Synthesis of new arylazopyrazoles as apoptosis inducers: Candidates to inhibit proliferation of MCF-7 cells

Article abstract of DOI:10.1002/ardp.202000214

New 4-arylazo-3,5-diamino-1H-pyrazole derivatives substituted in the 4-aryl ring with the acetyl moiety were designed and synthesized. The antiproliferative activity of the novel arylazopyrazoles was examined against the MCF-7 cell line. Among all target compounds, 8b (IC₅₀ 3.0 μM) and 8f (IC₅₀ 4.0 μM) displayed higher cytotoxicity as compared with the reference standard imatinib (IC₅₀ 7.0 μM). Further studies to explore the mechanism of action were performed on the most active hit of our library, 8b, via anti-CDK2 kinase activity. It demonstrated good inhibitory effects for CDK2 (IC₅₀ 0.24 μM) with 62.5% inhibition, compared with imatinib. The cell cycle analysis in the MCF-7 cell line revealed apoptosis induction by 8b and cell cycle arrest at the S phase. Docking in the CDK2 active site and pharmacophore modeling confirmed the affinity of 8b to the CDK2 active site. Absorption, distribution, metabolism, and excretion studies revealed that our target compounds are orally bioavailable, with no permeation through the blood–brain barrier.

Full text of DOI:10.1002/ardp.202000214

Received: 26 June 2020
Revised: 15 August 2020
Accepted: 20 August 2020
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DOI: 10.1002/ardp.202000214
F U L L P A P E R
Synthesis of new arylazopyrazoles as apoptosis
inducers: Candidates to inhibit proliferation of MCF‐7 cells
Magda M. F. Ismail | Dalia H. Soliman | Rehab Sabour | Amel M. Farrag
Department of Pharmaceutical Chemistry,
Faculty of Pharmacy (Girls), Al‐Azhar
University, Cairo, Egypt
Abstract
New 4‐arylazo‐3,5‐diamino‐1H‐pyrazole derivatives substituted in the 4‐aryl ring
Correspondence
with the acetyl moiety were designed and synthesized . The antiproliferative activity
Amel M. Farrag, Department of
of the novel arylazopyrazoles was examined against the MCF‐7 cell line. Among all
Pharmaceutical Chemistry, Faculty of
Pharmacy, Al‐Azhar University, Nasr City,
Cairo 11754, Egypt.
target compounds, 8b (IC50 3.0 µM) and 8f (IC50 4.0 µM) displayed higher cyto-
toxicity as compared with the reference standard imatinib (IC50 7.0 µM). Further
studies to explore the mechanism of action were performed on the most active hit of
our library, 8b, via anti‐CDK2 kinase activity. It demonstrated good inhibitory ef-
fects for CDK2 (IC50 0.24 µM) with 62.5% inhibition, compared with imatinib. The
cell cycle analysis in the MCF‐7 cell line revealed apoptosis induction by 8b and cell
cycle arrest at the S phase. Docking in the CDK2 active site and pharmacophore
modeling confirmed the affinity of 8b to the CDK2 active site. Absorption, dis-
tribution, metabolism, and excretion studies revealed that our target compounds are
orally bioavailable, with no permeation through the blood–brain barrier.
Email: amelfarrag@ymail.com
K E Y W O R D S
antiproliferative agents, breast cancer cells, MCF‐7, pyrazole, synthesis
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1
INTRODUCTION
EGFR, CDK2, telomerase, ROS receptor tyrosine kinase, and Aurora‐A
kinase. The most important role of aryl pyrazoles is as an antitumor
Cancer is appraised among the prominent causes of ailment and
deaths across the board.[1,2] Breast cancer is one of the pronounced
widespread cancers in females and the next most common reason for
malignancy‐related mortality. It has also obtruded to be one of the
most common cancers in 2016, amidst other cancers.[l,4] In the face of
the advances in the treatment of solid tumors (e.g., breast and
ovarian cancers), the survival rates are stock‐still markedly low.[5]
This emphasizes the urgent need to develop effective and more se-
lective chemotherapeutic agents. Cancer is usually figured to be a
disease of the cell cycle. Intrinsically, it is not astonishing that the cell
cycle deregulation is one of the most prevalent alterations during
tumor progression. Progression of the cell cycle is a well‐ordered and
strongly controlled process that includes diverse checkpoints that
assess cell size, extracellular growth signals, and DNA integrity.[5]
Pyrazole derivatives play an influential role among antitumor
agents.[6] These compounds have been validated to be useful candidates
agent, with two drugs being available in the market, ruxolitinib
(Jakavi®);
3‐[4‐(7H‐pyrrolo[2,3‐d]pyrimidin‐4‐yl)‐1H‐pyrazol‐1‐yl]‐3‐
cyclopentylpropanenitrile)[7] and crizotinib (XALKORI®; 3‐[(1R)‐1‐(2,
6‐dichloro‐3‐fluorophenyl)ethoxy]‐5‐[1‐(4‐piperidinyl)‐1H‐pyrazol‐4‐yl]‐
2‐pyridinamine).[8] Kryštof et al.[9] investigated big series of ar-
ylazopyrazoles, the most potent inhibitor, 4‐[(3,5‐diamino‐1H‐pyrazol‐4‐
yl)diazenyl]phenol (lead compound, CAN508), and revealed that it
reduced the frequency of the S phase cell line HT‐29 in antiproliferation
assays. Depending on CAN508 results, many anticancer arylazopyr-
azoles were synthesized via a molecular modification to improve cyto-
toxicity, CDK activity, and their antiproliferative potential against the
MCF‐7 cancer cell line.[10,11]
On the basis of the literature and as an extension to our search
about new therapy to breast adenocarcinoma,[12] the synthesis of a
new series of 4‐arylazo‐3,5‐diamino‐1H‐pyrazole bearing 4‐acetyl
function on the aryl moiety, developed from CAN508, was interest-
ing. Some optimization strategies will be followed, namely, bioisosteric
due to their strong inhibitory activities against BRAFV600E
,
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Arch Pharm. 2020;e2000214.
wileyonlinelibrary.com/journal/ardp
© 2020 Deutsche Pharmazeutische Gesellschaft
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replacement, chain extension, and variation of substituents, to explore
the effects of these structural modifications on the cytotoxic activity
(Figure 1). The antiproliferative activity of these candidates will be
evaluated against the MCF‐7 breast cancer cell line. Additionally, a
study of the inhibitory activity on CDK2 and cell cycle analysis will be
conducted for the most active compound. Finally, molecular docking
on the active site of CDK2 and three‐dimensional (3D) pharmaco-
phore model of the most active hit will be performed to investigate
the structural characteristics managing the antiproliferative
activities.
First, the intermediate 3‐[2‐(4‐acetylphenyl)hydrazono]pentane‐2,4‐
dione (5)[18] was prepared by the reaction of p‐aminoacetophenone
with acetyl acetone in the presence of sodium nitrite and sodium
acetate. Then, the target compounds 1‐{4‐[(3,5‐dimethyl‐1H‐pyrazol‐
4‐yl)diazenyl]phenyl}ethanone (6a) and 1‐{4‐[(3,5‐dimethyl‐1‐phenyl‐
1H‐pyrazol‐4‐yl)diazenyl]phenyl}ethanone (6b) were prepared by
cyclocondensation reaction of the intermediate 5 (0.01 mol) with
hydrazine hydrate and phenylhydrazine, respectively, in acetic acid
(Scheme 1).
Acylation of the 5‐amino functionality of 3a,b produced the
corresponding N‐acylated products, 7a,b. Acylation proceeded via
stirring of 3a,b with 2,4‐dichlorobenzoyl chloride or benzoyl chloride
in DMF for 4 hr.[19] Moreover, the synthesis of a series of Schiff's
bases 8a–h was achieved through the reaction with various sub-
stituted aromatic aldehydes or acetophenones in ethanol,[20] in the
presence of a basic medium (fused AcONa; Scheme 2).
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2
RESULTS AND DISCUSSION
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2.1
Chemistry
The route adopted for the synthesis of new pyrazole derivatives
is illustrated in Schemes and 2. The diazonium salt of
1
p‐aminoacetophenone was coupled with malononitrile[13–15] in ethanol
in the presence of sodium acetate. The resulting coupled product was
formulated as 2‐[(4‐acetylphenyl)diazenyl]malononitrile 2. Aminopyr-
azoles 3a,b were obtained by refluxing 2 with hydrazine hydrate in
ethanol,[15,16] whereas 3c was obtained by using phenylhydrazine in
glacial acetic acid.[14] Regarding the synthesis of compounds 4, when the
diazonium salt of compound 3a was coupled with 3‐phenyl‐1H‐pyrazol‐
5(4H)‐one,[17] the corresponding arylazo derivative 4a was afforded.
Similarly, the diazonium salt of 3b was coupled with
β‐naphthol to yield the arylazo derivative 4b in a good yield.
2.2
Antitumor studies
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2.2.1
In vitro antitumor activity against human
breast cancer cells (MCF‐7)
The in vitro antitumor activity against human breast cancer cells
(MCF‐7) of the 17 target compounds was assessed in the cell culture
lab, College of Pharmacy, Al‐Azhar University, Cairo, Egypt. Imatinib
and CAN508 were used, which showed IC50 values of 6.00 and
62.00 μM. The cytotoxicity of the target compounds on the MCF‐7
profile clarified variable activities, compared with imatinib and
CAN508, as presented in Table 1.
However, bioisosteric replacement of 3,5‐diamino substituents
with 3,5‐dimethyl groups, as in arylazopyrazoles 6a,b, was attempted.
FIGURE 1 The design of the target
compounds

