Synthesis and biological evaluation of some novel N-arylpyrazole derivatives as cytotoxic agents

Article abstract of DOI:10.1007/s11164-013-1051-1

A series of novel N-arylpyrazole derivatives, 5a-5i, were achieved from substituted phenylacetic acid via Vilsmeier-Haack reaction, hydrolysis, condensation, and aromatic substitution reaction. Their chemical structures were confirmed by ¹H NMR

Full text of DOI:10.1007/s11164-013-1051-1

Res Chem Intermed
DOI 10.1007/s11164-013-1051-1
Synthesis and biological evaluation of some novel
N-arylpyrazole derivatives as cytotoxic agents
•
Shenghui Li Shengjie Xu Shan Ding
•
•
•
Jinchao Zhang Shuxiang Wang Xiaoliu Li
•
Received: 25 August 2012 / Accepted: 4 January 2013
Ó Springer Science+Business Media Dordrecht 2013
Abstract A series of novel N-arylpyrazole derivatives, 5a–5i, were achieved from
substituted phenylacetic acid via Vilsmeier–Haack reaction, hydrolysis, condensa-
tion, and aromatic substitution reaction. Their chemical structures were confirmed
1
by H NMR, 13C NMR, FTIR, HRMS, and elemental analysis. The newly syn-
thesized compounds were tested for their in vitro cytotoxic activity against Bel-
7402, KB, HL-60, and BGC-823 cell lines and found to possess moderate activity.
Keywords Synthesis ꢀ N-arylpyrazole ꢀ Cytotoxicity ꢀ Cancer cell lines
Introduction
Heterocyclic compounds have played very important roles in the development of
medicines and pesticides in recent years and have become the new trend [1, 2].
Pyrazole derivatives in general are well-known nitrogen-containing heterocyclic
compounds, and various procedures have been developed for their synthesis [3–5].
The chemistry of pyrazole derivatives has been the subject of much research due to
their importance in various applications and their widespread potential biological
and pharmacological activities such as antimicrobial [6, 7], anti-cancer [8, 9],
anticonvulsant [10], antihistaminic [11], anti-inflammatory [12], antipyretic [13],
anti-infective [14], anti-virus [15], anti-anxiety [16], pain relief [17], etc. [18, 19].
S. Li (&) ꢀ S. Xu ꢀ S. Ding ꢀ J. Zhang (&) ꢀ S. Wang ꢀ X. Li
Chemical Biology Key Laboratory of Hebei Province, Key Laboratory of Medicinal Chemistry
and Molecular Diagnosis, Ministry of Education, College of Chemistry & Environmental Science,
Hebei University, Baoding 071002, People’s Republic of China
e-mail: lish@hbu.edu.cn
J. Zhang
e-mail: jczhang6970@yahoo.com.cn
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S. Li et al.
Scheme 1 Synthetic routine of the novel N-arylpyrazole derivatives 5a–5i. Reagents and conditions:
1 DMF, POCl3, 90–95 °C; 2 NaClO4; 3 NaOH, 90 °C; 4 NH2NH2ꢀH2O, r.t.; 5 p-Fluoronitrobenzene,
t-BuOK, DMSO, 72 °C, 2 h
Among pyrazole derivatives, N-arylpyrazole structures are often encountered in
pharmaceuticals and agrochemicals [20]. For example, N-arylpyrazoles have been
found to be a main pharmacophore in many non-steroidal anti-inflammatory drugs
such as the famous anti-inflammatory drugs Celecoxib and SC-558 [21, 22].
Recently, Melhaoui et al. synthesized two N-arylpyrazoles derivatives, 1-(4-
nitrophenyl)-3, 5-dimethylpyrazole and 1,10-bis(4-nitrophenyl)-5,50-diisopropyl-3,
30-bipyrazole, and investigated their cytotoxity against Hep cell line (human
laryngeal carcinoma). The results showed that these two compounds showed good
cytotoxic activity against the Hep cell line, with IC50: 8.25 and 10.20 mg mL-1
[23]. In this paper, in order to find better anticancer compounds, we design and
synthesize some new N-arylpyrazole derivatives by N-arylation reactions for the
first time. The cytotoxicity was tested by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-
nyltetrazolium bromide (MTT) assay. Compounds 5d and 5f exhibit the best
cytotoxicity among the nine compounds; moreover, their cytotoxicity is better than
that of cisplatin against Bel-7402 cell line (Scheme 1).
