Design, synthesis, cytotoxicity and molecular modeling studies of some novel fluorinated pyrazole-based heterocycles as anticancer and apoptosis-inducing agents

Article abstract of DOI:10.1007/s11030-018-9865-9

3,5-Diamino-4-(3-trifluoromethylphenyldiazenyl)-1H-pyrazole was used as a starting scaffold for the synthesis of new pyrazole-based heterocycles to study their effects on the proliferation of three human cancer cell lines; human liver carcinoma cell line (HepG-2), colon cancer cell line (HCT-116) and human breast cancer cell line (MCF-7) using MTT assay. The synthesized compounds were characterized on the basis of IR, ¹H NMR, ¹³C NMR, mass spectral data and elemental analysis results. Cytotoxicity assay results revealed that some of the compounds showed potent growth inhibition against all the cell lines tested, with IC50 values in the range of 0.64–7.73?μg/mL. Breast cancer cells were used for further detailed studies to understand the mechanism of cell growth inhibition and apoptosis-inducing effect of the most active compounds. The results indicated that compounds 3a, 10b and 11a arrested MCF-7 cells at G2/M phase of the cell cycle and might induce apoptosis via caspase-3-dependent pathway. Molecular modeling and binding mode analysis of the most active compounds to caspase 3 active site further provide a synergistic mechanism for their pro-apoptotic effects. In order to explore the structural requirements controlling the observed cytotoxic properties, 3D pharmacophore model was generated.

Full text of DOI:10.1007/s11030-018-9865-9

Molecular Diversity
https://doi.org/10.1007/s11030-018-9865-9
ORIGINAL ARTICLE
Design, synthesis, cytotoxicity and molecular modeling studies
of some novel fluorinated pyrazole-based heterocycles as anticancer
and apoptosis-inducing agents
Eman A. Fayed1 · Sally I. Eissa2,3 · Ashraf H. Bayoumi4 · Nirvana A. Gohar1 · Ahmed B. M. Mehany5 ·
Yousry A. Ammar6
Received: 5 March 2018 / Accepted: 2 August 2018
© Springer Nature Switzerland AG 2018
Abstract
3,5-Diamino-4-(3-trifluoromethylphenyldiazenyl)-1H-pyrazole was used as a starting scaffold for the synthesis of new
pyrazole-based heterocycles to study their effects on the proliferation of three human cancer cell lines; human liver car-
cinoma cell line (HepG-2), colon cancer cell line (HCT-116) and human breast cancer cell line (MCF-7) using MTT assay.
The synthesized compounds were characterized on the basis of IR, 1H NMR, 13C NMR, mass spectral data and elemental
analysis results. Cytotoxicity assay results revealed that some of the compounds showed potent growth inhibition against all
the cell lines tested, with IC50 values in the range of 0.64–7.73 μg/mL. Breast cancer cells were used for further detailed
studies to understand the mechanism of cell growth inhibition and apoptosis-inducing effect of the most active compounds.
The results indicated that compounds 3a, 10b and 11a arrested MCF-7 cells at G2/M phase of the cell cycle and might induce
apoptosis via caspase-3-dependent pathway. Molecular modeling and binding mode analysis of the most active compounds to
caspase 3 active site further provide a synergistic mechanism for their pro-apoptotic effects. In order to explore the structural
requirements controlling the observed cytotoxic properties, 3D pharmacophore model was generated.
Keywords Synthesis · Pyrazole · Pyrazolo[1,5-a]pyrimidines · Anti-proliferative activity · Apoptosis
Introduction
of death after cardiovascular disorders throughout the world
[1]. Resistance to chemotherapeutic agents remains a key
challenge in the fight against cancer. Another challenge for
chemotherapy is lack of selectivity. Generally anticancer
drugs destroy normal cells as well as cancer cells and often
causeseriousadverseeffects. Manyeffortshavebeenmadeto
develop safe and effective ways of treating this disease and
to search for novel chemotherapeutic agents with minimal
side effects [2].
Cancer is considered a large group of diseases character-
ized by uncontrolled, rapid and pathological proliferation
of abnormally transformed cells. Despite recent advances
in cancer therapy, cancer is still the second leading cause
Sally I. Eissa
sarazizo2005@yahoo.com
B
1
Apoptosis is the programmed cell death which maintains
the healthy survival/death balance in normal cells. Disrup-
tion or inappropriate regulation of the process of apoptosis
results in several diseases including cancer. The unique char-
acteristics of apoptosis are the activation of various enzymes
that are cysteine protease families called caspases. Caspase 3
is considered the key protein of the caspase family, which are
highly conserved in multicellular organisms and function as
central regulators of apoptosis. Thus, targeting critical reg-
ulators of apoptosis with the goal of inducing apoptosis in
cancer cells has emerged as an attractive strategy in cancer
therapy [3–5].
Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy (Girls), Al-Azhar University, Cairo, Egypt
2
Pharmaceutical Chemistry Department, Faculty of Pharmacy,
Al-Azhar University (Girls), Cairo, Egypt
3
Faculty of Pharmacy, Al-Maarefa University for Science and
Technology, Riyadh, Kingdom of Saudi Arabia
4
Pharmaceutical Organic Chemistry Department, Faculty of
Pharmacy (Boys), Al-Azhar University, Cairo, Egypt
5
Department of Zoology, Faculty of Science, Al-Azhar
University, Cairo, Egypt
6
Organic Chemistry Department, Faculty of Science (Boys),
Al-Azhar University, Cairo, Egypt
123

Molecular Diversity
On the other hand, pyrazoles are an important class of
compounds for new drug development that attracted much
attention due to their broad spectrum of biological activities
[6–9]. Preliminary in vitro biological studies demonstrated
that (pyrazole-based heterocycles) are able to alter tumor
growth rates and modulate carcinogenesis by a variety of
reported pathways including induction of apoptosis with sig-
nificant cell cycle arrest at different phases of cell cycle in a
variety of transformed cell lines [10–12].
Moreover, several recent reports suggest pyrazole deriva-
tives as promising anticancer agents [13, 14]. Among the
anticancer pyrazoles, Ruxolitinib and Crizotinib have been
reported to be highly potent and efficient cytotoxic agents
[15].
duction of azo group enhances the biological activity of
heterocyclic compounds [29].
Development of new analogs based on known inhibitor
is an important strategy for the discovery of new anti-
cancer drugs with high efficiency and a pharmacophore
hybridization approach for the synthesis of new bioactive
compounds is an effective strategy in modern medicinal
chemistry. Prompted by these observations, and continuing
our work to search for novel anticancer agents, we designed
a novel class of pyrazoles and pyrazolopyrimidines to which
trifluoromethyl and azo moieties were appended into their
molecular framework to examine the effect of such an asso-
ciation on the anticancer activity of the system and produced
a favorable impact on cytotoxicity in the new materials.
Cl
N
NH2
Results and discussion
F
Cl
O
N
Chemistry
N
N
The synthetic pathways employed to prepare the new tar-
geted derivatives are depicted in Schemes 1, 2 and 3.
The3-trifluoromethyl phenyl hydrazono-malononitrile 2 was
synthesized by diazotization of 3-trifluoroaniline 1 followed
by coupling of its diazonium salt with malononitrile in the
presence of sodium acetate. The IR spectrum showed a peak
at 3227 cm−1 attributed to NH and a pronounced peak of
CN group at 2220 cm−1. The structure of compound 2 was
also confirmed through 1HNMR which showed the appear-
ance of aromatic protons as two doublets at δ 7.52 and
7.71 ppm with J=9.0 Hz and one triplet and one singlet at
δ 7.61 and 7.74 ppm, respectively, in addition to one singlet
D2O exchangeable peak at δ 8.70 ppm corresponding to NH
proton. Condensation of 2 with hydrazine hydrate, phenyl-
hydrazine and thiosemicarbazide afforded the corresponding
pyrazole derivatives 3a–c (Scheme 1). The structure was
characterized and confirmed on the basis of analytical and
spectral data. Structure 3a was taken as a representative
example; and its IR spectrum (KBr/cm−1) showed strong
absorption bands at 3404, 3295 and 3200 corresponding to
NH2 and NH groups. Its 1H NMR spectrum (ppm) showed
two singlets at δ 5.96 and 6.48 ppm due to 2NH2 protons
and a singlet signal at δ 10.79 ppm attributed to NH. In addi-
tion, there were two doublets at δ 7.48 (J=9.0 Hz), 7.92
(J=9.0 Hz), triplet and a singlet signal at δ 7.55 and 8.01 ppm
due to four aromatic protons. 13C NMR spectrum was char-
acterized by signals at 66.7 and 154.05 assigned 3C-pyrazole
and a signal at 116.36 due to CF3 in addition to the aromatic
carbons. The mass spectrum exhibited a molecular ion peak
at m/z=270 [M·+] corresponding to the molecular formula
C10H9F3N6.
N
N
N
N
H
N
HN
Crizotinib
Ruxolitinib
More recently, various pyrazolopyrimidines have been
reported to have potential anti-tumor/anti-proliferative activ-
ity against different cancers [16, 17]. A literature survey
revealed that pyrazolo[1,5-a]pyrimidines are of consider-
able chemical and pharmacological importance due to the
structural similarity of the pyrazolopyrimidines scaffold with
some of the naturally occurring moieties such as purines, by
which they easily interact with biomolecules of living sys-
tems [18].
Additionally, it was found that compounds 7-amino-
6-cyano-2-(4-methoxyphenylamino)-5-(naphthalen-1-
yl)-pyrazolo[1,5-a]pyrimidine-3-carboxamide
exerted
promising anticancer activity against breast cancer cell line
MCF-7 (IC50 ꢀ0.085 μM; Doxorubicin IC50 ꢀ96.41 μM)
and also 2-(4-methoxyphenylamino)-5,7-dimethyl-N-
phenylpyrazolo[1,5-a]pyrimidine-3-carboxamide is another
example of a pyrazolo[1,5-a]pyrimidine derivative that
exhibited promising anticancer activity in Ehrlich ascites
carcinoma [19–21].
Furthermore, fluorine-building blocks are important
because of their extensive use in the synthesis of drugs cov-
ering a wide variety of therapeutic areas [22]. Incorporation
of trifluoromethyl group, being one of the most lipophilic
groups, exerts a variety of dramatic effects on the phar-
macological properties of the molecule [23–27]. It makes
the absorption of fluorinated molecule into cell membranes
much easier and hence increases the selectivity, efficacy and
bioavailability [28]. It is also well documented that the intro-
Compound 3a was used as starting material for preparing
bioactive compounds. Thus, acetylation of 3a with warm-
123

