Design and synthesis of some new tri-substituted pyrazole derivatives as anticancer agents

Article abstract of DOI:10.1007/s11164-016-2502-2

A new series of tri-substituted pyrazole derivatives were designed as anti-cancer agents and synthesized, starting with the formylation of semicarbazone via the Vilsmeier–Haack reaction to give 3-(4-bromophenyl)-1H-pyrazole-4-carbaldehyde I which was the

Full text of DOI:10.1007/s11164-016-2502-2

Res Chem Intermed
DOI 10.1007/s11164-016-2502-2
Design and synthesis of some new tri-substituted
pyrazole derivatives as anticancer agents
Hoda H. Fahmy1 Aladdin M. Srour1
•
•
Mohamed A. Ismail2 Mai A. Khater1
Rabah A. Serrya2 May A. El-Manawaty3
•
•
•
Received: 16 January 2016 / Accepted: 29 February 2016
Ó Springer Science+Business Media Dordrecht 2016
Abstract A new series of tri-substituted pyrazole derivatives were designed as
anti-cancer agents and synthesized, starting with the formylation of semicarbazone
via the Vilsmeier–Haack reaction to give 3-(4-bromophenyl)-1H-pyrazole-4-car-
baldehyde I which was the precursor of compounds 1–9. The new chemical entities
were screened for their anti-cancer activity on various human cancer cell lines,
namely: hepatocellular carcinoma HepG2, breast cancer MCF-7, lung carcinoma
A549 and prostatic cancer PC3. Most of the synthesized compounds showed
remarkable activity on the tested cell lines, while compound 2 had the highest
potency against the HepG2 cell line with an IC50 of 9.13 lM compared with
doxorubicin (IC50 = 34.24 lM), the reference standard used in this study, and
compound 7 was the most active on the rest of the three cell lines; MCF-7, A549
and PC3 (IC50 = 16.52, 6.52 and 9.13 lM, respectively) relative to IC50 = 20.85,
5.93 and 38.02 lM of the standard. Thus, some of the synthesized tri-substituted
pyrazole derivatives, specially 2 and 7, have the potential to be developed into
potent anticancer agents.
Electronic supplementary material The online version of this article (doi:10.1007/s11164-016-2502-2)
contains supplementary material, which is available to authorized users.
&
Aladdin M. Srour
dr_aladdinms@yahoo.com
1
2
3
Department of Therapeutical Chemistry, National Research Centre, El-Behoos Street, Dokki,
Cairo 12622, Egypt
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Cairo,
Egypt
Pharmacognosy Department, National Research Centre, El-Behoos Street, Dokki, Cairo 12622,
Egypt
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H. H. Fahmy et al.
Keywords Tri-substituted pyrazole Á Vilsmeier–Haack Á Antiproliferative Á
Anticancer
Introduction
Despite decades of research that have resulted in an enormous leap in cancer
therapy, cancer continues to be one of the major causes of death all over the world
and its incidence is rising in the developing as well as in the developed countries.
The latest WHO cancer statistics reported that 8.2 million people worldwide died
from cancer in 2012, and it is expected that annual cancer cases will rise from 14
million in 2012 to 22 million in the next two decades [1, 2]. It is worth mentioning
that 60 % of world’s total new annual cases occur in Africa, Asia and Central and
South America [3].
Cancer is a compilation of changes in genetic expression leading to uncontrolled
proliferation of somatic or germinal cells [4]. Normal cells lose control over their
regulatory functions causing abnormal growth and division. There are currently
more than 200 different types of cancer resulting from various cellular defects [5].
Meanwhile, pyrazole derivatives represent an important class of bio-active
heterocycles attracting a progressive interest of many researchers due to their widely
observed biological and pharmacological properties such as analgesic [6], anti-
oxidative [7], antihypertensive [8], antiviral [9], anti-inflammatory [10], antiprolif-
erative [11] and anticancer [12–15] activities. Ruxolitinib ‘‘(3R)-3-cyclopentyl-3-
[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)pyrazol-1-yl]propanenitrile’’ and Crizotinib
‘‘3-[(1R)-1-(2,6-dichloro-3-fluorophenyl)ethoxy]-5-(1-piperidin-4-ylpyrazol-4-yl)
pyridin-2-amine’’ are representative examples of commercially available anticancer
drugs containing pyrazole scaffolds (Fig. 1) [16, 17].
On the basis of the afore-mentioned findings, the need for new anticancer agents
is high and persistent.
