Design, synthesis, and biologic evaluation of some novel N-arylpyrazole derivatives as cytotoxic agents

Article abstract of DOI:10.1007/s00044-013-0552-1

A novel series of N-arylpyrazole derivatives (5a-5d, 7a-7c) has been designed and synthesized via aromatic substitution reaction of N-nonsubstituted pyrazoles with 4-fluoronitrobenzene in the presence of base. The structures of these compounds were establ

Full text of DOI:10.1007/s00044-013-0552-1

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0552-1
ORIGINAL RESEARCH
Design, synthesis, and biologic evaluation of some novel
N-arylpyrazole derivatives as cytotoxic agents
Shengjie Xu
Shuxiang Wang
•
Shenghui Li
•
•
Yonghe Tang
•
•
Jinchao Zhang
•
Chuanqi Zhou
Xiaoliu Li
Received: 29 October 2012 / Accepted: 18 February 2013
Ó Springer Science+Business Media New York 2013
Abstract A novel series of N-arylpyrazole derivatives
5a–5d, 7a–7c) has been designed and synthesized via
Keywords Pyrazole ꢀ N-Arylpyrazole ꢀ
(
Aromatic substitution ꢀ Cytotoxicity ꢀ Cancer cell lines
aromatic substitution reaction of N-nonsubstituted pyra-
zoles with 4-fluoronitrobenzene in the presence of base.
The structures of these compounds were established on the
basis of elemental (C, H, and N) and spectral analysis
Introduction
1
13
(
H NMR, C NMR, HRMS, and FT-IR). All the com-
Pyrazoles represent a class of nitrogen containing five mem-
bered heterocyclic compounds of significant importance and
are considered as extremely versatile building blocks in
organic chemistry. Owing to their easy preparation, interesting
chemical property and rich biologic activity, pyrazole, and its
derivatives are widely used in pharmaceuticals, agrochemi-
cals, food additives, dyes, materials, and cosmetic coloring
agents (Katritziky et al., 1996; Schmidt and Dreger, 2011).
In addition, much interest has been given to the che-
motherapeutic activity of N-arylpyrazole as antiparasitic
(Rathelot et al., 2002), antifungal (Prakash et al., 2008;
Sivaprasad et al., 2006), antibacterial (Finn et al., 2003),
antiviral (Elgemeie et al., 2005), and antidiabetic
(Bebernitz et al., 2001) agents. It has been also reported that
some N-arylpyrazole derivatives have shown several biologic
activities as seen in cyclooxygenase-2 (Bing and Lomnicka,
pounds were tested for their cytotoxic activity in vitro
against four human tumor cell lines: carcinoma (Bel-7402),
nasopharyngeal carcinoma (KB), immature granulocyte
leukemia (HL-60), and gastrocarcinoma (BGC-823) by the
3
-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide assay. The results showed that most of the obtained
compounds exhibited promising cytotoxicity against tested
carcinoma cell lines with low IC50 values. The bis-pyrazole
derivative 7c, bearing alkoxy group on the 5-position of
phenyl ring, was the most effective one. It is inhibition of
cell growth of Bel-7402 cells was 1.5-fold higher than that
found for cisplatin. And, also mono-pyrazole derivatives 5a
and 5b, decorated with trifluoromethyl group on the phenyl
ring, displayed better cytotoxicity than that of cisplatin
against Bel-7402 cell line.
2002;Kurumbailetal.,1996;Habeebetal.,2001;Chowdhury
et al., 2009), p38 MAP kinase (Regan et al., 2002), estrogen
receptor (Stauffer et al., 2000), and non-nucleoside HIV-1
reverse transcriptase inhibitory activity (Genin et al., 2000).
Moreover, many N-arylpyrazole derivatives exhibited prom-
ising antiproliferative properties against several kinds of
human tumor cell lines (Balbi et al., 2011; Farag et al., 2008;
Insuasty et al., 2010; Joksovi c´ et al., 2010; Rostom et al.,
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-013-0552-1) contains supplementary
material, which is available to authorized users.
