2H), 4.08–4.01 (dd, J = 16.8, 12.4 Hz, 2H),
3.39–3.33 (dd, J = 16.8, 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO, ppm): d 152.1
(2C), 143.9 (2C), 143.3 (2C), 142.8 (2C), 137.0 (2C), 130.2 (4C), 129.6 (2C), 129.3
(4C), 128.2 (2C), 127.2 (4C), 127.1 (4C), 124.5 (4C), 120.0 (2C), 118.9 (2C), 113.2
(2C), 62.9 (2C), 40.5 (2C). Anal. Calcd. for C44H34N4: C, 85.41; H, 5.54; N, 9.05.
Found: C, 85.39; H, 5.35; N, 9.02.
1,4-Bis(1-phenyl-3-(thiophen-3-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene
(4b)
Yield: 85 %. M.p. 279–281 °C. IR (KCl) v/cm-1: 3315, 3097, 1596, 1498, 1361,
1
1103, 997, 746, 692. H NMR (400 MHz, CDCl3, ppm): d 7.59–7.56 (dd, J = 4.8,
3.6 Hz, 2H), 7.30 (s, 4H), 7.24–7.22 (dd, J = 3.6, 1.2 Hz, 2H), 7.14 (t, J = 8.0 Hz,
6H), 6.90 (t, J = 7.2 Hz, 4H), 6.73 (t, J = 7.2 Hz, 2H), 5.39–5.35 (dd, J = 12.4,
6.4 Hz, 2H), 3.88–3.3.83 (dd, J = 17.4, 12.0 Hz, 2H), 3.10–3.02 (dd, J = 17.2,
6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3, ppm): d 153.0 (2C), 145.1 (2C), 144.8
(2C), 142.2 (2C), 129.3 (4C), 128.3 (2C), 128.1 (2C), 127.7 (4C), 126.8 (2C), 117.9
(2C), 110.8 (4C), 62.9 (2C), 40.5 (2C). Anal. Calcd. for C32H26N4S2: C, 72.42; H,
4.94; N, 10.56. Found: C, 72.39; H, 4.91; N, 10.48.
1,4-Bis(1-phenyl-3-(thiophen-2-yl)-4,5-dihydro-1H-pyrazol-5-yl)benzene
(4c)
Yield: 82 %. M.p. 231–233 °C. IR (KCl) v/cm-1: 3284, 3021, 1594, 1500, 1367,
1
1132, 998, 740, 690. H NMR (400 MHz, CDCl3, ppm): d 7.58–7.56 (dd, J = 4.8,
3.6 Hz, 2H), 7.29 (s, 4H), 7.21 (t, J = 3.6 Hz, 2H), 7.13 (t, J = 8.0 Hz, 4H),
7.09–7.07 (m, 2H), 6.90 (d, J = 7.2 Hz, 4H), 6.71 (t, J = 7.2 Hz, 2H), 5.45–5.40
(dd, J = 12.0, 6.4 Hz, 2H), 3.90–3.87 (dd, J = 17.2, 12.0 Hz, 2H), 3.10–3.08 (dd,
J = 17.2, 6.4 Hz, 2H). 13C NMR (100 MHz, CDCl3, ppm): d 163.3 (2C), 144.5
(2C), 144.1 (2C), 142.0 (2C), 129.3 (4C), 128.2 (2C), 128.0 (2C), 127.9 (4C), 127.0
(2C), 119.1 (2C), 113.2 (4C), 63.3 (2C), 44.1 (2C). Anal. Calcd. for C32H26N4S2: C,
72.42; H, 4.94; N, 10.56. Found: C, 72.35; H, 4.89; N, 10.51.
