Novel pyrazole derivatives as anticancer and radiosensitizing agents

Article abstract of DOI:10.1055/s-0031-1297252

The present article describes the synthesis of some novel pyrrole, pyrazolo[4,3-d]oxazole, pyrrolo[2,3-b]pyridine, 1,2,3-triazole and oxoazetidin derivatives incorporating pyrazole moiety, the structures of which were confirmed by elemental analyses and s

Full text of DOI:10.1055/s-0031-1297252

Original Article 105
Novel Pyrazole Derivatives as Anticancer and
Radiosensitizing Agents
Authors
H. M. Aly1, M. G. El-Gazzar2
Affiliations
1 Department of Chemistry, Faculty of Science (Girl’s), Al-Azhar University, Nasr city, Cairo, Egypt.
2 Department of Drug Radiation Research, National Center for Radiation Research and Technology (NCRRT), Atomic Energy
Authority, Nasr City, Cairo, Egypt
Key words
Abstract
▼
γ-radiation (radiosensitizing evaluation). The
results of in-vitro anticancer evaluation showed
that compounds 3 and 16a were the most potent
compounds on HEPG2 (IC50 =2.6 and 4.2μg/ml)
and compounds 2 and 10 were the most potent
on HCT (IC50 =2.7 and 3.9μg/ml) compared to
vinblastine (IC50 =4.6 on HEPG2 and 2.6μg/ml
on HCT), while, the activity of the most potent
compounds increased after combination with
γ-radiation and they showed no toxicity on nor-
mal hepatocytes and colon cells at their effective
concentrations.
▶
● pyrazoles
▶
● anticancer
The present article describes the synthesis
of some novel pyrrole, pyrazolo[4,3-d]oxa-
zole, pyrrolo[2,3-b]pyridine, 1,2,3-triazole and
oxoazetidin derivatives incorporating pyrazole
moiety, the structures of which were confirmed
by elemental analyses and spectral data. All the
target compounds were subjected to in-vitro
antitumor activity against liver and colon human
tumor cell lines (HEPG2 and HCT), furthermore,
the most potent compounds were evaluated for
their ability to enhance the cell killing effect of
▶
● radiosensitizing
Introduction
▼
kinase activity [12,13]. Some other protein
kinases are targets of pyrazole based derivatives,
some aryl- and heteroaryl-substituted pyrazole
act as inhibitors of the transforming growth fac-
tor beta type I receptor kinase domain [14]. The
anticancer effects of some pyrazoleamide deriva-
tives are mediated by inhibition of microtubule
depolymerization [15]. Based on the above infor-
mations and due to our interest in pyrazole as a
biologically active pharmacophore [16–21], we
synthesized novel pyrazole derivatives containing
oxazole, pyridine, triazole, oxoazetidin starting
from 4-aminoantipyrine to explore their antican-
Pyrazole showed promising anticancer effects
and in the 1960s, it was evaluated in phase I
studies as an antitumor agent in man, but, even
in doses of 0.15mmol/kg/day it proved too toxic
for human use because of development of signs
of hepato-toxicity [1]. Trying to overcome this
toxicity, 1-carboxamidopyrazole and 1-thiocar-
bamoylpyrazole were synthesized and they
showed significant anticancer effects on animal
experiments, but failed to pass the clinical evalu-
ation [2,3]. In search for better antitumor treat-
ment, a large series of pyrazole derivatives were
synthesized and tested over the years, the use of
this powerful pharmacophore being very popu-
lar and modern [4–6]. In the last decade several
pyrazole derivatives proved to have potent anti-
cancer action by the inhibition of the cyclin-
dependent kinases (CDKs). CDKs are members of
the large family of protein kinases and are
responsible for the eukariotic cell cycle regula-
tion; they are intensively studied for their cancer
implication [7–11]. Based on the model of 1-car-
boxamidopyrazole, a series of pyrazole deriva-
tives containing an urea scaffold proved to have
antiproliferative effects by inhibiting Aurora
received 14.09.2011
accepted 17.10.2011
Bibliography
DOI http://dx.doi.org/
10.1055/s-0031-1297252
Published online:
January 19, 2012
Arzneimittelforschung 2012;
62: 105–112
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
▶
cer and radiosensiting activities (● Fig. 1) .
Correspondence
M. G. El-Gazzar
Materials and Methods
▼
Chemistry
Department of Drug Radiation
Research, National Center
for Radiation Research and
Technology (NCRRT)
Atomic Energy Authority
PO box 29
Nasr City
Cairo
Egypt
Melting points (°C, uncorrected) were deter-
mined in open capillaries on a Gallenkemp
melting point apparatus (Sanyo Gallenkemp,
Southborough, UK). Precoated silica gel plates
(silica gel 0.25mm, 60G F 254; Merck, Germany)
were used for thin layer chromatography, dichlo-
romethane/methanol (9.5:0.5ml) mixture was
used as a developing solvent system at room
Tel.: +20/222/749 298
Fax: +20/222/749 298
marwagalalgazzar@yahoo.com
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

106 Original Article
Fig. 1 Structures of antitumor pyrazoles.
O
N
N
N
N
O
N
H
N
H
N
O
S
NH2
1-thiocarbamoyl pyrazole
AT9283 (Aurora kinase inhibitor)
O
Cl
O
NH
HN
N
N
Cl
NH
N
H
O
H
N
N
H
PNU (CDK2/cyclin A inhibitor)
AT7519 (CDK inhibitor)
temperature and the spots were visualized by ultraviolet light
and/or iodine. Infrared spectra were recorded in KBr discs using
IR-470 Shimadzu spectrometer (Shimadzu, Tokyo, Japan). 1H
NMR spectra (in DMSO-d6) were recorded on Bruker Ac-300
ultra shield NMR spectrometer (Bruker, Flawil, Switzerland,
ppm) at 300MHz, using TMS as internal standard. Electron
impact Mass Spectra were recorded on Shimadzu GCMS-QP-
1000EX mass spectrometers at 70eV. (Shimadzu, Tokyo, Japan).