F
Cl
NC
Cl
NH
N
O
N
N
N
N
H2N
N
N
NH
Ruxolitinib
Crizotinib
Fig. 1 Chemical structures of anticancer drugs containing pyrazole moiety available in the market
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Design and synthesis of some new tri-substituted pyrazole…
Thus, our current study is dealing with the rational design and synthesis of some
new tri-substituted pyrazole-containing derivatives expecting potential activity with
no or minimal side effects towards four aggressive cancer cell lines, namely:
hepatocellular carcinoma HepG2, breast cancer MCF7, lung carcinoma A549 and
prostatic cancer PC3.
Experimental
Chemistry
Melting points were recorded on a Stuart SMP30 melting point apparatus. IR spectra
(KBr) were recorded on a JASCO 6100 spectrophotometer. NMR spectra were
recorded on a JEOL AS 500 (DMSO-d6, 1H: 500 MHz, 13C: 125 MHz),
spectrometer. Chemical shifts (dH) are reported relative to TMS as the internal
standard. All coupling constant (J) values are given in Hertz. Chemical shifts (dC)
are reported relative to DMSO-d6 as internal standards. Mass spectra were recorded
on a Shimadzu GCMS-QP 1000 EX (EI, 70 eV) spectrometer. Elemental
microanalyses were recorded on a Vario El Elementar analyzer.
Synthesis of 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde (1)
A mixture of the carbaldehyde I (3 g, 11 mmol) and anhydrous potassium carbonate
(2 g, 14 mmol) was stirred in DMF (10 mL) for 2 h. Iodoethane (0.9 mL, 11 mmol)
was added dropwise, and stirring was continued overnight at room temperature,
25–30 °C. The reaction mixture was poured onto ice-cold water while stirring, the
solid form was collected, washed with water, dried and crystallized from petroleum
ether 60–80 to give 1.
Colorless crystals; yield (70 %); mp 90–92 °C; IR: mmax/cm-1: 3027 (CH,
aromatic), 2984, 2857 (CH, aliphatic), 1669 (C=O); 1H NMR: d 1.40 (t, J = 7.7 Hz,
3H), 4.21 (q, J= 7.7 Hz, 2H,), 7.63 (d, J = 8 Hz, 2H), 7.79 (d, J = 8 Hz, 2H), 8.58
(s, 1H), 9.82 (s, 1H); 13C NMR: d 15.5, 47.5, 115.9, 122.2, 129.4, 131.5, 132.3,
135.7, 150.7, 188.1; MS: m/z (%) 278 (M-1, 100); Anal. Calcd. For C12H11BrN2O
(279): C, 51.63; H, 3.97; N, 10.04. Found: C, 51.85; H, 3.74; N, 10.15 %.
Synthesis of (Z)-3-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-5-yl)-1-(4-chlorophenyl)
prop-2-en-1-one (2)
To a mixture of 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde (1) (2.8 g,
10 mmol) and 4-chloroacetophenone (0.8 mL, 10 mmol), sodium ethoxide
(10 mmol, 0.23 g Na in EtOH absolute 20 mL) was added portionwise. The
reaction mixture was stirred overnight at room temperature, 25–30 °C. The
precipitate formed was filtered, dried and crystallized from ethanol to give 2.
Yellow crystals; yield (58 %); mp 139–141 °C; IR: mmax/cm-1 3067 (CH,
1
alkene), 1658 (C=O); H NMR: d 1.43 (t, J = 7.2 Hz, 3H), 4.20 (q, J = 7.2 Hz,
2H), 7.47 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 8.6 Hz, 2H), 7.60 (d, J = 5 Hz, 1H),
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H. H. Fahmy et al.
7.62 (d, J = 5 Hz, 1H), 7.67 (d, J = 7.7 Hz, 2H), 8.05 (d, J = 8.6 Hz, 2H), 8.65 (s,
1H); 13C NMR: dd 15.5, 47.5, 115.9, 120.3, 122.2, 129.4, 130.6, 130.7, 131.5,
132.3, 135.8, 136.9, 138.4, 150.7, 188.1; MS: m/z (%) 416.09 (M ? 1, 16); Anal.
Calcd. For C20H16BrClN2O (415.7): C, 57.78; H, 3.88; N, 6.74. Found: C, 57.56; H,
3.65; N, 6.93 %.