1,4-Bis(3-(furan-2-yl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzene (4d) Yield:
80 %. M.p. 246–248 °C. IR (KCl) v/cm-1: 3122, 2915, 1594, 1504, 1394, 1257,
1
1130, 1027, 829, 748, 690. H NMR (400 MHz, CDCl3, ppm): d 7.52–7.51 (dd,
J = 3.2, 1.6 Hz, 2H), 7.29 (s, 4H), 7.20–7.17 (m, 4H), 7.05–7.03 (m, 4H), 6.81 (t,
J = 6.8 Hz, 2H), 6.57 (t, J = 3.2 Hz, 2H), 6.50–6.48 (m, 2H), 5.26–5.23 (dd,
J = 12.4, 4.8 Hz, 2H), 3.82–3.74 (dd, J = 16.8, 2.4 Hz, 2H), 3.11–3.05 (dd,
J = 17.0, 6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3, ppm): d 149.8 (2C), 139.6
(2C), 129.3 (4C), 129.1 (2C), 127.0 (2C), 119.1 (2C), 118.8 (2C), 113.3 (4C), 112.5
(4C), 112.4 (2C), 111.3 (2C), 62.6 (2C), 43.3 (2C). Anal. Calcd. for C32H26N4O2: C,
77.09; H, 5.26; N, 11.24. Found: C, 77.01; H, 5.19; N, 11.18.
1,4-Bis(1,3-diphenyl-4,5-dihydro-1H-pyrazol-5-yl)benzene (4e) Yield: 93 %. M.p.
224–226 °C. IR (KCl) v/cm-1: 3100, 1594, 1498, 1380, 1317, 1228, 1122, 1106,
1
827, 748, 709, 690. H NMR (400 MHz, DMSO, ppm): d 7.74–7.71 (dd, J = 7.6,
2.4 Hz, 4H), 7.43–7.39 (m, 6H), 7.29 (s, 4H), 7.14 (t, J = 7.6 Hz, 4H), 6.98 (d,
J = 7.6 Hz, 4H), 6.73–6.69 (dd, J = 12.2, 6.8 Hz, 2H), 5.45–5.40 (dd, J = 12.4,
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Synthesis and anticancer activities of 1,4-phenylene-bis-N…
6.8 Hz, 2H), 3.93–3.85 (dd, J = 17.4 12.4 Hz, 2H), 3.10–3.04 (dd, J = 17.4,
6.8 Hz, 2H). 13C NMR (100 MHz, CDCl3, ppm): d 147.6 (2C), 144.5 (2C), 142.2
(2C), 132.6 (2C), 129.3 (4C), 129.1 (4C), 129.0 (4C), 127.0 (2C), 126.1 (4C), 119.1
(2C), 113.3 (4C), 63.3 (2C), 43.4 (2C). Anal. Calcd. for C36H30N4: C, 83.37; H,
5.83; N, 10.80. Found: C, 83.29; H, 5.79; N, 10.78.
1,4-Bis(1-phenyl-3-(p-tolyl)-4,5-dihydro-1H-pyrazol-5-yl)benzene
(4f) Yield:
76 %. M.p. 278–280 °C. IR (KCl) v/cm-1: 3018, 2919, 1596, 1498, 1386, 1321,
1
1122, 817, 746, 690. H NMR (400 MHz, CDCl3, ppm): d 7.64–7.63 (dd, J = 7.4,
4.4 Hz, 4H), 7.29 (s, 4H), 7.22–7.20 (m, 8H), 7.08 (d, J = 7.6 Hz, 4 H), 6.83–6.75
(m, 2H), 5.26–5.21 (dd, J = 12.2, 7.2 Hz, 2H), 3.85–3.77 (dd, J = 15.0, 2.0 Hz,
2H), 3.15–3.09 (dd, J = 16.8, 7.2 Hz, 2H), 2.40 (s, 6H). 13C NMR (100 MHz,
DMSO, ppm): d 147.6 (2C), 143.5 (2C), 142.2 (2C), 137.1 (2C), 132.6 (2C), 129.9
(2C), 129.3 (4C), 129.0 (4C), 127.0 (2C), 126.1 (4C), 123.5 (2C), 113.3 (4C), 63.3
(2C), 43.4 (2C), 20.1 (2C). Anal. Calcd. for C38H34N4: C, 83.48; H, 6.27; N, 10.25.
Found: C, 83.37; H, 6.19; N, 10.15.