Experimental
Chemistry
RPMI-1640 medium, 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),
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Synthesis and biological evaluation
HL-60 (immature granulocyte leukemia), and BGC-823 (gastrocarcinoma) were
obtained from the American type culture collection. Mps were measured on a XT-4
microscopic melting-point spectrometer and are uncorrected. The 1H NMR and 13
C
NMR spectra were obtained from solution in DMSO-d6 with tetramethylsilane
(TMS) as internal standard using a Bruker AVIII 600 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 (HRMS) were recorded on a Bruker Apex
Ultra 7.0T FTMS mass spectrometer. Elemental analysis was determined on an
Elementar Vario EL III elemental analyzer.
General method for the preparation of pyrazole compounds 4a–4i
POCl3 (30.0 mmol) was added dropwise to 11.3 mL of dry DMF at 5–10 °C with
constant stirring. The mixture was stirred for an additional hour at room
temperature. Then, substituted phenylacetic acid (10.0 mmol) 1a–1i 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 onto crushed ice.
After decomposition of the excess Vilsmeyer reagent, a saturated solution of 4.3 g
NaClO4ꢀH2O was added with stirring. The resulting nearly white crystalline deposit
of the perchlorate salt 2a–2i was filtered and washed with two 4-mL portions of
water. This compound was used in the next step without further purification, in
which 2.422 g of the perchlorate salt 2a–2i were added to a warm solution of 1.0 g
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–3i. Then, 1.5 mL hydrazine hydrate (80 %) was added at once
with stirring, which effects dissolution of 3 after 15–20 min stirring at room
temperature. The pyrazole compounds 4a–4i deposit as thin white needles. The
mixture was left overnight and the precipitate was collected by filtration and oven-
dried in vacuum.
4-phenyl-1H-pyrazole (4a)
1
White solid; yield: 56 %; 200 °C sublimation; H NMR (DMSO-d6, 600 MHz) d:
12.95 (brs, 1H, NH), 8.05 (brs, 2H, PzH), 7.60 (d, J = 7.2 Hz, 2H, Ar–H2,6), 7.35 (t,
J = 7.2 Hz, 2H, Ar–H3,5), 7.18 (t, J = 7.2 Hz, 1H, Ar–H4); MS (ESI): 145.0
[M?H]?.
4-(4-fluorophenyl)-1H-pyrazole (4b)
1
White solid; yield: 68 %; 130 °C sublimation; H NMR (DMSO-d6, 600 MHz) d:
12.92 (brs, 1H, NH), 8.02 (brs, 2H, PzH), 7.62 (dd, J1 = 9.0 Hz, J2 = 5.4 Hz, 2H,
Ar–H2,6), 7.16 (t, J = 9.0 Hz, 2H, Ar–H3,5); MS (ESI): 163.0 [M?H]?.
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4-(4-chlorophenyl)-1H-pyrazole (4c)
Light yellow solid; yield: 64 %; 165 °C sublimation; 1H NMR (DMSO-d6,
600 MHz) d: 8.10 (s, 2H, PzH), 7.64 (d, J = 8.4 Hz, 2H, Ar–H2,6), 7.40 (d,
J = 8.4 Hz, 2H, Ar–H3,5); MS (ESI): 179.0 [M?H]?.
4-(1H-pyrazol-4-yl)phenol (4d)
1
White solid; yield: 59 %; mp: 222–224 °C; H NMR (DMSO-d6, 600 MHz) d:
12.78 (brs, 1H, NH), 9.33 (brs, 1H, OH), 7.88 (brs, 2H, PzH), 7.39(d, J = 8.4 Hz,
2H, Ar–H3,5), 6.75 (d, J = 8.4 Hz, 2H, Ar–H2,6); MS (ESI): 161.0 [M?H]?.