Synthesis of 2-amino-4-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-1,6-dihydro-
6-oxopyrimidine-5-carbonitrile (3)
To a mixture of 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde (1) (2.8 g,
10 mmol) and ethyl cyanoacetate (1 mL, 10 mmol), aqueous sodium hydroxide
solution (40 %, 2 g NaOH in 5 mL H2O) was added portionwise. The reaction
mixture was stirred in ethanol (15 mL) at room temperature, 25–30 °C. After
15 min, guanidine sulfate (2.1 g, 10 mmol) was added to the reaction mixture and
refluxed for an additional 11 h. The reaction mixture was poured onto ice-cold
water while stirring, neutralized with dil. HCl, and the precipitate formed was
filtered, washed with water, dried and crystallized from ethanol to give 3.
Yellow powder; yield (72 %); mp 242–247 °C; IR: mmax/cm-1 3423, 3226 (NH2,
1
NH), 2217 (C:N), 1646 (C=O); H NMR: d 1.41 (t, J = 7.2 Hz, 3H), 4.31 (q,
J = 7.2 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.70 (d, J = 7.6 Hz, 2H), 8.74 (s, 1H),
13.69 (br. s, 1H); 13C NMR: d 15.7, 47.5, 99.8, 112.6, 117.2, 123.1, 131.0, 131.3,
132.5, 132.7, 145.8, 153.4, 164.2; MS: m/z (%) 385.22 (M ? 2, 36.3); Anal. Calcd.
For C16H13BrN6O (385.22): C, 49.89; H, 3.40; N, 21.82. Found: C, 49.64; H, 3.25;
N, 21.96 %.
General procedure for synthesis of (Z)-5-((3-(4-bromophenyl)-1-ethyl-1H-pyrazol-
4-yl)methylene)-3-aryl-2-thioxoimidazolidin-4-one (4a–c)
A mixture of 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde (1) (1.9 g,
7 mmol), glycine (0.5 g, 7 mmol) and aryl isothiocyanate (7 mmol) was refluxed in
glacial acetic acid (15 mL) for 10 h. The precipitate formed was filtered, washed
with water, dried and crystallized from n-butanol to give 4a–c.
(Z)-5-((3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)methylene)-3-phenyl-2-thioxoim-
idazolidin-4-one (4a)
Yellow crystals; yield (70 %); mp 253–255 °C; IR: mmax/cm-1 3419 (NH), 3107
1
(CH, aromatic), 1649 (C=O), 1170 (C=S); H NMR: d 1.46 (t, J = 7.2 Hz, 3H),
4.21 (q, J = 7.2 Hz, 2H), 6.43 (s, 1H), 7.33 (d, J= 6.3 Hz, 2H), 7.44 (d, J = 6.3 Hz,
2H), 7.42-7.49 (m, 5H), 8.76 (s, 1H), 12.40 (br. s, 1H); 13C NMR: d 15.5, 47.5,
104.7, 111.6, 122.0, 125.0, 129.3, 129.4, 130.8, 131.9, 132.2, 132.4, 133.8, 150.8,
163.9, 177.8; MS: m/z (%) 454.3 (M ? 1, 45); Anal. Calcd. For C21H17BrN4OS
(453.35): C, 55.64; H, 3.78; N, 12.36; S, 7.07. Found: C, 55.89; H, 3.84; N, 12.44; S,
7.19 %.
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Design and synthesis of some new tri-substituted pyrazole…
(Z)-5-((3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)methylene)-3-(4-fluorophenyl)-
2-thioxoimidazolidin-4-one (4b)
Yellow crystals; yield (59 %); mp 263–266 °C; IR: mmax/cm-1 3425 (NH), 3121
1
(CH, aromatic), 1720 (C=O), 1159 (C=S); H NMR: d 1.37 (t, J = 7.2 Hz, 3H),
4.19 (q, J = 7.2 Hz, 2H), 6.65 (s, 1H), 7.32-7.69 (m, 8H), 8.90 (s, 1H), 12.27 (br. s,
1H); 13C NMR: d 15.8, 47.5, 110.6, 112.1, 116.1, 116.3, 122.3, 126.0, 131.2, 131.6,
132.2, 132.8, 151.8, 161.0, 162.0, 163.0, 174.6; MS: m/z (%) 472.35 (M ? 1, 100);
Anal. Calcd. For C21H16BrFN4OS (471.35):C, 53.51; H, 3.42; N, 11.89; S, 6.8.
Found: C, 53.78; H, 3.29; N, 11.65; S, 7.1 %.
