Design, synthesis, and anticancer screening for repurposed pyrazolo[3,4-d]pyrimidine derivatives on four mammalian cancer cell lines

Article abstract of DOI:10.3390/molecules26102961

The present study reports the synthesis of new purine bioisosteres comprising a pyra-zolo[3,4-d]pyrimidine scaffold linked to mono-, di-, and trimethoxy benzylidene moieties through hydrazine linkages. First, in silico docking experiments of the synthesized compounds against Bax, Bcl-2, Caspase-3, Ki67, p21, and p53 were performed in a trial to rationalize the observed cytotoxic activity for the tested compounds. The anticancer activity of these compounds was evaluated in vitro against Caco-2, A549, HT1080, and Hela cell lines. Results revealed that two (5 and 7) of the three synthesized compounds (5, 6, and 7) showed high cytotoxic activity against all tested cell lines with IC50 values in the micro molar concentration. Our in vitro results show that there is no significant apoptotic effect for the treatment with the experimental compounds on the viability of cells against A549 cells. Ki67 expression was found to decrease significantly following the treatment of cells with the most promising candidate: drug 7. The overall results indicate that these pyrazolopy-rimidine derivatives possess anticancer activity at varying doses. The suggested mechanism of action involves the inhibition of the proliferation of cancer cells.

