Some pyrazole and pyrazolo[3,4-d]pyrimidine derivatives: Synthesis and anticancer evaluation

Article abstract of DOI:10.1002/ardp.201400064

5-Amino-1-p-tolyl-1H-pyrazole-4-carbonitrile (1) was used for the preparation of some novel pyrazoles and pyrazolo[3,4-d]pyrimidines 2-10. Moreover, the cytotoxicity and in vitro anticancer activities of the prepared compounds were also assessed against the MCF-7 breast cancer, HepG2 liver cancer, and A549 lung carcinoma cell lines, along with investigation of the effect of the synthesized compounds on the expression of urokinase plasminogen activator (uPA). The tested compounds exhibited remarkable cytotoxic activity against MCF-7 and HepG2 cells. Among the tested compounds, 2 and 9 revealed promising anticancer activity compared to the activity of the commonly used anticancer drug, doxorubicin, by inhibiting the expression of uPA.

Full text of DOI:10.1002/ardp.201400064

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
1
Full Paper
Some Pyrazole and Pyrazolo[3,4-d]pyrimidine Derivatives:
Synthesis and Anticancer Evaluation
Ahmed H. Shamroukh1,2, Aymn E. Rashad1,3, Randa E. Abdel-Megeid1,4, Hatem S. Ali5,
and Mamdouh M. Ali6
1
Photochemistry Department, National Research Center, Dokki, Cairo, Egypt
Chemistry Department, Faculty of Science, Hail University, Kingdom of Saudi Arabia
Chemistry Department, Faculty of Science and Human Studies, Huraiymla, Shaqra University, Kingdom of
2
3
Saudi Arabia
4
Chemistry Department, Faculty of Education, Afif, Shaqra University, Kingdom of Saudi Arabia
Food Science and Nutrition Department, College of Food Science and Agriculture, King Saud University,
5
Kingdom of Saudi Arabia
6
Biochemistry Department, Division of Genetic Engeneering and Biotechnology, National Research Center,
Dokki, Cairo, Egypt
5-Amino-1-p-tolyl-1H-pyrazole-4-carbonitrile (1) was used for the preparation of some novel pyrazoles
and pyrazolo[3,4-d]pyrimidines 2–10. Moreover, the cytotoxicity and in vitro anticancer activities of the
prepared compounds were also assessed against the MCF-7 breast cancer, HepG2 liver cancer, and A549
lung carcinoma cell lines, along with investigation of the effect of the synthesized compounds on the
expression of urokinase plasminogen activator (uPA). The tested compounds exhibited remarkable
cytotoxic activity against MCF-7 and HepG2 cells. Among the tested compounds, 2 and 9 revealed
promising anticancer activity compared to the activity of the commonly used anticancer drug,
doxorubicin, by inhibiting the expression of uPA.
Keywords: Anticancer / Pyrazole / Pyrazolopyrimidine / Pyrazolothiazolopyrimidine / Urokinase
Received: February 19, 2014; Revised: March 21, 2014; Accepted: April 2, 2014
DOI 10.1002/ardp.201400064
Introduction
Urokinase plasminogen activator (uPA) is a serine protease
that functions in the conversion of the circulating plasmino-
gen to the active, broad-spectrum serine protease, plasmin.
uPA is secreted as an inactive single-chain proenzyme by
many different cell types and exists in a soluble or cell-
associated form by binding to a specific membrane uPA
receptor (uPAR) [2, 3]. The uPA is involved in many
physiological functions and, along with members of the
matrix metalloproteinases (MMPs) family, it has been
implicated in cancer invasion and metastasization [4–6].
Besides the proteolytic function, upon binding to uPAR, uPA is
involved in initiating versatile intracellular signal pathways
that regulate cell proliferation, adhesion, and migration
through its interaction with various integrins and vitronec-
tin [7]. Urokinase is implicated in a large number of
malignancies, e.g. cancers of breast, lung, bladder, cervix,
kidney, stomach, and brain [8, 9]. Also, the expression of
urokinase is associated with tumor growth and invasion
and may be a useful prognostic factor for hepatocellular
Targeted therapies have a high specificity toward tumor cells,
providing a broader therapeutic window with less toxicity.
They are also often useful in combination with cytotoxic
chemotherapy or radiation to produce additive or synergistic
anticancer activity because their toxicity profiles often do not
overlap with traditional cytotoxic chemotherapy. Thus,
targeted therapies represent a new and promising approach
to cancer therapy, one that is already leading to beneficial
clinical effects. There are multiple types of targeted therapies
available, including monoclonal antibodies, inhibitors of
tyrosine kinases, and antisense inhibitors of growth factor
receptors [1].
Correspondence: Dr. Ahmed H. Shamroukh, Photochemistry Depart-
ment, National Research Center, Dokki, Cairo 112312, Egypt
E-mail: ahshamroukh@yahoo.com
Fax: þ20 2 03370931
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim

2
A. H. Shamroukh et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
carcinoma [10]. The role of uPA in human cancer progression
is further supported by clinical evidences demonstrating that
high tissue levels of its components correlate with a poor
prognosis in different types of cancer such as breast and
gastrointestinal cancers [11].
enhancees its biological activities. Keeping this in mind,
the aim of this work is to prepare some novel pyrazole and
pyrazolopyrimidine derivatives bearing a p-tolyl moiety for
the purpose of developing their biological effect as anticancer
agents.