Molecular Diversity
Scheme 1 Synthesis of compounds 3a–c
ing acetic anhydride afforded the acetamide derivative 4. In
addition, the reaction of compound 3a with acetylacetone in
glacial acetic acid under reflux temperature afforded the 5,7-
dimethylpyrazolo[1,5-a]pyrimidine 5. The structure of 5 was
confirmed on the basis of their analytical and spectral data,
and its IR spectrum showed strong absorption bands at 3281,
3200 cm−1 corresponding to NH2 and a band at 1632 cm−1
group followed by intramolecular cyclization by attack of
the endocyclic NH function on the α, β-unsaturated sys-
tem and spontaneous auto-oxidation through the loss of a
hydrogen molecule. The structure of 9 was elucidated based
on their analytical and spectral data. Thus, its IR spec-
trum (KBr/cm−1) showed bands at 3423 and 3307 cm−1
for (NH2), 1623 cm−1 for C=N and 1555 cm−1 for N=N
groups, respectively. Its 1H NMR spectrum (δ/ppm) revealed
the presence of two singlet signals at δ 7.40 and 7.89 ppm cor-
responding to NH2 and CH-pyrimidine and multiplet showed
at δ 7.59–8.40 ppm attributed to the aromatic protons. The
mass spectrum of this compound showed an ion peak at
m/z: 492 (74.86%) corresponding for a molecular formula
C25H16ClF3N6 with a base peak at m/z 225.
1
due to C=N group. Its H NMR spectrum showed two sin-
glets at δ 2.48 and 2.55 ppm due to two CH3 of the pyrimidine
nucleus, and a signal at δ 7.00 ppm due to the H-6 proton
of the pyrimidine nucleus. Also, there is a singlet signal at
δ 7.33 ppm assigned for NH2 which was D2O exchange-
able. Multiplet, doublet and singlet signals were present at δ
7.64–7.72, 8.05 and 8.13 ppm due to four aromatic protons.
Its 13C NMR spectrum was characterized by signals at 16.33
and 24.04 assigned to two CH3 carbon, a signal at 117.53
corresponding to CF3 carbon, signals at 66.48, 151.87 and
153.38 due to 3C-pyrazole and signals at 110.47, 145.53 and
160.55 due to 3C-pyrimidine. The formation of compounds 5
was therefore assumed to proceed via the initial attack of the
exocyclic amino group of 3a on the keto group of acetylace-
tone followed by intramolecular cyclization via elimination
of water (Scheme 2).
This thesis was extended to cover the reactivity of
aminopyrazoles toward activated double bonds on the hope
of obtaining many derivatives pyrazolo[1,5-a]pyrimidines.
Thus, interaction of the aminopyrazole derivative 3a
with 3-(4-chlorophenyl)-1-phenylprop-2-en-1-one afforded
a single product which was analyzed on the basis of
spectral data as 2-amino-7-(4-chlorophenyl)-5-phenyl-3-[(3-
trifluoromethyl phenyl)diazenyl][1,5-a]pyrimidine (9). The
formation to proceed via initial condensation of the exo-
cyclic amino function of compound 3a with the carbonyl
Furthermore, reaction of compound 3a with arylidene-
malononitrile in ethanol containing a few drops of piperidine,
the 7-aminopyrazolo[1,5-a]pyrimidine 10 seemed possible
product for such reaction on 1H NMR spectra which revealed
a signal corresponding to the amino group at δ >7.00 ppm.
De-shielding of 7-aminopyrazolopyrimidine protons by ring
nitrogen anisotropy has been previously observed [21, 30,
31]. Compound 10 proposed to form via initial addition of
the exocyclic amino group in 3a to the activated double bond
and cyclization through addition of the pyrazole NH to the
cyano group and spontaneous auto-oxidation through the loss
of the H2 molecule. Structure 10 was assigned upon com-
1
patible elemental analyses and spectral data. The H NMR
spectrum (DMSO-d6, δ/ppm) of 10a as example revealed
the presence of two singlet signals at δ 7.20, 8.57 ppm corre-
sponding for 2NH2 protons which confirm its structure. Mass
spectrum of compound 10a exhibited a molecular ion peak at
m/z 422 (47.49%) corresponding to C20H13F3N8 with a base
peak at m/z 222. The fragmentation pattern for 10a–c showed
123

Molecular Diversity
F3C
N
N
NHCOCH3
Ac2O
H3COCHN
NH
4
N
F3C
F3C
CH3
O
N N
H2N
N
CH3
N
N
N
CH3COCH2COCH3
CH3COOH
N
N
H2N
NH
F3C
N
CH3
H3C
N
N
NH2
NH
5
F3C
H2N
N
N N
N
CH3
3a
N
6
H2N
N
CH3COCH2COPh
CH3COOH
F3C
N N
H2N
N
X
7
N
N
CH3
F3C
F3C
CH3
CH3COCHRCOOC2H5
CH3COOH
N N
H2N
N
N
C
H
N
N
N
R
3
R
N
H
NH
H2N
N
O
O
C2H5O
F3C
8
N N
H2N
N
CH
3
N
N
R
OH
8 a, R=H
b, R=CH3
Scheme 2 Synthesis of compounds 5–8
three fragments for 10a at m/z 103 (6.4%) corresponding for
as a reaction product on the basis of spectral data. Compound
11 proposed to form via initial addition of the exocyclic
amino group in 3a to the unsaturated system and cycliza-
tion through nucleophilic attack of the pyrazole NH to the
ester with elimination of ethanol molecule and spontaneous
auto-oxidation through the loss of the H2 molecule. The
structure 11 was inferred from its spectral data. Thus, the IR
spectrum of showed absorption bands at 3412, 3266, 3150
and 2215 cm−1 corresponding to OH, NH2 and CN for 11a,
1H NMR for compound 11b showed two signals at δ 2.41,
6.82 ppm for CH3 and NH2, a broad signal was observed at
δ 8.25 ppm for OH, and the aromatic protons were observed
·+
PhCN ·+, for 10b at m/z 117 (3.94%) for CH3C6H4CN
and for 10c at m/z 133 (7.79%) for CH3OC6H4CN ·+ which
confirm the structure. 13C NMR spectrum of 10b showed
signals at 21.46 (CH3), 76.38 (C4, pyrazole), 117.52 (CN),
124.51 (CF3), 147.04 (C5, pyrazole), 129.11, 129.37, 130.83,
130.76, 140.75, 149.31, 152.48 (C, aromatic) and 153.56,
161.68 (2C=N).
In a similar manner interaction of the aminopyrazole 3a
withethylcyanocinnamatesinethanolcontainingafewdrops
of piperidine, two products the 7-hydroxy 11 or 7-amino 12
seemed to be possible products for such reaction. However,
the 7-hydroxypyrazolo[1,5-a]pyrimidine 11 was established
123