S. Xu ꢀ S. Li (&) ꢀ Y. Tang ꢀ J. Zhang (&) ꢀ S. Wang ꢀ
C. Zhou ꢀ X. Li
Key Laboratory of Chemical Biology of Hebei Province,
Key Laboratory of Medicinal Chemistry and Molecular
Diagnosis, Ministry of Education, College of Chemistry and
Environmental Science, Hebei University, Baoding 071002,
People’s Republic of China
2
003). Therefore, the use of N-arylpyrazole motif is believed
to be a powerful tool for novel compounds with potential
pharmacological and biologic activities.
e-mail: lish@hbu.edu.cn
Much effort has been made to construct such an N-aryl-
pyrazole structural unit. Important methods involve
J. Zhang
e-mail: jczhang6970@yahoo.com.cn
123

Med Chem Res
approaches based on the condensation of N-arylhydrazines
with a variety of 1,3-carbonyl compounds or its related
compounds (Chang et al., 2002; Giacomelli et al., 2003;
Peruncheralathan et al., 2005) and the transition metal-cata-
lyzed N-arylation of 1H-pyrazoles (Klapars et al., 2001; Son
et al., 2004; Zhang et al., 2005). In addition, aromatic sub-
stitution of N-nonsubstituted pyrazoles with activated halo-
genated benzenes is also a powerful method for synthesis of
N-arylpyrazoles(Wang etal., 2000; Bouabdallahetal., 2007).
Based on the previous findings and with the aim of obtaining
new compounds with antitumor activity, we synthesized a
novel series of N-arylpyrazole derivatives (5a–5d, 7a–7c) via
aromatic substitution reaction of N-nonsubstituted pyrazoles
with 4-fluronitrobenzene in the presence of base. And, the
cytotoxic activity of the obtained compounds was evaluated
by MTT assay against four different human tumor cell lines
stirred for an additional hour at room temperature. Then,
substituted phenylacetic acid (10.0 mmol) 1a–1d was
added at once and the clear solution formed was stirred for
4 h at 90–95 °C and then at room temperature overnight.
The resulting black mixture was poured on crushed ice.
After decomposition of the excess Vilsmeyer reagent, a
saturated solution of 4.3 g (30 mmol) NaClO ꢀH O was
4
2
added with stirring. The resulting nearly white crystalline
deposit of the perchlorate salt 2a–2d was filtered and
washed with two 4 mL portions of water. This compound
was used in the next step without further purification.
The perchlorate salt 2a–2d (10 mmol) was added to a
warm solution of 0.8 g (20 mmol) NaOH in 6 mL water,
and the mixture was heated with stirring for 15 min (bath
temperature 90 °C) until total dissolution of the organic salt
was observed. The yellow-colored mixture was cooled to
room temperature, diluted with 1.5 mL water, and 10 %
HCl solution was added to pH = 5, leading to precipitation
of compound 3a–3d. Then, 1.5 mL hydrazine hydrate
(80 %) was added at once with stirring, which effects dis-
solution of 3 and after 15–20 min stirring at room temper-
ature the pyrazole compounds 4a–4d deposits as light
yellow solid. The mixture was left overnight and the pre-
cipitate was collected by filtration and oven-dried in vacuo.
(
Bel-7402, KB, HL-60, and BGC-823).
Experimental
Materials and methods
All chemicals and reagents were of analytical grade. RPMI-
1
640medium, trypsin, and fetal bovine serum were purchased
from Gibco. MTT, benzylpenicillin, and streptomycin were
from sigma. Four different human carcinoma cell lines:
Bel-7402 (liver carcinoma), KB (nasopharyngeal carcinoma),
HL-60 (immature granulocyte leukemia), and BGC-823
4-(4-(Trifluoromethyl)phenyl)-1H-pyrazole 4a
Light yellow solid, yield 52 %; 145 °C sublimation;
1
H NMR (DMSO-d , 600 MHz) d: 13.11 (brs, 1H, NH),
6
(
gastrocarcinoma) were obtained from American Type Cul-
8.37 (s, 1H, PzH), 8.05 (s, 1H, PzH), 7.84 (d, J = 7.8 Hz,
1
2H, Ar–H3,5), 7.69 (d, J = 7.8 Hz, 2H, Ar–H2,4). C NMR
3
ture Collection. Mps were measured on a XT-4 microscopic
1
melting-point spectrometerandare uncorrected.The H NMR
(DMSO-d , 150 MHz) d: 137.66, 137.15, 127.01, 126.11,
6
1
3
and C NMR spectra were obtained from solution in DMSO-
d6 with tetramethylsilane (TMS) as internal standard using a
Bruker AVIII 600, Bruker DMX300, and Bruker Avance
DMX400 NMR spectrometer. The IR spectra were recorded
using KBr pellets and a Perkin–Elmer Model-683 spectro-
photometer. The mass spectra were measured by LC–MS
apparatus Agilent 1200-6310. High-resolution mass spectra
126.08, 125.88, 120.31. HRMS (ESI) m/z calcd. for
?