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NH2
O
1
a
O
N
CN
CN
H
N
Cl
N
N
N
O
b
N
e
O
O
O
5
2
c
c
R
N
R
N
H2N
N
N
N
N
N
N
NH2
a; R = H, X = O
b; R = H, X = NNH2
X
O
a; R = H
b; R = Ph
6a,b
3a–c
c; R = Ph, X = O
a
Ar
N
H
N
N
R
N
-
+
N
N
N
N
Cl
d
N
N
N
NH2
N
NH2
4a,b
X
Ph
a; X = O,
Ar =
Ar =
N
O
X
N
H
b;
HO
X = NNH2
,
SCHEME 1 The synthesis of compounds 6a,b and 4a,b. Reagents and conditions: (a) NaNO2/HCl/0°C, stirring; (b) CH2(CN)2/NaOAc/EtOH,
stirring 30 min/cooling; (c) RNHNH2/EtOH, reflux, 4 hr; (d) 3‐phenyl‐1H‐pyrazol‐5(4H)‐one or β‐naphthol/sodium acetate/EtOH, stirring
30 min/cooling; (e) acetylacetone/NaOAc/EtOH, stirring 30 min/cooling
Noticeably, most of the target compounds bearing 4‐acetyl
functionality on the aryl moiety (3a, 4a, 6b, 7a, 8b, and 8f) showed a
potent to moderate antiproliferative activity against the MCF‐7 cell
line (IC50 3.08–38.7 µM; Figure 2), whereas most of their counter-
parts carrying the 4‐hydrazonoethyl group lacked activity. Thus, the
variation of substituent has a crucial role in cytotoxicity. In diamino
arylazopyrazole 3a (IC50 = 26.86 μM), when NH of pyrazole is sub-
stituted with the phenyl moiety (N‐Ph), as in 3c (IC50 = > 100 μM), the
activity is significantly reduced, which means that the NH group of
pyrazole is detrimental for the activity. On the contrary, compound 6
where bioisosteric replacement occurs in 3,5‐dimethyl instead of 3,5‐
diamino of the lead compound, it is interesting to note that 6b
(R1 = –N(Ph)–) showed moderate cytotoxicity (IC50 = 38.7 μM),
whereas 6a (R1 = –NH–) was inactive. This means that the lipophilic
criterion in this type of compound may be necessary.
Regarding extension at p‐5 via adding an extra arylazo group, a
moderate anticancer activity (IC50 = 34.7 μM) was noticed for the
coupling product 4a , which bears pyrazolinyl‐azo moiety, whereas
substituent variation (2‐naphthol‐1‐yl) in 4b abolished the activity.
Other compounds underwent an extension tactic via acylation of
5‐amino group such as acylation of 3a with 2,4‐dichlorobenzoyl
chloride to yield 7a with a good anticancer activity (IC50 = 13.8 μM);
however, N‐benzoylated derivative, 7b, displayed a weak activity
against the MCF‐7 cell line. As a continuation for the synthesis of
new analogs with chain extension at 5‐amino of pyrazole, Schiff bases
8a–h were furnished through a reaction with various aldehydes or
ketones. It was clear that 8b (IC50 3.08 µM) showed two times more

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R
N
H2N
N
N
N
NH2
a; X = O,
b; X = NNH2, R = H
R = H
X
3a,b
b
a
R2
O
R1
R1
H
N
H
N
HN
N
N
N
N
N
N
N
NH2
NH2
X
X
8
8a–h
R2
7a,b
R1
Cl
X
a
b
c
H
NNH2
a; X = O,
b; X = NNH2,
R1 = 2,4-(Cl)2Ph
R1 = Ph
CH3
H
CH3
H
CH3
H
Cl
O
NNH2
CH3
CH3
OCH3
OCH3
OH
OH
NNH2
d
e
f
g
NNH2
O
NNH2
CH3
NNH2
h
SCHEME 2 The synthesis of compounds 7a,b and 8a–h. Reagents and conditions: (a) RCOCl, DMF, stirring; (b) aromatic aldehydes or
ketones, EtOH, fused Na acetate, reflux, 3 hr
potent cytotoxicity than imatinib (IC50 6.00 µM). Another promising
analog, 8f (IC50 4.03 µM), elicited 1.5‐fold activity than imatinib. It is
interesting to note that 8b and 8f bearing 4‐acetyl on aryl moiety
were the most potent hits among our study. Both have R2 = CH3, so
condensation with acetophenones is preferable than aldehydes
among all prepared Schiff bases. It is worth mentioning that in the
case of 8b, R1 = Cl, whereas in the case of 8f, R1 = OCH3, which
means that the electronic effect is not essential for activity. Among
all inactive analogs having hydrazono‐ethyl instead of 4‐acetyl on the
aryl moiety, 8d (R1, R2 = CH3) showed a moderate activity (IC50
38.9 μM) and 8g (R1 = OH, R2 = H) elicited mild cytotoxicity (IC50
69.1 μM).
MCF‐7 cells to 8b resulted in an interference with the normal cell
cycle distribution of this cell line. The cells in the S phase, control
group, accounted for about 23.59%, whereas after cells were treated
with compound 8b, the ratio was approximately 33.08%. This shows
that the cells were arrested in the
S phase, as shown in
Table 2. Such an increase came with a reduction of cells at both the
G0/G1 and G2/M phases of the cell cycle. Moreover, treatment with
8b produced a significant increase in pre‐G1 phase percent by four-
fold, which resulted in interference with the normal cell cycle and was
indicative of apoptosis. The results were represented by a bar chart
that shows the percentage of MCF‐7 cells at each stage of the cell
cycle in control cells and 8b‐treated cells (Figure 3a,b and Table 2).
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2.2.2
Effect on cell cycle arrest on MCF‐7 cells
2.2.3
Annexin V‐fluorescein isothiocyanate (FITC)
apoptosis assay
The most active compound 8b was selected for more study for its
effect on cell cycle progression in the MCF‐7 cell line. The MCF‐7 cells
were incubated for 24 hr with 8b concentrations of IC50, and their
effect on the normal profile of the cell cycle was analyzed. Exposure of
Apoptosis induction by 8b following S‐phase arrest was assessed by
the Annexin V‐FITC apoptosis assay. One of the earliest cellular
changes evident during apoptosis is the translocation of