On the other hand, the chemistry of pyrazole ring system
has attracted much attention as it shows diverse proper- Results and discussion
ties [12–14]. These compounds are not only important from
a
viewpoint of biological activities as potential anti-
Chemistry
microbial [15, 16], anti-inflammatory [17], antiviral [18], and
anticancer agents [19], but also useful as starting compounds
for preparation of other fused pyrazolopyrimidine derivatives
of considerable chemical and biological importance as purine
analogs [20, 21]. In addition, it was reported that several
pyrazolopyrimidines have xanthine oxidase inhibitory activi-
ty [22–24], and showed significant antimicrobial [25, 26],
antitumor [27], and antiviral activity [28, 29]. For small
organic molecules, simple nitrogen-containing heterocycles
receive a large amount of attention in the literature; of these
heterocycles, the synthesis, reactions, and biological activities
of azole- and azoloazines-containing molecules stand as an
ever expanding area of research in hetero aromatic chemis-
try [30–32].
5-Amino-1-p-tolyl-1H-pyrazole-4-carbonitrile (1) [38] was used
as the key compound for this study. Thus, when compound 1
was stirred at room temperature with acetic anhydride,
it gave the N-acetyl-2H-pyrazol-3-yl-acetamide derivative 2
(Scheme 1). The structure of the latter compound was
confirmed on the basis of its spectral data (cf. Experimental
section, Scheme 1). The IR spectrum of compound 2 showed
ꢀꢀ
ꢀꢀ
ꢀꢀ
absorption bands assignable to the CꢀꢀN and two C
O
ꢀꢀ
groups. While carrying out the same reaction under reflux in
the presence of glacial acetic acid, annulations took place to
give 6-methyl-1-p-tolyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-
one (3). The absence of absorption band characteristic for
CꢀꢀN group and the presence of C O in the IR and 13C NMR
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
spectra of compound 3 confirmed its structure. Also, the
1H NMR spectrum of the pyrimidinone derivative 3 showed
signals at 9.42 and 2.64 ppm for NH and C6-CH3, respectively
(cf. Experimental section).
In previous reports [33–37], replacement of the nitrogen
proton in position-1 of the pyrazole ring in pyrazolopyrimi-
dine derivatives by some other substituents drastically
Scheme 1. Synthesis of compounds 2–6.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
Anticancer Evaluation of Pyrazole and Pyrazolopyrimidine Derivatives
3
However, treatment of compound 1 with a mixture of
HCl/AcOH at room temperature gave the [1,3]oxazine
derivative 4 [39]. The IR spectrum of the latter compound
showed the absence of absorption band characteristic for
derivative 6 showed signals at (d, ppm) 10.10 and 8.55 ppm
for NH and C6-H, respectively (cf. Experimental section).
On the other hand, treatment of compound 1 with carbon
disulfide afforded the thione derivative 7. The 1H NMR
spectrum of the latter compound showed the presence of the
SH and NH groups at 4.50 and 11.30 ppm (exchangeable with
CꢀꢀN group and the presence of imine NH at 3210 cmꢀ1, and
ꢀꢀ
ꢀꢀ
the 1H NMR spectrum showed signals at 12.10 and 2.32 ppm
ꢀꢀ
ꢀꢀ
for NH and C -CH , respectively (cf. Experimental section).
D O) in addition to the peak corresponding to the C S group
ꢀꢀ
ꢀꢀ
6
3
2
Moreover, heating of the oxazine derivative 4 in a mixture of
HCl/AcOH for 2 h gave compound identical in all respects
to derivative 3.
at 170.23 ppm in its 13C NMR spectrum (cf. Experimental
section). Compound 7 was treated with chloroacetic acid
and 4-methoxybenzaldehyde in glacial acetic acid/acetic
anhydride in the presence of sodium acetate to give the
pyrazolothiazolopyrimidine derivative 8 (Scheme 2). More-
over, on stirring of compound 1, in dimethyl formamide, with
p-chlorophenyl isothiocyanate at room temperature afforded
the corresponding thiourea derivative 9. The MS spectrum of
the latter compound gave fragments of the isotopic pattern
due to the presence of chlorine atom (cf. Experimental
section). Moreover, on heating of compound 9 in dry dimethyl
formamide, it cyclized to pyrazolo[3,4-d]pyrimidine-6(5H)-
thione derivative 10. The latter compound was also obtained
Meanwhile, treatment of the 5-amino-1-p-tolyl-1H-pyrazole-
4-carbonitrile (1) with formic acid, at room temperature, gave
the N-formyl derivative 5 (Scheme 1). The IR and 13C NMR
ꢀꢀ
spectra of compound 5 showed absorption bands for CꢀꢀN and
ꢀꢀ
ꢀꢀ
HC O groups. While, pyrazolo[3,4-d]pyrimidin-4-one 6 was
ꢀꢀ
obtained either by heating of compound 1 or compound 5
with formic acid. The absence of absorption band character-
ꢀꢀ
ꢀꢀ
ꢀꢀ
istic for CꢀꢀN group and the presence of C O group in the IR
ꢀꢀ
and 13C NMR spectra of compound 6 confirmed its structure.