4-(4-toyl)-1H-pyrazole (4e)
1
Yellow solid; yield: 49 %; 160 °C sublimation; H NMR (DMSO-d6, 600 MHz) d:
12.91 (brs, 1H, NH), 8.01 (s, 2H, PzH), 7.49 (d, J = 8.4 Hz, 2H, Ar–H2,6), 7.16 (d,
J = 8.4 Hz, 2H, Ar–H3,5), 2.29 (s, 3H, CH3-H); MS (ESI): 159.1 [M?H]?.
4-(4-methoxyphenyl)-1H-pyrazole (4f)
Light yellow solid; yield: 45 %; 180 °C sublimation; 1H NMR (DMSO-d6,
600 MHz) d: 12.87 (brs, 1H, NH), 7.96 (s, 2H, PzH), 7.52 (d, J = 8.4 Hz, 2H, Ar–
H
2,6), 6.92 (d, J = 8.4 Hz, 2H, Ar–H3,5); MS (ESI): 175.1 [M?H]?.
4-(3-methoxyphenyl)-1H-pyrazole (4g)
Light yellow solid; yield: 48 %; mp: 140–141 °C; 1H NMR (DMSO-d6, 600 MHz)
d: 12.94 (brs, 1H, NH), 8.21 (s, 1H, PzH), 7.93 (s, 1H, PzH), 7.26 (t, J = 7.8 Hz,
1H, Ar–H5), 7.18 (d, J = 7.8 Hz, 1H, Ar–H4), 7.17 (s, 1H, Ar–H2), 6.76 (d,
J = 7.8 Hz, 1H, Ar–H6), 3.78 (s, 3H, –OCH3); MS (ESI): 175.1 [M?H]?.
4-(3,4-dimethoxyphenyl)-1H-pyrazole (4h)
Light yellow solid; yield: 55 %; mp: 160–161 °C; 1H NMR (DMSO-d6, 600 MHz)
d: 12.85 (brs, 1H, NH), 8.12 (s, 1H, PzH), 7.17 (d, J = 1.8 Hz, 1H, PzH), 7.12 (dd,
J1 = 8.4 Hz, J2 = 1.8 Hz,1H, Ar–H6), 6.92 (d, J = 8.4 Hz, 1H, Ar–H5), 3.81 (s,
3H, –OCH3), 3.75 (s, 3H, –OCH3); MS (ESI): 205.1 [M?H]?.
4-(3,4,5-trimethoxyphenyl)-1H-pyrazole (4i)
1
White solid; yield: 63 %; mp: 157–159 °C; H NMR (DMSO-d6, 600 MHz) d:
12.91 (brs, 1H, NH), 8.19 (s, 1H, PzH), 7.94 (s, 1H, PzH), 6.89 (s, 2H, Ar–H2,6),
3.83 (s, 6H, 3,5-OCH3), 3.65 (s, 3H, 4-OCH3); MS (ESI): 235.1 [M?H]?.
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Synthesis and biological evaluation
General method for the synthesis of N-arylpyrazole compounds 5a–5i
To a solution of compounds 4a–4i (1.25 mmol) in dimethylsulphoxide (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 filtration and oven-dried in vacuum. The
residue was recrystallizated by ethanol to give N-arylpyrazole derivative 5a–5i.
1-(4-nitrophenyl)-4-phenyl-1H-pyrazole (5a)
1
Yellow solid; yield: 91 %; mp: 197–198 °C; H NMR (DMSO-d6, 600 MHz) d:
9.22(s, 1H, PzH), 8.40(d, J = 9.0 Hz, 2H, ArNO2-H), 8.39 (s, 1H, PzH), 8.16 (d,
J = 9.0 Hz, 2H, ArNO2-H), 7.76 (d, J = 7.8 Hz, 2H, Ar–H), 7.44 (d, J = 7.8 Hz,
2H, Ar–H), 7.30 (t, J = 7.8 Hz, 1H, Ar–H); 13C NMR (DMSO-d6, 150 MHz)
d:145.24, 144.41, 141.04, 131.62, 129.41, 127.54, 125.97, 125.93, 125.91, 125.82,
118.75; IR (KBr) m: 3,081 (mC–H, Pz), 1,593 (mC=N), 1,519 (maNO2), 1,340 (msNO2
)
cm-1; Anal. calcd for C15H11N3: C 67.92 H 4.18 N 15.84; Found C 68.01 H 4.16 N
15.81; HRMS (ESI) m/z calcd for C15H12N3O2: 266.0924 [M?H]?, found:
266.0924.