Elemental analyses were performed on Carlo Erba 1108 Elemen-
tal Analyzer (Heraeus, Hanau, Germany). All compounds were
within±0.4% of the theoretical values.
2,3,5,8-Tetramethyl-1-phenyl-1,2,3,5,8,9-
hexahydropyrazolo[4,3 : 4`,5`] imidazolo[2,1: 3`,4`]
[1,2,4]triazolo[4,3-b][1,2,4]triazole (6)
A mixture of 3 (2.47g, 0.01mol) and hydrazine hydrate (0.02mol,
80%) in acetic acid (20ml) was refluxed for 5h. The H2S liberated
during the reaction was detected on a lead acetate paper. The
reaction mixture was allowed to cool and the obtained solid was
filtered off, washed with water, dried and recrystallized from
acetic acid to give 6 as pale yellow crystals (88% yield), m.p.
138–139 °C; IR (KBr, cm−1): 3287 (NH), 3191 (CH arom.), 2954
(CH aliph.), MS (m/z): 311 (M++1, 12.66%), 55 (100% base peak),
1HNMR (DMSO-d6) δ: 1.7, 1.9, 2.0 (s, 9H, 3CH3), 2.8 (s, 3H,
N-CH3), 4.2(s, 1H, NH), 7.2–7.5 ppm (m, 8H, Ar-H). Anal. Calcu-
lated for C16H21N7: C, 61.72; H, 6.80; N, 31.49; Found: C, 61.42;
H, 6.50; N, 31.29.
4-Amino-1,5-dimethyl-2-phenyl-1,2-dihydropyrazole-3-
thione (2)
A mixture of compound 1 (2.03g, 0.01mol), and P2S5 (3g,
0.01mol) in pyridine (50ml) was heated under reflux for 10h.
The solvent was removed under reduced pressure and the
obtained residue was washed with 2N HCl, and recrystallized
from dioxane to give gray crystals 2. (69% yield), m.p. 210–
212°C; IR(KBr, cm−1): 3398, 3338 (NH2), 3155 (CH arom.), 2967
(CH aliph.), 1555 (C=S), MS(m/z): 219 (M+-1, 5.86%), 63(100%
base peak), 1HNMR (DMSO-d6) δ: 1.9 (s, 3H, CH3), 3.4 (s, 3H,
N-CH3), 7.2–7.5 (m, 5H, Ar-H), 9.0 ppm (s, 2H, NH2). Anal. Calcu-
lated for C11H13N3S: C, 60.24; H, 5.97; N, 19.16; S, 14.62; Found:
C, 60.04; H, 5.57; N, 19.06; S, 14.42.
Ethyl-2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-ylamino) acetate (7)
A mixture of 1 (2.03g, 0.01mol), anhydrous potassium carbon-
ate (2.7g, 0.02mol) and ethyl bromoacetate (1.85g, 0.015mol) in
dry acetone (50ml) was refluxed for 24h. The excess solvent was
removed and the solid product was filtered off, and recrystal-
lized from benzene to give 7 as colorless crystals; (74% yield),
m.p. 167–169°C; IR (KBr, cm−1): 3210 (NH), 3156 (CH arom.),
2950, 2848(CH aliph.), 1728, 1668 (2C=O), 1HNMR (DMSO-d6)
δ: 1.2 (s, 3H, CH3), 1.4 (t, 3H, CH2CH3), 2.4 (s, 2H, NH), 3.3 (s, 3H,
N-CH3), 4.0 (q, 2H, CH2CH3), 4.6 (s, 2H, CH2CO), 7.2–7.5 ppm (m,
5H, Ar-H). Anal. Calculated for C15H19N3O3: C, 62.27; H, 6.62; N,
14.52; Found: C, 62.07; H, 6.32; N, 14.32.
2,3-Dimethyl-1-phenyl-1,2-dihydro-4H-pyrazolo[4,3-d]
oxazole-5(6aH)-thione (3)
To a warmed ethanolic potassium hydroxide solution [prepared
by dissolving (0.40g, 10mmol) potassium hydroxide in ethanol
(50mL)] of compound 1 (2.03g, 0.01mol) excess carbon disul-
phide (10mL) was added. The mixture was heated on a water
bath at 80°C under reflux for 7h, then allowed to cool to r.t.
poured into water (100mL), and neutralized by dilute HCl; the
formed precipitate was filtered off and dried. The product was
crystallized from benzene to give white crystals 3. (70% yield),
m.p.>280°C; IR(KBr, cm−1): 3328 (NH), 3102 (CH arom.),
2917(CH aliph.), 1145 (C=S), MS (m/z): 247 (M, 3.59%), 55
2-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
ylamino)acetic acid (9)
A mixture of 1 (2.03g, 0.01mol), chloroacetatic acid (0.5g,
0.005mol) and anhydrous sodium acetate (2g, 0.02mol) in ace-
tic acid (10ml)/acetic anhydride (5ml) was refluxed for 6h. The
reaction mixture was allowed to cool and then poured onto
water, stirred for 1h and left overnight. The precipitated solid
was filtered off, dried and recrystallized from petroleum ether to
give 9 as white crystals (61% yield), m.p. 150–152 °C; IR (KBr,
cm−1): 3440, 3284 (br, OH/NH), 3025 (CH arom.), 2950, 2880
(CH aliph.), 1710, 1690 (2C=O), 1HNMR (DMSO-d6) δ: 1.8 (s, 3H,
CH3), 3.3 (s, 3H, N-CH3), 4.3(s, 2H, CH2CO), 7.2–7.7 (m, 6H,
1
(100% base peak), HNMR (DMSO-d6) δ: 1.9 (s, 3H, CH3), 3.4 (s,
3H, N-CH3), 7.3–7.5 (m, 5H, Ar-H), 9.88 ppm (s, H, NH). Anal.
calculated for C12H13N3OS: C, 58.28; H, 5.30; N, 16.99; S, 12.97;
Found: C, 58.08; H, 5.10; N, 16.69; S, 12.57.