In addition, the 1H NMR spectrum of the pyrimidinone
Scheme 2. Synthesis of compounds 7–10.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

4
A. H. Shamroukh et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
by refluxing compound 1, dissolved in dimethyl formamide,
with 4-chlorophenyl isothiocyanate for 6 h. The IR spectrum
60%), 2 (90, 87%), 3 (77, 63%), 4 (54, 48%), 5 (82, 67%), 6 (60,
56%), 7 (7, 5%), 8 (3, 5%), 9 (86, 83%), and 10 (9, 6%). From the
results, compound 2 exhibited a good activity in MCF-7 (90%)
and HepG2 cells (87%) similar to doxorubicin (91 and 88%,
respectively) (Table 1). In both MCF-7 and HepG2 cells, the
order of uPA activity inhibition of the tested compounds was
2, 9, 5, 3, 1, 6, 4, 10, 7, and 8 in a descending order, which is in
accordance with the cytotoxic activity.
ꢀꢀ
of compound 10 revealed the absence of the CꢀꢀN group as
ꢀꢀ
well as the presence of fragments showing the isotopic
pattern due to the presence of chlorine atom in MS spectrum
(cf. Experimental section).
Anticancer evaluation
In vitro cytotoxic activity
Taken together, these findings suggested that there is
correlation between the cytotoxicity of the tested compounds
and inhibition of the urokinase activity. The tested com-
pounds exert anti-carcinogenic activity in MCF-7 breast and
HepG2 liver cancer cells by inhibiting the activity of urokinase
enzyme, which may reduce the cell proliferation and result
in significant growth inhibition.
The cytotoxicity of the tested compounds 1–10 was tested
using SRB assay as described by Skehan et al. [40] in breast
cancer cell line MCF-7, liver cancer cell line HepG2, and lung
carcinoma cell line A549. For comparison, doxorubicin was
also tested and the results revealed that all the compounds
did not exert any activity against lung carcinoma cell line
A549. In case of breast cancer cell line MCF-7 and liver
cancer cell line HepG2 compound 2 (IC50: 2.60 ꢁ 0.27 and Conclusion
3.75 ꢁ 0.44 mg/mL, respectively) exhibited similar activity
to doxorubicin (IC50
:
2.80 ꢁ 0.24 and 3.75 ꢁ 0.35mg/mL,
In conclusion, the present results suggested that there are
correlations between the cytotoxic activity of the tested
compounds 1–10 and inhibition of the urokinase activity. The
tested compounds exert anti-carcinogenic activity in hepatic
HepG2 and breast MCF-7 cancer cell lines through down-
regulation of the activity of urokinase enzyme, which may
reduce cell proliferation and result in significant growth
inhibition, especially compounds 2 and 9 that revealed
promising activity compared to the activity of the commonly
used anticancer drug, doxorubicin. The structure–activity
relationship (SAR) of the tested compounds established that
the pyrazole derivative 1 (starting material) revealed moder-
ate activity, whereas substituted amino group as in
derivatives 2, 5, and 9 increased the activity. However, fusing
another ring (oxazine, pyrimidine, or fused pyrimidine) to the
respectively). The order of cytotoxic activity of the tested
compounds was 2, 9, 5, 3, 1, 6, 4, 10, 7, and 8 in a descending
order.
The level of uPA protein expression
To identify the mechanism of action responsible for the
cytotoxicity of tested compounds 1–10, the level of uPA
protein expressed in the two cell lines (breast cancer cell line
MCF-7 and liver cancer cell line HepG2) was estimated
quantitatively. The result revealed that the data of uPA
expression were inconsistent with the cytotoxic activity. In
case of breast cancer cell line MCF-7 and liver cancer cell line
HepG2, the level of uPA decreased in compounds by the
following percent in MCF-7 and HepG2, respectively: 1 (66,
Table 1. In vitro cytotoxic activity and the percent inhibition of uPA of the synthesized compounds on different cell lines.
IC50 (mg/mL)
% inhibition of uPAa)
HepG2
Compound
MCF-7
HepG2
A549
MCF-7
A549
Doxorubicin
DMSO
2.80 ꢁ 0.24
N.A.
3.90 ꢁ 0.37
N.A.
4.11 ꢁ 0.40
N.A.
91 ꢁ 3.70
N.A.
88 ꢁ 6.36
N.A.
82 ꢁ 6.35
N.A.
1
2
3
4
5
6
7
8
9
5.90 ꢁ 0.56
2.60 ꢁ 0.27
5.00 ꢁ 0.63
6.30 ꢁ 0.60
4.60 ꢁ 0.48
5.70 ꢁ 0.72
16.70 ꢁ 1.50
19.00 ꢁ 1.85
3.65 ꢁ 0.29
14.18 ꢁ 1.36
8.00 ꢁ 0.92
3.75 ꢁ 0.44
6.20 ꢁ 0.80
7.25 ꢁ 0.35
4.80 ꢁ 0.47
6.90 ꢁ 0.65
18.30 ꢁ 1.75
20.00 ꢁ 1.92
4.20 ꢁ 0.44
16.10 ꢁ 1.40
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
66 ꢁ 5.44
90 ꢁ 5.76
77 ꢁ 4.28
54 ꢁ 4.93
82 ꢁ 6.95
60 ꢁ 2.84
7 ꢁ 0.94
3 ꢁ 0.45
86 ꢁ 3.09
9 ꢁ 0.47
60 ꢁ 5.09
87 ꢁ 6.45
63 ꢁ 4.65
48 ꢁ 4.82
67 ꢁ 4.78
56 ꢁ 1.98
5 ꢁ 0.46
5 ꢁ 0.46
83 ꢁ 4.65
6 ꢁ 0.45
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
10
N.A. is no activity.
a)
The percentage changes as compared with control untreated cancer cells (DMSO treated).