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TABLE 1 IC50 values of compounds 3–8 against MCF‐7 cells
TABLE 2 Cell cycle distribution after treatment with compound 8b
Cpd no.
3a
R1
R2
–
X
IC50 (μM)
26.86 0.54
>100
Cell cycle distribution (%)
H
O
Pre‐G
3.09
G0/G1
69.37
63.54
S
G2/M
7.04
Cpd no.
3b
H
–
NNH2
O
Control
23.59
33.08
3c
Ph
–
>100
8b
12.64
3.38
4a
Antipyrin‐4‐yl
–
O
34.7 14.78
>100
4b
2‐Naphthol‐1‐yl
–
NNH2
O
usually performed. Annexin V/PI staining was performed in cells ex-
posed to 8b for 24 hr. The results revealed that the application of
compound 8b on MCF‐7 cells for 24 hr increases the early apoptosis
ratio (lower right quadrant of the cytogram) from 0.42% to 7.64% and
increases the late apoptosis ratio (higher right quadrant of the cyto-
gram) from 0.39% to 18.73%. This means that compound 8b induced
apoptosis almost up to 32.5‐fold, both early and late cellular apoptosis
when compared with the control. These data suggest that compounds
8b triggered apoptosis via the programmed cell death pathway rather
than a necrotic pathway (Table 3 and Figure 4).
6a
H
–
>100
6b
Ph
–
O
38.7 3.50
13.8 0.56
>100
7a
2,4‐(Cl)2Ph
Ph
–
O
7b
–
NNH2
NNH2
O
8a
Cl
H
>100
8b
Cl
CH3
H
3.08 0.15
>100
8c
CH3
CH3
OCH3
OCH3
OH
NNH2
NNH2
NNH2
O
8d
CH3
H
38.9 1.53
>100
8e
8f
CH3
H
4.03 0.28
69.1 10
>100
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2.2.4
CDK2/cyclin E kinase inhibitory activity
8g
NNH2
NNH2
To explore the mechanism of action of 8b, CDK2/cyclin E in vitro
assay was performed using imatinib and CAN508. The obtained re-
sults revealed that 8b showed a good inhibitory effect (IC50 0.24 µM)
on CDK2/cyclin E enzyme, compared with imatinib (IC50 0.15 µM),
which are both more potent than CAN508 (Table 4).
8h
OH
CH3
Imatinib
CAN508
6.00 0.53
62.00 8.00
phosphatidylserine (PS) from the inner to the outer side of the plasma
membrane.[21] Fluorescently labeled annexin V, which binds to phos-
phatidylserine,[22] can be used as a sensitive probe for PS in the outer
leaflet of the plasma membrane.[23] Also, propidium iodide (PI), a
fluorescent molecule binding to nucleic acids, but which does not enter
live cells, can be used to counterstain dead cells. To differentiate be-
tween apoptosis and necrosis, both annexin V‐FITC and PI staining are
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2.3
Molecular modeling studies
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2.3.1
Docking with CDK2
Table 3 displays docking scores and bond interactions of ligand X, the
3,5‐diaminopyrazole CDK2‐inhibitor, and the target compound 8b
with amino acids of CDK2 active site. Furthermore, Figure 5a,b re-
present interactions with the amino acids of CDK2 in 2D and 3D
styles, respectively.
In this study, the docked model of lead compound X with CDK2
showed that compound X bonded to the active site via three hy-
drogen bond donors with Ile10, Leu83, and Gln131. The pyrazole
moiety faces Lys 89 by arene–cation interaction. Moreover, hydro-
phobic interaction with Ile10 and Leu134 was observed.
Docking study of compound 8b showed higher affinity in the
active site of CDK2 (ΔG = −5.6 kcal/mol) than that of the ligand
(ΔG = −4.4 kcal/mol).
The binding mode of 8b was revealed in Figure 6a,b, where
pyrazole scaffold binds to the active site of CDK2 via three hydrogen
bonds. Two hydrogen bond donors are between 1N, 2N of pyrazole
and the CDK amino acids Leu83 and Glu81, respectively. Another
hydrogen bond acceptor is between 2N of pyrazole and amino acid
Leu83. Compound 8b has the same binding mode as previously re-
ported inhibitors with the same pyrazole core.
FIGURE 2 IC50 values of the promising derivatives against the
MCF‐7 cell line

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FIGURE 3 (a) The effect of 8b on DNA
ploidy of MCF‐7 cells by flow cytometric
analysis. (b) Bar chart showing the percentage
of MCF‐7 cells in control cells and 8b‐treated
cells at each stage of the cell cycle
The aryl moiety attached at position 5 of the pyrazole ring creates
hydrophobic interactions with the side chain of Ile10, Lys 89, and Gln131
residues. More hydrophobic interactions occurred between the acet-
ophenone moiety and the amino acids Asp145 and Leu134.
regression analysis, using the correlation of the geometric fit value
contrasted with the biological activity. The better the geometric fit,
the greater is the activity prediction of the compound. The fit func-
tion checks if the feature is mapped. It also contains a distance term,
which measures the distance breaking the feature on the molecule
from the centroid of the feature in the pharmacophore hypothesis.
Both terms are used to calculate the geometric fit value.[25]
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2.3.2
3D‐QSAR pharmacophore modeling
The 3D‐QSAR Pharmacophore Generation protocol (HypoGen pro-
tocol of CATALYST; 13) was applied using Discovery Studio 2.5
software to create 10 predictive pharmacophore models via aligning
different conformations in which the molecules were likely to bind
with the receptor pharmacophore models.[24]
Pharmacophore study results
In this study, pharmacophore models were generated using two hy-
drogen bond acceptors (HBA), hydrophobic (Hyd), and ring aromatic
(RA) features, and nine pharmacophore models were exported for
further studies. All the generated pharmacophore models contained
at least two chemical features. The best‐generated pharmacophore
contained two HBA features, 1 ring aromatic, and 1 hydrophobic
feature (Figure 7). The constraint distances and angles between the
different features of the generated top pharmacophore are pre-
sented in Table 5.
The given hypothesis was combined with known activity data to
create a 3D‐QSAR model that identifies overall aspects of molecular
structure governing the activity. During hypothesis generation, the
structure and activity correlations in the training set were rigorously
examined. HypoGen identifies features common to the active com-
pounds and excludes features common to the inactive compounds
within a conformationally allowable region of space. It further esti-
mates the activity of the newly synthesized and tested compounds
using regression parameters. The parameters were calculated by
The diazo linker and C═O group contribute to the pharmaco-
phore by two HBA features, and the methyl group of the
5‐ethylidene amino moiety represents a hydrophobic feature, in ad-
dition to the phenyl group of the acetophenone moiety, which acts as
an aromatic ring feature, as shown in Figure 8.
The best‐generated pharmacophore significance was 98%. The
top pharmacophore hypothesis generated was developed with a total
cost value of 104.77, null cost = 226.1, and fixed cost = 59.75. Further
evaluation of the generated pharmacophore models was based on
the correlation coefficient,[26] which was found to be 0.869, indicat-
ing the capability of the pharmacophore model to predict the activity
of the training set compounds. The predicted activities through the
pharmacophore model are represented in Table 6 as well as their fit
TABLE 3 The effect of 8b on annexin V‐fluorescein
isothiocyanate‐positive staining in MCF‐7 cells
Apoptosis in MCF‐7 cells
Total
28.36
1.79
Early
7.64
0.42
Late
18.73
0.39
Necrosis
1.99
Cpd no.
8b
Control
0.98