Molecular Diversity
O
F3C
F3C
N N
H2N
Cl
N
N
N
N
H
ethanol/ piperidine
N
NH
H
H2N
N
N
Cl
Cl
9
R
F3C
R
F3C
F3C
N
N
H
N
N
N
NH2
NH
R
CH=C(CN)2
N
N
N
H
N
ethanol/ piperidine
H2N
N
H2N
CN
H2N
NH
N
C
N
NH2
CN
N
3a
10
10 a, R= H
b, R= CH3
c, R= OCH3
F3C
R
N
N
N
11
11 a, R= H
N
CN
H2N
N
CN
OH
R
CH=C
b, R= CH3
COOC2H5
c, R= OCH3
ethanol/ piperidine
F3C
R
N
N
N
X
12
N
H2N
N
COOC2H5
NH2
Scheme 3 Synthesis of compounds 9–12
as three doublets at δ 7.33, 7.54 and 7.91 ppm and a triplet
at δ 7.59 ppm and a singlet signal at δ 7.97 ppm.
standard with (IC50 ꢀ4.69, 7.73 μg/mL) against HepG-2
and MCF-7, respectively, while showed one-third of activity
of Taxol against HCT-116 with (IC50 ꢀ6.63 μg/mL).
Based on our literature survey, it was revealed that
pyrazolo[1,5-a]pyrimidines are of considerable anticancer
activity, and our efforts were directed to predict the
scope for either keeping or enhancing the potency of 3,5-
diaminopyrazole compound 3a by appropriate chemical
modification using extension strategy of compound 3a on the
hope of obtaining new pyrazolo[1,5-a]pyrimidines deriva-
tives with considerable anticancer activities.
Anti-tumor activity
Ten of the newly pyrazole-based heterocyclic compounds 3a,
5, 6, 8, 9, 10a–c, 11a–b, were screened for their anticancer
activity against three cell lines which are human liver carci-
noma cell line (HepG-2), colon cancer cell line (HCT-116)
and human breast cancer cell line (MCF-7), the in vitro anti-
tumor activity was achieved in the cell culture laboratory,
College of Pharmacy, Al-Azhar University, Cairo, Egypt,
Taxol was used as a reference standard, and it showed IC50
of 4.3, 2.9, 4.7 μg/mL, respectively.
Out of the newly synthesized pyrazolo[1,5-a]pyrimidines,
it was found that 2-amino-5-methyl-7-phenyl-3-(3-
trifluoromethylphenyldiazenyl)pyrazolo[1,5-a]pyrimidine
(6) showed promising anticancer activity ranging from
The anticancer activity profile suggested that the tested
compounds showed variable activities compared to reference
drug as shown in (Table 1 and Fig. 1).
Out of the 3,5-diaminopyrazole series, compound 3,5-
diamino-4-(3-trifluoromethylphenyldiazenyl)-1H-pyrazole
3a, was chosen to be our starting compound that was found
to be approximately equipotent of activity to the reference
(0.5–0.33) of activity of the reference standard (IC50
ꢀ
18.3, 7.64, 11.4 μg/mL) against HepG-2, HCT-116 and
MCF-7, respectively; while its counterparts (that contain
7-methyl (compound-5), 5,7-diphenyl (compound-9) or
5,6-dimethyl substituents (compound-8) instead of 7-phenyl
123

Molecular Diversity
Table 1 IC50 of the tested
compounds against human
hepatocellular carcinoma cell
line (HepG-2), human colon
cancer cells (HCT-116) and
human breast cancer cells
(MCF-7)
Compd. no.
IC50 (μg/mL)
HepG-2 cells
IC50 (μg/mL)
HCT-116 cells
IC50 (μg/mL)
MCF-7 cells
3a
4.69
36.0
18.3
45.1
39.0
24.8
0.64
10.3
2.41
8.47
4.3
6.63
11.6
7.64
38.0
19.6
22.6
1.89
22.5
2.39
12.0
2.9
7.73
14.2
11.4
43.0
23.6
23.5
2.79
23.8
3.09
11.2
4.7
5
6
8
9
10a
10b
10c
11a
11b
Taxol
Fig. 1 IC50 of the tested
compounds against human hep-
atocellular carcinoma cell line
(HepG-2), human colon can-
cer cells (HCT-116) and human
breast cancer cells (MCF-7)
in (compound-6) showed remarkable decreased in anticancer
activity.
a]pyrimidine-6-carbonitrile 11b showed promising
anticancer activity, and it showed promising cytotoxic
Surprisingly, it is worth to mention that the pyra-
zolopyrimidine scaffold 10b and 11a that bearing amino
and cyano substituent in 2 and 6 positions, respectively,
remarkably induced lowest cell growth promotion when
compared with its pyrazolopyrimidine counterparts or the
reference drug Taxol, and it also exhibited broad spec-
trum cell growth inhibition against all the three tested
cell lines. It worth to mention that 2,7-diamino-5-p-
tolyl-3-((3-(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-6-carbonitrile 10b showed 7 times higher
activity than Taxol against HepG-2, 1.5 higher activ-
ity than Taxol against HCT-116 and about two times
higher activity than Taxol against MCF-7 (IC50 ꢀ0.64,
1.89, 2.79 μg/mL, respectively). The non-substituted
phenyl or 4-methoxy-substituted analogs (10a, 10c)
lead to remarkable decrease in anticancer activity.
Similarly, Compound 2-amino, 7-hydroxy-5-p-tolyl-
3-((3-(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
activity on HepG-2, MCF-7 and HCT-116 with (IC50
8.47, 11.2 and 12 μg/mL), respectively.
ꢀ
Cell cycle analysis
In response to DNA damage, cells arrest primarily at
the G1/S and G2/M boundaries. G1/S arrest blocks entry
of damaged DNA into the S phase, and G2/M arrest pro-
hibits entry of damaged DNA into mitosis. To analyze the
possible mechanism underlying the anti-proliferative effect
exerted by our new compounds, MCF-7 cell cycle progres-
sion was assessed following treatment with compounds 3a,
10b and 11a (compounds that had the marked highest anti-
proliferative activity on the human MCF-7 cell line for 24
using propidium iodide flow cytometry analysis [33, 34]. The
apoptosis-inducing activity of 3a, 10b and 11a was also char-
acterized by flow cytometric analysis of the DNA profile in
MCF-7 (Table 2, Figs. 2, 3).
123