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
Anticancer Evaluation of Pyrazole and Pyrazolopyrimidine Derivatives
5
pyrazole structure (compounds 4, 6, 7, 8, and 10) decreased the
anticancer activity.
The reaction mixture was poured into icewater; the formed
solid was filtered off and washed several times with water to give
compound 4 in 78% yield; mp 180–182°C; IR (KBr) n 3210 (NH)
cmꢀ1; 1H NMR (DMSO-d6): d 12.10 (s, 1H, NH, D2O exchangeable),
8.22 (d, 2H, Ar-H, J ¼ 8.6 Hz), 7.60 (s, 1H, C3-H), 7.32 (d, 2H, Ar-H,
J ¼ 8.6 Hz), 2.42 (s, 3H, CH3), 2.32 (s, 3H, CH3). 13C NMR (DMSO-d6):
Experimental
Chemistry
ꢀꢀ
d 160.10 (C NH), 155.99 (C-6), 140.98 (C-3); 122.22 (C-7a);
ꢀꢀ
All melting points are uncorrected and were measured using an
Electrothermal IA 9100 apparatus, Shimadzu (Japan). The IR
spectra (KBr) were recorded on a Perkin–Elmer 1650 spectropho-
tometer, National Research Centre, Cairo, Egypt. Microanalytical
data were measured by Vario El-Mentar apparatus, Organic
Microanalysis Section, National Research Centre, Cairo, Egypt,
and the results were found to be in agreement with the calculated
values (ꢁ0.3). 1H and 13C NMR spectra were determined on a Jeol
300 MHz in dimethyl sulfoxide (DMSO-d6), National Research
Centre, and the chemical shifts were expressed in ppm relative to
TMS as internal reference. Mass spectra were recorded on 70 eV EI
Ms-QP 1000 EX (Shimadzu, Japan), National Research Centre,
Cairo, Egypt. Compound 1 (mp 167°C; literature mp 167–
167.5°C) was prepared according to a reported method [38].
104.51 (C-3a); 134.19, 130.40, 127.16, 113.42 (Ar-C), 24.20
(CH3), 25.41 (CH3); MS, m/z (%): 240 (Mþ, 59); Anal. calcd. for
C
13H12N4O: C, 64.99; H, 5.03; N, 23.32. Found: C, 64.95; H, 4.95;
N, 23.35.
N-(4-Cyano-2-p-tolyl-2H-pyrazol-3-yl)formamide 5
A solution of compound 1 (0.002 mol) in formic acid (10 mL, 85%)
was stirred at room temperature for 6 h. The reaction mixture
was poured into water; the formed solid was filtered off,
washed several times with water, dried, and recrystallized from
ethanol to give compound 5 in 71% yield; mp 150–152°C; IR (KBr):
n 2233 (CꢀꢀN), 1723 (C O) cmꢀ1; 1H NMR (DMSO-d6): d 8.45 (bs, 1H,
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
NH, D2O exchangeable), 8.26 (s, 1H, CHO), 8.20 (d, 2H, Ar-H,
J ¼ 8.6 Hz), 7.61 (s, 1H, C3-H), 7.33 (d, 2H, Ar-H, J ¼ 8.6 Hz), 2.42
13
(s, 3H, CH3). C NMR (DMSO-d ): d 161.30 (C O), 148.05 (C-5);
6
N-Acetyl-N-(4-cyano-2-p-tolyl-2H-pyrazol-3-yl)acetamide 2
A solution of compound 1 (0.002 mol) in acetic anhydride (10 mL)
was stirred at room temperature for 3 h. The solvent was removed
under reduced pressure and the obtained solid was recrystallized
ꢀꢀ
139.72 (C-3); 134.09, 130.29, 127.09, 113.35 (Ar-C), 119.8 (CꢀꢀN),
ꢀꢀ
94.79 (C-4); 25.55 (CH3); MS, m/z (%): 226 (Mþ, 62); Anal. calcd. for
C12H10N4O: C, 63.71; H, 4.46; N, 24.77. Found: C, 63.65; H, 4.49;
N, 24.79.
from dry ethanol to give compound 2 in 71% yield; mp 150–
1
152°C; IR (KBr): n 2233 (CꢀꢀN), 1741, 1720 (C O) cmꢀ1; H NMR
ꢀꢀ
ꢀꢀ
ꢀꢀ
ꢀꢀ
1-(p-Tolyl)-1,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-4-one 6
Method A: A solution of compound 1 (0.01 mol) in formic acid
(20 mL, 85%) was heated under reflux for 10 h. The reaction
mixture was cooled and poured into water, and the formed solid
was filtered off, dried, and recrystallized from dioxane to give
compound 6 in 76% yield.