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FIGURE 4 The effect of 8b on MCF‐7 cell death
TABLE 4 CDK2 inhibitory activity of compound 8b
HypoGen selects the best hypotheses by applying a cost analysis. The
overall cost of each hypothesis is calculated by summing three cost
factors—a weight cost, an error cost, and a configuration cost.
HypoGen also calculates two theoretical costs, the null and fixed
costs, which can be used to determine the significance of the selected
hypotheses. The cost values of the optimized hypotheses should lie
between these two costs. A larger difference between the fixed and
null costs than that between the fixed and total costs signifies the
quality of a pharmacophore model. The closer the cost value to
the fixed cost and the further away it is from the null cost, the more
significant the hypothesis might be.
Cpd no.
Imatinib
CAN508
8b
IC50 (µM)
0.15 5.92
20 6.00
0.24 9.55
values. Furthermore, compound 8b was mapped into the best‐
generated pharmacophore model, using a ligand pharmacophore
mapping protocol, with fit value = 6.85.
Fischer validation is another method for pharmacophore model
validation. This validation method checks the correlation between
chemical structures and biological activity. It also generates phar-
macophore hypotheses using the same parameters as those used to
Validation of 3D‐QSAR pharmacophore
Validation of the obtained pharmacophore model was carried out
using cost analysis and Fischer validation test for activity prediction.
FIGURE 5 The proposed ligand X binding mode docked in the active CDK2 site. (a) Two‐dimensional ligand–receptor interactions and
(b) three‐dimensional ligand–receptor interactions (C atoms are gray, N atoms are blue, and O atoms are red). The important amino acid
residues are shown together with their respective numbers. Compound X formed three hydrogen bonds with Ile10, Leu83, and Gln131

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FIGURE 6 The proposed 8b binding mode docked in the active CDK2 site. (a) Two‐dimensional ligand–receptor interactions and
(b) three‐dimensional ligand–receptor interactions (C atoms are gray, N atoms are blue, and O atoms are red). The important amino acid
residues are shown together with their respective numbers. Compound 8b formed three hydrogen bonds with Leu83 and Glu81
develop the original pharmacophore hypothesis by randomizing the
activity data of the training set compounds. The Fischer validation
confidence level chosen was 98%. In addition to cost analysis and
Fischer validation, the pharmacophore model was validated through
activity prediction of the synthesized structures as the training set.
data using SwissADME,[27] Molsoft,[28] and Pre‐ADME[29] websites,
and DataWarrior software.[30] With respect to physicochemical
properties (Table 7), all the synthesized compounds have zero vio-
lations for Lipinski's rule for oral drugs. Also, according to the
screening process with Veber rules, all the hits meet the criteria of
drug‐likeness, except compound 4a, which did not obey Veber rules
regarding TPSA (<140). Additionally, absorption (%ABS) was esti-
mated by using the following equation: %ABS = 109 − (0.345 ×
TPSA),[33] and it was found that the calculated % ABS of all these hits
ranged between 52.87885% and 88.43455%, demonstrating that
these synthesized derivatives may have the required cell membrane
permeability and bioavailability. All compounds have rotatable bonds
between 3 and 6, which indicates molecular flexibility to their bio-
target. Curiously, all the screened derivatives are not substrates of
P‐gp protein, so they have a minor chance of efflux out of the cell,
thus resulting in a maximum effect. Furthermore, all the screened
analogs have no permeation to the blood–brain barrier, except
compound 6b, thus ensuring that these systemically targeted mole-
cules will have low to no side effects on the central nervous system.
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2.3.3
In silico evaluation of physicochemical and
absorption, distribution, metabolism, and excretion
(ADME) properties
A computational study of the synthesized compounds was performed
to evaluate physicochemical properties, ADME, and ligand efficiency
TABLE 5 Constraint distances and angles between features of the
generated top pharmacophore model
Constraint distances (Å)
HBA1–HBA2: 7.18
RA–HBA1: 3.77
RA–HBA2: 3.69
Hyd–RA: 5.20
Constraint angles (°)
HBA1, HBA2, RA: 45.59
HBA1, Hyd, RA: 74.78
HBA2, Hyd, RA: 21.38
FIGURE 7 Constraint distances and angles between features of
the generated top pharmacophore model with the features
considered hydrogen bond acceptors (HBA1 and HBA2) colored in
green, aromatic ring (RA) colored in orange, and hydrophobic colored
in cyan
Hyd–HBA1: 4.13
Hyd–HBA2: 8.46

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TABLE 6 Fit values and estimated activities for the synthesized
compounds mapped with the generated three‐dimensional
pharmacophore model
Predicted activity,
IC50 (µM)
Experimental activity,
IC50 (µM)
Cpd. no.
3a
Fit values
5.21
4.20
5.22
5.59
4.77
5.22
5.22
5.74
4.47
4.70
6.85
4.69
5.65
4.96
6.88
5.68
5.66
140.01
1.42
26.6
3b
3c
1,120.6
485.6
239.9
246.6
107
139.46
58.98
386.25
137.95
138.18
41.44
771.88
460.31
3.20
4a
4b
6a
6b
7a
38.5
FIGURE 8 The best‐generated pharmacophore hypothesis with
the features considered hydrogen bond acceptors (HBA1 and HBA2)
colored in green, ring aromatic (RA) colored in orange, hydrophobic
(Hyd) colored in cyan, and the synthesized structure 8b fitted in the
pharmacophore with fit value 6.85
13.6
7b
8a
680.3
380
8b
8c
3.1
468.06
51.01
248.36
3.00
425.3
39.3
Bioavailability is an index of the amount of drug present in the
plasma and is considered as the most crucial factor affecting ab-
sorption.[34] Interestingly, it has been found that all the screened
derivatives have high bioavailability scores. Synthetic accessibility
scores of all the analogs were found to be between 2.47 and 4.24,
indicating that they can be readily synthesized on a large scale.
In pan assay interference compounds (PAINS) structural alert, all
the compounds were found to have only one structural alert (azo
group). Although PAINS are important features to be considered
while developing drugs to avoid false‐positive results, yet over-
estimation and blind use of these filters might only lead to the ex-
clusion of promising hits based on phantom PAINS.[35]
8d
8e
1,248.3
4.1
8f
8g
47.47
50.62
69
8h
154.9
closely correlated to increased binding promiscuity and diminished safety
thresholds. Ligand efficiency (LE) and lipophilic ligand efficiency (LLE) are
two parameters describing the balance between potency and molecular
size, which is related to various pharmacokinetic and toxicological para-
meters.[37,38] LE (= 1.37 [pIC50]/non‐hydrogen atoms)[39] measures the
average binding energy per non‐hydrogen atom instead of considering
the binding affinity of the whole molecule. Thus, it allows comparing and
prioritizing ligands corrected for their sizes, and LLE (= pIC50 − cLogP) is a
measure of how efficiently a ligand can exploit its lipophilicity to bind to
its target.[40] So LLE monitoring highlights the price paid in ligand lipo-
philicity at the expense of its potency. Herein, LE and LLE values of the
most active compound 8b were calculated on the basis of its IC50 values
against the screened enzyme assays (CDK2/cyclin E). The results
(Table 7) indicated that 8b had an acceptable limit, 0.33572, >0.3.
Additionally, 8b showed an accepted LLE value, 3.0375. Collectively, and
based on the estimated physicochemical properties, ADME, and LE pre-
dictors, these active compounds considered as a pharmacologically active
framework should be considered for developing further potential hits.
Additionally, Molsoft software was used to estimate the solubi-
lity score of the screened derivatives, and it was found that these
derivatives fulfilled the requirements of the solubility with values in
the range of 14.04–2,993.99 mg/l (more than 0.0001 mg/l)
The Pre‐ADME server has been used to assess the intestinal ab-
sorption, identify potential drugs for oral delivery, and give information
regarding the therapeutic drugs in the central nervous system and
plasma–protein binding (PPB) model in its disposition and efficacy.[36]
The in silico assessment results of pharmacokinetic parameters,
Caco2 permeability coefficient, HIA, and PPB of the selected mole-
cules, were obtained from the Pre‐ADMET server. As shown in Ta-
ble 7, all the screened compounds showed medium cell permeability
in the Caco‐2 cell model, with values between 6.13254 and
31.9615 nm/s, except 3a and 8f, which display low cell permeability,
0.43128 and 1.27469 nm/s, respectively. Also, they revealed high
human intestinal absorption values (78.427541–97.802862%), in-
dicating very well‐absorbed compounds. According to the analysis of
the PPB abilities, all compounds showed high protein binding
(90.456917–99.845118%), except 3a and 8f, which displayed low
protein binding, 69.716397 and 82.451914, respectively.
|
3
CONCLUSION
In the process of anticancer drug discovery, to find new potential
antibreast cancer agents, we synthesized a novel series of pyrazole
derivatives. The antiproliferative activity of new derivatives was
It has been established that increased molecular weight and lipo-
philicity result in improved binding potency, but in the meantime, they are