Full text of DOI:10.3390/molecules26102961

molecules
Article
Design, Synthesis, and Anticancer Screening for Repurposed
Pyrazolo[3,4-d]pyrimidine Derivatives on Four Mammalian
Cancer Cell Lines
Eman M. Othman 1 , Amany A. Bekhit , Mohamed A. Anany 3,4, Thomas Dandekar 1 , Hanan M. Ragab 5,*
,2
2
6
,
and Ahmed Wahid *
1
2
3
4
5
6
Department of Bioinformatics, Biocenter, University of Wuerzburg, Am Hubland, 97074 Würzburg, Germany;
eman@toxi.uni-wuerzburg.de (E.M.O.); dandekar@biozentrum.uni-wuerzburg.de (T.D.)
Department of Biochemistry, Faculty of Pharmacy, Minia University, Minia 61519, Egypt;
amany_bkeet@mu.edu.eg
Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wuerzburg,
97080 Würzburg, Germany; mohamed_M@klinik.uni-wuerzburg.de
Division of Genetic Engineering and Biotechnology, Department of Microbial Biotechnology,
National Research Centre, Giza 12622, Egypt
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21500, Egypt
Department of Pharmaceutical Biochemistry, Faculty of Pharmacy, Alexandria University,
Alexandria 21500, Egypt
*
Correspondence: hanan.ragab@alexu.edu.eg (H.M.R.); ahmed.wahid@alexu.edu.eg (A.W.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Abstract: The present study reports the synthesis of new purine bioisosteres comprising a pyrazolo[3,4-
d]pyrimidine scaffold linked to mono-, di-, and trimethoxy benzylidene moieties through hydrazine
linkages. First, in silico docking experiments of the synthesized compounds against Bax, Bcl-2,
Caspase-3, Ki67, p21, and p53 were performed in a trial to rationalize the observed cytotoxic activity
for the tested compounds. The anticancer activity of these compounds was evaluated in vitro against
Citation: Othman, E.M.; Bekhit,
A.A.; Anany, M.A.; Dandekar, T.;
Ragab, H.M.; Wahid, A. Design,
Synthesis, and Anticancer Screening
for Repurposed
Pyrazolo[3,4-d]pyrimidine
Caco-2, A549, HT1080, and Hela cell lines. Results revealed that two (5 and 7) of the three synthesized
Derivatives on Four Mammalian
Cancer Cell Lines. Molecules 2021, 26,
compounds ( , and ) showed high cytotoxic activity against all tested cell lines with IC50 values
5
,
6
7
in the micro molar concentration. Our in vitro results show that there is no significant apoptotic
effect for the treatment with the experimental compounds on the viability of cells against A549 cells.
Ki67 expression was found to decrease significantly following the treatment of cells with the most
2
961. https://doi.org/10.3390/
molecules26102961
promising candidate: drug 7. The overall results indicate that these pyrazolopyrimidine derivatives
possess anticancer activity at varying doses. The suggested mechanism of action involves the
inhibition of the proliferation of cancer cells.
Academic Editor: Margherita Brindisi
Received: 8 April 2021
Accepted: 10 May 2021
Published: 16 May 2021
Keywords: pyrazolo[3,4-d]pyrimidine; anticancer activity; apoptosis; Ki67
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1
. Introduction
Cancer is considered a serious health hazard problem. Indeed, cancer is ranked the
second cause of recorded deaths in the world. More than 9 million deaths were recorded
due to cancer in 2018 [1].
About 68,800 novel cancer cases are recorded in Egypt every year. Liver cancer is
Copyright: © 2021 by the authors. prevalent in Egypt. This is most likely due to the spread of the hepatitis C virus among
Licensee MDPI, Basel, Switzerland.
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distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
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the Egyptian population. Other types of cancer that exist in Egypt include bladder, non-
Hodgkin lymphoma, leukaemia, lung, and prostate cancers [2,3].
The cost of anticancer drugs is increasingly being recognized as a global problem.
The price of cancer treatment in Egypt is exorbitant and may delay both diagnosis and
treatment. A study was performed in order to conduct cost analysis to assess expenditures
for acute myeloid leukaemia (AML) treatment in Egypt from 1999 to 2002. In the study, the
4
.0/).
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https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2961
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median total cost of therapy per patient was $16,701 (or 33,158 LE, comparatively high for
the average household therapy) [4].
Purines are an important scaffold for the design of potent antitumour agents. They
were modified using the bioisostere pyrazolo [3,4-d] pyrimidine as the central nucleus
of the synthesized compounds [
d]pyrimidines substituted with a diazole moiety bearing different aromatic nuclei (com-
pounds of the general formula A; Figure 1) [ ]. Some of the synthesized compounds
5]. Ghorab et al. synthesized a group of pyrazolo[3,4-
6
showed moderate cytotoxic activity. A 2-phenylaminopyrimidine called imatinib is specifi-
cally used for the treatment of gastrointestinal stromal tumours and chronic myelogenous
leukaemia (CML) [
hibitor, which has been studied for the treatment of B-cell cancers [
modulates many signal pathways that regulate cell proliferation, growth, and apoptosis [
7
]. In addition, ibrutinib is a pyrazolopyrimidine-based kinase in-
]. Pyrazolopyrimidine
].
8
9
Figure 1. Chemical structure of previously reported compounds (
design of the newly synthesized compounds (5–7).
A–C) [10] as a scaffold for the
Furthermore, the abovementioned findings led to the design and synthesis of a group
of compounds that comprise the pyrazolo[3,4-d]pyrimidine scaffold linked to a piper-
azine moiety, bearing different aromatic nuclei through different amide linkages (com-
pounds of the general formulas B and C; Figure 1), as well as to tests of their anticancer
activity [10]. Finally, further screening of literature revealed that the introduction of a
3
,4-dimethoxybenzylidene moiety to the hydrazine group at the 4-position of the above-
mentioned nucleus resulted in a new compound ( ; Figure 1): this was capable of inhibiting
6
BCR-ABL tyrosine kinase, an enzyme highly implicated in cell proliferation leading to
the development of tumours. In addition, the inhibitors may bind to the ATP-binding
pocket and restrain the ATP-binding capacities of other kinases, which may diminish their
proliferative effects [11]. However, the study tested the effect of such an action on treating
type 2 diabetes mellitus (T2DM).
Thus, it was the goal of this study to design and synthesize a group of compounds
that comprise the N-aryl pyrazolo[3,4-d]pyrimidine scaffold having different arylidene-
hydrazino substitution at the 3-, 4- and 5-positions (compounds 5–7) and to test their