C H F N : 213.0634 [M?H] , found: 213.0633.
8
8 3 2
4-(3,5-Bis(trifluoromethyl)phenyl)-1H-pyrazole 4b
1
Light yellow solid, yield 68 %; 150 °C sublimation; H NMR
(DMSO-d , 600 MHz) d: 13.18 (br s, 1H, NH), 8.45 (s, 2H,
6
1
3
(
HRMS) were recorded on a Bruker Apex Ultra 7.0T FTMS
PzH), 8.31 (s, 2H, Ar–H ), 7.84 (s, 1H, Ar–H ); C NMR
2,6 4
mass spectrometer. Elemental analyses were determined on
anElementarVarioELIIIelementalanalyzer. Theprogressof
reactions was monitored by thin layer chromatography (TLC)
analyses and TLC visualization was checked with UV lamp.
(DMSO-d , 150 MHz) d: 136.46, 131.47, 131.25, 125.69,
6
124.81, 123.00, 119.10, 119.02. HRMS (ESI) m/z calcd. for
?
C H F N : 281.0507 [M?H] , found: 281.0509.
1
1 7 6 2
4-(4-Nitrophenyl)-1H-pyrazole 4c
1
Synthesis
Orange solid, yield 63 %; 190 °C sublimation; H NMR
DMSO-d , 600 MHz) d: 13.24 (br s, 1H, NH), 8.45 (br s,
1H, PzH), 8.21 (d, J = 9.0 Hz, 2H, Ar–H3,5), 8.13 (br s,
(
6
General method for the preparation of pyrazole
1
3
compounds 4a–4d
1H, PzH), 7.90 (d, J = 9.0 Hz, 2H, Ar–H2,6); C NMR
DMSO-d , 150 MHz) d: 145.46, 140.64, 125.98, 124.70,
119.91. MS (ESI): 190.1 [M?H] HRMS (ESI) m/z calcd.
(
6
?
POCl (30.0 mmol) was added dropwise to 11.3 mL of dry
3
?
DMF at 5–10 °C with constant stirring. The mixture was
for C H N O : 190.0611 [M?H] , found: 190.0609.
9 8 3 2
1
23

Med Chem Res
4
-(1H-Pyrazol-4-yl)pyridine 4d
General method for the synthesis of N-arylpyrazole
compounds 5a–5d
1
Orange solid, yield 28 %. m.p. 195–198 °C. H NMR
DMSO-d , 600 MHz) d: 13.19 (br s, 1H, NH), 8.49 (dd,
J = 4.8 Hz, J = 1.2 Hz, 2H, Ar–H), 8.43 (brs, 1H, PzH),
(
To a solution of compounds 4a–4d (1.25 mmol) in
dimethylsulfoxide (DMSO) (1.5 mL) solid potassium tert-
butoxide (1.38 mmol) was added followed by the addition
of 4-fluoronitrobenzene (1.31 mmol) in DMSO (0.5 mL)
through a syringe. The resulting mixture was heated to
72 °C and kept at this temperature for 2 h. Then, the
mixture was cooled to room temperature and quenched
with water (10 mL). The precipitate was collected by fil-
tration and oven-dried in vacuo. The crude product was
recrystallized from ethanol to give N-arylpyrazole deriva-
tive 5a–5d in good to excellent yields.
6
1
2
8
2
1
.12 (brs, 1H, PzH), 7.61 (dd, J = 4.8 Hz, J = 1.2 Hz,
1 2
1
3
H, Ar–H); C NMR (DMSO-d , 150 MHz) d: 150.46,
6
?
40.76, 120.12, 119.22. MS (ESI): 146.1 [M?H] . HRMS
?
ESI) m/z calcd. for C H N : 146.0712 [M?H] , found:
(
8
8 3
1
46.0710.