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ISMAIL ET AL.
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TABLE 7 In silico physicochemical properties, ADME (absorption, distribution, metabolism, and excretion), and ligand efficiency data of the
active compounds
Compd. no.
3a
4a
6b
7a
8b
8f
SWISS ADME
MW
244.25
415.41
318.37
417.25
380.83
376.41
MlogP
0.42
1.59
2.76
2.4
3.06
2.27
HBD
3
3
0
3
2
2
HBA
4
8
4
5
5
6
Lipinski's violation
No. of rotatable bonds
TPSA
0
0
0
0
0
0
3
6
4
6
5
6
122.51
66.73405
0
162.67
52.87885
1
59.61
88.43455
0
125.59
65.67145
0
108.85
71.44675
0
118.08
68.2624
0
%ABS
Veber rule
P‐gp substrate
BBB
No
No
No
No
No
No
No
No
Yes
No
No
No
Bioavailability score
Synthetic accessibility
PAINS
0.55
2.47
1
0.55
4.24
1
0.55
2.9
0.55
2.99
1
0.55
3.35
1
0.55
3.44
1
1
Molsoft
S (mg/l)
2,993.99
92.57
15.33
52.42
14.04
36.36
Pre‐ADME
Caco2a
HIAb
0.43128
6.13254
31.9615
13.9876
14.7366
1.27469
83.800246
69.716397
78.427541
92.399604
97.802862
90.456917
89.232413
99.845118
91.575738
95.372135
90.257977
82.451914
PPBc
DataWarrior
LEd(CDK2/cyclin E)
LLEe
–
–
–
–
–
–
–
–
0.33572
3.0375
–
–
Abbreviations: %ABS, percentage of absorption; BBB, blood–brain barrier; Caco2, permeability through cells derived from human colon adenocarcinoma;
HBA, the number of hydrogen bond acceptors; HBD, the number of hydrogen bond donors; HIA, percentage human intestinal absorption; LE, ligand
efficiency; LLE, lipophilic ligand efficiency; LogP, logarithm of compound partition coefficient between n‐octanol and water; MW, molecular weight; PPB,
plasma–protein binding; S, aqueous solubility; TPSA, topological polar surface area.
aCaco2 values < 4 nm/s (low permeability), values ranged from 4 to 70 nm/s (medium permeability), and values >70 nm/s (high permeability).
bHIA values ranged from 0% to 20% (poorly absorbed), values ranged from 20% to 70% (moderately absorbed), and ranged from 70% to 100% (well‐
absorbed).
cPPB values <90% (poorly bound) and values >90% (strongly bound).
dThe lowest acceptable limit is 0.3.[31]
eValues ≥3 are acceptable for lead compounds, whereas drug‐like candidates record values ≥5.[32]
|
EXPERIMENTAL
examined against MCF‐7 breast cancer using the 3‐(4,5‐dimethylthiazole‐
2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) technique. Compound 8b
was the most active member against MCF‐7 cells, showing an IC50 value
of 3.08 µM. Biological assessment using flow cytometric analysis revealed
that it induced apoptosis and cell cycle arrest at S phase and exhibited
62.5% inhibition of CDK2/cyclin E as compared with imatinib. Moreover,
3D‐QSAR pharmacophore modeling showed the importance of the diazo
linker and C═O of acetophenone moiety as 2HBA features; ring aromatic
was also important. Additionally, the methyl group of ethylidene amino
moiety was an essential hydrophobic feature. Finally, a combination of 3D
pharmacophore modeling and cytotoxicity results would provide an ef-
fective technique for understanding the observed pharmacological
properties, which might, thus, be adopted for developing effective lead
structures.
4
|
4.1
Chemistry
|
4.1.1
General
All chemicals were bought from VWR International Merck, Germany,
or Sigma‐Aldrich and utilized without further purification. Melting
points were determined with an open capillary tube method using
Stuart SMP3 Melting Point apparatus, and they were uncorrected.
Elemental microanalysis was carried out at the Regional Center for
Mycology and Biotechnology, Al‐Azhar University. Infrared spectra
were recorded on a Shimadzu infrared spectrometer IR Affinity‐1
(FTIR‐8400S‐Kyoto‐Japan) and expressed as wavenumber (cm−1),