Molecular Diversity
Table 2 MCF-7 cell cycle
distribution of compounds 3a,
10b and 11a
Compd. no.
G0/G1%
S %
G2/M %
Apoptosis %
Necrosis %
3a/MCF7
61.72
54.47
45.88
69.16
21.04
22.68
20.35
26.58
8.92
12.63
19.41
3.69
8.32
10.22
14.36
0.57
0.85
0.96
2.12
0.22
10b/MCF7
11a/MCF7
cont.MCF7
Fig. 2 Percentage of each cell
cycle phase with various treat-
ments or with control
Caspase 3 enzyme assay
11a may induce antineoplastic activity of MCF-7 cell via a
caspase-dependent pathway.
Disruption of apoptotic pathway has been established as a
prominent hallmark of several cancers [5]. Consequently,
the activation of apoptosis is emerging as a key approach
to treat different tumors. The unique characteristics of apop-
tosis are the activation of various enzymes that are cysteine
protease families called caspases [35]. Caspase 3 is consid-
ered the key protein of the caspase family, which are highly
conserved in multicellular organisms and function as central
regulators of apoptosis [36–40]. Clarifying the role of cas-
pases in newly synthesized derivatives-induced cancer cell
death is important for understanding molecular mechanisms
of the antineoplastic effect of those derivatives. To elucidate
a pathway leading to new derivatives-induced cell death, we
determined the ability of the most potent compounds 3a, 10b
and 11a to activate caspase 3 in cancer cell line (MCF-7).
Compared with the control, we tested the increase in caspase
3 activities in the tested cell in response 3a, 10b and 11a treat-
ment at its IC50. As shown in Table 3, Fig. 4, the activities of
caspase 3 were increased following the treatment of MCF-7
cell with 3a, 10b and 11a. In details, treatment of MCF-7
cells with 3a, 10b caused a significant increase in caspase 3
level by about fivefold, while treatment of MCF-7 cells with
11a caused increase in caspase 3 level by about 3.6 folds,
compared to control,. These results suggest that 3a, 10b and
Molecular modeling studies
Docking analysis
In order to analyze the binding mode of the most potent com-
pounds 3a, 10b and 11a into caspase 3 active site, the binding
affinities of these compounds to binding sites in addition to
the hydrogen bonds formed were determined using Accel-
rys Discovery studio 2.5 software, (Caspase X, PDB code:
3KJF).
From the docking results, it could be seen that all the three
docked compounds lie in the right chain (Chain B), where
CDOCKER energy score of compound 3a was 38,67 and
formed two hydrogen bonds interaction one of them between
hydrogen of NH2 and SER 205, the second hydrogen bond
between F of CF3 and PHE 250. Compound 10b, formed
two hydrogen bond, one of them between the hydrogen of
amino group and SER 205 and second between CF3 and
PHE 250 with CDOCKER energy score 29.16. Finally, Com-
pound 11a, formed three hydrogen bond, the first of them
between the hydrogen of amino group and ARG 207, the
second between oxygen of hydroxyl group and ARG 207
and the third hydrogen bond formed between CF3 and PHE
123

Molecular Diversity
Fig. 3 Effects of 3a, 10b and 11a on MCF-7 cell cycle profile at its
IC50 compared to untreated cell using flow cytometry data for 24 h; a
untreated MCF-7 cell; (A1) MCF-7 cell treated with 3a; (A2) MCF-7
cell treated with 10b; (A3) MCF-7 cell treated with 11a; (B-B3) tested
compounds Induced apoptosis in human MCF-7 cell as assayed by
annexin V staining compared to control cell using flow cytometry for
24 h; b control cell, (B1), (B2) and (B3) Representative scatter plots of
PI (y axis) vs annexin V (x axis) showing the effects of 3a, 10b and
11a, respectively, on MCF-7 cell apoptosis
123

Molecular Diversity
Table 3 Caspase 3 enzyme assay of the most active compounds against
MCF-7
pharmacophore features used are hydrogen bond acceptor
(HBA), hydrogen bond donor (HBD), hydrophobic (HYP),
ring aromatic (RA) and negative ionizable features. Fisher
validation was applied and set to 95% significance. Pharma-
cophores were then generated in HypoGen module, and the
top ten scoring hypotheses were exported. HypoGen identi-
fies features common to the active compounds and excludes
features common to the inactive compounds within con-
formational allowable regions of space. It further estimates
the activity of our newly synthesized and tested compounds
using regression parameters. The parameters were computed
by regression analysis using the relationship of geometric
fit value versus the activity. The better the geometric fit the
greater the activity prediction of the compound.
Compd. no.
Caspase 3 conc. Pg/mL
3a
564
10b
542.5
392.7
110.1
11a
Cont. MCF-7
Pharmacophore study results
Ten predictive pharmacophore models (hypothesis) were
generated via aligning different conformations of the rep-
resented training set ligands to bind with the generated
pharmacophore models. For the three tested human cell lines,
liver carcinoma cell line (HepG-2), colon cancer cell line
(HCT-116) and human breast cancer cell line (MCF-7), the
top pharmacophore model generated, Hypothesis 2, 1, 7,
respectively, all of the generated pharmacophore models con-
tained at least three chemical features one HBD, one HYP
(except HepG-2 that contain two HYP), and one RA feature
Figs. 6, 7 and 8.
It is worth to be mentioned that the results of anticancer
activity were consistent with pharmacophore fit score val-
ues where it was observed that the most potent anticancer
compounds showed high fit values. From the results, it is
important to note that in the best mapping conformations
of the ligand compound 10b and 11a showed that the free
amino group (NH2) of pyrazole is mostly fits with feature
of hydrogen bond (HBD) while the phenyl moiety of Cf3-
C6H5 fits with RA feature. In addition the trifluoromethyl
group of Cf3-C6H5 fits with (HYP), and the terminal methyl
group of CH3-C6H5 fits with the second (HYP_2) in HepG-2
pharmacophore model.
Fig. 4 Effect of 3a, 10b and 11a on caspase 3 activity in MCF-7 cells
measured by ELISA-based assay. The experiment was done in triplicate.
Data are mean SEM. * significantly different from control at p <0.05
252 with CDOCKER energy score 29.1628.82 These results
were in consistency with the binding mode of the original
ligand compound with CDOCKER energy score 56.05 and
four hydrogen bond interactions with ARG 207, SER 209
and SER 205; at the same time the docking results came
in agreement with the results of our caspase enzyme assay
(Fig. 5).
Modeling study (Pharmacophore modeling)
Physical methods such as pharmacophore modeling and
docking studies, where the interpretability is much more
explicit, could be used for preliminary SAR interpretation.
In such a way pharmacophore methods can provide a much
more direct view into structural features contributing to the
SAR.
In this study, pharmacophore generation was performed
using Discovery Studio 2.5 software. The 3D QSAR Pharma-
cophore Generation protocol (Catalyst HypoGen algorithm)
was used to derive structure activity relationship hypothesis
models (3D QSAR pharmacophore models) from a set of lig-
ands with known activity values on a given biological target.
The training set is composed of ten synthesized compounds
from the present study with measured IC50 for the against
three human cell lines for the generation of pharmacophore
models considered as the certainty value. The uncertainty
value was set as 1.5 instead of the default value 3.0. The
The previous preliminary analysis and the above dis-
cussion for the observed anticancer activity could be used
to point out several structural requirements for the newly
synthesized compounds as anticancer agents which can be
summarized as follow;
• For the newly synthesized compounds either pyrazole
or pyrazolopyrimidine-based heterocycles, the free polar
(NH2) group in either position 3 of non-fused pyrazole
scaffold or position 2 in pyrazolopyrimidine containing
compounds might be requisite for anticancer activity (as
concluded from pharmacophore models generation and
123

Molecular Diversity
Fig. 5 Docking simulation of original ligand (a), and the most active compounds; compound 3a (b), compound 10b (c) and compound 11a (d) on
the caspase-3 active site
docking results in which such the free amino (NH2) is
mostly fits with feature of hydrogen bond.
• For all pharmacophore models, the trifluoromethyl moi-
ety was mostly fitted with the hydrophobic feature (HYP),
which emphasizes on the importance of fluorine-building
blocks in pyrazolopyrimidine scaffold substitution; this
result was consistent with our previous literature survey
with relevant to fluorinated molecule.
• Pyrazolopyrimidine compounds containing CN group at
position 6 displayed highest anticancer activity profile,
whereas the un-substituted position 6 of pyrazolopyrimi-
dine scaffold or any other substitution than CN group leads
to decrease in anticancer activity.
• Substitution at position 5 of pyrazolopyrimidine scaffold
with phenyl or substituted phenyl group has a good impact
on anticancer activity profile than any other substitution,
which might be due to increasing binding affinity through
hydrophobic binding involvement.
It is important to realize that the choice of modeling tech-
nique can influence to what extent and in how much detail
an SAR can be explored. Further strong predictive model
for structure activity relationship will be planned for future
studies.
123