1,2-Bis(3,5-dimethyl-1H-pyrazol-4-yl)benzene (6a),
1,3-bis(3,5-dimethyl-1H-pyrazol-4-yl)benzene (6b)
0
and 4,4 -(5-(octyloxy)-1,3-phenylene)bis(3,5-dimethyl-
1
H-pyrazole) (6c) were synthesized to the published
methods (Ramirez et al., 1967; Li et al., 2006)from
the corresponding aromatic dialdehyde: ortho-
phthalaldehyde, isophthalaldehyde and 5-
1-(4-Nitrophenyl)-4-(4-(trifluoromethyl)phenyl)-1H-
pyrazole 5a
1
(
octyloxy)isophthalaldehyde
Yellow solid, yield 97 %. m.p. 173–174 °C. H NMR
(
DMSO-d , 600 MHz) d: 9.41 (s, 1H, PzH), 8.51 (s, 1H,
6
1
,2-Bis(3,5-dimethyl-1H-pyrazol-4-yl)benzene 6a
PzH), 8.42 (d, J = 9.0 Hz, 2H, ArNO –H), 8.18 (d,
2
J = 9.0 Hz, 2H, ArNO –H), 7.98 (d, J = 8.2 Hz, 2H, Ar–
2
1
13
H), 7.80 (d, J = 8.2 Hz, 2H, Ar–H). C NMR (DMSO-d6,
white solid, yield 21 %. m.p.[300 °C. H NMR (d6-
DMSO, 300 MHz), d: 7.28–7.31 (m, 2H), 7.19–7.22 (m,
150 MHz) d: 145.51, 144.23, 141.27, 135.90, 127.07,
126.42, 126.34, 126.32, 125.97, 124.43, 118.99. IR (cm ):
1
3
-1
2
1
1
2
H), 1.74 (s, 12H). C NMR (DMSO-d , 150 MHz) d:
6
34.09, 131.87, 126.91, 117.57, 13.46–11.67 (br s),
3127, 1596, 1519, 1403, 1334, 1267, 1176, 1118, 1064,
954, 854, 750, 686, 597, 503. Anal. calcd. for
C H N O Fꢀ0.25H O: C, 56.89; H, 3.13; N, 12.44.
1.07–9.58 (br s). HRMS (ESI) m/z calcd. for C H N :
1
6 19 4
?
67.1604 [M?H] , found: 167.1600.
1
6
10
3
2
2
Found: C, 56.81; H, 3.11; N, 12.10. HRMS (ESI) m/z calcd.
?
for C H F N O : 334.0797 [M?H] , found: 334.0797.
1
,3-Bis(3,5-dimethyl-1H-pyrazol-4-yl)benzene 6b
1
6 11 3 3 2
1
White solid, yield 24 %. m.p. 288–289 °C. H NMR (d -
4-(3,5-Bis(trifluoromethyl)phenyl)-1-(4-nitrophenyl)-1H-
pyrazole 5b
6
DMSO, 400 MHz) d: 12.29 (br s, 2H, –NH) 7.40 (t,
J = 7.5 Hz, 1H, ArH-5), 7.14 (d, J = 7.5 Hz, 2H, ArH-
1
,6), 7.13 (s, 1H, ArH-2), 2.21 (s, 12H); C NMR (DMSO-
3
1
4
Yellow solid, yield 98 %. m.p. [250 °C. H NMR
d , 100 MHz) d: 134.55, 129.42, 128.96, 126.48, 117.30,
1
(DMSO-d , 600 MHz) d: 9.59 (s, 1H, PzH), 8.71 (s, 1H,
6
6
?
1.81–12.26 (br s); MS (MALDI-TOF): 267.1 [M?H] ;
0
0
PzH), 8.48 (s, 2H, Ar–H2 ,6 ), 8.44 (d, J = 9.0 Hz, 2H,
ArNO –H), 8.16 (d, J = 9.0 Hz, 2H, ArNO –H), 7.97 (s,
Anal. calcd. for C H N : C, 72.15; H, 6.81; N, 21.04.
1
6
18
4
2
2
1
3
Found: C, 71.71; H, 6.92; N, 21.16.