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ISMAIL ET AL.
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using potassium bromide discs at the Armed Forces Laboratories. 1
H
4‐({4‐[(4‐Acetylphenyl)diazenyl]‐3‐amino‐1H‐pyrazol‐5‐yl}diazenyl)‐
3‐phenyl‐1H‐pyrazol‐5(4H)‐one (4a)
NMR (nuclear magnetic resonance) spectra were recorded on a
Varian Gemini 300‐MHz spectrophotometer; the spectra were run at
300 MHz in deuterated dimethylsulfoxide (DMSO‐d6) at the Armed
Forces Laboratories. 13C NMR spectra were recorded on a Varian
Gemini 300 MHz spectrophotometer, and the spectra were run at
300 and 400 MHz in DMSO‐d6 at the Armed Forces Laboratories.
Chemical shifts were expressed in δ units and were related to that of
the solvents. As for the proton magnetic resonance, D2O was carried
out for NH and OH exchangeable protons. Mass spectra (MS) were
recorded using a Shimadzu gas chromatograph mass spectrometer‐
Qp 2010 plus (Japan) and were carried out at the Regional Center for
Mycology and Biotechnology, Al‐Azhar University. All the reactions
were followed by thin‐layer chromatography using silica gel F254
plates (Merck) and were visualized by an ultraviolet (UV) lamp. The
intermediates 2 and 5 are prepared according to the reported
method,[41,42] as well as compounds 3a,b.[12]
M.p. >300°C; yield 50%; IR (KBr, cm−1): 3,400–3,200 (br. NH2, NH),
1,690 (CO), 1,666 (CON), and 1,594 (N═N); 1H NMR (300 MHz,
DMSO‐d6) δ (ppm): 2.64 (s, 3H, CH3), 7.05 (s, 2H, NH2, D2O‐
exchangeable), 7.40–8.17 (m, 9H, 4Ar‐H, 5Ph‐H), 12.27 (s, 1H, NH,
D2O‐exchangeable), 12.39 (s, 1H, NH, D2O‐exchangeable), and 12.89
(s, 1H, NH, D2O‐exchangeable); 13C NMR (100 MHz, DMSO‐d6)
δ (ppm): 26.70, 99.40, 118.89, 121.81, 126.58, 126.82, 128.58,
129.51, 130.05, 144.58, 160.00, and 196.55; MS [m/z, %]: 415 [M+,
0.22]; Anal. calcd. for C20H17N9O2 (%): C, 57.83; H, 4.12; N, 30.35.
Found: C, 57.96; H, 4.17; N, 30.49.
1‐[4‐({3‐Amino‐5‐[(2‐hydroxynaphthalen‐1‐yl)diazenyl]‐1H‐pyrazol‐
4‐yl}diazenyl) phenyl]ethanone (4b)
M.p. 147–150°C; yield 55%; IR (KBr, cm−1): 3,385–2,740 (OH, NH, NH2),
1,625 (C═N), 1,598 (N═N); 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 2.57
(s, 1H, OH, D2O‐exchangeable), 2.61 (s, 3H, CH3), 6.73–8.50 (m, 12H,
10Ar‐H, NH2), and 15.87 (s, 1H, NH, D2O‐exchangeable); MS [m/z, %]:
413 [M+, 5.00]; Anal. calcd. for C21H19N9O (%): C, 61.01; H, 4.63; N,
30.49. Found: C, 61.17; H, 4.70; N, 30.72.
The InChI codes of the investigated compounds, together
with some biological activity data, are provided as Supporting
Information.
|
4.1.2
Synthesis of 1‐{4‐[(3,5‐diamino‐1‐phenyl‐
|
1H‐pyrazol‐4‐yl)diazenyl]phenyl}ethanone (3c)
4.1.4
General procedure for the synthesis
of compounds 6a,b
A mixture of equimolar amounts of 2‐[(4‐acetylphenyl)diazenyl]
malononitrile 2 and phenylhydrazine was refluxed in ethanol (95%,
30 ml) for 4 hr and then poured after cooling into ice‐cold water to
produce 3c. The solid product so formed was filtered off and crys-
tallized from ethanol. M.p. 218–220°C; yield 64%; IR (KBr, cm−1):
3,466, 3,414, 3,346, 3,280 (2NH2), 1,672 (C═O), 1,625 (C═N), and
1,596 (N═N); 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 2.58 (s, 3H,
CH3), 5.95 (s, 2H, NH2, D2O‐exchangeable), 6.76–7.49 (m, 3H, Ph‐H),
7.56 (d, 2H, Ph‐H2,6, J = 8.4 Hz), 7.74 (d, 2H, Ph‐H3,5, J = 8.4 Hz), 7.82
(d, 2H, Ph‐H), and 9.30 (s, 1H, NH, D2O‐exchangeable); 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 21.81, 72.40, 112.64, 112.96, 118.70,
120.52, 122.08, 125.63, 129.31, 137.81, 138.44, 140.24, 146.00,
152.55, and 196.96; MS [m/z, %]: 320 [M+, 16.03]; Anal. calcd. for
C17H16N6O (%): C, 63.74; H, 5.03; N, 26.23. Found: C, 63.88; H, 5.11;
N, 26.49.
A mixture of 5 (0.01 mol) and hydrazine hydrate 98% or phenylhy-
drazine (0.015 mol) in glacial acetic acid (15 ml) was refluxed for
4–5 hr. The resulting mixture was concentrated and allowed to cool.
Then the resulting solid so formed was filtered, washed, dried, and
crystallized from ethanol.
1‐{4‐[(3,5‐Dimethyl‐1H‐pyrazol‐4‐yl)diazenyl]phenyl}ethanone (6a)
M.p. 109–110°C; yield 65%; IR (KBr, cm−1): 3,322 (NH), 1,651 (C═O),
and 1,555 (N═N); 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 1.82 (s, 6H,
2CH3), 2.58 (s, 3H, COCH3), 7.79 (d, 2H, Ph‐H3,5, J = 8.4 Hz), 8.07
(d, 2H, Ph‐H2,6, J = 8.4 Hz), and 9.64 (s, 1H, NH, D2O‐exchangeable);
13C NMR (100 MHz, DMSO‐d6) δ (ppm): 19.58, 20.88, 27.27, 121.79,
129.90, 135.15, 137.18, 156.07, 168.40, and 197.72; MS [m/z, %]:
242 [M+, 100]; Anal. calcd. for C13H14N4O (%): C, 64.45; H, 5.82;
N, 23.13. Found: C, 64.62; H, 5.89; N, 23.21.
|
4.1.3
General procedure for the synthesis
1‐{4‐[(3,5‐Dimethyl‐1‐phenyl‐1H‐pyrazol‐4‐yl)diazenyl]phenyl}-
ethanone (6b)
of compounds 4a,b
M.p. 82–85°C; yield 64%; IR (KBr, cm−1): 1,653 (C═O), 1,592 (C═N),
and 1,558 (N═N); 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 2.27 (s, 3H,
CH3), 2.44 (s, 3H, CH3), 2.65 (s, 3H, COCH3), 7.68–7.74 (m, 5H, Ph‐H),
7.79 (d, 2H, Ph‐H3,5, J = 8.4 Hz), and 8.07 (d, 2H, Ph‐H2,6, J = 8.4 Hz);
13C NMR (100 MHz, DMSO‐d6) δ (ppm): 20.34, 20.40, 26.85, 121.31,
121.43, 129.40, 134.66, 136.70, 155.58, 167.89, 173.95, and 197.22;
MS [m/z, %]: 318 [M+, 0.50]; Anal. calcd. for C19H18N4O (%): C, 71.68;
H, 5.70; N, 17.60. Found: C, 71.85; H, 5.76; N, 17.83.
A solution of β‐naphthol or 3‐phenyl‐1H‐pyrazol‐5(4H)‐one (0.1 mol)
in ethanol (100 ml) was treated with a suspension of sodium acetate
(0.1 mol in 50 ml of H2O). A solution of diazonium salt of 3a or 3b
(0.1 mol) in acetic acid (50 ml) was then added, with stirring the
previous mixture. The solid products, obtained on standing, were
collected by filtration and washed several times with hot water and
recrystallized from ethanol.