Molecules 2021, 26, 2961
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anticancer activity. Synthesized compounds are expected to retard the progression of ma-
lignancies. The substitution pattern of the synthesized compounds was carefully selected
so as to confer different electronic and lipophilic properties to molecules.
Accordingly, we investigated if the newly synthesized pyrazolo[3,4-d]pyrimidine
derivatives could significantly improve cancer therapy. We also identified the primary
mechanism of action of the new drugs.
Furthermore, in silico docking experiments of the synthesized compounds against
Bax, Bcl-2, Caspase-3, Ki67, P21, and P53 were executed, aiming at providing insights into
the observed cytotoxic and antiproliferative activities for the tested compounds.
2. Results
2.1. Chemistry of the Compounds under Investigation in the Current Study
The synthetic strategies employed for the synthesis of the target compounds in the
current study are illustrated in (Figure 2). Compounds
with the methods described in the literature [12 13]. Compounds
) with the appropriate aromatic aldehyde. The
1–
4
were synthesized in accordance
,
5–7
were obtained through
condensation of the hydrazine compound (
4
1
H-NMR spectra lacked the two D O-exchangeable protons corresponding to the NH
2
2
and revealed instead the appearance of a benzylidene CH proton at its expected chemical
shift. Furthermore, the spectra indicated the appearance of additional aromatic protons
at δ 7–8 ppm corresponding to the aromatic aldehyde aromatic protons together with the
13
methoxy protons at their expected chemical shifts. Finally, C-NMR of the synthesized
derivatives indicated the appearance of a signal corresponding to the benzylidene carbons
in addition to the methoxy carbons at their expected chemical shift.
Figure 2. Synthetic pathway for the synthesis of the target compounds
5–7; reagents and reaction
conditions: i: HCONH , reflux, 10 h; ii: POCl , reflux, 4 h; iii: NH NH , EtOH, reflux, 6 h; iv: ArCHO,
2
3
2
2
EtOH, reflux, 3 h.
.2. In Vivo Toxicity Evaluation
Before the assessment of the anticancer activity of the compounds, in the present study,
2
we examined their toxicity in vivo on normal tissues. The treated rats with 20 mg/kg/day
did not show significant toxicity in comparison to the control group (Table 1). The results
indicate the possible future application of the studied compounds in vivo with lower
side effects.

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Table 1. Measurement of the change in the hepatic and renal biochemical parameters in rats treated
with the investigated compounds.
Control Group
5
6
7
Cholesterol (mg/dL)
Triglyceride (mg/dL)
Bilirubin (mg/dL)
Albumin (mg/dL)
ALT (U/L)
AST (U/L)
ALP (U/L)
70 ± 3.1
74 ± 5.1
71 ± 4.8
72 ± 8.6
69 ± 7.8
75 ± 6.5
71 ± 5.8
77 ± 7.5
0.12 ± 0.13
4.5 ± 0.12
44 ± 4.1
0.11 ± 0.12
4.4 ± 0.37
43 ± 5.2
0.12 ± 0.12
4.6 ± 1.8
44 ± 5.7
0.11± 0.32
4.6 ±1.1
42 ± 7.2
103 ± 4.6
1.92 ± 0.47
5.8 ± 0.23
28 ± 2.5
102 ± 8.7
2.1 ± 0.52
5.7 ± 0.98
27 ± 4.2
100 ± 9.8
2 ± 0.12
103 ± 8.8
1.8 ± 0.88
5.88 ± 0.90
28 ± 3.86
Urea (U/L)
Creatinine (mg/dL)
5.8 ± 0.80
29 ± 3.86
2.3. Docking Analysis
The docking simulation for the ligand was carried out using molecular operating
environment (MOE) software supplied by the Chemical Computing Group, Inc., Montreal,
QC, Canada [14]. Six different protein targets were examined for the binding site; the
selected proteins are the most common proteins in the cytotoxicity process, resembling
different mechanisms for cytotoxicity. Studying the ligand interactions of all six proteins, it
was found that tightly bound conserved water molecules are of importance in the protein–
ligand complex binding sites of three of them (Caspase-3, Bax, and P53). Accordingly, their
docking simulations were done without the removal of the water chains.
2.3.1. Docking of the Test Compounds (5–7) in the Binding Site of Ki67 Protein
Docking studies were carried out using the enzyme parameters obtained from exam-
ining the structure of Ki67 (Protein Data Bank (PDB) ID: 2aff) [15].
The binding modes for the test compounds synthesized in our study (
analysed, and the following results were obtained.
5–7) were
In the case of the binding mode of compound
5 (Figure 3a), the results revealed a
binding score equal to ( 4.5039). The X-ray crystal structure revealed the presence of
−
two arene–cation bonds between Arg245 and the phenyl and pyrazole rings, in addition to
various hydrophobic interactions with other amino acids.
The binding mode of
6
(Figure 3b) showed a binding score equal to ( 4.4699). The
−
X-ray crystal structure revealed two arene–H bonds, one between the pyrimidine ring
and Lys257 and the second between the phenyl and Asp258, in addition to hydrophobic
interactions with several amino acids.
Figure 3c shows the binding mode of
7, indicating its superior binding score of
(
−
6.5176) relative to and 4.5039 and 4.4699, respectively). The X-ray crystal struc-
5
6
(
−
−
ture revealed hydrogen bonding between the pyrazole unsubstituted-N and Gln248 and
an arene–H bonding between the N-phenyl and Glu251, in addition to the hydrophobic
interactions with several other amino acids.
2.3.2. Docking of the Test Compounds (5–7) in the Binding Site of Caspase-3 Protein
The docking study was carried out using the enzyme parameters obtained from
Protein Data Bank of the structure of caspase-3, having the PDB ID: 6bdv [16].
Running the binding simulation for the tested compounds ( ) gave the following results
In the case of binding mode (Figure 4a), the results revealed a binding score equal to
5.838). The X-ray crystal structure revealed only various hydrophobic interactions with
other different amino acids.
The binding mode of
5–7
5
(
−
6
(Figure 4b) showed a binding score equal to ( 5.0599). The
−
X-ray crystal structure revealed arene–H binding between the phenyl ring and Asn208,
in addition to hydrophobic interactions with several amino acids. Figure 4c shows the
binding mode of
7
, indicating a binding score of ( 5.7119). Furthermore, it indicates the
−
presence of an arene–cation bonding between Arg64 and the pyrazolo-phenyl ring together
with a hydrogen bonding between the pyrazolo-N and Arg207. Furthermore, two water