1,4-Bis(3-(4-methoxyphenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzene (4g)
Yield: 79 %. M.p. 265–267 °C. IR (KCl) v/cm-1: 3007, 1733, 1716, 1698, 1683,
1
1558, 1540, 414. H NMR (400 MHz, CDCl3, ppm): d 7.67–7.66 (m, 4H), 7.31 (s,
4H), 7.23 (t, J = 7.6 Hz, 4H), 7.04 (d, J = 7.2 Hz, 4H), 6.94–6.91 (m, 4H), 6.79 (t,
J = 7.2 Hz, 2H), 5.23–5.18 (dd, J = 12.2, 7.2 Hz, 2H), 3.85–3.80 (m, 8H),
3.14–3.10 (dd, J = 17.0, 7.2 Hz, 2H). 13C NMR (100 MHz, DMSO, ppm): d 163.2
(2C), 147.7 (2C), 145.0 (2C), 142.2 (2C), 129.3 (4C), 127.7 (4C), 127.0 (2C), 125.2
(4C), 118.7 (2C), 114.5 (4C), 113.1 (4C), 63.2 (2C), 56.0 (2C), 43.6 (2C). Anal.
Calcd. for C38H34N4O2: C, 78.87; H, 5.92; N, 9.68. Found: C, 78.81; H, 5.89; N,
9.59.
1,4-Bis(3-(4-chlorophenyl)-1-phenyl-4,5-dihydro-1H-pyrazol-5-yl)benzene
(4h)
Yield: 84 %. M.p. 284–286 °C. IR (KCl) v/cm-1: 1594, 1502, 1488, 1386, 1321,
1
1132, 1087, 1010, 821, 742, 690. H NMR (400 MHz, DMSO, ppm): d 7.70 (d,
J = 8.0 Hz, 4H), 7.41 (d, J = 8.0 Hz, 4H), 7.26 (s, 4H), 7.11 (t, J = 7.2 Hz, 4H),
6.96 (d, J = 6.8 Hz, 4H), 6.69 (t, J = 6.8 Hz, 2H), 5.44–5.40 (dd, J = 12.0,
6.4 Hz, 2H), 3.90–3.82 (dd, J = 17.4, 12.0 Hz, 2H), 3.08–3.02 (dd, J = 17.4,
6.4 Hz, 2H). 13C NMR (100 MHz, DMSO, ppm): d 150.7 (2C), 144.4 (2C), 140.0
(2C), 133.5 (2C), 133.0 (2C), 130.1 (2C), 129.9 (4C), 129.1 (4C), 128.6 (4C), 127.7
(4C), 113.2 (2C), 106.1 (2C), 63.4 (2C), 43.2 (2C). Anal. Calcd. for C36H28Cl2N4:
C, 73.59; H, 4.80; N, 9.54. Found: C, 73.52; H, 4.79; N, 9.48.
Anticancer activity
Cell culture
Antiproliferative activities of the synthesized compounds were investigated on C6
(rat brain tumor cells) and HeLa (human uterus carcinoma) cells using a
proliferation BrdU ELISA assay [33, 34]. Cells were grown in Dulbecco’s modified
Eagle’s medium (DMEM, Sigma), supplemented with 10 % (v/v) fetal bovine
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serum (Sigma, Germany) and PenStrep solution (Sigma, Germany) at 37 °C in 5 %
CO2 humidified atmosphere.
Cell proliferation assays
Cells were plated in 96-well culture plates (COSTAR, Corning, USA) at density of
3 9 104 cells per well. The activities of samples were investigated at eight
concentrations. 5-FU was used as standard compound. The cells were then
incubated overnight before applying the BrdU Cell Proliferation ELISA assay
reagent (Roche, Germany) according to the manufacturer’s procedure. The amount
of cell proliferation was determined as A450 nm by using a microplate reader
(Awareness Chromate, USA). Results are reported as percentage inhibition of cell
proliferation, where the optical density measured from vehicle-treated cells was
considered to be 100 % proliferation. Percentage of inhibition of cell proliferation
was calculated as follows: [1 - (Atreatment/Avehicle control)] 9 100.
Stock solution of tested compounds was prepared in dimethyl sulfoxide (DMSO),
and diluted with DMEM. The final DMSO concentration was below 0.1 % in all
tests. IC50 and IC75 values were determined using ED50 plus v1.0. The results of the
in vitro investigation are presented as mean standard deviation (SD) of six
measurements. Differences between groups were tested with analysis of variance
(ANOVA) with p values \0.01 considered significant, analyzed by SPSS (version
11.5 for Windows 2000, SPSS Inc.).
Acknowledgments The authors are indebted to the Gaziosmanpasa University Scientific Research
Projects Commission (Project No. BAP2011/95) for financial support.
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