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ISMAIL ET AL.
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|
4.1.5
General procedure for the synthesis
134.96, 135.36, 154.97, 156.37, 158.34, 163.81, and 187.57;
MS [m/z, %]: 380 [M+, 1.63]; Anal. calcd. for C18H17ClN8 (%): C, 56.77;
H, 4.50; N, 29.42. Found: C, 56.94; H, 4.57; N, 29.60.
of compounds 7a,b
Equimolar amounts of 3a or 3b reacted with benzoyl chloride or 2,4‐
dichlorobenzoyl chloride by stirring in DMF (15 ml) at room tem-
perature for 2 hr, and then the mixture was poured into water (30 ml)
and neutralized with few drops of HCl. The resulted solid was then
filtered and dried.
1‐{4‐[(3‐Amino‐5‐{[1‐(4‐chlorophenyl)ethylidene]amino}‐1H‐pyrazol‐
4‐yl)diazenyl]phenyl}ethanone (8b)
M.p. 213–215°C; yield 60%; IR (KBr, cm−1): 3,394, 3,299, 3,190 (NH,
NH2), 1,662 (C═O), 1,613 (C═N), and 1,561 (N═N); 1H NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.02 (s, 3H, N═C–CH3), 2.56 (s, 3H,
CH3), 6.34 (s, 2H, NH2, D2O‐exchangeable), 7.50–7.64 (m, 4H, Ar‐H),
7.70 (d, 2H, Ph‐H3,5, J = 8.4 Hz), and 7.92 (d, 2H, Ph‐H2,6, J = 8.4 Hz);
13C NMR (100 MHz, DMSO‐d6) δ (ppm): 15.15, 27.08, 114.82,
120.57, 120.70, 125.54, 127.66, 128.72, 128.91, 129.73, 134.44,
134.90, 135.81, 138.36, 142.34, 152.92, 158.31, and 197.36; MS
[m/z, %]: 380 [M+, 6.18]; Anal. calcd. for C19H17ClN6O (%): C, 59.92;
H, 4.50; N, 22.07. Found: C, 60.04; H, 4.57; N, 22.19.
N‐{4‐[(4‐Acetylphenyl)diazenyl]‐3‐amino‐1H‐pyrazol‐5‐yl}‐2,4‐
dichlorobenzamide (7a)
M.p. 118–120°C; yield 44%; IR (KBr, cm−1): 3,421, 3,344 (NH2), 3,172
(NH), 1,718 (Ar–C═O), 1,641 (CH3–CO), 1,612 (C═N), and 1587
(N═N); 1H NMR (300 MHz, DMSO‐d6) δ (ppm): 2.56 (s, 3H, CH3), 6.37
(s, 2H, NH2, D2O‐exchangeable), 7.58–7.76 (m, 4H, Ar‐H), 7.84–8.00
(m, 3H, Ar‐H), and 8.40 (s, 1H, NH, D2O‐exchangeable); 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 27.22, 76.18, 121.61, 127.60, 129.73,
129.91, 130.33, 130.88, 134.50, 136.40, 147.13, 149.44, 152.40,
156.14, 157.36, 161.16, 161.55, and 197.36; MS [m/z, %]: 417 [M++1,
0.23]; Anal. calcd. for C18H14Cl2N6O2 (%): C, 51.81; H, 3.38; N, 20.14.
Found: C, 51.99; H, 3.46; N, 20.42.
{4‐[1‐(Hydrazonoethyl)phenyl]diazenyl}(4‐methylbenzylidene)‐1H‐
pyrazole‐3,5‐diamine (8c)
M.p. 218–220°C; yield 54%; IR (KBr, cm−1): 3,397, 3,280, 3,178 (NH,
NH2), 1,605 (C═N), and 1,559 (N═N); 1H NMR (300 MHz, DMSO‐d6)
δ (ppm): 2.37 (s, 3H, tolyl‐H), 2.40 (s, 3H, CH3), 5.99 (s, 2H, NH2, D2O‐
exchangeable), 7.29 (d, 2H, H3,5 4‐CH3–C6H4, J = 7.8 Hz), 7.70 (d, 2H,
Ph‐H3,5, J = 8.4 Hz), 7.76 (d, 2H, H2,6, 4‐CH3–C6H4, J = 7.8 Hz), 7.94 (d,
2H, Ph‐H2,6, J = 8.4 Hz), and 8.48 (1H, CH═ N); 13C NMR (100 MHz,
DMSO‐d6) δ (ppm): 21.59, 25.81, 115.97, 120.52, 127.86, 128.59,
128.63, 129.08, 129.91, 129.99, 132.29, 135.28, 141.31, 152.60,
155.58, 158.18, 163.93, and 175.54; MS [m/z, %]: 360 [M+, 18.14];
Anal. calcd. for C19H20N8 (%): C, 63.32; H, 5.59; N, 31.09. Found:
C, 63.46; H, 5.65; N, 31.31.
N‐(3‐Amino‐4‐{[4‐(1‐hydrazonoethyl)phenyl]diazenyl}‐1H‐pyrazol‐
5‐yl)benzamide (7b)
M.p. 107–110°C; yield 42%; IR (KBr, cm−1): 3,330, 3,260 (br., 2NH2,
2NH), 1,676 (C═O), 1,604 (C═N), and 1,576 (N═N); 1H NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.42 (s, 3H, CH3), 7.42–7.64 (m, 7H,
Ar‐H), 7.91 (d, 2H, Ph‐H2,6), and 12.86 (s, 1H, NH, D2O‐
exchangeable); 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 26.32,
125.07, 126.84, 128.40, 128.59, 129.14, 129.23, 130.69, 132.75,
132.87, and 167.23; MS [m/z, %]: 362 [M+, 9.77]; Anal. calcd. for
C
18H18N8O (%): C, 59.66; H, 5.01; N, 30.92. Found: C, 59.80; H, 5.12;
4‐{[4‐(1‐Hydrazonoethyl)phenyl]diazenyl}(1‐p‐tolylethylidene)‐1H‐
pyrazole‐3,5‐diamine (8d)
N, 31.14.
M.p. 207–210°C; yield 54%; IR (KBr, cm−1): 3,393, 3,296, 3,172 (NH,
NH2), 1,614 (C═N), and 1,557 (N═N); 1H NMR (300 MHz, DMSO‐d6)
δ (ppm): 1.63 (s, 3H, N═C–CH3), 2.03 (s, 3H, tolyl‐H), 2.49 (s, 3H,
CH3), 6.32 (s, 2H, NH2, D2O‐exchangeable), 7.56–7.71 (m, 6H, Ph‐
H2,6, H3,5 and H2,6, 4‐CH3–C6H4), and 7.92 (d, 2H, Ph‐H2,6); 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 21.35, 25.75, 27.05, 114.95, 115.72,
116.81, 120.45, 120.54, 125.54, 126.88, 127.57, 129.44, 129.73,
134.21, 138.16, 142.47, 153.03, 155.18, 157.59, and 175.87; MS
[m/z, %]: 374 [M+, 0.83]; Anal. calcd. for C20H22N8 (%): C, 64.15; H,
5.92; N, 29.93. Found: C, 64.28; H, 6.01; N, 30.18.
|
4.1.6
General procedure for the synthesis
of compounds 8a–h
An equimolar amount of 3a or 3b (0.01 mol) was refluxed with dif-
ferent aromatic aldehydes or aromatic acetophenones in ethanol
(20 ml)/fused sodium acetate (0.2 g) for 3–5 hr, and then the formed
solid product was collected by filtration and washed with hot ethanol.
4‐Chlorobenzylidene‐4‐{[4‐(1‐hydrazonoethyl)phenyl]diazenyl}‐1H‐
pyrazole‐3,5‐diamine (8a)
M.p. 210–213°C; yield 50%; IR (KBr, cm−1): 3,453, 3,388, 3,277 (NH,
NH2), 1,613 (C═N), and 1562 (N═N); 1H NMR (300 MHz, DMSO‐d6)
δ (ppm): 2.44 (s, 3H, CH3), 6.19 (s, 4H, 2NH2, D2O‐exchangeable),
7.51 (d, 2H, H3,5, 4‐Cl–C6H4, J = 8.1 Hz), 7.69 (d, 2H, H2,6, 4‐Cl–C6H4,
4‐{[4‐(1‐Hydrazonoethyl)phenyl]diazenyl}(4‐methoxybenzylidene)‐
1H‐pyrazole‐3,5‐diamine (8e)
M.p. 180–183°C; yield 41%; IR (KBr, cm−1): 3,446, 3,292, 3,178 (NH,
NH2), 1,637 (C═N), and 1,595 (N═N); 1H NMR (300 MHz, DMSO‐d6)
δ (ppm): 2.42 (s, 3H, CH3), 3.83 (s, 3H, OCH3), 7.68–8.01 (m, 8H,
Ar‐H), 8.48 (s, 1H, CH═N), and 12.24 (s, 1H, NH, D2O‐exchangeable)
ppm; 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 25.76, 55.90, 82.47,
114.84, 120.38, 126.80, 128.90, 129.73, 130.93, 131.12, 152.60,
157.66, 162.90, 168.80, and 175.55; MS [m/z, %]: 376 [M+, 1.68];
J = 8.1 Hz), 7.89 (d, 2H, Ph‐H3,5, J = 7.5 Hz), 7.95 (d, 2H, Ph‐H2,6
,
J = 7.5 Hz), 8.52 (s, 1H, –CH═N), and 10.80 (s, 1H, NH, D2O‐
exchangeable); 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 14.60,
115.19, 120.11, 120.25, 127.45, 128.84, 129.06, 129.64, 133.33,