Molecules 2021, 26, 2961
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molecules were involved in a bond between the phenyl ring and Phe250, in addition to the
hydrophobic interactions with several other amino acids.
2.3.3. Docking of the Test Compounds (5–7) in the Binding Site of p53 Protein
This docking study was carried out using the enzyme parameters obtained from the
Protein Data Bank structure of p53 (PDB ID: 5mct) [17].
To predict the binding modes for the test compounds (
performed, and the following results were obtained.
5–
7), docking simulations were
In the case of the binding mode of compound
5 (Figure 5a), the results revealed a
binding score equal to (−5.2404). The X-ray crystal structure revealed that the compound
forms four arene–H bonds with the protein, the first between the pyrazole ring and Gly112
and the second between the pyrimidine ring and Arg110, in addition to two bonds between
the N-phenyl and Hist115 and Phe113. Furthermore, various hydrophobic interactions
with other amino acids were observed.
(
a)
(b)
(
c)
Figure 3. Two-dimensional binding mode of the test compounds (
a)
6, (
b
)
5, and (
c)
7
in the binding site of Ki67 protein
(
PDB ID: 2aff) using MOE software.
The binding mode of
6
(Figure 5b) showed a binding score equal to ( 4.9425). The
−
X-ray crystal structure revealed that the compound forms two arene–H bonds with the
protein, the first between the pyrimidine ring and Arg110 and the second between the
N-phenyl and Phe113, in addition to hydrophobic interactions with several amino acids.
Figure 5c shows the binding mode of
7
, indicating a superior binding score of ( 5.3192)
−
relative to and 5.2404 and 4.9425, respectively). Furthermore, it showed hydrogen
5
6
(
−
−
bonding between the pyrimidine-N and Gln104 and an arene–H bonding between the
N-phenyl and Tyr126, in addition to the hydrophobic interactions with several other
amino acids.

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(
a)
(b)
(
c)
Figure 4. Two-dimensional binding mode of the test compounds (
PDB ID: 6bdv) using MOE software.
a) 6, (b) 5, and (c) 7 in the binding site of caspase-3 protein
(
2.3.4. Docking of the Test Compounds (5–7) in the Binding Site of the Bax Protein
A docking study was carried out using the enzyme parameters obtained from the
structure of Bax (PDB ID: 6eb6) [18].
Performing the binding modes for the test compounds (5–7) revealed to the following
In the case of the binding mode of compound
5 (Figure 6a), the results revealed a
binding score equal to (4.0295). The X-ray crystal structure revealed that the N-phenyl ring
formed an arene–H bonding with Asn104, while its pyrazolo unsubstituted-N accepted a
hydrogen bonding from Asn106. Furthermore, hydrophobic interactions were observed
with other amino acids.
The binding mode of 6 (Figure 6b) showed a binding score equal to (4.7846). The X-ray
crystal structure revealed an arene–H bonding with Asn106, in addition to hydrophobic
interactions with several amino acids.
Figure 6c shows the binding mode of
7
, indicating a superior binding score of ( 5.1502)
−
relative to and 4.0295 and 4.7846, respectively). Furthermore, three arene–H bondings
5
6
(
−
between the methoxy and Phe105, the pyrimidine and Asn106, and finally the pyrazole
ring and Asn106, in addition to the hydrophobic interactions with several other amino
acids. Surprisingly enough, water molecules were not involved in any of the bonding
between the protein and any of the tested compounds, despite its involvement in the
binding between the protein and the reported ligand.