1H, Ar–H4 ). C NMR (DMSO-d , 150 MHz) d: 145.63,
0
6
1
44.13, 141.43, 134.67, 131.61, 131.40, 127.79, 126.29,
-1
0
4,4 -(5-(Octyloxy)-1,3-phenylene)bis(3,5-dimethyl-1H-
pyrazole) 6c
126.01, 123.15, 122.94, 119.00. IR (cm ): 3118, 1596,
1521, 1336, 1288, 1180, 1128, 1033, 948, 893, 854, 794,
7
50, 701, 680. Anal. calcd. for C H N O F ꢀ0.5H O: C,
1
7
9
3
2
2
2
1
White solid, yield 51 %. m.p. 167–168 °C. H NMR
CDCl , 300 MHz) d: 6.75 (s, 3 H, ArH-2,4,6), 4.01 (t,
J = 6 Hz, 2H), 2.37 (s, 12H, –CH ), 1.82–1.80 (m, 2H),
49.77; H, 2.46; N, 10.24. Found: C, 49.53; H, 2.37; N, 9.81.
(
HRMS (ESI) m/z calcd for C H F N O : 402.0671
17 10 6 3 2
?
[M?H] , found: 402.0676.
3
3
1
3
1
.48 (m, 2H), 1.29 (m, 8H), 0.88 (br s, 3H). C NMR
(
CDCl , 75 MHz): d 159.17, 141.94, 135.02, 122.77,
4-(4-Nitrophenyl)-1-(4-nitrophenyl)-1H-pyrazole 5c
3
1
1
18.40, 113.32, 68.09, 31.83, 29.41, 29.37, 29.26, 26.67,
?
4.12, 11.82. MS (MALDI-TOF): 395.1 [M?H] . Anal.
1
Red brown solid, yield 93 %. m.p. [250 °C. H NMR
calcd. for C H N O: C, 73.06; H, 8.69; N, 14.20. Found:
2
(DMSO-d , 600 MHz) d: 9.50 (s, 1H, PzH), 8.57 (s, 1H,
4
34
4
6
C, 72.87; H, 8.76; N, 13.96.
PzH), 8.44 (d, J = 9.0 Hz, 2H, Ar–H), 8.31 (d, J = 9.0 Hz,
123

Med Chem Res
2
H, Ar–H), 8.19(d, J = 9.0 Hz, 2H, Ar–H), 8.04 (d,
3
854, 752, 692, 516; Anal. calcd. for C H N O ꢀ0.25H O:
2
8
24
6
4
2
1
J = 9.0 Hz, 2H, Ar–H). C NMR (DMSO-d , 150 MHz) d:
C, 65.55; H, 4.81; N, 16.38. Found: C, 65.34; H, 4.78; N,
6
?
16.18. MS (ESI): 531.5 [M?Na] ; 547.2 [M?K] . HRMS
?
1
1
1
6
1
46.39, 145.66, 144.11, 141.53, 138.72, 127.83, 126.62,
-
1
?
(ESI) m/z calcd. for C H N O : 509.1931 [M?H] ,
found: 509.1932.
25.99, 124.83, 123.90, 119.12. IR (cm ): 3133, 1592,
513, 1405, 1344, 1268, 1211, 1108, 1031, 952, 852, 750,
84, 561. Anal. calcd. for C H N O : C, 58.07; H, 3.25; N,
2
8 25 6 4
1
5 10 4 4
8.06. Found: C, 57.93; H, 3.30; N, 17.78. HRMS (ESI) m/
?
1,3-Bis(3,5-dimethyl-1-(4-nitrophenyl)-pyrazol-4-
yl)benzene 7b
z calcd. for C H N O : 311.0774 [M?H] , found:
1
5 11 4 4
3
11.0779.
-(1-(4-Nitrophenyl)-1H-pyrazol-4-yl)pyridine 5d
1
Light yellow solid, yield 89 %. m.p. 235–236 °C. H NMR
DMSO-d , 600 MHz) d: 8.39 (d, J = 9.0 Hz, 4H, ArNO –
H), 7.91 (d, J = 9.0 Hz, 4H, ArNO –H), 7.60 (t, J =
4
(
6
2
2
1
Yellow solid, yield: 82 %. m.p. 228–229 °C. H NMR
DMSO-d , 600 MHz) d: 9.46 (s, 1H, PzH), 8.60 (d,
J = 5.1 Hz, 2H, Pyridine), 8.55 (s, 1H, PzH), 8.43(d,
7
2
6
1
1
1
1
.8 Hz, 1H, Ar–H ), 7.38 (dd, J = 7.8 Hz, J = 1.5 Hz,
5 1 2
(
6
H, Ar–H4,6), 7.36 (d, J = 1.5 Hz, 1H, Ar–H ), 2.47 (s,
2
13
H, CH ), 2.33 (s, 6H, CH ). C NMR (DMSO-d6,
3
3
J = 9.0 Hz, 2H, ArNO –H, 8.17(d, J = 9.0 Hz, 2H,
2
1
ArNO –H), 7.74 (d, J = 5.1 Hz, 2H, Pyridine). C NMR
50 MHz) d: 149.18, 145.86, 144.93, 136.61, 133.29,
30.71, 128.92, 128.20, 124.80, 123.87, 122.86, 12.81,
-1
2.60. IR (cm ): 3086, 2919, 1594, 1506, 1429, 1340,
3
2
(
DMSO-d , 150 MHz) d: 150.70, 145.61, 144.12, 141.33,
6
-
1
1
3
9
39.08, 127.68, 125.97, 123.35, 120.39, 119.10. IR (cm ):
067, 1594, 1517, 1407, 1334, 1275, 1222, 1110, 1035,
95, 961, 943, 854, 824, 748, 684, 526, 490. Anal. calcd.