Molecular Diversity
Fig. 6 Best generated pharmacophore model for HepG-2 cell line where
(1-A) showed synthesized structure compound (10b) fitted in the phar-
macophore with fit value 6.62, (1-B) showed synthesized structure
compound (11a) fitted in the pharmacophore with fit value 5.47, with
the features considered hydrogen bond donor (HBD) colored in violet,
ring aromatic (RA) colored in orange, hydrophobic (HYP) colored in
cyan. (Color figure online)
Fig. 7 Best generated pharmacophore model for HCT-116 cell line
where (2-A) showed synthesized structure compound (10b) fitted in the
pharmacophore with fit value 6.14, (2-B) showed synthesized structure
compound (11a) fitted in the pharmacophore with fit value 6.13, with
the features considered hydrogen bond donor (HBD) colored in violet,
ring aromatic (RA) colored in orange, hydrophobic (HYP) colored in
cyan. (Color figure online)
Pharmacophore validation
(99.32, 61.02), respectively, forHypothesis(2, 1, 7);alsothey
were chosen as the best generated pharmacophore hypothe-
sis as they are characterized by the lowest total cost the best
correlation coefficient which indicates the capability of the
pharmacophore model to predict the activity of the training
set compounds. The pharmacophore models (hypothesis 2, 1,
7) were also validated through activity prediction of the syn-
thesized structures as training set. It should be noted that the
predicated IC50 by the 3D Pharmacophore QSAR model was
close to those experimentally observed, indicating that these
models can be safely applied for prediction of anticancer
activity of newly synthesized compounds (Table 4).
The pharmacophore models generated should be statistically
significant, abletopredicttheactivitiesofnewchemicalcom-
pounds and retrieve active compounds from the database.
The selection of the generated pharmacophore models 2, 1,
7 for (HepG-2), colon cancer cell line (HCT-116) and human
breast cancer cell line (MCF-7) was based on its valida-
tion using two methods: cost analysis and activity prediction.
HypoGen selects the best hypothesis by applying a cost anal-
ysis. The quality of the generated pharmacophore hypothesis
was evaluated by considering the cost functions calcula-
tion by HypoGen module during hypothesis generation. In
detail, the null cost and fixed cost of the three pharmacophore
hypothesis were equal to (129.21, 61.89), (104.68, 61.11) and
123

Molecular Diversity
Fig. 8 Best generated pharmacophore model for MCF-7 cell line where
(3-A) showed synthesized structure compound (10b) fitted in the phar-
macophore with fit value 5.65, (2-B) showed synthesized structure
compound (11a) fitted in the pharmacophore with fit value 6.18, with
the features considered hydrogen bond donor (HBD) colored in violet,
ring aromatic (RA) colored in orange, hydrophobic (HYP) colored in
cyan
Table 4 Fit values and predicted activities the synthesized compounds mapped with the generated 3D-pharmacophore models
Compound
HepG-2
HCT-116
MCF-7
Predicted
activity
Exp.
activity
IC50
Fit value
Predicted
activity
Exp.
activity
IC50
Fit value
Predicted
activity
Exp.
activity
IC50
Fit value
(mg/mL)
(mg/mL)
(mg/mL)
3a
5
17.96
16.65
14.31
17.27
19.42
14.69
0.99
4.69
36
5.36
5.46
5.46
5.46
5.46
5.44
6.62
5.68
5.47
5.58
11.65
17.62
9.588
22.04
10.429
14.80
4.02
6.63
11.6
7.64
38
6.03
5.95
6.11
6.11
6.14
5.88
6.14
6.07
6.13
6.07
16.77
17.03
17.51
20.20
20.94
16.54
3.91
7.73
14.2
11.4
43
5.55
5.65
5.63
5.57
5.56
5.66
5.65
5.65
6.18
6.10
6
18.3
45.1
39
8
9
19.6
22.6
1.89
22.5
2.39
12
23.6
23.5
2.79
23.8
3.09
11.2
10a
10b
10c
11a
11b
24.8
0.64
10.3
2.41
8.47
3.57
13.82
4.00
16.89
5.024
9.45
13.79
6.09
9.541
Conclusion
At the same time compound 11a showed promising cytotoxic
activity; nearly two times of activity of the reference standard
against HepG-2, 0.75 of activity of the reference standard
against MCF-7 and 1.3 time of activity of Taxol against HCT-
116 (IC50 ꢀ2.41, 2.39 and 3.09 μg/mL), respectively.
Furthermore, we observed the combined effect on cell
apoptosis, cell cycle and caspase assays to elucidate the
possible mechanism of 3a, 10b and 11a effects on MCF-
7 cell. The data indicated that compounds 3a, 10b and 11a
inhibited tumor proliferation by inducing cell cycle arrest
in the G2/M phase of MCF-7 cells and induced cancer
cell apoptosis, probably, via caspase 3-dependent pathway.
The combination of docking studies and 3D-pharmacophore
modeling provides as an effective technique for understand-
ing the observed pharmacological properties and thus could
be adopted for developing effective lead structures. These
In an effort to develop potent anticancer agents, the present
work demonstrated the synthesis and quantitative evalua-
tion of a series of novel pyrazole-based heterocycles that
might be efficient anti-tumor agents. The cytotoxicity of
the newly synthesized compounds and the reference stan-
dard Taxol were determined by MTT method using HepG-2
cell line, HCT-116 and MCF-7. Cytotoxicity assay results
revealed that some of the compounds specifically, 3a, 10b,
and 11a, showed potent growth inhibition against all the cell
lines tested. It was observed that pyrazolo[1,5-a]pyrimidine-
6-carbonitrile 10b was the most potent anticancer agent; it
showed 7 times higher activity than Taxol against HepG-2,
1.5 times higher activity than Taxol against HCT-116 and
about two times higher activity than Taxol against MCF-7.
123