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2.3.5. Docking of the Test Compounds (5–7) in the Binding Site of Bcl-2 Protein
A docking study was carried out using the enzyme parameters obtained from the
Protein Data Bank of the structure of Bcl-2, having the PDB ID: 5whh [19] for the synthesized
compounds (5–7) showing the following results.
(
a)
(b)
(
c)
Figure 5. Two-dimensional binding mode of the test compounds (
a)
6
, (
b)
5, and (
c)
7
in the binding site of P53 protein
(
PDB ID: 5mct) using MOE software.
In the case of the binding mode of compound
binding score equal to (
acid Lys116 donates two hydrogen bondings, one to the hydrazono-N and the other to
the pyrimidine-N, while the pyrazolo nucleus forms an arene–H bonding with Arg11, in
addition to various hydrophobic interactions with other amino acids.
5
(Figure 7a), the results revealed a
−
5.9126). The X-ray crystal structure revealed that the amino
The binding mode of
6
(Figure 7b) showed a binding score equal to ( 5.6649). The
−
X-ray crystal structure revealed that the pyrazolo nucleus forms two arene–H bondings
with Val112 and Thr 114, in addition to hydrophobic interactions with several amino acids.
Figure 7c shows the binding mode of
7
, indicating a superior binding score of ( 6.5306)
−
relative to and 5.9126 and 5.6649, respectively). Furthermore, the pyrazolo ring
5
6
(
−
−
accepts a hydrogen bonding from Asp113 and an arene–H bonding between the pyrimidine
ring and Ile23, in addition to the hydrophobic interactions with several other amino acids.
2.3.6. Docking of the Test Compounds (5–7) in the Binding Site of P21 Protein
The docking study was carried out using the enzyme parameters obtained from the
Protein Data Bank of the structure of P21, having the PDB ID: 6nzv [20].

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In a trial to predict the binding modes for the test compounds (
simulation has been performed, and the following results were obtained.
5–
7), a docking
(
a)
(b)
(
c)
Figure 6. Two-dimensional binding mode of the test compounds (
a
)
6
, (
b)
5
, and (
c)
7
in the binding site of Bax protein
(
PDB ID: 6eb6) using MOE software.
In the case of the binding mode of compound
binding score equal to (
ring formed an arene–H bonding with Thr1042. Furthermore, the Gln1041 formed an
arene–H bonding to the hydrazono-NH, in addition to various hydrophobic interactions
with other amino acids.
5
(Figure 8a), the results revealed a
−
6.6203). The X-ray crystal structure revealed that the pyrimidine
The binding mode of
6
(Figure 8b) showed a binding score equal to ( 6.4258). The
−
X-ray crystal structure revealed a hydrogen bonding between Glu1013 and hydrazono-NH,
in addition to hydrophobic interactions with several amino acids.
Figure 8c shows the binding mode of
7
, indicating a binding score of ( 6.3091). Fur-
−
thermore, it showed two hydrogen bondings, one between the hydrazono-N and Lys1136
and the other between the pyrimidine-N and Gly1137, in addition to the hydrophobic
interactions with several other amino acids.
2.4. Cell Viability with MTT
A (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium) MTT assay was used for the
determination of the cytotoxic effects of three compounds (
Hela, HT1080, Caco-2, and A549 cells were treated with various concentrations of the
compounds employed in the current study (0, 1, 10, 50, 100, 500 M) for 48 h. Compound
did not show any significant decrease in the cell viability measured in the tested cell lines
data not shown). However, compound showed anticancer activity against HT1080, Hela,
Caco-2, and A549 cells with IC50 values of 96.25, 74.8, 76.92, and 148 M, respectively, and
5, 6, and 7) in vitro (Figure 9).
µ
6
(
5
µ