281, 1110, 1018, 854, 790, 750, 690, 509, 347; Anal.
calcd. for C H N O : C, 66.13; H, 4.76; N, 16.53. Found:
8 24 6 4
2
C, 66.25; H, 4.70; N, 16.52. HRMS (ESI) m/z calcd. for
?
C H N O : 509.1931 [M?H] , found: 509.1935.
for C H N O ꢀ0.25H O: C, 62.10; H, 3.91; N, 20.69.
1
4
10
4
2
2
2
8
25 6 4
Found: C, 62.53; H, 3.72; N, 20.27. HRMS (ESI) m/z calcd.
?
for C H N O : 267.0876 [M?H] , found: 267.0871.
1
4 11 4 2
0
4,4 -(5-(Octyloxy)-1,3-phenylene)bis(3,5-dimethyl-1-(4-
nitrophenyl)-pyrazole) 7c
General method for the synthesis of N-arylpyrazole
compounds 7a–7c
1
Light yellow solid, yield 92 %. m.p. 165–166 °C. H NMR
DMSO-d , 600 MHz) d: 8.39 (d, J = 9.0 Hz, 4H,
ArNO –H), 7.90 (d, J = 9.0 Hz, 4H, ArNO –H), 6.91 (t,
(
To a solution of compounds 6a–6c (1.25 mmol) in dimeth-
ylsulfoxide (DMSO) (3.0 mL) solid potassium tert-butoxide
6
2
2
J = 1.2 Hz, 1H, Ar–H ), 6.90 (d, J = 1.2 Hz, 2H, Ar–
2
(
2.76 mmol) was added followed by the addition of
-fluoronitrobenzene (2.62 mmol) in DMSO (1.0 mL)
through a syringe. The resulting mixture was heated to
2 °C and kept at this temperature for 2 h. Then, the mixture
was cooled to room temperature and quenched with water
10 mL). The precipitate was collected by filtration and
H4,6), 4.07 (t, J = 6.8 Hz, 2H), 2.47 (s, 6H, CH ), 2.32 (s,
4
3
6
0
H, CH ), 1.76 (m, 2H), 1.45 (m, 2H), 1.25–1.37 (m, 8H),
3
1
.86 (t, J = 6.8 Hz, 3H). C NMR (DMSO-d , 150 MHz)
3
7
6
d: 159.45, 149.18, 145.85, 144.94, 136.61, 134.40, 124.79,
1
2
2
6
1
23.85, 123.16, 122.92, 114.41, 68.28, 31.81, 29.38, 29.33,
(
-
1
9.24, 26.11, 22.65, 14.08, 12.84, 12.63. IR (cm ): 3095,
919, 1594, 1506, 1428, 1340, 1193, 1110, 1018, 854, 750,
90; Anal. calcd. for C H N O : C, 67.91; H, 6.33; N,
oven-dried in vacuo. The residue was recrystallized from
ethanol to give N-arylpyrazole derivative 7a–7c.
3
6 40 6 5
3.20. Found: C, 67.90; H, 6.32; N, 13.20. HRMS (ESI)
?
1,2-Bis(3,5-dimethyl-1-(4-nitrophenyl)-pyrazol-4-
yl)benzene 7a
m/z calcd. for C H N O : 637.3132 [M?H] , found:
36 41
6 5
637.3131.