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

Original Article 107
Ar-H+NH), 9.4ppm (s, 1H, OH). Anal. Calculated for C13H15N3O3:
C, 59.76; H, 5.79; N, 16.08; Found: C, 59.46; H, 5.69; N, 15.78.
5-Amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
pyrazol-4-yl)-1H-1,2,3-triazole-4-carbonitrile (14)
A mixture of compound 13 (2.99g, 0.01mol) and malononitrile
(1.4g, 0.02mol) was refluxed in ethanol (50mL) containing
sodium ethoxide (0.5g, 0.01mol) for 5h. The reaction mixture
was cooled and acidified with diluted HCl. The separated crys-
tals were recrystallized from petroleum ether to give brown
crystals 14: (95% yield), m.p.>280°C; IR (KBr, cm−1): 3331,
3245 (NH2), 3056 (CH arom.), 2980, 2854 (CH aliph.), 2220
(C≡N),1668 (C=O), MS(m/z): 295 (M+1, 3.82%), 261 (100% base
1,5-Dimethyl-4-(2-oxo-2-phenylethylamino)-2-phenyl-1,2-
dihydropyrazol-3-one (10)
A mixture of 1 (2.03g, 0.01mol) and phenacyl bromide (1.99g,
0.01mol) in ethanol (50mL) was refluxed for 3h. After cooling
the formed precipitate was filtered, washed with absolute etha-
nol and dried to give the final product as white crystals (80%
yield), m.p. 138–139°C; IR (KBr, cm−1): 3292 (NH), 3058 (CH
arom.), 2923(CH aliph.), 1699, 1668 (2C=O), 1HNMR (DMSO-d6) δ:
1.7 (s, 3H, CH3), 3.3 (s, 3H, N-CH3), 3.9 (s, 2H, CH2CO), 6.5 (s, 1H,
NH) 7.0–7.7ppm (m, 10H, Ar-H). Anal. Calculated for C19H19N3O2:
C, 71.01; H, 5.96; N, 13.08; Found: C, 71.31; H, 6.26; N, 13.38.
1
peak), HNMR (DMSO-d6) δ: 1.8 (s,3H, CH3), 2.8 (s, 3H, N-CH3),
6.5 (s, 2H, NH2), 7.0–7.8ppm (m, 5H, Ar-H). Anal. calculated for
C14H13N7O: C, 56.94; H, 4.44; N, 33.20; Found: C, 56.64; H, 4.14;
N, 33.00.
4,6-Diamino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-
1H-pyrazol-4-yl)-3-phenyl-1H-pyrrolo[2,3-b]pyridine-5-
carbonitrile (11)
General procedure for the synthesis of schiff base
derivatives (16a,b)
A mixture of compound 1 (2.03g, 0.01mol) and 4-nitro and/or
4-florobenzaldehyde (0.02mol) in the presence of anhydrous
K2CO3 (0.5g) was refluxed in dioxane (50mL) containing tri-
ethylamine (0.01mol) for 5h. The reaction mixture was cooled
and acidified with diluted HCl. The separated crystals were
recrystallized from ethanol to give yellow crystals 16a,b, respec-
tively.
A mixture of compound 10 (3.21g, 0.01mol) and malononitrile
(1.4g, 0.02mol) was refluxed in ethanol (50mL) containing
sodium ethoxide (0.5g, 0.01mol) was refluxed for 5h. The reac-
tion mixture was cooled and acidified with diluted HCl. The
separated crystals were recrystallized from petroleum ether to
give brown crystals 11: (85% yield), m.p. 126–127°C; IR(KBr,
cm−1): 3331, 3287 (br,2NH2), 3056 (CH arom.), 2924, 2858 (CH
aliph.), 2201 (C≡N),1668 (C=O), MS(m/z): 435 (M+, 2.36%), 77
(100% base peak), 1HNMR (DMSO-d6) δ: 1.5 (s,3H, CH3), 3.4
(s, 3H, N-CH3), 6.3, 6.5 (2s, 2H, 2NH2), 7.0–7.5ppm (m, 10H,
Ar-H). Anal. Calculated for C25H21N7O: C, 68.95; H, 4.86; N, 22.51;
Found: C, 68.65; H, 4.56; N, 22.31.
1,5-Dimethyl-4-(4-nitrobenzylideneamino)-2-phenyl-1,2-
dihydro-pyrazol-3-one (16a)
Yellow crystals: (90% yield), m.p 156–158°C; IR(KBr, cm−1):
3071(CH arom.), 2932(CH-aliphatic), 1663 (C=O), 1615 (C=N),
MS(m/z): 336 (M++1, 45.8%), 56 (100% base peak), 1HNMR
(DMSO-d6) δ: 2.4 (s,3H, CH3), 3.3 (s, 3H, N-CH3), 7.3,7.4 (2d, 9H,
Ar-H AB system), 8.3 ppm (s, 1H, N=CH). Anal. Calculated for
C18H16N4O3: C, 64.28; H, 4.79; N, 16.66; Found: C, 64.58; H, 4.99;
N, 16.86.