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ISMAIL ET AL.
|
Anal. calcd. for C19H20N8O (%): C, 60.63; H, 5.36; N, 29.77. Found:
C, 60.79; H, 5.44; N, 29.92.
supplemented with 100 mg/ml of streptomycin, 100 units/ml of
penicillin, and 10% of heat‐inactivated fetal bovine serum, in a
humidified 5% (v/v) CO2 atmosphere at 37°C.
1‐{4‐[(3‐Amino‐5‐{[1‐(4‐methoxyphenyl)ethylidene]amino}‐1H‐
pyrazol‐4‐yl)diazenyl]phenyl}ethanone (8f)
M.p. 199–201°C; yield 45%; IR (KBr, cm−1): 3,388, 3,293, 3,174 (NH,
NH2), 1,663 (C═O), 1,612 (C═N), and 1,558 (N═N); 1H NMR
(300 MHz, DMSO‐d6) δ (ppm): 2.03 (s, 3H, N═C–CH3), 2.56 (s, 3H,
CH3), 3.83 (s, 3H, OCH3), 6.34 (s, 2H, NH2, D2O‐exchangeable),
7.62–7.96 (m, 8H, Ar‐H), and 10.89 (s, 1H, NH, D2O‐exchangeable);
13C NMR (100 MHz, DMSO‐d6) δ (ppm): 15.01, 27.06, 55.71, 114.21,
114.27, 114.89, 116.69, 120.50, 120.58, 125.54, 127.59, 128.49,
129.73, 134.31, 138.25, 142.39, 152.98, 157.51, 175.69, and 197.34;
MS [m/z, %]: 376 [M+, 1.90]; Anal. calcd. for C20H20N6O2 (%):
C, 63.82; H, 5.36; N, 22.33. Found: C, 63.97; H, 5.42; N, 22.52.
4.2.2
Cell growth inhibitory assay
|
Cytotoxicity was determined using the MTT method. Exponentially
growing cells from the MCF‐7 cancer cell line were trypsinized,
counted, and seeded at the appropriate densities (2,000–1,000 cells/
0.33 cm2 well) in 96‐well microtiter plates. Cells then were incubated
in a humidified atmosphere at 37°C for 24 hr. Then, cells were ex-
posed to different concentrations of compounds (0.1, 10, 100, and
1,000 µM) for 72 hr. Then the viability of treated cells was de-
termined using MTT technique as follows: Media were removed, cells
were incubated with 200 μl of 5% MTT solution/well (Sigma‐Aldrich,
MO), and they were allowed to metabolize the dye into colored in-
soluble formazan crystals for 2 hr. The remaining MTT solution was
discarded from the wells and the formazan crystals were dissolved in
200 µl/well of acidified isopropanol for 30 min, covered with alumi-
num foil, and with continuous shaking using a MaxQ 2000 plate
shaker (Thermo Fisher Scientific Inc., MI) at room temperature.
Absorbance was measured at 570 nm using a Stat FaxR 4200 plate
reader (Awareness Technology, Inc., FL). The cell viability was ex-
pressed as a percentage of control and the concentration that in-
duces 50% of maximum inhibition of cell proliferation (IC50) was
determined using GraphPad Prism version 5 software (GraphPad
Software Inc., CA).[43,44]
4‐[({3‐Amino‐4‐[4‐(1‐hydrazonoethyl)phenyl]diazenyl}‐1H‐pyrazol‐
5‐yl)imino]methyl}phenol (8g)
M.p. 278–280°C; yield 62%; IR (KBr, cm−1): 3,441–3,156 (OH, NH,
NH2), 1,608 (C═N), and 1,554 (N═N); 1H NMR (300 MHz, DMSO‐d6)
δ (ppm): 2.45 (s, 3H, CH3), 6.00 (s, 2H, NH2, D2O‐exchangeable), 6.60
(s, H, OH, D2O‐exchangeable), 6.79 (d, 2H, H3,5, 4‐OH–C6H4),
7.62–7.71 (m, 4H, Ph‐H2,6 and H2,6, 4‐OH–C6H4), 7.91 (d, 2H, Ph‐
H3,5), and 8.40 (1H, CH═N); 13C NMR (100 MHz, DMSO‐d6) δ (ppm):
25.71, 87.60, 115.85, 116.72, 120.50, 127.66, 130.54, 135.69,
145.50, 155.32, 159.01, 162.92, 175.26; MS [m/z, %]: 362 [M+,
28.94]; Anal. calcd. for C18H18N8O (%): C, 59.66; H, 5.01; N, 30.92.
Found: C, 59.81; H, 5.09; N, 31.13.
4‐[1‐({3‐Amino‐4‐[4‐(1‐hydrazonoethyl)phenyl]diazenyl}‐1H‐pyrazol‐
5‐yl)imino]ethyl}phenol (8h)
|
4.2.3
Cell cycle analysis (DNA flow cytometry
M.p. 215–218°C; yield 58%; IR (KBr, cm−1): 3,393–3,187 (OH, NH,
NH2), 1,612 (C═N), and 1,559 (N═N); 1H NMR (300 MHz, DMSO‐d6)
δ (ppm): 1.66 (s, 3H, N═C–CH3), 2.43 (s, 3H, CH3), 6.00 (s, H, OH,
D2O‐exchangeable), 6.35 (s, 2H, NH2, D2O‐exchangeable), 6.81
analysis)
MCF‐7 cells at a density of 4 × 106 cells/T‐75 flask were exposed to
8b at its IC50 concentration for 24 hr. The cells then were collected
by trypsinization, washed in phosphate‐buffered saline (PBS), and
fixed in ice‐cold absolute alcohol. Thereafter, cells were stained using
Cycle TEST™ PLUS DNA Reagent Kit (BD Biosciences, San Jose, CA)
according to the manufacturer's instructions. Cell cycle distribution
(d, 2H, H3,5, 4‐OH–C6H4), 7.58–7.91 (m, 4H, Ph‐H2,6 and H2,6
,
4‐OH–C6H4), 7.93 (d, 2H, Ph‐H3,5), and 11.00 (s, 1H, NH, D2O‐
exchangeable); 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 14.56, 25.38,
114.50, 116.38, 120.01, 125.03, 126.31, 126.51, 127.06, 129.21,
133.66, 135.26, 137.62, 141.95, 152.56, 154.71, 157.13, and 175.21;
MS [m/z, %]: 376 [M+, 3.13]; Anal. calcd. for C19H20N8O (%): C, 60.63;
H, 5.36; N, 29.77. Found: C, 60.79; H, 5.41; N, 29.89.
was established using
Biosciences).
a
FACSCalibur flow cytometer (BD
|
4.2.4
Annexin V‐FITC assay
|
4.2
In vitro antitumor assay
MCF‐7 cells were seeded in a six‐well plate (1 × 105 cells/well), in-
cubated for 24 hr, and then treated with vehicle (0.1% DMSO) or
3.08 µM of compound 8b for 24 hr. The cells were then harvested,
washed using PBS, stained for 15 min at room temperature in the
dark using annexin V‐FITC and PI in binding buffer (10mM 4‐(2‐
hydroxyethyl)‐1‐piperazineethanesulfonic acid, 140 mM NaCl, and
2.5 mM CaCl2 at pH 7.4), and then analyzed by the flow
cytometer.[45]
|
4.2.1
Methodology: cell culture
Cancer cells from the breast cancer cell line (MCF‐7, human
breast adenocarcinoma) were purchased from American Type
Cell Culture Collection (Manassas) and grown in an appropriate
growth medium, Dulbecco's modified Eagle's medium (DMEM) or
Roswell Park Memorial Institute (RPMI)‐1640 medium,

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|
4.2.5
Determination of CDK2/cyclin E kinase
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sured using serine/threonine‐protein kinase CDK2/Cyclin E, ADP‐
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15 min at 1,000g, 2–8°C, and assayed immediately according to the
manufacturer's instructions. Shortly, the assay was performed using
100 ml of supernatant in cells in each well, which were incubated for
2 hr at 37°C before starting the assay procedure. Finally, the lumi-
nescence was recorded (Integration time 0.5–1 s).
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ACKNOWLEDGMENTS
The authors are grateful to all members of the cell culture lab, Col-
lege of Pharmacy, Al‐Azhar University, for carrying out the antitumor
activity and to confirmatory diagnostic unit VACSERA‐EGYPT for
carrying out the CDK2 assay.
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CONFLICTS OF INTERESTS
The authors declare that there are no conflicts of interests.
ORCID
Amel M. Farrag
http://orcid.org/0000-0002-8207-0859
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