1
Light yellow solid, yield 86 %. m.p. 117–119 °C. H NMR
(
CDCl , 600 MHz) d: 8.35 (d, J = 9.0 Hz, 2H, ArNO –H),
Determination of in vitro cytotoxic activity
3
2
8
.32 (d, J = 9.0 Hz, 2H, ArNO –H), 7.67 (d, J = 9.0 Hz,
2
2
H, ArNO –H), 7.62 (d, J = 9.0 Hz, 2H, ArNO –H), 7.51
2
Cell culture
2
(
m, 2H, Ar–H), 7.40 (m, 2H, Ar–H), 2.17 (s, 3H, CH3),
2
.14 (s, 3H, CH ), 2.08 (s, 3H, CH ), 2.06 (s, 3H, CH ).
3
Four different human carcinoma cell lines: Bel-7402, KB,
HL-60, and BGC-823 were cultured in RPMI-1640
medium supplemented with 10 % fetal bovine serum, 100
units/mL of penicillin, and 100 mg/mL of streptomycin.
Cells were maintained at 37 °C in a humidified atmosphere
3
3
1
3
C NMR (DMSO-d , 150 MHz) d: 148.85, 148.83,
6
1
1
1
3
45.61, 145.56, 144.98, 144.92, 137.62, 137.48, 132.88,
32.85, 132.23, 128.22, 125.34, 125.32, 124.00, 123.95,
-
22.32, 122.28, 12.69, 12.64, 12.57, 12.51. IR (cm ):
086, 2928, 1596, 1519, 1436, 1338, 1110, 1033, 1007,
1
of 5 % CO in air.
2
1
23

Med Chem Res
Scheme 1 Reagents and
conditions: (i) DMF, POCl
R
R
R
-
3
,
i
ii
ClO4
iii
iv
9
0–95 °C; (ii) NaClO
4
;
RCH COOH
2
(
(
(
iii) NaOH, 90 °C;
iv) NH
v) p-Fluoronitrobenzene,
.
CH3 2N
O
O
N NH
2
NH
2
2
H O, r.t.;
(
)
N CH
3
( )
2
1
4
t-BuOK, DMSO, 72 °C, 2 h
2
3
5
5
a:R=
b:R=
CF
3
R
v
CF3
N
N
CF3
NO2
5
5
c:R=
d:R=
NO2
5
N
v
N
N
N
N
N
N
N
H
N
H
NO2
O N
2
6a
7a
R1
R1
7
b: R =H
1
v
N
N
N
N
N
7
c: R =OC H
8 17
1
N
N
N
H
H
NO2
O N
2
6
b, 6c
7b, 7c
Solutions
were added to the wells to achieve final concentrations.
Control wells were prepared by addition of culture med-
ium. Wells containing culture medium without cells were
used as blanks. All experiments were performed in quin-
tuplicate. The MTT assay was performed as described by
Mosmann (1983). Upon completion of the incubation for
48 h, stock MTT dye solution (20 mL, 5 mg/mL) was
added to each well. After 4-h incubation, 2-propanol
The compounds were dissolved in DMSO at a concentra-
tion of 5 mM as stock solution, and diluted in culture
medium at concentrations of 1.0, 10, 100, and 500 mM as
working-solution. To avoid DMSO toxicity, the con-
centration of DMSO was less than 0.1 % (v/v) in all
experiments.
(100 mL) was added to solubilize the MTT formazan. The
Cytotoxicity analysis
optical density (OD) of each well was measured on a
microplate spectrophotometer at a wavelength of 570 nm.
The IC50 value was determined from plot of % viability
against dose of compounds added.
The cells harvested from exponential phase were seeded
equivalently into a 96-well plate and then the compounds
123

Med Chem Res
Results and discussion
Accordingly, for mono-pyrazole derivatives 5a–5d, the
structure–activity relationships are summarized as follows:
(1) Phenyl substitution at 4-position of pyrazole ring makes
the obtained compounds more toxic than pyridyl substitu-
tion. Compound 5a–5c exhibited better activity than
compound 5d against Bel-7402, KB, HL-60, and BGC-823
cells. (2) The introduction of different electron withdraw-
ing groups on the phenyl ring could obviously affect the
cytotoxicity. Compounds 5a and 5b exhibited better
activity than compound 5c against Bel-7402 cells, but they
showed weaker activity than compound 5c against KB,
HL-60, and BGC-823 cells. For bis-pyrazole derivatives
7a–7c, the structure–activity relationships are summarized
as follows: (1) meta bis-pyrazole derivatives are more toxic
than ortho bis-pyrazole derivative. Compounds 7b and 7c
exhibited better activity than compound 7a against
Bel-7402, KB, HL-60, and BGC-823 cells. (2) For meta
bis-pyrazole derivatives, the alkoxy group on the 5-position
of phenyl ring indeed influence its cytotoxicity. Compound
7c decorated with octyloxy group on the 5-position of the
phenyl ring showed better activity than compound 7b.