General procedure for diazodisation and Coupling of
antipyrine 1
To a mixture of 1 (0.5g, 0.002mol), water (7ml) and conc. HCl
(3ml), NaNO2-solution (0.20g in 1ml water, 0.002mol) was
added dropwise at 0°C. After 30min in an ice bath, the mixture
was filtered and the diazonium salt solution was added drop-
wise to cold solution of the corresponding nucleophile (sodium
azide and ethyl cyanoacetate). The reaction mixture was stirred
for 30min, extracted with Et2O, dried with Na2SO4 and evapo-
rated. The obtained products were purified by column chroma-
tography to give 13 and 15, respectively.
4-(4-Fluorobenzylideneamino)-1,5-dimethyl-2-phenyl-1,2-
dihydropyrazol-3-one (16b)
Brown crystals: (70% yield), m.p 90–92°C; IR(KBr, cm−1):
3051(CH arom.), 2927 (CH-aliphatic), 1665 (C=O), MS(m/z):
309 (M+, 4.62%), 56 (100% base peak), 1HNMR (DMSO-d6) δ: 2.4
(s,3H, CH3), 3.2 (s, 3H, N-CH3), 7.5,7.6 (2d, 9H, Ar-H AB system),
8.1ppm (s, 1H, N=CH). Anal. Calculated for C18H16FN3O: C,
69.89; H, 5.21; N, 13.58; Found: C, 69.59; H, 5.01; N, 13.38.
4-Azido-1,5-dimethyl-2-phenyl-1,2-dihydropyrazol-3-one
(13)
4-(3-Chloro-2-(4-nitrophenyl)-4-oxoazetidin-1-yl)-1,5-
dimethyl-2-phenyl-1,2-dihydropyrazol-3-one (17)
Yellow crystals: (84% yield), m.p. >280°C; IR(KBr, cm−1): 3066
(CH arom.), 2927 (CH-aliphatic), 2112 (N3), 1700 (C=O),
MS(m/z): 229 (M+-1, 1.47%), 55 (100% base peak), 1HNMR
(DMSO-d6) δ: 2.3 (s,3H, CH3), 3.2 (s, 3H, N-CH3), 7.0–7.5ppm (m,
5H, Ar-H). Anal. Calculated for C11H11N5O: C, 57.63; H, 4.84; N,
30.55; Found: C, 57.93; H, 4.94; N, 30.75.
The mixture of 1,5-dimethyl-4-(4-nitrobenzylideneamino)-2-
phenyl-1,2-dihydro pyrazol-3-one 16a (0.01mol) and chloro-
acetyl chloride (0.01mmol) was dissolved in ethanol (50ml)
containing piperidine (0.01mol). The reaction mixture was then
stirred for 3h and left at room temperature for 48h. The product
obtained was purified by column chromatography using 30%
ethyl acetate: 70% benzene as an eluent to give 2-azetidinones
17 as yellow crystals: (87% yield), m.p 160–162°C; IR(KBr,
cm−1): 3075(CH-arom.), 2928(CH-aliphatic), 1764 (C=O of
β-lactam), 1665 (C=O), MS(m/z): 412 (M+, 4.2%), 56 (100% base
Ethyl-2-cyano-2-((1,5-dimethyl-3-oxo-2-phenyl-2,3-
dihydro-1H-pyrazol-4-yl)diazenyl)acetate (15)
Brown crystals: (88% yield), m.p. 162–164 °C; IR(KBr, cm−1):
3207 (NH), 3180 (CH arom.), 2931(CH aliph.), 2221
(C≡N),1774,1681 (2C=O), 1HNMR (DMSO-d6) δ: 1.4 (t, 3H,
CH2CH3),1.9 (s,3H, CH3), 2.2 (s, 3H, N-CH3), 4.0 (q, 2H, CH2CH3),
6.4(s, H, NH), 7.0–7.8ppm (m, 5H, Ar-H). Anal. Calculated for
C16H17N5O3: C, 58.71; H, 5.23; N, 21.39; Found: C, 58.51; H, 5.03;
N, 21.19.
1
peak), HNMR (DMSO-d6) δ: 2.3 (s,3H, CH3), 3.3(s, 3H, N-CH3),
5.3 (s, 1H, N-CH), 5.3(s,1H, CH–Cl), 7.9,8.0 ppm (2d, 9H, Ar-H AB
system) Anal. Calculated for C20H17ClN4O4: C, 58.19; H, 4.15; N,
13.57; Found: C, 58.39; H, 4.45; N, 13.77.