4-(4-fluorophenyl)-1-(4-nitrophenyl)-1H-pyrazole (5b)
Golden yellow solid; yield: 95 %; mp: 240–241 °C; 1H NMR (DMSO-d6,
600 MHz) d: 9.22 (s, 1H, PzH), 8.41 (d, J = 9.0 Hz, 2H, ArNO2-H), 8.38 (s, 1H,
PzH), 8.15 (d, J = 9.0 Hz, 2H, ArNO2-H), 7.80 (dd, J1 = 9.0 Hz, J2 = 5.4 Hz, 2H,
ArF-H), 7.29 (t, J = 9.0 Hz, 2H, ArF-H); 13C NMR (DMSO-d6, 150 MHz)
d:162.59, 160.97, 145.26, 144.37, 141.00, 128.20 128.18, 127.91 127.86, 125.95
125.80, 124.96, 118.74, 116.34 116.20; IR (KBr) m: 3,081 (mC–H, Pz), 1,592 (mC=N),
1,502 (maNO2), 1,330 (msNO2) cm-1; Anal. calcd for C15H10N3F: C 63.60 H 3.56 N
14.83; Found C 63.35 H 3.77 N 14.74; HRMS (ESI) m/z calcd for C15H11N3O2F
284.0835 [M?H]?, found: 284.0829.
4-(4-chlorophenyl)-1-(4-nitrophenyl)-1H-pyrazole (5c)
1
Yellow solid; yield: 94 %; mp: 226–228 °C; H NMR (DMSO-d6, 600 MHz) d:
9.27 (s, 1H, PzH), 8.40 (m, 3H, PzH, ArNO2-H), 8.14 (d, J = 8.4 Hz, 2H, ArNO2-
H), 7.78 (d, J = 7.8 Hz, 2H, Ar–H), 7.50 (d, J = 7.8 Hz, 2H, Ar–H); 13C NMR
(DMSO-d6, 150 MHz) d:145.37, 144.32, 141.07, 131.93, 130.63, 129.42, 127.65,
126.22, 125.99, 124.73, 118.84; IR (KBr) m: 3,116 (mC–H, Pz), 1,596 (mC=N), 1,515
(maNO2), 1,328 (msNO2) cm-1; Anal. calcd for C15H10N3O2Cl: C 60.11 H 3.36 N
14.02; Found C 59.52 H 3.46 N 13.43; HRMS (ESI) m/z calcd for C15H11N3O2Cl
300.0534 [M?H]?, found: 300.0539.
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4-(1-(4-nitrophenyl)-1H-pyrazol-4-yl)phenol (5d)
1
Yellow solid; yield: 85 %; mp: [250 °C; H NMR (DMSO-d6, 600 MHz) d: 9.53
(brs, 1H, OH), 9.06 (s, 1H, PzH), 8.40 (d, J = 9.0 Hz, 2H, ArNO2-H), 8.14 (d,
J = 9.0 Hz, 2H, ArNO2-H), 8.26(s, 1H, PzH), 7.56 (d, J = 8.4 Hz, 2H, ArOH-H),
6.83 (d, J = 8.4 Hz, 2H, ArOH-H); 13C NMR (DMSO-d6, 150 MHz) d: 157.18,
145.01, 144.51, 140.77, 127.31, 126.21, 125.91, 124.47, 122.42, 118.50, 116.19; IR
(KBr) m: 3,350 (mO–H), 3,115 (mC–H, Pz), 1,594 (mC=N), 1,504 (maNO2), 1,334 (msNO2
)
cm-1; Anal. calcd for C15H11N3O3: C 64.05 H 3.94 N 14.94; Found C 64.26 H
3.97 N 14.90; HRMS (ESI) m/z calcd for C15H12N3O3 282.0873 [M?H]?, found:
282.0867.