(Z)-5-((3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)methylene)-3-(4-methoxyphenyl)-
2-thioxoimidazolidin-4-one (4c)
Brown crystals; yield (56 %); mp 255–259 °C; IR: mmax/cm-1 3417 (NH), 3095
1
(CH, aromatic), 1716 (C=O), 1164 (C=S); H NMR: d 1.37 (t, J = 7.1 Hz, 3H),
3.77 (s, 3H), 4.20 (q, J = 6.7 Hz, 2H), 6.64 (s, 1H), 7.00 -7.70 (m, 8H), 8.90 (s,
1H), 12.20 (br. s, 1H); 13C NMR: d 15.5, 48.0, 55.9, 104.5, 110.4, 112.2, 114.4,
126.3, 130.4, 130.8, 131.2, 131.9, 132.2, 151.8, 159.7, 162.2, 164.1, 174.7; MS: m/z
(%) 484.37 (M ? 1, 100); Anal. Calcd. For C22H19BrN4O2S (483.38): C, 54.66; H,
3.96; N, 11.59; S, 6.63. Found: C, 54.47; H, 3.72; N, 11.73; S, 6.71 %.
Synthesis of 5-((3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)methylene)pyrimidine-
2,4,6(1H,3H,5H)-trione (5)
To a mixture of 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde (1) (1.9 g,
7 mmol) and barbituric acid (0.9 g, 7 mmol), few drops of piperidine were added.
The reaction mixture was refluxed in ethanol (20 mL) for 5 h. The precipitate
formed was filtered, dried and crystallized from glacial acetic acid to give 5.
Dark yellow powder; yield (77 %); mp[300 °C; IR: mmax/cm-13441 (NH), 3185
1
(CH, aromatic), 1708 (2 C=O), 1650 (C=O); H NMR: d 1.41 (t, J = 7.1 Hz, 3H),
4.30 (q, J = 5.7 Hz, 2H), 7.44 (d, J = 7.65 Hz, 2H), 7.72 (d, J = 7.65 Hz, 2H),
8.04 (s, 1H), 9.30 (s, 1H), 11.24 (d, 2H); 13C NMR: d 15.6, 47.7, 113.2, 132.2,
131.0, 131.9, 132.3, 132.6, 133.0, 144.5, 150.8, 156.5, 163.2, 164.4; MS: m/z (%)
389.2 (M, 68.6); Anal. Calcd. For C16H13BrN4O3 (389.2): C 49.38; H 3.37; N 14.40.
Found: C 49.68; H 3.11; N 14.15 %.
General procedure for synthesis of 5-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-
2H-1,2,4-triazols (6a, b)
A mixture of 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde (1) (1.9 g,
7 mmol), semicarbazides (7 mmol) and anhydrous sodium acetate (0.8 g, 10 mmol)
in ethanol (20 mL) was refluxed for 7 h. The reaction mixture was poured onto ice-
cold water, and the solid formed was collected, washed with water, dried and
crystallized from ethyl acetate affording 6a, b.
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H. H. Fahmy et al.
5-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-2H-1,2,4-triazol-3(4H)-one (6a)
Faint yellow powder; yield (90 %); mp 150–152 °C; IR: mmax/cm-1 3471, 3235 (2
NH), 3165 (CH, aromatic), 1714 (C=O); 1H NMR: d 1.38 (t, J = 7.2 Hz, 3H), 4.13
(q, J = 7.2 Hz, 2H), 7.47 (d, J = 8.6 Hz, 2H), 7.61 (d, J = 8.6 Hz, 2H), 7.84 (br. s,
1H), 8.30 (s, 1H), 9.94 (br. s, 1H). 13C NMR: d 15.7, 47.5, 115.6, 121.7, 129.9,
130.4, 132.0, 133.1, 148.1, 157.2, 170.4; MS: m/z (%) 335.28 (M ? 1, 30); Anal.
Calcd. For C13H12BrN5O (334.17): C, 46.72; H, 3.62; N, 20.96. Found: C, 46.49; H,
3.82; N, 21.14 %.
5-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-2H-1,2,4-triazole-3(4H)-thione (6b)
Faint brown crystals; yield (97 %); mp 100–102 °C; IR: mmax/cm-1 3397, 3237 (2
1
NH), 1181 (C=S); H NMR: d 1.39 (t, J = 7.2 Hz, 3H), 4.13 (q, J = 7.2 Hz, 2H),
7.49 (d, J = = 8.6 Hz, 2H), 7.62 (d, J = 8.6 Hz, 2H), 8.10 (s, 1H), 8.37 (s, 1H),
11.17 (s, 1H); 13C NMR: d 15.6, 47.5, 115.0, 121.9, 130.3, 130.4, 132.0, 132.4,
136.5, 148.9, 177.7; MS: m/z (%) 353.21 (M ? 3, 13.49); Anal. Calcd. For
C13H12BrN5S (350.24): C, 44.58; H, 3.45; N, 20.0; S, 9.16 Found: C, 44.39; H, 3.22;
N, 20.18; S, 9.22 %.