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

108 Original Article
4-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
ylamino)-4-oxobutanoic acid (18)
with 5% CO2 for a period of 48h. 3 wells were used for each con-
centration of the test sample. Control cells were incubated with-
out test sample and with or without DMSO. The little percentage
of DMSO present in the wells (maximal 0.1%) has no effect on
the cell line. After incubation of the cells for 24h at 37°C, various
concentrations of sample (50, 25, 12.5, 6.25, 3.125 & 1.56μg)
were added, and the incubation was continued for 48h and via-
ble cells yield was determined by a colorimetric method. In brief,
after the end of the incubation period, media were aspirated and
the crystal violet solution (1%) was added to each well for at
least 30min. The stain was removed and the plates were rinsed
using tap water until all excess stain is removed. Glacial acetic
acid (30%) was then added to all wells and mixed thoroughly,
and then the absorbance of the plates were measured after gen-
tly shaken on Microplate reader (TECAN, Inc.), using a test wave-
length of 490nm. All results were corrected for background
absorbance detected in wells without added stain. Treated sam-
ples were compared with the cell control in the absence of the
tested compounds. All experiments were carried out in tripli-
cate. The relation between surviving fraction and drug concen-
tration was plotted to get the survival curve of each tumor cell
line after the specified time. The concentration required for 50%
inhibition of cell viability (IC50) was calculated and compared
with the reference drug vinblastine and the results are given
A mixture of compound 1 (2.03g, 0.01mol) and succinic anhy-
dride (1g, 0.01mol) was refluxed in ethanol (50mL) for 5h, the
solid obtained was poured onto cold water and acidified with
diluted HCl. The separated crystals were recrystallized from
dioxane to give white crystals 18: (66% yield), m.p. 184–186°C;
IR (KBr, cm−1): 3410, 3352 (br, OH/NH), 3062 (CH arom.), 2936
1
(CH aliph.), 1764,1741,1664 (3C=O), HNMR (DMSO-d6) δ: 1.8
(s,3H, CH3), 2.4, 3.3 (2t, 4H, 2CH2), 3.1 (s, 3H, N-CH3), 7.3–7.5ppm
(m, 5H, Ar-H), 9.1(s,1H,NH), 12.4(s,1H,OH). Anal. Calculated for
C15H17N3O4: C, 59.40; H, 5.65; N, 13.85; Found: C, 59.70; H, 5.85;
N, 13.95.
1-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-
yl)-3-methylenepyrrolidine-2,5-dione(19)
A mixture of compound 1(2.03g, 0.01mol) and 3-methylene-
dihydrofuran-2,5-dione (1.12g, 0.01mmol) was fused in an oil
bath at 250°C for 15min, the product was triturated in dimethyl
formamide and poured onto cold water, the solid obtained was
recrystallized from ethanol to give black crystals 19: (74% yield),
m.p. 162–164°C; IR (KBr, cm−1): 3144 (CH arom.), 2951 (CH
aliph.), 1744,1706,1669 (3C=O), MS(m/z): 297 (M+1, 1.66%),
1
50 (100% base peak), HNMR (DMSO-d6) δ: 2.3 (s,3H, CH3), 2.7
▶
(s, 2H, CO-CH2-C), 3.3 (s, 3H, N-CH3), 5.5(d,2H,CH2 =C), 7.3–
7.8ppm (m, 5H, Ar-H). Anal. Calculated for C16H15N3O3: C, 64.64;
H, 5.09; N, 14.13; Found: C, 64.34; H, 2.9; N, 14.03.
in● Table 1.
Radiosensitizing evaluation
The most potent compounds resulted from the previous experi-
ment; compounds 3, 16a, 2 and 10, were selected to be evalu-
ated again for their in vitro anticancer activity alone and in
combination with γ-radiation. This study was conducted to eval-
uate the ability of these compounds to enhance the cell killing
effect of γ-radiation and to prove the efficacy of combining radi-
otherapy with chemotherapy to reduce the dosage and toxicity
of both. Cells were subjected to a single dose of γ-radiation at a
dose level of 8Gy with a dose rate of 2 Gy/min. Irradition was
performed in the National Cancer Institute, Cairo University,
using Gamma cell-40 (60CO) source. The surviving fractions
were expressed as means±standard error and were analysed
In-vitro anticancer evaluation
Materials and methods
The human tumor cell lines (HEPG2 and HCT) were available at
the regional center for mycology & biotechnology at Al-Azhar
University, Cairo, Egypt. Mammalian cell lines: HEPG2 and HCT
cells (human cell line of a well differentiated hepatocellular and
colon carcinoma isolated from a liver colon biopsy of a male
Caucasian aged 15 years) were obtained from the American Type
Culture Collection (ATCC). Chemicals Used: Dimethyl sulfoxide
(DMSO), crystal violet and trypan blue dye were purchased from
Sigma (St. Louis, Mo., USA). Fetal Bovine serum, DMEM, RPMI-
1640, HEPES buffer solution, L-glutamine, gentamycin and
0.25% Trypsin-EDTA were purchased from Lonza. Crystal violet
stain (1%): It composed of 0.5% (w/v) crystal violet and 50%
methanol then made up to volume with ddH2O and filtered
through a Whatmann No.1 filter paper.
Table 1 In-vitro anticancer evaluation against human tumor liver and colon
cell lines (HEPG2 and HCT).
IC50 a,b (μg/ml)
Cpd. No.
HEPG2
HCT
Procedure
2
8.8
2.6
2.7
9.4
The cells were propagated in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat-inactivated fetal
bovine serum, 1% L-glutamine, HEPES buffer and 50μg/ml gen-
tamycin. All cells were maintained at 37°C in a humidified
atmosphere with 5% CO2 and were subcultured 2 times a week.
Cell toxicity was monitored by determining the effect of the test
samples on cell morphology and cell viability.
Cytotoxicity evaluation using viability assay [22, 23]: For cyto-
toxicity assay, the cells were seeded in 96-well plate at a cell con-
centration of 1×104 cells per well in 100μl of growth medium.