1-(4-nitrophenyl)-4-p-tolyl-1H-pyrazole (5e)
1
Yellow solid; yield: 92 %; mp: 165–168 °C; H NMR (DMSO-d6, 600 MHz) d:
9.19 (s, 1H, PzH), 8.40 (d, J = 9.0 Hz, 2H, ArNO2-H), 8.35 (s, 1H, PzH), 8.16 (d,
J = 9.0 Hz, 2H, ArNO2-H), 7.65 (d, J = 7.8 Hz, 2H, Ar–H), 7.25 (d, J = 7.8 Hz,
2H, Ar–H), 2.33 (s, 3H, –CH3); 13C NMR (DMSO-d6, 150 MHz) d: 145.12, 144.42,
140.92, 136.77, 129.93, 128.73, 125.89, 125.86, 125.3, 118.63, 21.22; IR (KBr) m:
3,132 (mC–H, Pz), 2,924 (mC–H, CH3), 1,598 (mC=N), 1,513 (maNO2), 1,330 (msNO2) cm-1
;
Anal. calcd for C16H13N3O2: C 68.81 H 4.69 N 15.05; Found C 67.98 H 4.70 N
14.59; HRMS (ESI) m/z calcd for C16H14N3O2 280.1081 [M?H]?, found:
280.1082.
4-(4-methoxyphenyl)-1-(4-nitrophenyl)-1H-pyrazole (5f)
1
Yellow solid; yield: 95 %; mp: 209–211 °C; H NMR (DMSO-d6, 600 MHz) d:
9.13 (s, 1H, PzH), 8.40 (d, J = 9.0 Hz, 2H, ArNO2-H), 8.32 (s, 1H, PzH), 8.15 (d,
J = 9.0 Hz, 2H, ArNO2-H), 7.68 (d, J = 8.4 Hz, 2H, Ar–H), 7.01 (d, J = 8.4 Hz,
2H, Ar–H), 3.79 (s, 3H, –OCH3); 13C NMR (DMSO-d6, 150 MHz) d: 158.96,
145.11, 144.49, 140.89, 127.24, 125.95, 125.79, 124.93, 124.07, 118.69, 114.87,
55.63; IR (KBr) m: 3,136 (mC–H, Pz), 2,994, 2,827 (mC–H, OCH3), 1,598 (mC=N), 1,504
(maNO2), 1,332 (msNO2) cm-1; Anal. calcd for C16H13N3O3ꢀ0.5H2O: C 63.64 H
4.54 N 13.81; Found C 63.91 H 4.43 N 13.35; HRMS (ESI) m/z calcd for
C16H14N3O3 296.1030 [M?H]?, found: 296.1033.
4-(3-methoxyphenyl)-1-(4-nitrophenyl)-1H-pyrazole (5g)
1
Yellow solid; yield: 94 %; mp: 191–193 °C; H NMR (DMSO-d6, 600 MHz) d:
9.25 (s, 1H, PzH), 8.40 (m, 3H, PzH, ArNO2-H), 8.16 (d, J = 9.0 Hz, 2H, ArNO2-
H), 7.34 (m, 3H, Ar–H2 ,4 ,6 ), 6.86 (m, 1H, Ar–H5 ); 13C NMR (DMSO-d6,
150 MHz) d:160.27, 145.20, 144.37, 141.14, 132.94, 130.44, 125.99, 125.90,
125.85, 118.69, 118.29, 113.12, 111.47, 55.61; IR (KBr) m: 3,120 (mC–H, Pz), 2,972,
2,841 (mC–H, OCH3), 1,596 (mC=N), 1,510 (maNO2), 1,338 (msNO2) cm-1; Anal. calcd for
C16H13N3O3ꢀ0.5H2O: C 63.64 H 4.54 N 13.81; Found C 63.74 H 4.39 N 13.63;
HRMS (ESI) m/z calcd for C16H14N3O3 296.1030 [M?H]?, found: 296.1030.