Synthesis of 4-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-6-(4-chlorophenyl)pyrim-
idin-2-amine (7)
A mixture of (E)-3-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-1-(4-chlorophe-
nyl)prop-2-en-1-one (2) (1.8 g, 43 mmol) and guanidine sulfate (0.46 g, 43 mmol)
was refluxed in ethanol (20 mL) for 2 h. then, aqueous NaOH (5 %, 6 mL) was
added portionwise, refluxing was continued for 12 h., the reaction mixture was then
poured onto ice-cold water and neutralized with dil. HCl. The solid formed was
collected by filtration, washed with water, dried and crystallized from ethanol to
give 7.
Yellow crystals; yield (52 %), mp 146–148 °C; IR: mmax/cm-1 3420, 3310 (NH2),
1
3195 (CH, aromatic); H NMR: d 1.42 (tt, J = 7.6 Hz, 3H), 4.20 (q, J = 7.6 Hz,
2H), 6.62 (s, 2H), 7.03 (s, 1H), 7.51 (d, J = 8.6 Hz, 2H), 7.55 (d, J = 8.6 Hz, 2H),
7.60 (d, J = 8.6 Hz, 2H), 7.94 (d, J = 8.6 Hz, 2H), 8.35 (s, 1H); 13C NMR: d 15.7,
47.5, 104.1, 119.1, 121.6, 128.8, 129.2, 131.3, 131.5, 132.8, 133.4, 135.6, 148.2,
161.8, 163.0, 164.3; MS: m/z (%) 454.17 (M, 100); Anal. Calcd. For C21H17BrClN5
(454.17): C, 55.46; H, 3.77; N, 15.4. Found: C, 55.64; H, 3.87; N, 15.64 %.
Synthesis of 4-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-6-(4-chlorophenyl)-
1,2-dihydro-2-oxopyridine-3-carbonitrile (8)
A mixture of (E)-3-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-1-(4-chlorophe-
nyl)prop-2-en-1-one (2) (1.2 g, 3 mmol), ethyl cyanoacetate (0.3 mL, 3 mmol) and
anhydrous ammonium acetate (1.8 g, 24 mmol) was refluxed in absolute ethanol
(15 mL) for 12 h. The reaction mixture was cooled and the precipitate formed was
filtered, dried and crystallized from glacial acetic acid to give 8.
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Design and synthesis of some new tri-substituted pyrazole…
Yellow crystals; yield (60 %); mp 345 °C; IR: mmax/cm-1 3436 (NH), 3114 (CH,
1
aromatic), 2216 (C:N), 1639 (C=O); H NMR: d 1.43 (t, J = 13.4 Hz, 3H), 4.23
(q, J = 7.65 Hz, 2H), 6.50 (s, 1H), 7.37-7.69 (m, 8H), 8.34 (s, 1H), 12.70 (br. s,
1H); 13C NMR: d 15.7, 47.5, 115.0, 121.8, 129.4, 129.9, 130.0, 132.1, 132.6, 132.7,
136.5, 148.0, 163.1, 172.9; MS: m/z (%) 480 (M ? 1, 3); Anal. Calcd. For
C23H16BrClN4O (479.76): C, 57.58; H, 3.36; N, 11.68. Found: C, 57.68; H, 3.62; N,
11.79 %.
Synthesis of 1-(5-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-3-(4-chlorophenyl)-
4,5-dihydropyrazol-1-yl)ethanone (9)
A mixture of (E)-3-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-1-(4-chlorophe-
nyl)prop-2-en-1-one (2) (2 g, 5 mmol) and hydrazine hydrate 99 % (0.4 mL,
8 mmol) was refluxed for 5 h. in glacial acetic acid (15 mL). The reaction mixture
was poured onto ice-cold water, and the solid formed was collected, washed with
water, dried and crystallized from ethanol to afford 9.