Method B: Compound 5 (0.01 mol) was heated under reflux in
formic acid (20 mL, 85%) for 4 h. The reaction mixture was cooled
and poured into water. The formed solid was filtered off, dried,
(DMSO-d6): d 8.19 (d, 2H, Ar-H, J ¼ 8.6 Hz), 7.60 (s, 1H, C3-H), 7.33 (d,
13
2H, Ar-H, J ¼ 8.6 Hz), 3.25 (s, 3H, CH3), 3.24 (s, 3H, CH3), 2.42 (s, 3H,
ꢀꢀ
CH3); C NMR (DMSO-d ): d 158.20 (2C O), 149.25 (C-5); 140.81
ꢀꢀ
6
ꢀꢀ
(C-3); 134.17, 130.38, 127.16, 113.51 (Ar-C), 120.7 (CꢀꢀN), 95.72
ꢀꢀ
(C-4); 25.55 (CH3), 20.19 (CH3), 20.15 (CH3); MS, m/z (%): 282
(Mþ, 80); Anal. calcd. for C15H14N4O2: C, 63.82; H, 5.0; N, 19.85.
Found: C, 63.98; H, 4.92; N, 19.89.
6-Methyl-1-p-tolyl-1,5-dihydro-pyrazolo[3,4-d]pyrimidin-4-
one 3
and recrystallized from dioxane to give compound 6 in 89% yield.
1
mp 275–277°C; IR (KBr) n 3150 (NH), 1674 (C O) cmꢀ1; H NMR
ꢀꢀ
ꢀꢀ
Method A: A mixture of compound 1 (0.01 mol) in acetic anhydride/
glacial acetic acid (20 mL, 1:1) was heated under reflux for 10 h.
The reaction mixture was cooled and poured into ice water; the
formed solid was filtered off, dried, and recrystallized from
dioxane to give compound 3 in 84% yield.
Method B: A mixture of compound 4 (0.01 mol) in hydrochloric
acid/glacial acetic acid (20 mL, 1:3) was heated under reflux for
5 h. The reaction mixture was poured into ice water; the formed
solid was filtered off, washed several times with water, dried, and
(DMSO-d6): d 10.10 (s, 1H, NH, D2O exchangeable), 8.55 (s, 1H,
C6-H), 8.20 (d, 2H, Ar-H, J ¼ 8.6 Hz), 7.59 (s, 1H, C3-H), 7.34 (d, 2H,
Ar-H, J ¼ 8.6 Hz), 2.43 (s, 3H, CH3); 13C NMR (DMSO-d6): d 167.20
(C O), 158.12 (C-6), 144.22 (C-3); 123.01 (C-7a); 103.22 (C-3a);
133.12, 130.59, 127.11, 113.21 (Ar-C), 24.18 (CH3); MS, m/z (%): 226
(Mþ, 90); Anal. calcd. for C12H10N4O: C, 63.71; H, 4.46; N, 24.77.
Found: C, 63.65; H, 4.50; N, 24.74.
6-Mercapto-1-p-tolyl-1,5-dihydro-pyrazolo[3,4-d]-
pyrimidine-4-thione 7
recrystallized from dioxane to give compound 3 in 89% yield.
1
mp 210–212°C; IR (KBr) n 3220 (NH), 1675 (C O) cmꢀ1; H NMR
ꢀꢀ
ꢀꢀ
A solution of compound 1 (0.01 mol) and 20% potassium
hydroxide solution (potassium hydroxide 1.68 g, 7 mL water) in
dimethylsulfoxide (20 mL) was stirred and carbon disulfide
(0.03 mol) was added in several portions for 30 min. After 1 h
at room temperature, the precipitate was collected, washed
several times with water, dried, and recrystallized from dimethyl
formamide to give compound 7 in 69% yield; mp 255–257°C; IR
(KBr) n 3100 (NH) cmꢀ1; 1H NMR (DMSO-d6): d 11.30 (s, 1H, NH, D2O
exchangeable), 8.21 (d, 2H, Ar-H, J ¼ 8.6 Hz), 7.60 (s, 1H, C3-H), 7.34
(d, 2H, Ar-H, J ¼ 8.6 Hz), 4.50 (s, 1H, SH, D2O exchangeable), 2.41 (s,
(DMSO-d6): d 9.42 (s, 1H, NH, D2O exchangeable), 8.20 (d, 2H, Ar-H,
J ¼ 8.6 Hz), 7.62 (s, 1H, C3-H), 7.34 (d, 2H, Ar-H, J ¼ 8.6 Hz), 2.64 (s,
13
3H, CH3), 2.41 (s, 3H, CH3); C NMR (DMSO-d ): d 165.10 (C O),
6
154.84 (C-6), 141.92 (C-3); 122.23 (C-7a); 102.32 (C-3a); 134.18,
130.39, 127.17, 113.52 (Ar-C), 24.19 (CH3), 25.40 (CH3); MS, m/z (%):
240 (Mþ, 67); Anal. calcd. for C11H10N4: C, 64.99; H, 5.03; N, 23.32.
Found: C, 65.12; H, 4.90; N, 23.39.
6-Methyl-1-p-tolyl-1,5-dihydro-pyrazolo[3,4-d][1,3]oxazin-
4-one 4
A mixture of compound 1 (0.01 mol) in hydrochloric acid/glacial
acetic acid (20 mL, 1:3) was stirred at room temperature for 5 h.