Molecular Diversity
results imply that the most active compounds might repre-
sent targets for further optimization.
4-((3-(Trifluoromethyl)phenyl)diazenyl)-1H-
pyrazole-3,5-diamine (3a)
Fluorescent yellowish crystals; yield: (78%); m.p.:
226–228 °C; IR (KBr, cm−1): 3404, 3295, 3200 (2NH2,
NH) and 1614 (C=N), 1563 (N=N); H NMR (DMSO-d6)
Experimental
1
δ/ppm: 5.96, 6.48 (2 s, 4H, 2NH2, D2O exchangeable), 7.48
(d, 1H, J=9 Hz, Ar H,), 7.55 (t, 1H, Ar–H), 7.92 (d, 1H,
J=9 Hz, Ar–H), 8.01 (s, 1H, Ar H),; 10.79 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO-d6) δ/ppm: 66.7, 154.05
(3C-pyrazole), 116.36 (CF3), 122.5, 124.33, 129.53, 129.69,
129.95, 130.37 (Ar–C); MS (m/z): 270 (M+., 18.55%),
213 (5.32%), 173 (3.85%), 145 (100%), 95 (24.42%), 42
(29.22%); Anal. Calc. for C10H9F3N6 (270.21): C, 44.45%;
H, 3.36, %; N, 31.40%. Found: C, 44.61%; H, 3.34%; N,
31.19%;
All melting points were taken on Electrothermal LA 9000
SERIES, Digital Melting point Apparatus and were uncor-
rected. IR Spectra were determined using KBr disk technique
on Nikolet IR 200 FT IR Spectrophotometer at Pharmaceu-
tical Analytical Unit, Faculty of Pharmacy (Boys), Al-Azhar
University, and values are represented in cm−1. The 1H
NMR Spectra were recorded on Gemini 300 MHz, and Mer-
cury 400 MHz and 13C NMR spectra at 100 MHz on NMR
Spectrometer at the Main Chemical Warfare Laboratories,
ChemicalWarfareDepartment, MinistryofDefense. DMSO-
d6 was used as a solvent; chemical shifts were measured in δ
ppm, relative to TMS as an internal standard. Mass Spectra
were recorded at 70 eV on DI-50 unit of Shimadzu GC/MS-
QP5050A Spectrometer at Regional Center for Mycology
and Biotechnology, Al-Azhar University. Microanalysis was
carried out at Regional Center for Mycology and Biotech-
nology, Al-Azhar University. Progress of the reaction was
monitored by using TLC sheets precoated with UV fluores-
centsilicagelMerck60F254platesandwerevisualizedusing
UV lamp.
1-Phenyl-4-((3-(trifluoromethyl)phenyl)diazenyl)-
1H-pyrazole-3,5-diamine (3b)
Yellow powder; yield: (91%); m.p.: 160–162 °C; IR (KBr,
cm−1): 3439, 3343, 3260, 3161 (2NH2), and 1610 (C=N),
1505 (N=N); 1H NMR (DMSO-d6) δ/ppm: 6.23, 6.93 (2 s,
4H, 2NH2, D2O exchangeable); 7.28–7.64 (m, 7H, Ar H,
Ar–H), 8.02 (d, 1H, J=9 Hz, Ar–H), 8.12 (s, 1H, Ar H); MS
(m/z): 346 (M+., 50.40%) and 77 (100%); Anal. Calc. for
C16H13F3N6 (346.31): C, 55.49%; H, 3.78, %; N, 24.27%.
Found: C, 55.72%; H, 3.81%; N, 24.52%.
(3-(Trifluoromethyl)phenyl)carbonohydrazonoyl
dicyanide (2)
3,5-Diamino-4-((3-
(trifluoromethyl)phenyl)diazenyl)-1H-pyrazole-
1-carbo-thioamide (3c)
To an ice-cooled solution of m-trifluoromethylaniline 1
(1.61 g, 0.01 mol) in hydrochloric acid (2.5 mL) and distilled
water (5 mL), a solution of sodium nitrite (0.69 g, 0.013 mol)
in distilled water (5 mL) was added. The cold diazotized
solution was then added portion-wise to a well-stirred cold
solution of malononitrile (0.66 g, 0.01 mol) in 50% aqueous
ethanol (10 mL) containing sodium acetate (0.9 g, 0.01 mol).
The reaction mixture was kept in ice for 2 h and then filtered.
The obtained solid was dried and crystallized from ethanol.
Yellow colored powder; yield: 77%; m.p.: 146–148 °C; IR
(KBr, cm−1): 3227 (NH), 3089 (CH-aromatic), 2220 (2CN),
1603 (N=C).
Orange powder; yield: (74%); m.p.: 265–267 °C; IR (KBr,
cm−1): 3361, 3269, 3174 (3NH2), 1620 (C=N) and 1524
(N=N); 1H NMR (DMSO-d6) δ/ppm: 4.46 (s, 2H, CSNH2,
D2O exchangeable), 6.99–7.24 (hump, 4H, 2NH2, D2O
exchangeable), 7.67 (d, H, J=9 Hz, Ar H), 7.96 (t, H, Ar–H),
8.31 (d, H, J=9 Hz, Ar–H), 8.40 (s, 1H, Ar H); MS (m/z): 329
(M+, 1.13%), 178 (100.0%); Anal. Calc. for C11H10F3N7S
(329.31): C, 40.12%; H, 3.06, %; N, 29.77%. Found: C,
40.12%; H, 3.11%; N, 29.89%.
N,N’-(4-((3-(trifluoromethyl)phenyl)diazenyl)-1H-
pyrazole-3,5-diyl)diacetamide (4)
4-((3-(Trifluoromethyl)phenyl)diazenyl)-1H-
pyrazoles-3,5-diamine (3a–c)
A solution of 3 (2.7 g, 0.01 mol) in acetic anhydride (10 mL)
was warmed on water bath for 2 h. The reaction mixture was
cooled and the solid product was collected and recrystallized
from ethanol.
Brown powder; yield: (72%); m.p.: 190–192 °C; IR (KBr,
cm−1): 3285, 3144 (3NH), 1680 (br, 2C=O), 1562 (N=N);
1H NMR (DMSO-d6) δ/ppm: 2.14, 2.19 (2 s, 6H, 2CH3),
A solution of 2 (2.38 g, 0.01 mol) and hydrazine hydrate,
phenyl hydrazine or thiosemicarbazide (0.01 mol) in ethanol
(30 mL) was heated under reflux for 5–10 h. The reaction
mixture was cooled, and the solid product was collected and
recrystallized from ethanol.
123

Molecular Diversity
6.75, 10.14 (2 s, 3H, 3NH; D2O exchangeable), 7.48 (d, 1H,
J=9 Hz, Ar H), 7.56 (t, 1H, J=9 Hz, Ar–H), 8.10 (d, 1H,
J=9 Hz, Ar–H), 8.15 (s, 1H, Ar H); MS (m/z): 354 (M+,
5.91%) and 43 (100.0%); Anal. Calc. for C14H13F3N6O2
(354.11): C, 47.46%; H, 3.70, %; N, 23.72%. Found: C,
47.62%; H, 3.68%; N, 23.93%.
Calc. for C20H15F3N6 (396.37): C, 60.40%; H, 3.81, %; N,
21.20%. Found: C, 60.37%; H, 3.81%; N, 21.47%.
2-Amino-5-methyl-3-(3-
trifluoromethylphenyldiazenyl)pyrazolo[1,5-
a]pyrimidine-7-oles (8a, b)
5-Methyl-7-(methyl or phenyl)-3-(3-
trifluoromethylphenyldiazenyl)pyrazolo[1,5-
a]pyrimidine-2-amino (5, 6)
To a solution of 3a (0.01 mol) in acetic acid (20 mL),
ethylacetoacetateorethylmethylacetoacetate(0.01mol)was
added. The reaction mixture was heated under reflux for 6 h.
The reaction mixture was cooled, and the solid product was
collected and crystallized from ethanol.
To a solution of 3a (0.01 mol) in acetic acid (20 mL),
acetylacetone or benzoylacetone (0.01 mol) was added. The
reaction mixture was heated under reflux for 4 h. The reac-
tion mixture was cooled, and the solid product was collected
and recrystallized from ethanol.
2-Amino-5-methyl-3-(3-
trifluoromethylphenyldiazenyl)pyrazolo[1,5-
a]pyrimidine-7-ol (8a)
5,7-Dimethyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidin-2-amine (5)
Orange powder; yield: (79%); m.p.: 280–282 °C; IR (KBr,
cm−1):3429, 3274(OH, NH2), 1629(C=N), 1600(N=N); 1H
NMR(DMSO-d6)δ/ppm:2.37(s, 3H, CH3), 5.93(s, 1H, CH-
pyrim.), 6.71, 10.28 (2 s, 3H, NH2&OH; D2O exchangeable),
7.68–7.79 (m, 2H, Ar H), 8.14 (d, 1H, J=9 Hz, Ar–H), 8.21
(s, 1H, Ar H); MS (m/z): 336 (M.+, 100.00%), 289 (3.71%),
252 (10.83%), 233 (6.05%), 212 (8.12%), 191 (94.67%),
186 (25.62%), 162 (12.95%), 145 (53.82%), 1135 (61.07%),
120 (13.07%), 108 (38.98%), 95 (19.18%),77 (2.89%),
67 (82.06%); Anal. Calc. for C14H11F3N6O (336.27): C,
50.00%; H, 3.30, %; N, 24.99%. Found: C, 50.16%; H,
3.28%; N, 25.23%.
Yellow powder; yield: (73%); m.p.: 249–251 °C; IR (KBr,
1
cm−1): 3281, 3200 (NH2), 1632 (C=N), 1567 (N=N); H
NMR (DMSO-d6) δ/ppm: 2.48, 2.55 (2 s, 6H, 2CH3), 7.00
(s, H, CH-pyrim.), 7.33 (s, 2H, NH2, D2O exchangeable),
7.64–7.72 (m, 2H, Ar H), 8.05 (d, 1H, J=9 Hz, Ar–H),
8.13 (s, 1H, Ar H); 13C NMR (DMSO-d6): 16.33, 24.04
(2CH3), 117.53 (CF3), 66.48, 151.87, 153.38 (3C-pyrazole),
110.47, 145.53, 160.55 (3C-pyrinidine), 124.30, 124.33,
129.20, 129.63, 129.80, 130.0 (Ar–C); MS (m/z): 334 (M+
, 39.39%), 292 (60.12%), 264 (22.84%), 255 (61.20%), 225
(100.0%), 189 (82.62%), 145 (23.34%), 129 (15.33%), 102
(10.06%), 95 (19.18%),77 (16.13%), 67 (14.03%); Anal.
Calc. for C15H3F3N6 (334.30): C, 53.89%; H, 3.92, %; N,
25.14%. Found: C, 54.02%; H, 3.97%; N, 25.32%.
2-Amino-5,6-dimethyl-3-(3-
trifluoromethylphenyldiazenyl)pyrazolo[1,5-
a]pyrimidine-7-ol (8b)
5-Methyl-7-phenyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidin-2-amine (6)
Red crystals; yield: (81%); m.p.: > 300 °C; IR (KBr, cm−1):
3426 (OH), 3283, 3195 (NH2) 1627 (C=N), 1570 (N=N);
1H NMR (DMSO-d6) δ/ppm: 2.06, 2.39 (2 s, 6H, 2CH3),
6.74 (s, 2H, NH2, D2O exchangeable), 7.65–7.73 (m, 2H,
Ar H), 8.10 (d, 1H, J=9 Hz, Ar–H), 8.19 (s, 1H, Ar H),
12.10 (s,1H, OH exchangeable by D2O); 13C NMR: 11.17,
17.68 (2 CH3), 117.67 (C–N=N), 123.29 (CF3), 124.60,
125.19, 126.00, 128.71, 130.28, 130.59, 130.63, 155.77,
172.46 (Ar–C). MS (m/z): 350 (M.+, 59.59%), 293 (6.73%),
266 (6.77%), 255 (1.22%), 225 (3.88%), 205 (56.62%),
191 (3.55%), 177 (38.33%), 161 (16.9), 149 (100.0%), 122
(29.98%), 95 (24.82%),77 (7.58%), 67 (76.72%); Anal. Calc.
for C15H13F3N6O (350.30): C, 51.43%; H, 3.74, %; N,
23.99%. Found: C, 51.70%; H, 3.81%; N, 24.17%.
Yellow powder; yield: (75%); m.p.: 290–292 °C; IR (KBr,
cm−1): 3398, 3271 (NH2), 1620 (C=N), 1557 (N=N); H
1
NMR (DMSO-d6) δ/ppm: 2.64 (s, 3H, CH3), 7.22 (s, H, CH-
pyrim.), 7.27 (s, 2H, NH2, D2O exchangeable), 7.58–8.15
(m, 9H, Ar H); 13C NMR (DMSO-d6) δ/ppm: 24.60 (CH3),
118.19 (CF3), 152.26, 153.81 (3C-pyrazole), 110.71, 145.31,
161.68 (3C-pyrimidine), 115.63, 118.23, 124.60, 124.84,
126.03, 128.87, 129.98, 130.23, 130.55, 130.76, 131.40,
148.16 (Ar–C); MS (m/z): 396 (M+, 52.00%), 368 (17.56),
326 (3.42), 251 (100), 223 (15.87), 196 (27.3%), 180
(7.02%), 168 (28.64%), 145 (17.17%), 129 (39.28%), 115
(4.47%), 102 (7.6%), 77 (12.97%), 67 (13.21%); Anal.
123