Chemistry
The synthetic protocol used to synthesize the title com-
pounds is outlined in Scheme 1. The synthetic route to
compounds 4a–4d began with a Vilsmeyer–Haack-type
reaction of substituted phenylacetic acid 1a–1d with
POCl /DMF to give 2a–2d, which was isolated as its
3
perchlorate salt. Hydrolysis of 2a–2d followed by treat-
ment of the resulting dialdehyde 3a–3d (not isolated) with
hydrazine furnished the pyrazole derivative 4a–4d as light
yellow solids (Lozan et al., 2007; Barton et al., 1980).
The synthesis of N-arylpyrazole derivatives 5a–5d and 7a–
7
c was performed by the literature method (Wang et al., 2000;
Bouabdallah et al., 2007) bythe direct N-arylation of pyrazole
derivatives with 4-fluoronitrobenzene using potassium tert-
butoxide as a base in DMSO at 72 °C. Products of the reaction
have been isolated, purified, and characterized by various
1
13
C
spectral techniques such as FT-IR, HRMS, H NMR,
NMR techniques, and elemental analysis.
Cytotoxicity
Conclusions
The in vitro cytotoxic activity of compounds 5a–5d and
a–7c against Bel-7402, KB, HL-60, and BGC-823 was
A series of N-arylpyrazole derivatives was synthesized and
7
1
13
their structures were characterized by H NMR, C NMR,
FT-IR, elemental analyses, and mass spectrometry. The
cytotoxicity of the obtained compounds against four human
cancer cell lines was studied and showed a variable extent of
IC50 values. The preliminary results have shown that modi-
fication of the substituents on phenyl ring with various
electron-withdrawing substituents indeed influenced the
cytotoxicity. Compounds 5a, 5b, and 7c exhibited better
cytotoxicity than that of cisplatin against Bel-7402. It is
important to point out that meta bis-pyrazole derivatives
bearing octyloxy group on the 5-position of the phenyl ring is
the most potent compound of the series against all cell lines.
Further structure–activity studies are required to clearly
elucidate the role of the substitution on the benzene ring.
evaluated by MTT assay. The IC50 values were listed in
Table 1 and the activities of Cisplatin were as a control.
The results showed that all of the target compounds
exhibited cytotoxicity against tested carcinoma cell lines
with a lower IC50 value. The bis-pyrazole derivative 7c,
bearing alkoxy group on the 5-position of phenyl ring, was
the most effective one. It’s inhibition of cell growth of Bel-
7
402 cells was 1.5-fold higher than that found for cisplatin
under the same experimental conditions. Among mono-
pyrazole derivatives, compounds 5a and 5b also displayed
better cytotoxicity than that of cisplatin against Bel-7402.
Table 1 The cytotoxicity of the compounds (5a–5d and 7a–7c)
against Bel-7402, KB, HL-60, and BGC-823
Acknowledgments This work was supported by the National Basic
Research 973 Program (Grant No. 2010CB534913), Hebei Provincial
Natural Science Foundation of China-Shijiazhuang Pharmaceutical
Group (CSPC) Foundation (B2011201174), Hebei Province Nature
Science Fund for Distinguished Young Scholars (B2011201164), the
Nature Science Fund of Hebei Province (B2011201135), the Key Basic
Research Special Foundation of Science Technology Ministry of Hebei
Province (11966412D, 12966418D), and Open Fund of Key Laboratory
of Chemical Biology of Hebei Province (No. 09265631D-7).
Compounds
IC50 (lL)
Bel-7402
KB
HL-60
BGC-823
5
5
5
5
7
7
7
a
b
c
7.15
7.39
21.34
11.99
8.40
17.33
11.40
8.78
13.79
18.52
11.92
20.75
25.36
11.92
8.40
9.48
d
a
b
c
23.17
19.86
11.75
5.22
19.16
25.58
10.07
6.31
17.80
16.18
10.62
7.91
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