3H, CH3); 13C NMR (DMSO-d ): d 170.23 (C S), 159.52 (C-6), 143.12
(C-3); 123.25 (C-7a); 104.62 (C-3a); 134.19, 130.30, 127.09, 113.35
ꢀꢀ
ꢀꢀ
6
(Ar-C), 24.19 (CH3); MS, m/z (%): 274 (Mþ, 90); Anal. calcd. for
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

6
A. H. Shamroukh et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
C12H10N4S2: C, 52.53; H, 3.67; N, 20.42; S, 23.37. Found: C, 52.42;
H, 3.70; N, 20.48; S, 23.41.
Eagle’s (DMEM) medium was provided from Cambrex (NJ, USA).
DMSO, doxorubicin, penicillin, and streptomycin were obtained
from Sigma Chemical Company (Saint Louis, MO, USA). The level
of uPA protein was determined using Assay Max human
urokinase (uPA) ELISA kit (Assaypro, USA).
7-(4-Methoxy-benzylidene)-4-thioxo-1-p-tolyl-1,4-dihydro-
pyrazolo[3,4-d]thiazolo[3,2-a]pyrimidin-6-one 8
A mixture of compound 7 (0.002 mol), chloroacetic acid (10 mol),
4-methoxybenzaldehyde (0.002 mol), and anhydrous sodium
acetate was heated under reflux in glacial acetic acid (15 mL)
and acetic anhydride (10 mL) for 5 h. The reaction mixture was
cooled and poured into cold water (100 mL), the deposited
precipitate was filtered off and recrystallized from dioxane to
produce 8 in 56% yield; mp 250–252°C; 1H NMR (DMSO-d6): d 8.36
Cell lines and culturing
Anticancer activity screening was performed for the tested
compounds utilizing three different human tumor cell lines
including breast cancer cell line MCF-7, liver cancer cell line
HepG2, and lung carcinoma cell line A549 obtained from the
American Type Culture Collection (Rockville, MD, USA). The
tumor cells were maintained in Dulbecco’s modified Eagle’s
medium (DMEM) supplemented with 10% heat-inactivated fetal
calf serum (GIBCO), penicillin (100 U/mL), and streptomycin
(100 mg/mL) at 37°C in humidified atmosphere containing 5%
CO2. Cells at a concentration of 0.50 ꢂ 106 were grown in a 25 cm2
flask in 5 mL of complete culture medium.
ꢀꢀ
(s, 1H, C -H), 8.20 (d, 2H, Ar-H, J ¼ 8.5 Hz), 8.12 (s, 1H, CH), 7.51–
ꢀꢀ
3
7.80 (m, 4H, Ar-H), 7.34 (d, 2H, Ar-H, J ¼ 8.5 Hz), 3.92 (s, 3H, OCH3),
2.41 (s, 3H, CH3); MS, m/z (%): 432 (Mþ, 45); Anal. calcd. for
C22H16N4O2S2: C, 61.09; H, 3.73; N, 12.95; S, 7.40. Found: C, 61.40;
H, 3.72; N, 12.88; S, 7.46.
1-(4-Chlorophenyl)-3-(4-cyano-2-p-tolyl-2H-pyrazol-3-yl)-
thiourea 9
In vitro cytotoxicity assay
The cytotoxicity activity was measured in vitro using the Sulfo-
Rhodamine-B stain (SRB) assay according to the previously
reported standard procedure [40]. Cells were inoculated in 96-
well microtiter plate (104 cells/well) for 24 h before treatment
with the tested compounds to allow attachment of cells to the
wall of the plate. Test compounds were dissolved in DMSO at
1 mg/mL immediately before use and diluted to the appropriate
volume just before addition to the cell culture. Different
concentrations of tested compounds and doxorubicin were added
to the cells. Triplicate wells were prepared for each individual
dose. Monolayer cells were incubated with the compounds for
48 h at 37°C and in atmosphere of 5% CO2. After 48 h, cells were
fixed, washed, and stained for 30 min with 0.4% w/v SRB dissolved
in 1% acetic acid. Unbound dye was removed by four washes with
1% acetic acid, and attached stain was recovered with Tris–EDTA
buffer. Color intensity was measured in an ELISA reader. The
relation between surviving fraction and drug concentration was
plotted to get the survival curve for each cell line after the
specified time. The concentration required for 50% inhibition of
cell viability (IC50) was calculated, and the results are given
in Table 1. The results were compared to the antiproliferative
effects of the reference control doxorubicin.
A mixture of compound 1 (0.002 mol), p-chlorophenyl isothio-
cyanate (0.002 mol), and triethylamine (2 drops) in dimethyl
formamide (10 mL) was stirred at room temperature for 3 h. The
reaction mixture was poured onto ice water; the deposited solid
was filtered off and washed several times with water to give
compound 9 in 66% yield; mp 152–154°C; IR (KBr) n 3359, 3176
(NH), 2215 (CꢀꢀN) cmꢀ1; 1H NMR (DMSO-d6): d 8.28–8.21 (m, 4H, Ar-
ꢀꢀ
ꢀꢀ
H), 7.90 (s, 1H, NH, D2O exchangeable), 7.62 (s, 1H, C3-H), 7.41–7.33
(m, 4H, Ar-H), 6.50 (s, 1H, NH, D2O exchangeable), 2.41 (s, 3H, CH3);
MS, m/z (%): 369 (Mþþ2, 37Cl, 39.98), 367 (Mþ, 35Cl, 100); Anal.
calcd. for C18H14ClN5S: C, 58.77; H, 3.84; N, 19.04; S, 8.72. Found:
C, 58.52; H, 3.79; N, 19.18; S, 8.49.