Faint pink fluffy powder; yield (84 %); mp 158–160 °C; IR: mmax/cm-1 3124
(CH, aromatic), 1659 (C=O); 1H NMR: d 1.31 (t, J = 7.2 Hz, 3H), 2.24 (t,
J = 6.5 Hz, 3H), 3.77 (q, J = 6.5 Hz, 2H), 4.03 (q, J = 7.2 Hz, 2H), 5.59-5.60 (m,
1H), 7.49 (d, J = 7.7 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H), 7.62 (d, J = 7.7 Hz, 2H),
7.72 (d, J = 8.6 Hz, 3H); 13C NMR: d 15.8, 22.3, 39.5, 43.5, 51.2, 120.8, 121.1,
128.8, 129, 129.3, 130.2, 131.9, 133.0, 134.0, 146.2, 153.6, 167.9; MS: m/z (%)
472.32 (M ? 1, 100); Anal. Calcd. For C22H20BrClN4O (471.78): C, 56.01; H,
4.27; N,11.88. Found: C, 56.22; H, 4.34; N, 11.94 %.
In-vitro antitumor screening
Compounds 1–9 were screened against human cancer cell lines for anticancer
activity using a cell-based approach [22–25]. Antitumor potency was tested on five
human tumor cell lines obtained from the National Research Centre (Cairo, Egypt),
namely: hepatocellular carcinoma HepG2, breast cancer MCF-7, lung carcinoma
A549 and prostatic cancer PC3. Cell viability was assessed by the mitochondrial-
dependent reduction of yellow MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide] to purple Formosan [26, 27].
The in vitro antitumor screening was performed by adopting previously reported
procedures [25–27]. Cells were suspended in RPMI-1640 medium for HepG2 and
DMEM for MCF-7, A549 and PC3 in addition to 1 % antibiotic–antimycotic
mixture (10,000 lg/ml potassium penicillin, 10,000 lg/ml streptomycin sulfate and
25 lg/ml amphotericin B) and 1 % L-glutamine at 37 °C, under 5 % CO2 and 95 %
humidity. Cells were seeded at a concentration of 10 9 103 cells/well in fresh
complete growth medium in 96-well microtiter plates for 24 h. Media were
aspirated, fresh medium (without serum) was added and cells were incubated with
different concentrations of the sample to give a final concentration of (100, 50, 25
and 12.5 lM). An amount of 0.5 % DMSO was used as the negative control and
doxorubicin was used as the positive control. A MTT assay was used for assessment
of cytotoxicity [25–27]. After 72 h of incubation, the medium was aspirated, 40 ll
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H. H. Fahmy et al.
MTT salt (2.5 mg/ml) were added to each well and incubated for a further 4 h. To
stop the reaction and dissolve the formed crystals, 200 ll of 10 % sodium dodecyl
sulfate (SDS) in deionized water were added to each well and incubated overnight at
37 °C. The absorbance was then measured at 595 nm and a reference wave length of
620 nm. The cell surviving fraction was calculated as follows:
Surviving fraction ¼ Optical density ðO:D:Þ of treated cells/O:D: of control cells:
The IC50 (concentration required to produce 50 % inhibition of cell growth
compared to the control experiment) was determined using Graph-Pad PRISM v.5
software. Statistical calculations for the determination of the mean and standard
error values were determined by SPSS 11 software. IC50 results are mean values of
three separate experiments using a probit analysis in the SPSS 11 program.
Results and discussion
Chemistry
The synthetic pathway employed to obtain some new tri-substituted pyrazole
derivatives was started by the formylation of semicarbazone via Vilsmeier–Haack
reaction to give 3-(4-bromophenyl)-1H-pyrazole-4-carbaldehyde I according to the
earlier reported approach [18]. The formed carbaldehyde I was treated with
iodoethane in DMF at room temperature in the presence of anhydrous potassium
carbonate to give the corresponding N–alkylated analog 3-(4-bromophenyl)-1-ethyl-
1H-pyrazole-4-carbaldehyde 1 (Scheme 1). The IR spectrum of 1 reveals the
presence of a strong stretching carbonyl vibration band at m = 1669 cm-1 while, no
1