Molecular Diversity
7-(4-Chlorophenyl)-5-phenyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-2-amine (9)
(s, 2H, NH2 exchangeable by D2O), 7.36 (d, 3H, J=9 Hz,
Ar–H), 7.69 (t, 1H, Ar–H), 7.77 (d, 2H, J=6 Hz, Ar–H),
8.05 (d, 1H, J=6 Hz, Ar–H), 8.15 (s, 1H, Ar–H), 8.56 (s, 2H,
NH2 exchangeable by D2O);13C NMR: 21.46 (CH3), 117.52
(C≡N), 124.51(CF3), 76.38, 129.11, 129.37, 130.83, 130.76,
140.75, 147.04, 149.31, 152.48, 153.56, 161.68 (Ar–C);
MS (m/z): 436 (M.+, 41.82%), 408 (9.63%), 347 (2.99%),
291 (100.0%), 263 (76.99%), 236 (65.49%), 209 (13.59%),
167 (11.39%), 145 (22.97%), 140 (23.21%), 139 (5.51%),
118 (16.77%), 91 (18.20%); Anal. Calc. for C21H15F3N8
(436.39): C, 57.80%; H, 3.46, %; N, 25.68%. Found: C,
58.02%; H, 3.48%; N, 25.89%.
To a mixture of compound 2a (2.7 g, 0.01 mol) and 3-(4-
chlorophenyl)-1-phenylprop-2-en-1-one (2.42 g, 0.01 mol)
in ethanol (30 mL), three drops of triethylamine were added.
The resulting mixture was heated under reflux for 4 h and
allowed to cool and diluted with cooled water. The formed
solid product was collected by filtration and crystallized from
ethanol.
Yellow powder; yield: (74%); m.p.: 225–227 °C; IR (KBr,
cm−1): 3423, 3307 (NH2) 1623 (C=N), 1555 (N=N); H
1
NMR (DMSO-d6) δ/ppm: 7.40 (s, 2H, NH2, D2O exchange-
able),7.89 (s, 1H, CH-pyrim.), 7.59–8.40 (m, 13H, Ar–H);
MS (m/z): 492 (M.+, 74.86%), 494 (26.12%), 359 (19.87%),
347 (28.83%), 292 (18.17%), 266 (18.25%), 255 (57.42%),
246 (81.15%), 225 (100.0%), 189 (25.93%), 145 (14.4%),
129 (25.25%), 102 (11.67%), 77 (34.05%); Anal. Calc. for
C25H16ClF3N6 (492.88) C, 60.92%; H, 3.27, %; N, 17.05%.
Found: C, 61.08%; H, 3.26%; N, 17.18%.
2,7-Diamino-5-(4-methoxyphenyl)-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-a]-
pyrimidine-6-carbonitrile (10c)
Brown powder; yield: (73%); m.p.: 299–300 °C; IR (KBr,
cm−1): 3435, 3307, 3230, 3172 (2NH2), 2214 (CN), 1608
1
(C=N); H NMR (DMSO-d6) δ/ppm: 3.87 (s, 3H, OCH3),
7.11 (m, 5H, 3H, Ar–H and 2H, NH2 exchangeable by D2O),
7.70 (d, 2H, J=6 Hz, Ar–H), 7.74 (m, 2H, Ar–H), 8.05 (d,
2H, J=6 Hz, Ar–H), 8.13 (s, 1H, Ar–H), 8.43 (s, 2H, NH2
exchangeable by D2O); 13C NMR: 55.85 (OCH3), 116.74
(C≡N), 124.52 (CF3), 75.95, 114.225, 117.47, 129.72,
130.59, 130.82, 130.88, 147.03, 149.38, 152.50, 153.58,
161.13, 161.55 (Ar–C); MS (m/z): 452 (M.+, 20.98%), 405
(1.7%), 307 (44.23%), 279 (62.73%), 252 (70.63%), 236
(36.51%), 208 (100.0%), 167 (5.73%), 145 (65.58%), 140
(20.82%), 139 (17.11%), 118 (22.64%), 91 (20.55%); Anal.
Calc. for C21H15F3N6O (452.39): C, 55.75%; H, 3.34, %; N,
24.77%. Found: C, 55.97%; H, 3.41%; N, 25.01%.
2,7-Diamino-5-aryl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidines-6-carbonitrile (10a–c)
A suspension of compound 3a (2.7 g, 0.01 mol) and aryliden-
emalononitriles (0.01 mol) in ethanol (30 mL) was treated
with piperidine (0.5 mL). The mixture was heated under
reflux for 6 h and allowed to cool. The solid product was
collected by filtration and crystallized from ethanol.
2,7-Diamino-5-phenyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-6-carbonitrile (10a)
2-Amino-7-hydroxy-5-aryl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-6-carbonitriles (11a–c)
Yellow powder; yield: (76%); m.p.: > 300 °C; IR (KBr,
cm−1): 3437, 3314, 3176 (2NH2), 2214 (CN), 1611 (C=N);
1H NMR (DMSO-d6) δ/ppm: 7.20 (s, 2H, NH2 exchangeable
by D2O), 7.56 (d, 2H, Ar–H), 7.71 (d, 1H, J=9 Hz, Ar–H),
7.86 (t, 4H, Ar–H), 8.05 (d, 1H, J=9 Hz, Ar–H), 8.16 (s,
1H, Ar–H), 8.57 (s, 2H, NH2 exchangeable by D2O); MS
(m/z): 422 (M.+, 47.49%), 222 (100.0%); Anal. Calc. for
C20H13F3N8 (422.37): C, 56.87%; H, 3.10, %; N, 26.53%.
Found: C, 57.04%; H, 3.07%; N, 26.81%.
A suspension of compound 3a (2.7 g, 0.01 mol) and aryli-
denecyanoacetate (0.01 mol) in ethanol (30 mL) was treated
with piperidine (0.5 mL). The mixture was heated under
reflux for 8 h and allowed to cool. The formed solid product
was collected by filtration and crystallized from ethanol.
2-Amino-7-hydroxy-5-phenyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-6-carbonitrile (11a)
2,7-Diamino-5-p-tolyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-6-carbonitrile (10b)
Yellow powder; Yield: (65%); m.p.: >300 °C; IR: υ/cm−1
ꢀ3412, 3266, 3150 (OH&NH2), 3050 (CH-aromatic), 2215
(C≡N), 1618 (C=N); (DMSO-d6) δ/ppmꢀ6.87 (s, 2H, NH2
exchangeable by D2O), 7.51–7.97 (m, 9H, Ar–H); 8.10
(s, 1H, OH exchangeable by D2O); MS (m/z): 423 (M.+,
32.35%), 278 (89.82%), 223 (51.69%), 195 (34.37%), 145
Orange powder; yield: (78%); m.p.: > 300 °C; IR (KBr,
cm−1): 3450, 3307, 3242, 3183 (2NH2), 2214 (CN), 1614
(C=N); 1H NMR (DMSO-d6) δ/ppm: 2.41 (s, 3H, CH3), 7.20
123