5-(4-Chlorophenyl)-4-imino-1-(4-methylphenyl)-1,4,5,7-
tetrahydro-6H-pyrazolo[3,4-d]pyrimidin-6-thione 10
Method A: A mixture of 9 (0.002 mol) and triethylamine (2 drops)
in dimethyl formamide (10 mL) was refluxed for 5 h. The reaction
mixture was poured onto ice water; the deposited solid was
filtered off and washed several times with water to give compound
10 (68% yield).
Method B:
A mixture of compound 1 (0.002 mol) and
p-chlorophenyl isothiocyanate (0.002mol) in dimethyl formamide
(20 mL) was refluxed for 6 h. The reaction mixture was poured
onto ice water and the solid formed was separated by filtration
and washed several times with water to give compound 10 (61%
yield). mp 175–177°C; IR (KBr) n 3272, 3190 (2NH) cmꢀ1; 1H NMR
(DMSO-d6): d 10.45 (s, 1H, NH, D2O exchangeable), 8.41 (s, 1H, C3-
H), 8.20–8.29 (m, 4H, Ar-H), 7.4–7.3 (m, 4H, Ar-H), 6.42 (s, 1H, NH,
D2O exchangeable), 2.42 (s, 3H, CH3); 13C NMR (DMSO-d6): d 171.95
Determination of the level of uPA protein expression
The level of uPA protein expression was determined using Assay
Max human urokinase (uPA) ELISA kit (Assaypro, USA) according
to manufacturer’s instructions. The prepared compounds as well
as standard drug doxorubicin were incubated for 48 h with MCF7,
HepG2, and A549 cells at a concentration of 1/10 of the IC50 values
of each compound shown in Table 1.
After 48 h from compounds treatment, medium was collected
and centrifuged at 2000 ꢂ g for 10 min to remove cellular
debris; 50 mL of the cell extract was added per well and
incubated for 2 h. Wells were washed with 200 mL of wash
buffer; then 50 mL of biotinylated uPA antibody was added to
each well and incubated for 1 h at 25°C. After washing, plates
were incubated with 50 mL of streptavidin–peroxidase conju-
gate per well and incubated for 30 min; then the microplate was
washed as described above; 50 mL of chromogen substrate was
added per well and incubated for about 10 min or till the
optimal blue color density developed; 50 mL of stop solution was
ꢀ
(C S), 160.99 (C-4), 143.01 (C-3); 123.25 (C-7a); 104.62 (C-3a);
ꢀ
134.20, 132.12, 129.05, 128.60, 128.12, 127.29, 126.83, 125.21
(Ar-C), 24.25 (CH3); MS, m/z (%):369 (Mþþ2, 37Cl, 39.98), 367
(Mþ, 35Cl, 100); Anal. calcd. for C18H14ClN5S: C, 58.77; H, 3.84;
N, 19.04; S, 8.72. Found: C, 58.65; H, 3.81; N, 18.98; S, 8.62.
Anticancer evaluation
Chemicals
Fetal bovine serum (FBS) and L-glutamine were obtained from
Gibco Invitrogen Company (Scotland, UK). Dulbecco’s modified
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 1–7
Anticancer Evaluation of Pyrazole and Pyrazolopyrimidine Derivatives
7
added to each well. The color changed from blue to yellow. The
absorbance was read on a microplate reader at a wavelength of
450 nm immediately, the concentrations of uPA in the samples
were determined, and the percentage of uPA inhibition for
each compound was calculated as compared with control
cancer cells (DMSO treated).
[18] S. R. Shih, T. Y. Chu, G. R. Reddy, S. N. Tseng, H. L. Chen,
W. F. Tang, M. S. Wu, J. Y. Yeh, Y. S. Chao, J. T. Hsu, H. P. Hsieh,
J. T. Horng, J. Biomed. Sci. 2010, 17, 13–22.
[19] A. M. Farag, A. S. Mayhoub, S. E. Barakat, A. H. Bayomi, Bioorg.
Med. Chem. 2008, 16, 881–889.
[20] E. I. El-Afaleq, S. A. Abubshait, Molecules 2001, 6, 621–638.
[21] V. Massey, H. Komai, G. Palmer, G. B. Elion, J. Biol. Chem. 1970,
245, 2837–2844.
Statistical analysis
The results were reported as mean ꢁ SE. Statistical differences
were analyzed according to one-way ANOVA test Student’s t-test
wherein the differences were considered to be significant at
p < 0.05.
[22] S. Gupta, L. M. Rodrigues, A. P. Esteves, A. M. F. Oliveira-
Campos, M. S. José Nascimento, N. Nazareth, H. Cidade,
M. P. Neves, E. Fernandes, M. Pinto, N. M. F. S. A. Cerqueira,
N. Brás, Eur. J. Med. Chem. 2008, 43, 771–780.
[23] R. K. Robins, J. Am. Chem. Soc. 1956, 78, 784–790.
The authors extend their appreciation to the Deanship of Scientific
Research at King Saud University for supporting this work through the
research group project no. RGP-VPP-032.