assignable band due to NH stretching vibration was detected. H NMR reveals the
presence of a triplet signal at d 1.40 ppm (J = 7.7 Hz) and a quartet signal at d
4.21 ppm (J = 7.7 Hz) for CH3 and CH2, respectively, 13C NMR reveals the
presence of two peaks at d 15.5 and 47.5 ppm representing CH3 and CH2,
respectively, EI-MS spectrum reveals peak at m/z 278 (M-1). Compound 1, the
precursor of the target compounds 2–9 (Scheme 2), was treated via Claisen–
Schmidt condensation with 4-chloroacetophenone in the presence of sodium
ethoxide to give the corresponding chalcone (E)-3-(3-(4-bromophenyl)-1-ethyl-1H-
Br
Br
O
O
N
N
H
NH2
OHC
OHC
NH2CONHNH2.HCl
NaOAc
CH3CH2I
K2CO3
POCl3/DMF
NaOH/H2O
N
N
N
N
H
Br
Br
Ι
1
Scheme 1 Synthesis of the starting compound 3-(4-bromophenyl)-1-ethyl-1H-pyrazole-4-carbaldehyde
1
123

Design and synthesis of some new tri-substituted pyrazole…
O
O
R
HN
Ar1
N
Ar1
NH
O
N
H
O
S
Br
4
d
5
c
a, R= H
b, R=F
c, R=OCH3
OHC
N
X
HN
e
b
Ar1
CN
N
NH
N
Ar1
1
N
H2N
N
H
O
6
a, X=O
b, X=S
a
3
NH2
Ar2
O
h
f
Cl
Ar1
N
N
N
Ar1
N
Ar1
Ar2
2
7
O
g
9
Ar2
Ar1
Br
HN
CN
O
Ar1=
Cl
Ar2=
N
N
Scheme 2 Synthesis route of compounds 2–9. Reagents and conditions: (a) 4-Chloroacetophenone,
EtONa, stirring rt; (b) ethylcyano acetate, guanidine sulfate, NaOH/H2O, EtOH/reflux 11 h.; (c) glycine,
arylisothiocyanates, gl. AcOH/reflux 10 h.; (d) barbituric acid, piperidine, EtOH/ reflux 5 h.;
(e) semicarbazides, NaOAc, EtOH/reflux 7 h.; (f) guanidine sulfate, NaOH/H2O, EtOH/reflux 12 h.;
(g) ethyl cyanoacetate, amm. acetate, EtOH/reflux 12 h.; (h) hydrazine hydrate 99 %, gl. AcOH/reflux 5 h
pyrazol-4-yl)-1-(4-chlorophenyl)prop-2-en-1-one 2. The IR spectrum of 2 revealed
1
the presence of a strong stretching CH=CH vibration band at m = 3067 cm-1. H
NMR spectrum shows a triplet signal at d 1.43 ppm (J = 7.2 Hz) and a quartet
signal at d 4.20 ppm (J = 7.2 Hz) representing CH3 and CH2, respectively, as well
as a, b-unsaturated ketone’s protons, CH=CH, appeared as two doublets at
7.60 ppm (J = 5 Hz) and 7.62 ppm (J = 5 Hz). The 13C NMR spectrum revealed
the presence of peaks at d 15.5, 47.5, 122.2, 138.4 and 188.1 ppm representing CH3,
CH2, HC=CH and C=O, respectively. 2-amino-4-(3-(4-bromophenyl)-1-ethyl-1H-
pyrazol-4-yl)-1,6-dihydro-6-oxopyrimidine-5-carbonitrile 3 was obtained upon
treatment of 1 with ethyl cyanoacetate and guanidine sulfate in 40 % aqueous
123

H. H. Fahmy et al.
NaOH. In addition, compound 1 was heated under reflux in glacial acetic acid with
aryl isothiocyanate derivatives for the proper time, and afforded the corresponding
aryl thioxo imidazolidinone derivatives 4a–c. The chemical shift of the hydantoin’s
CH proton of compounds 4a–c are d 6.43, 6.65 and 6.64 ppm, respectively, which
indicate that the cis form is afforded [19–21]. Furthermore, the reaction of 1 and
barbituric acid in ethanol with catalytic amount of piperidine gave the correspond-
ing 5-((3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)methylene)pyrimidine-2,4,6(1H,
3H,5H)-trione (5). 5-(3-(4-bromophenyl)-1-ethyl-1H-pyrazol-4-yl)-2H-1,2,4-triazols
6a,b were obtained via reaction of 1 with semicarbazide derivatives and anhydrous
sodium acetate (Scheme 2). On the other hand, cyclocondensation of the chalcone 2
with guanidine sulfate in refluxing ethanol in the presence of 40 % aqueous NaOH
solution gave 4-chlorophenyl)pyrimidin-2-amine 7, while with ethyl cyanoacetate and
anhydrous ammonium acetate in refluxing ethanol or hydrazine hydrate in glacial
acetic acid afforded the corresponding 1,2-dihydro-2-oxopyridine-3-carbonitrile 8 and
4,5-dihydropyrazol-1-yl)ethanone 9 derivatives, respectively (Scheme 2) (cf. ‘‘Ex-
perimental’’ section, and Figs. 5–52 of Supplementary material).