Fresh medium containing different concentrations of the test
sample was added after 24h of seeding. Serial 2-fold dilutions of
the tested chemical compound were added to confluent cell
monolayers dispensed into 96-well, flat-bottomed microtiter
plates (Falcon, NJ, USA) using a multichannel pipette. The micro-
titer plates were incubated at 37°C in a humidified incubator
3
6
12.6
36.4
20.6
12.5
43.8
9.8
8.2
8
24.8
6.4
9
10
3.9
11
7.4
13
9.5
14
23.5
30.2
4.2
12.6
25.9
9.4
15
16a
16b
16.8
13.2
32.4
22.9
4.6
9.6
17
13.8
24.6
19.6
2.6
18
19
Vinblastine
a IC50, compound concentration required to inhibit tumor cell proliferation by 50%
b Values are means of 3 experiments
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

Original Article 109
Table 2 In-vitro anticancer evaluation of compounds 3, 16a against human liver cell line (HEPG2) and compounds 2, 10 against human colon cell line (HCT)
after subjection to radiation.
Cpd.No
Cell line
Irradiation (8Gy)
Cpd.Concentration (μg/ml)+ irradiation(8Gy)
IC50(μg/ml)
IC50(μg/ml)
(normal cells)
0.5
1
1.5
3
Survival fractionb ±standard error
3
16a
2
HEPG2
HCT
0.927±0.02*
0.927±0.02*
0.927±0.02*
0.927±0.02*
0.126±0.01*
0.097±0.01*
0.134±0.02
0.099±0.04*
0.092±0.01*
0.076±0.03*
0.096±0.03*
0.081±0.01*
0.084±0.02*
0.055±0.04*
0.077±0.05*
0.067±0.01*
0.052±0.03*
0.029±0.03*
0.058±0.02*
0.036±0.01*
0.12
0.13
0.14
0.12
47.7
NAc
35.9
NAc
10
a Each value is the mean of 3 experiments±standard error
b Each value is the mean of 3 experiments
c No activity
*Significant difference from control group at p<0.05
Fig. 2 Synthetic pathways for compounds 2, 3
and 6.
N
N
P2S5
Ph
NH2
N
S
2
N
Ph
NH2
O
1
N
CS2
N
Ph
NH
S
O
3
N2H4.H2O
N
N
N
N
N
N
Ph
Ph
Ph
NH
S
N
N
N
N
N
O
N
N
H
N
H2N
HN
4
5
6
▶
using 1-way ANOVA test. The results are given in ● Table 2. For
better predicting the selectivity of these compounds to cancer
cells rather than normal cell, the 4 compounds were tested on
normal hepatocytes (cpd. 3 and 16a) and colon cells (cpd. 2 and
agents. Thus, when 4-aminoantipyrine 1 heating with carbon
disulphide in ethanolic potassium hydroxide gave pyrazolo[4,3-
d]oxazole derivative 3. Moreover, on reacting pyrazolo[4,3-d]
oxazole derivative 3 with hydrazine hydrate in boiling ethanol
was recovered unchanged. When the reaction was repeated in
boiling acetic acid yielded the corresponding pyrazole derivative
6 and the other expected pyrrolo[3,2-c]pyrazoluredo derivative
4 and 5 were ruled out on the basis of analytical and spectral
data. Structure of compound 6 was confirmed on the basis of
▶
10) and their IC50 was calculated and is shown in● Table 2.
Results and Discussion
▼
Chemistry
▶
elemental analysis and spectral data (● Fig. 2) .
The synthetic procedures adopted to obtain the target new com-
pounds are showed in Schemes 1–4. The 4-amino-1,5-dimethyl-
2-phenyl-1,2-dihydropyrazol-3-one 1 was obtained from Sigma
Company and used without further purification. 4-aminophena-
zone 1 was reacted successfully with phosphorus pentasulfide
to afford the 4-amino-1,5-dimethyl-2-phenyl-1,2-dihydro-
pyrazole-3-thione 2. (Scheme 1). On the other hand,
pyrazolo[4,3–d]oxazole derivative 3 was prepared by treatment
of compound 1 with bifunctional one carbon donor cyclizing
The reactivity of amino group in the 4-position for antipyrine 1
towards halogenated compounds have been investigated. Thus,
the treatment of antipyrine 1 with ethyl bromoacetate affords
the N-alkylated product 7 and attempts to cyclizing the pyrazol-
4-yl-aminoacetate derivative 7 by refluxing in ethanol containing
piperidine and/ or pyridine to afford 2,3-dimethyl-1-phenyl-
1,2-dihydropyrazolo[3,4-b] [1,4]oxazin-6-one 8 was unsuccess-
ful on the basis of analytical and spectral data. The structure of
compound 7 was confirmed by their IR, 1HNMR and Mass spec-
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

110 Original Article
O
O
Br
N
N
Br
N
N
O
Ph
Ph
O
Ph
N
H
1
N
Ph
H
O
O
O
O
10
O
7
Cl
CN
OH
–EtOH
CN
N
OH
N
NH2
N
N
N
Ph
N
N
H
O
CN
Ph
O
N
O
N
Ph
9
O
O
Ph
8
CN
CN
NH2
Ph
CN
N
N
N
Ph
N
NH2
O
11
Fig. 3 Synthetic pathways for compounds 7, 9–11.