[24] R. Helmut, A. Martina, S. Frank, Molecules 2007, 12, 563–575.
[25] A. H. Shamroukh, A. E. Rashad, H. S. Ali, F. M. E. Abdel-
Megeid, J. Heterocyclic Chem. 2013, 50, 758–765.
[26] L. Ballell, R. A. Field, G. A. C. Chung, R. J Young, Bioorg. Med.
Chem. Lett. 2007, 17, 1736–1740.
The authors have declared no conflict of interest.
[27] M. M. Ghorab, F. A. Ragab, S. I. Alqasoumi, A. M. Alafeefy,
References
S. A. Aboulmagd, Eur. J. Med. Chem. 2010, 45, 171–178.
[28] A. E. Rashad, M. I. Hegab, R. E. Abdel-Megeid, N. A. Fatahala,
F. M. E. Abdel-Megeid, Eur. J. Med. Chem. 2009, 44, 1535–
1556.
[1] A. Arora, E. M. Scholar, J. Pharm. Exp. Ther. 2005, 315, 971–979.
[2] L. Yuli, P. Nana, L. Jianping, Z. Hongqin, H. Zi-Chun, Mol.
Cancer 2013, 12, 54–65.
[29] K. S. Gudmundsson, B. A. Johns, J. Weatherhead, Bioorg. Med.
Chem. Lett. 2009, 19, 5689–5692.
[3] T. Syrovets, T. Simmet, Cell Mol. Life Sci. 2004, 61, 873–885.
[4] T. Tamura, E. Morita, S. Kawai, R. Okada, M. Naito, K. Wakai,
[30] S. Maddila, S. B. Jonnalagadda, Lett. Org. Chem. 2013, 10, 374–
Y. Hori, T. Kondo, N. Hamajima, Gene 2013, 524, 363–367.
379.
[5] P. Mignatti, D. B. Rifkin, Physiol. Rev. 1993, 73, 161–195.
[6] K. Bdeir, A. Kuo, B. S. Sachais, Blood 2003, 102, 3600–3608.
[7] F. Blasi, P. Carmeliet, Nat. Rev. Mol. Cell Biol. 2002, 3, 932–943.
[31] S. Maddila, A. S. Kumar, S. Gorle, M. Singh, P. Lavanya,
S. B. Jonnalagadda, Lett. Drug Des. Discov. 2013, 10, 186–
193.
[32] S. Maddila, S. B. Jonnalagadda, Arch. Pham. 2012, 345, 163–
[8] P. A. Andreasen, L. Kjoller, L. Christensen, M. J. Duffy, Int. J.
Cancer 1997, 72, 1–22.
168.
[33] B. S. Holla, M. S. Mahalinga, M. S. Karthekyan, P. M. Akberali,
[9] T. Meyer, I. R. Hart, Eur. J. Cancer 1998, 34, 214–221.
N. S. Shetty, Bioorg. Med. Chem. 2006, 14, 2040–2047.
[10] N. N. Al-Hassan, A. Behzadian, R. Caldwell, V. S. Ivanova,
[34] T. E. Ali, Eur. J. Med. Chem. 2009, 44, 4385–4392.
V. Syed, K. Motamed, N. A. Said, Neoplasia 2012, 14, 259–270.
[35] A. F. Eweas, S. A. Swelam, O. A. Fathalla, N. M. Fawzy, I. Sh,
[11] M. J. Duffy, C. Duggan, Clin. Biochem. 2004, 37, 541–548.
Abdel-Moez, Med. Chem. Res. 2012, 21, 3848–3857.
[12] R. E. Khidre, H. A. Mohamed, B. F. Abdel-Wahab, Turk. J. Chem.
2013, 37, 1–35.
[36] S. B. Yewale, S. B. Ganorkar, K. G. Baheti, R. U. Shelke, Bioorg.
Med. Chem. Lett. 2012, 22, 6616–6620.
[13] J. Prabhashankar, V. K. Govindappa, A. K. Kariyappa, Turk. J.
Chem. 2013, 37, 853–857.
[37] A. M. Vijesh, A. M. Isloor, S. Telkar, S. K. Peethambar, S. Rai,
N. Isloor, Eur. J. Med. Chem. 2011, 46, 3531–3536.
[14] M. Chauhan, R. Kumar, Bioorg. Med. Chem. 2013, 21, 5657–
[38] C. C. Cheng, R. K. Robins, J. Org. Chem. 1956, 21, 1240–
5668.
1256.
[15] T. E. Ali, S. A. A. Abdel-Ghaffar, K. M. El-Mahdy, S. M. Abdel-
Karim, Turk. J. Chem. 2013, 37, 160–169.
[39] F. M. E. Abdel-Megeid, N. A. Hassan, M. A. Zahran,
A. E. Rashad, Sulfur Lett. 1996, 21, 269–284.
[16] S. M. Gomha, H. M. E. Hassaneen, Molecules 2011, 16, 6549–
6560.
[40] P. Skehan, R. Storeng, D. Scudiero, A. Monks, J. McMahon,
D. Vistica, J. T. Warren, J. Natl. Cancer Inst. 1990, 82, 1107–
1112.
[17] D. Raffa, B. Maggio, F. Plescia, S. Cascioferro, M. V. Raimondi,
S. Plescia, M. G. Cusimano, Arch Pharm. 2009, 342, 321–326.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com