0
0
0
0
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Synthesis and biological evaluation
4-(3,4-dimethoxyphenyl)-1-(4-nitrophenyl)-1H-pyrazole (5h)
1
Yellow solid; yield: 93 %; mp: 141–143 °C; H NMR (DMSO-d6, 600 MHz) d:
9.16 (s, 1H, PzH), 8.41(d, J = 9.0 Hz, 2H, ArNO2-H), 8.36 (s, 1H, PzH), 8.15 (d,
0
J = 9.0 Hz, 2H, ArNO2-H), 7.34 (d, J = 1.8 Hz, 1H, Ar–H2 ), 7.29 (dd,
0
0
J1 = 8.4 Hz, J2 = 1.8 Hz, 1H, Ar–H6 ), 7.01 (d, J = 8.4 Hz, 1H, Ar–H2 ), 3.86
(s, 3H, –OCH3), 3.78 (s, 3H, –OCH3); 13C NMR (DMSO-d6, 150 MHz) d:149.63,
148.59, 145.06, 144.47, 141.00, 126.10, 125.92, 125.04, 124.35, 118.53, 118.25,
112.70, 110.03, 56.12, 56.02; IR (KBr) m: 3,123 (mC–H, Pz), 2,933, 2,834 (mC–H,
OCH3), 1,596 (mC=N), 1,504 (maNO2), 1,334 (msNO2) cm-1; Anal. calcd for
C17H15N3O4: C 62.76 H 4.65 N 12.92; Found C 62.21 H 4.58 N 12.49; HRMS
(ESI) m/z calcd for C17H16N3O4 326.1135 [M?H]?, found: 326.1135.
1-(4-nitrophenyl)-4-(3,4,5-trimethoxyphenyl)-1H-pyrazole (5i)
1
Yellow solid; yield: 95 %; mp: 198–199 °C; H NMR (CDCl3, 600 MHz) d: 8.38
(d, J = 9.0 Hz, 2H, ArNO2-H), 8.23 (s, 1H, PzH), 8.04 (s, 1H, PzH), 7.96 (d,
0
J = 9.0 Hz, 2H, ArNO2-H), 7.29 (dd, J1 = 8.4 Hz, J2 = 1.8 Hz, 1H, Ar–H6 ), 7.01
0
0
0
(d, J = 8.4 Hz, 1H, Ar–H2 ,6 ), 6.77 (s, 2H, Ar–H2 ), 3.96 (s, 6H, -OCH3), 3.91 (s,
3H, -OCH3); 13C NMR (DMSO-d6, 150 MHz) d:153.84, 145.19, 144,42, 141.27,
137.22, 127.23, 126.20, 125.98, 125.74, 118.63, 103.57, 60.57, 56.52; IR (KBr) m:
3,122 (mC–H, Pz), 2,937, 2,841 (mC–H, OCH3), 1,592 (mC=N), 1,511 (maNO2), 1,332
(msNO2) cm-1; Anal. calcd for C18H17N3O5ꢀ0.5H2O: C 59.34 H 4.98 N 11.53; Found
C 59.42 H 4.61 N 11.28; HRMS (ESI) m/z calcd for C18H18N3O5 356.1241
[M?H]?, found: 356.1238.
Cell culture
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 of 5 % CO2 in air.
Solutions
The obtained N-arylpyrazole derivatives, 5a–5i, were dissolved in DMSO at a
concentration 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 concentration of DMSO was less than 0.1 % (v/v) in all experiments.
Cytotoxicity analysis
The cells harvested from exponential phase were seeded equivalently into a 96-well
plate, and then the compounds were added to the wells to achieve final
concentrations. Control wells were prepared by addition of culture medium. Wells
containing culture medium without cells were used as blanks. All experiments were
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performed in quintuplicate. The MTT assay was performed as described by
Mosmann [24]. Upon completion of the incubation for 44 h, stock MTT dye
solution (20 mL, 5 mg/mL) was added to each well. After 4 h incubation,
2-propanol (100 mL) was added to solubilize the MTT formazan. The 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.
Results and discussion
Chemistry
The synthetic route to compounds 4a–4i began with a Vilsmeyer–Haack-type
reaction of substituted phenylacetic acid 1a–1i with POCl3/DMF to give 2a–2i,
which was isolated as its perchlorate salt. Hydrolysis of 2a–2i followed by treatment
of the resulting dialdehyde 3a–3i (not isolated) with hydrazine furnished the
pyrazole derivative 4a–4i as thin white needles [25]. The synthesis of N-
arylpyrazole derivatives 5a–5i was performed using the literature method [26] by
the 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 spectral techniques such as
1
FTIR, HRMS, H NMR, 13C NMR, and elemental analysis.