Biological evaluations
In vitro antitumor screening
A cell-based approach was used for the in vitro antitumor screening [22–25] against
4 cancer cell lines, namely: hepatocellular carcinoma HepG2, breast cancer MCF-7,
lung carcinoma A549 and prostatic cancer PC3. For cytotoxicity assessment, MTT
assay was used [25–27], and DMSO and doxorubicin were used as negative and
positive controls, respectively. Compounds 1–9 showed very good inhibitory effects
on the four cell lines. IC50 values were calculated for compounds showing high
antiproliferative activity using Graph-Pad PRISM v.5 software and the SPSS 11
program (Table 1), and three replicates were used for each tested concentration. The
IC50 (potency) of the tested compounds were determined due to the attached dose
response curves possessing standard error values for each data point (tested
concentration) (Figs. 1–4 of Supplementary material). Most of our synthesized
compounds; 3, 4c, 5, 6b, 7, 8 and 9 showed higher activity than the standard
reference doxorubicin (IC50 = 34.24 lM) on the HepG2 cell line with IC50 of
18.70, 21.74, 11.30, 24.57, 9.78, 10.44 and 11.74 lM, respectively, with compound
2 being the most potent with an IC50 of 9.13 lM, whereas compounds 4a and 6a
showed low activity with IC50 values of 86.96 and 81.09 lM respectively. 4b is the
only inactive compound on the same cell line. As for the MCF-7 cell line,
compound 7 is the most active (IC50 = 16.52 lM) in contrast to 20.85 lM for the
standard on the same cell line. Compound 5 also showed higher activity
(IC50 = 20 lM) than doxorubicin, while compounds 2, 9, 3c and 6b had moderate
activities with IC50 values of 29.35, 52.17, 36.67 and 28.91 lM, respectively. The
rest of the compounds showed minimal or no activity on MCF-7. Concerning the
lung carcinoma cell line A549, compound 7 (IC50 = 6.52 lM) had an activity very
123

Design and synthesis of some new tri-substituted pyrazole…
Table 1 Antiproliferative
Compound
IC50 lM
activity of compounds 1–9 four
cell lines: hepatocellular
carcinoma (HepG2), breast
cancer (MCF7), lung carcinoma
(A549) and prostate cancer
(PC3) cell lines
HepG2
MCF-7
A549
PC3
1
[100
9.13
[100
29.35
[100
[100
91.78
36.67
20.00
99.11
28.91
16.52
[100
52.17
20.85
[100
21.11
[100
[100
22.17
[100
[100
[100
28.48
6.52
[100
15.22
[100
[100
[100
[100
29.78
[100
53.04
9.13
2
3
18.70
86.96
[100
21.74
11.30
81.09
24.57
9.78
4a
4b
4c
5
6a
6b
7
8
10.44
11.74
34.24
12.39
15.44
5.93
[100
82.89
38.02
IC50 concentration which gives
50 % growth inhibition; N.B.
IC50 values are the mean SD of
three separate experiments
9
Doxorubicin
close to doxorubicin (IC50 = 5.93 lM), also compounds 2, 4a, 6b, 8 and 9 had high
activity of 21.11, 22.17, 28.48, 12.39 and 15.44 lM, respectively. Compounds 1, 3,
4a, 4c, 5 and 6a had no activity on the A549 cell line. Finally, the prostatic cancer
PC3 cell line which exhibited resistance towards most of our compounds, except for
compounds 2, 5 and 7 which had more potent activity (IC50 = 15.22, 29.78 and
9.13 lM) than our standard reference (IC50 = 38.02 lM). Compounds 6b and 9 had
low activity with IC50 values of 53.04 and 82.89 lM, respectively.
Conclusion
In conclusion, a series of new tri-substituted pyrazole derivatives 1–9 were
designed, synthesized, and screened for their anticancer activity on various cancer
human cell lines, namely; hepatocellular carcinoma HepG2, breast cancer MCF-7,
lung carcinoma A549 and prostatic cancer PC3. Many of the newly synthesized
compounds showed remarkable activity on the tested cell lines, while compounds 2,
6b, 7 and 9 showed antiproliferative activity on the four cell lines. Compound 2 had
the highest activity on the HepG2 cell line (IC50 = 9.13 lM) compared to the
reference standard doxorubicin (IC50 = 34.24 lM). Compound 7 was the most
potent of the synthesized compounds, with IC50 values of 16.52, 6.52 and 9.13 lM,
(doxorubicin IC50 = 20.85, 5.93 and 38.02 lM) on MCF-7, A549 and PC3 cell
lines, respectively. Accordingly, the newly synthesized tri-substituted pyrazole
derivatives can serve as a starting point for the development of potent anti-cancer
agents.
123

H. H. Fahmy et al.
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