tra. Furthermore, treatment of 1 with chloroacetic acid in the
presence acetic acid/acetic anhydride and anhydrous sodium
acetate yielded pyrazol-4-ylaminoacetic acid derivative 9, which
showed evolution of CO2 gas with aqueous sodium carbonate
solution indicating the presence of carboxylic group. In a similar
manner, the reaction of antipyrine 1 with equimolar amount of
phenacyl bromide in refluxing ethanol afforded the correspond-
ing phenylethylamine derivative 10. Both of compounds 9 and
10 were elucidated by analytical and spectral data. Compound
10 was allowed to react with malononitrile in sodium ethoxide
as a catalyst yielded the corresponding pyrrolo[2,3-b]pyridine
2-(4-nitrophenyl)-4-oxoazetidin-1-yl)-1,5-dimethyl-2-phenyl-
▶
1,2-dihydropyrazol-3-one 17 ( ● Fig. 4). The structure of
compound 17 was confirmed by IR, 1H NMR, MS spectra, and
elemental analysis. To explain the outcome of this reaction, we
postulated the reaction mechanism of azetidinone formation
from chloroacetyl chloride and imines. We concluded that the
atom carrying a free pair of electrons would form a loose bond
with carbon and generate a 5 membered transition state (I). This
would ease the formation of azetidinone (II) by bringing the
appropriate carbon atoms C-3 and C-4 in (II) close enough to
▶
form a bond (● Fig. 5) [27,28]. Finally, the behavior of ami-
▶
derivative 11 (● Fig. 3) .
noantipyrine towards the anhydride derivatives was also inves-
tigated. Thus, the reaction of antipyrine 1 with succinic
anhydride in ethanol yielded the corresponding pyrazole deriva-
tive 18. While, treatment of antipyrine 1 with 3-methylene-
dihydrofuran-2,5-dione under condition of fusion, gave the
corresponding 1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-
Diazodization of 4-aminoantipyrine 1 with sodium nitrite and
hydrochloric acid at 5–10°C followed by coupling with the cor-
responding nucleophile (such as sodium azide and ethyl cyanoa-
cetate) furnished novel 4-azidopyrazole 13 [24] and hydrazone
derivatives 15, respectively. Interaction of compound 13 with
malononitrile in sodium ethoxide as a catalyst yielded the cor-
▶
pyrazol-4-yl)-3-methylenepyrrolidine-2,5-dione 19 (● Fig. 4) .
▶
responding 1,2,3-triazole derivative 14 [25] (● Fig. 4) .
S c h i ff base derivatives were reported to possess significant bio-
logical activities and new series have been tested for their anti-
tumor, antimicrobial, and antiviral activities [26]. In the light of
these facts, we report the synthesis of 1,5-dimethyl-4-(4-substi-
tuted-benzylideneamino)-2-phenyl-1,2-dihydropyrazol-3-one
16a,b by the condensation reaction of compound 1 with 4-nitro
and/or 4-florobenzaldehyde in presence of anhydrous K2CO3.
The azomethine group in 16a underwent cycloaddition with
chloroacetyl chloride in dimethylformamide afforded 4-(3-chloro-
In-vitro anticancer evaluation
The synthesized compounds were evaluated for their in vitro
cytotoxicity against human liver and colon cancer cell lines
(HEPG2 and HCT), vinblastine, which is one of the most potent
anticancer agents, is the reference drug used in this study. From
▶
the results obtained in ● Table 1, concerning HEPG2, SAR of the
tested compounds showed that the pyrazolooxazole 3 was the
most active compound (IC50 =2.6μg/ml) and was found to be
more active than the reference drug (IC50 =4.6μg/ml), while, its
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

Original Article 111
Fig. 4 Synthetic pathways for compounds 12–19.
O
N
N
O
N
N
CH2
OH
N
Ph
Ph
N
H
O
O
O
CH2
O
O
O
18
19
O
O
O
O
1
N
N
Ph
N
N2 Cl
N
CH–Ar
N
Ph
O
12
O
16a,b
16a, Ar = 4-NO2-C6H4
16b, Ar = 4-F-C6H4
CN
COOC2H5
NaN3
Cl
N
N
N
Cl
COOC2H5
CN
16a
N
N3
Ph
O
CH
N
N
Ph
O
N
N
O
N
H
13
NO2
Ph
N
C
CN
CN
O
COOC2H5
Cl
O
N
Ph
N N
C
H
17
CN
O
N
15
N
N
Ph
N
N
O
H2N
14
CN
double cyclization to the 4-ringed 6 yielded in a decrease in
activity, the thione 2 and azide 13 derivatives showed moderate
activity (IC50 =8.8 and 9.8μg/ml), while, cyclization of the azide
to the corresponding triazole derivative 14 resulted in a decrease
in the activity. The Schiff base 16a showed high activity similar
to that of the reference drug (IC50 =4.2μg/ml), while, a decrease
in activity was observed upon cyclization to the corresponding
lactam 17. Compound 10 showed moderate activity
(IC50 =12.6μg/ml), while, its cyclization to give 11 yielded in a
drop in activity. Compounds 5, 8, 9 18 and 19 were the least
potent compounds on HEPG2 with their IC50 ranging from 20.6–
36.4μg/ml concerning HCT cell line, most of the compounds
showed better activity than on HEPG2, the thione 2 was the
most active (IC50 =2.7μg/ml) then comes compounds 10 and 9
(IC50 =3.9 and 6.4μg/ml). On the contrary to their results on
HEPG2, compound 6 showed higher activity (IC50 =8.2μg/ml)
than its starting material 3. On the other hand, the azide 13 and
the Schiff bases 16a,b showed moderate activities (IC50 =9.5, 9.4,
9.6μg/ml, respectively) and they were found to be more active
than the triazole 14 and the lactam derivative 17. Finally, the
least potent compounds on HCT were compounds 5, 8, 18 and 19
with their IC50 ranging from 19.6–25.9μg/ml.