Pyrazole NH proton peaks in the 1H NMR spectra of compounds 4a–4i.
1
characteristically appear around 12.78–12.95 ppm, which disappear in the H NMR
spectra of compounds 5a–5i. Pair of 1H doublets at *8.14 and *8.40 ppm strongly
suggest para-disubstituted phenyl ring in the 1H NMR spectra of compounds5a–5i. The
IR spectra of compounds 5a–5i showed characteristic absorption at 1,502–1,519 cm-1
and 1,328–1,340 cm-1, attributable to the nitro group. From above, it is evident that the
nitophenyl group has been successfully introduced into the pyrazole skeleton.
Furthermore, the HRMS of compounds 5a–5i all showed a protonated molecular ion
peak [M?H]? in the positive mode. The mass spectrum of compound 5a showed a
molecular ion peak at m/z = 266.0924, corresponding to molecular formula
C15H12N3O2. This was evidenced from HRMS analysis of the other compounds, 5b–5i.
Cytotoxic activity
The in vitro cytotoxic activity of compounds 5a–5i against Bel-7402, KB, HL-60,
and BGC-823 was evaluated by MTT assay. The IC50 values are listed in Table 1
and the activities of Cisplatin were a control. It can be seen that most of the obtained
compounds exhibited cytotoxic activity and were found to be more toxic against
Bel-7402 than KB, HL-60, and BGC-823 cells. Compound 5f, bearing the methoxy
group on the 40-position of the phenyl ring, was the most effective one. Its inhibition
of cell growth of Bel-7402 cells was 1.6-fold higher than that found for Cisplatin
under the same experimental conditions. Compound 5d, with the hydroxyl group on
the phenyl ring, was also more toxic to Bel-7402 cells than Cisplatin. The improved
123

Synthesis and biological evaluation
Table 1 The cytotoxicity of the compounds 5a–5i against Bel-7402, KB, HL-60, and BGC-823
Compounds
IC50 (lL)
Bel-7402
KB
HL-60
BGC-823
5a
11.67
22.53
10.55
7.09
12.40
26.22
12.58
9.71
15.62
23.83
28.54
12.24
26.71
15.30
13.04
44.71
22.94
2.29
10.30
20.51
15.19
12.02
15.14
16.24
26.88
22.56
21.24
6.48
5b
5c
5d
5e
9.59
11.24
6.53
5f
4.93
5g
13.19
23.81
12.24
8.12
20.62
52.83
21.54
2.65
5h
5i
Cisplatin
cytotoxic activity of 5d, 5e, and 5f hinted that electron-donating groups on the
benzene ring could enhance the cytotoxicity of N-arylpyrazole derivatives on
comparing the activities with 5b and 5c with electron-withdrawing groups. The
location of the substituent also had an important effect on cytotoxic activity. For
example, compound 5f, with the methoxy group at the 40-position of the phenyl ring,
showed stronger cytotoxic activity than compound 5g, with the methoxy group on
the 30-position.
Conclusions
A series of N-arylpyrazole derivatives were designed and synthesized, and their
cytotoxicity was evaluated in vitro. The results showed a number of the title
compounds exhibiting potent activity against four human cancer cell lines. The
compounds 5d and 5f exhibited better cytotoxicity than that of cisplatin against Bel-
7402. The results suggest that the electronic parameters, location, and number of the
methoxy group on the benzene ring had important effects on cytotoxic activity.
These results may be helpful for the design of new antitumor agents.
Acknowledgments We gratefully thank the National Basic Research 973 Program (Grant no.
2010CB534913), the Pharmaceutical Joint Research Foundation of the Natural Science Foundation of
Hebei Province and China Shiyao Pharmaceutical Group Co. Ltd. (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) and Open Fund of Laboratory of Chemical Biology of Hebei
Provincem (No. 09265631D-7).
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