Molecular Diversity
(100.0%), 140 (38.67%), 128 (42.08%), 77 (32.67%), 67
(86.39%); Anal. Calc. for C20H12F3N7O (423.35): C, 56.74;
H, 2.86; N, 23.16. Found: C, 56.98; H, 2.88; N, 23.42.
Evaluation of the anti-tumor activity by MTT assay
Viability of control and treated cells were evaluated using
the MTT assay in triplicate. MTT assay is a laboratory test,
and a standard colorimetric assay (an assay which mea-
sures changes in color) for measuring cellular growth, yellow
MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, a tetrazole] was reduced to purple formazan in the
mitochondria of living cells.
2-Amino-7-hydroxy-5-p-tolyl-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo[1,5-
a]pyrimidine-6-carbonitrile (11b)
Yellow powder; Yield: (63%); m.p.: >300 °C; IR: υ/cm−1
ꢀ3417, 3276 (OH&NH2), 3050 (CH-aromatic), 2958 (CH-
aliphatic), 2215 (C≡N), 1617 (C=N), 1570 (N=N); 1H NMR
(DMSO-d6) δ/ppmꢀ2.41 (s, 3H, CH3), 6.82 (s, 2H, NH2
exchangeable by D2O), 7.33 (d, 3H, J=9 Hz, Ar–H), 7.54
(d, 2H, J=9 Hz, Ar–H), 7.59 (t, 1H, Ar–H); 7.91 (d, 1H,
J=9 Hz, Ar–H); 7.97 (s, 1H, Ar–H); 8.25 (s, 1H, OH
exchangeable by D2O); 13C NMR: 21.49 (CH3), 117.52
(C≡N), 124.51(CF3), 72.71, 129.11, 129.37, 130.83, 130.76,
140.75, 147.04, 149.31, 152.48, 153.56, 161.6,8 (Ar–C); MS
(m/z): 438 (M+1, 15.84%), 437 (M.+, 57.90%), 292 (48.32%),
237 (30.72%), 209 (32.07%), 170 (19.72%), 145 (100%),
143 (33.37%), 142 (51.59%), 140 (45.95%); Anal. Calc. for
C21H14F3N7O (437.38): C, 57.67; H, 3.23; N, 22.42. Found:
C, 57.89; H, 3.30; N, 22.74.
A solubilizing solution dimethyl sulfoxide (DMSO) was
added to dissolve the insoluble purple formazan product into
a colored solution. Briefly, three tumor cell lines were seeded
in 96-well plates containing 100 μL of the growth medium at
adensityof 1 × 104 cells/well. Cells werepermittedtoadhere
for 24 h till confluence, washed with PBS and then treated
with different concentration of compounds in fresh mainte-
nance medium from 50 to 1.56 μg and incubated at 37 °C for
24 h. A control of untreated cells was made in the absence
of test compounds. Untreated cells used as negative control.
Serial twofold dilutions of the tested compounds were added
into a 96-well tissue culture plate using multichannel pipette
(Eppendorf, Germany). After treatment (24 h), the culture
supernatant was replaced by fresh medium. Then, the cells
in each well were incubated at 37 °C with 100 μL of MTT
solution (5 mg/mL) for 4 h. After the end of incubation, the
MTT solution was removed, and then 100 μL of DMSO was
added to each well. The absorbance was detected at 570 nm
using a microplate reader (SunRise TECAN, Inc, USA). The
absorbance of untreated cells was considered as 100%. The
results were determined by three independent experiments
(Liu et al. 1997) [32].
2-Amino-7-hydroxy-5-(4-methoxyphenyl)-3-((3-
(trifluoromethyl)phenyl)diazenyl)pyrazolo [1,5-
a]pyrimidine-6-carbonitrile (11c)
Yellow powder; Yield: (70%); m.p.: >300 °C; IR: υ/cm−1
ꢀ
3411, 3260, 3174 (OH&NH2), 2210 (C≡N), 1610 (C=N); 1H
NMR (DMSO-d6) δ/ppmꢀ3.84 (s, 3H, OCH3), 6.72 (s, 2H,
NH2 exchangeable by D2O), 7.04 (d, 2H, J=9 Hz, Ar–H),
7.49 (d, 1H, J=9 Hz, Ar–H), 7.58 (t, 1H, Ar–H), 7.81 (d,
2H, J=9 Hz, Ar–H), 7.88 (d, 1H, J=9 Hz, Ar–H), 7.94 (s,
1H, Ar–H); 8.00 (s, 1H, OH exchangeable by D2O); MS
(m/z): 454 (M+1, 4.52%), 453 (M.+, 15.57%), 308 (58.08%),
253 (38.89%), 216 (35.75%), 209 (61.10%), 186 (70.82%),
158 (56.73%), 145 (100%), 67 (68.59%), 40 (64.23%); Anal.
Calc. for C21H14F3N7O2 (453.38): C, 55.63; H, 3.11; N,
21.63. Found: C, 55.81; H, 3.49; N, 21.84.
Caspase 3 enzyme assay
Cells were obtained from American Type Culture Collection,
and cells were grown in RPMI 1640 containing 10% fetal
bovine serum at 37 °C, stimulated with the compounds to be
tested for caspase 3, and lysed with Cell Extraction Buffer.
This lysate was diluted in standard diluent buffer over the
range of the assay and measured for human active caspase
3 content (cells are plated in a density of 1.2–1.8×10,000
cells/well in a volume of 100 μL complete growth medium+
100 uL of the tested compound per well in a 96-well plate
for 48 h before the enzyme assay).
Cell lines
Data analysis
Hepatocellular (HepG-2), human colon (HCT-116) and
breast (MCF-7) cancer cells were obtained from the Ameri-
can Type Culture Collection (ATCC, Rockville, MD, USA).
The cells were grown in RPMI-1640 medium, supplemented
with 10% inactivated fetal calf serum and 50 μg/mL gen-
tamycin. The cells were maintained at 37 °C in a humidified
atmosphere with 5% CO2.
The percentage cell viability was calculated using the
Microsoft Excel®. Percentage cell viability was calculated
as follows: according to the following calculation: the per-
centage of cell viabilityꢀ[1−(ODt/ODc)]×100%, where
ODt is the mean optical density of wells treated with the
tested compounds and ODc is the mean optical density of
123

Molecular Diversity
untreated cells. The test compounds were compared using the
IC50 value, i.e., the concentration of an individual compound
leading to 50% cell death that was estimated from graphical
plots of surviving cells versus compound concentrations.
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