CH2Cl
COCl
CH2
C
HC
N
Cl
CH
Ar
Ar
N
N
Cl
Cl
N
O
N
N
O
O
Ph
Ph
Cl
Cl
-
CH
Ar
CH Ar
HC
C
H2C
Et3N
N
N
N
Ph
N
N
N
C
O
O
O
(I)
O
Ph
Cl
HC
H
C
H
C
Cl
Cl
Ar
O
Ar
CH2
C
N
N
C
N
N
O
O
N
N
O
Ph
Ph
(II)
Ar= 4–NO2–C6H4 , 4–F–C6H4
Radiosensitizing evaluation
The rationale for combining chemotherapy and radiotherapy is
based mainly on 2 ideas, one being spatial cooperation, which is
effective if chemotherapy is sufficiently active to eradicate sub-
clinical metastases and if the primary local tumor is effectively
treated by radiotherapy. In this regard, no interaction between
radiotherapy and chemotherapy is required. The other idea is
the enhancement of radiation effects. Cytotoxic agents can
enhance radiation effects by direct enhancement of the initial
radiation damage by incorporating drugs into DNA, inhibiting
Fig. 5 Postulated mechanism for the formation of compound 17.
cellular repair, accumulating cells in a radiosensitive phase or
eliminating radioresistant phase cells, eliminating hypoxic cells
or inhibiting the accelerated repopulation of tumor cells [29,30].
Consequently, the ability of the most active 4 compounds, com-
pounds 3, 16a, 2 and 10, to enhance the cell killing effect
of γ-irradiation was studied. From the results obtained in
Aly HM, El-Gazzar MG. Novel Pyrazole Derivatives as… Arzneimittelforschung 2012; 62: 105–112

112 Original Article
10 Squires MS, Feltell RE, Wallis NG et al. Biological characterization of
AT7519, a small-molecule inhibitor of cyclin-dependent kinases, in
human tumor cell lines. Mol Cancer Ther 2009; 8: 324–332
11 Moravcová D, Kry-stof V, Havlícek L et al. Pyrazolo[4,3-d]pyrimidines
as new generation of cyclin-dependent kinase inhibitors. Bioorg Med
Chem Lett 2003; 13: 2989–2992
12 Regan J, Breitfelder S, Cirillo P et al. Pyrazole urea-based inhibitors of
p38 MAP kinase: from lead compound to clinical candidate. J Med
Chem 2002; 45: 2994–3008
13 Howard S, Berdini V, Boulstridge JA et al. Fragment-based discovery
of the pyrazol-4-yl urea (AT9283), a multitargeted kinase inhibitor
with potent aurora kinase activity. J Med Chem 2009; 52: 379–388
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▶
● Table 2, we can observe that their IC50 synergistically
decreased after irradiation. Concerning HEPG2, compounds
3 and 16a showed IC50 of 2.6 and 4.2μg/ml, while after irradia-
tion decreased to be 0.12 and 0.13, respectively, similarly, on
HCT cell line, compounds 2 and 10 their IC50 decreased from 2.7
and 3.9μg/ml to become 0.14 and 0.12μg/ml after irradiation.
On the other hand, concerning the test of toxicity on normal
cells, compound 3 showed IC50 =47.7μg/ml on normal hepato-
cytes, while, compounds 16a showed no activity on the same
cells. Compound 2 showed IC50 =35.9μg/ml on normal colon
cells, while, compounds 10 showed no activity on the same cells
which indicates their selectivity on cancer cells rather than nor-
mal cells when given at their effective dosages.
16 Ghorab MM, Ragab FA, Heiba HI et al. Synthesis of some new pyrazole
and pyrimidine derivatives carrying a sulfonamide moiety of expected
antitumor activity and study of the synergistic effect of gamma-irra-
diation. Arzneimittelforschung 2010; 60 (1): 48–55
17 Ghorab MM, Heiba HI, Hassan AA et al. Antimicrobial evaluation
of novel pyrrole, pyrazole, pyrimidine and pyrrolo[2,3-d]pyrimi-
dine derivatives bearing sulfonamide moiety. J Am Sci 2011; 7
(1): 1063–1073
18 Ghorab MM, Ragab FA, Heiba HI et al. Synthesis of Novel Pyrazole and
Pyrimidine derivatives bearing Sulfonamide moiety as antitumor and
radiosensitizing agents. Med Chem Res doi:10.1007/s00044-011-9653-x
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and thienopyrimidine derivatives. Phosphorous Sulfur and Silicon
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Conclusion
▼
From the above results, we can conclude that administration of
the tested compounds on human liver and colon (HEPG2 and
HCT) cell lines showed promising cytotoxic activity especially on
colon cell line, while, combining the most potent compounds
with radiation at lower concentrations enhances their activity
which demonstrates the importance of the combination therapy
for the patients with cancer to decrease the side effects of both
drugs and radiation. On the other hand, the test of toxicity
showed that the tested compounds were safe on normal hepato-
cytes and colon cells which indicate their selectivity to cancer
cells.
21 Aly HM, Saleh NM , Elhady HA, Design and Synthesis of Some New
Thiophene, Thienopyrimidine and Thienothiadiazine Derivatives of
Antipyrine as Potential Antimicrobial Agents, Eur J Med Chem 2011;
46: 4566
22 Mosmann T. Rapid colorimetric assay for cellular growth and sur-
vival: application to proliferation and cytotoxicity assays. J Immunol
Methods 1983; 65: 55–63
Conflict of Interest
▼
The authors declare that they have no conflict of interest with
respect to this paper.
23 Vijayan P, Raghu C, Ashok G et al. Antiviral activity of medicinal plants
of Nilgiris. Indian J Med Res 2004; 120: 24–29
24 Dong HS, Wang B. The synthesis and crystal structure of new
3,6-bis[1-(4-ethoxyphenyl)-5-methyl-1,2,3- triazol-4-yl]-s-triazolo[3,4-
b]-1,3,4-thiadiazole. J Chem Cryst 2005; 35 (1): 61–65
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