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with significant reduction in cell viability upon treatment with lower concentrations than
the IC50 (50, 50, 100, and 50 µM, respectively).
(
a)
(b)
(
c)
Figure 7. Two-dimensional binding mode of the test compounds (
a)
6, (
b)
5, and (c)
7
in the binding site of Bcl-2 protein
(
PDB ID: 5whh) using MOE software.
Compound
3.75, 17.50, 73.08, and 68.75
upon treatment with lower concentrations than the IC50 (25, 12.5, 1, and 1
7
showed anticancer activity against the same cells with IC50 values of
M, respectively, with significant reduction in cell viability
M, respectively).
4
µ
µ
For the first application of the in silico results to biological system, two main proteins
cleaved by caspase-3 were examined for their expression in the cell lines treated with
compound
7, as this was the most promising candidate in the studied compounds. Further
assessment of the detailed mechanism will be conducted in the future studies.
2.5. Apoptosis Assays
In order to exclude apoptotic effects as a mechanism for the observed anticancer
activity of the compounds and according to the in silico results, induction of apoptosis
by compound was examined using an annexin V flow cytometry assay (Figure 10a).
After incubation of the cells with compound for 24 h, the results showed that there is
7
7
no significant effect of the treatment with the compound on the viability of A549 cells
through apoptosis in comparison to the untreated group. This suggests that the mechanism
of action of this anticancer compound is not mediated via apoptosis. These results were

Molecules 2021, 26, 2961
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further confirmed by western blots. Our results show that there is no significant change in
the level of the apoptotic cleaved caspase-3 protein expression compared to the untreated
cells (Figure 10b).
(
a)
(b)
(
c)
Figure 8. Two-dimensional binding mode of the test compounds (
PDB ID: 6nzv) using MOE software.
a)
6
, (
b)
5, and (
c)
7
in the binding site of P21 protein
(
2
.6. Proliferation Assay
Ki67 expression was determined using a flow cytometry assay. Ki67 expression was
found to decrease significantly following the cells’ treatment using compound at IC50
7
concentration for 48 h (Figure 10c). The percentage of cells labelled using Ki67 antibody
in the control cells was 85.61%, whereas those labelled with the same antibody following
treatment with compound
7 was 68.06%. We demonstrated that Ki67 expression was
significantly reduced in the cells treated with compound 7.

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Figure 9. Normalised percentage cell viability inhibition of compounds
5
and
7
, measured by three
independent MTT assays in A549 and Caco-2 cell lines and by three independent experiments with
crystal violet staining in Hela and HT1080 cell lines. Cells were treated with different concentrations
(from 1 and up to 500
µM) of the compounds for 48 h, * = first concentration significant reduction in
cell viability in comparison to the control.

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(
a)
(
b)
(
c)
Figure 10.
IC50 concentration. Flowcytometry was conducted using Alexa Fluor 488 annexin V conjugate.
The percentage gating in the lower left quadrant was ( ) 97.8 in the control untreated cells and
) 97.58% in compound . ( ) Western blot analysis showing cleaved caspase-3 protein expression
level. The upper panel shows (1) control, (2) compound , (3) compound , and (4) compound
at IC50 concentration. The lower panel demonstrates B actin as loading control. ( ) Proliferation
analysis (Ki67) in A549 cells following treatment cells with compound at IC50 concentration. The
analysis was done using flow cytometry and the Ki-67 FITC antibody. ( ) shows the flow cytometric
analysis of the control, whereas (B) shows the flow cytometric analysis of compound 7.
(a) Apoptosis analysis in A549 cells following treatment of cells with compound 7 at
A
(B
7 b
5
6
7
c
7
A

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3. Discussion
Protein kinases have a fundamental role in cell signal transduction cascades that
possess several cellular activities (36). Recently, small molecules of protein kinase inhibitor
have been broadly tested for antineoplastic properties, such as midazo [1, 5-a] pyrazines,
benzotriazines, indoles, and pyrazolopyrimidines. Numerous pyrazolopyrimidines-based
compounds have been approved for the treatment of various types of malignancies due to
their effectiveness on signal cascades [21].
An MTT assay was used for the determination of the cytotoxic effects of three com-
pounds (
tions of the drugs employed in the current study were inoculated to the cells. According
to Brown et al. [ ], compound (the 3,4-dimethoxy derivative) was expected to show the
5, 6, 7) in vitro against Hela, HT1080, Caco-2, and A549 cells. Various concentra-
5
6
highest antitumour activity, where it was introduced in the aforementioned research as
a promising protein kinase inhibitor for the treatment of type 2 diabetes mellitus T2DM.
However, upon testing, it did not show any significant decrease in the cell viability mea-
surements against HT1080 and Hela cells, indicating that despite the fact that it is a protein
kinase inhibitor, it does not possess any anticancer activity against the selected cell lines.
Nevertheless, it is worth testing further whether it will be able to reduce the measurement
of cell viability against other cell lines that might have protein kinase as a part of their
survival mechanism, and it will be a part of a future study conducted in our laboratory.
Testing the other two derivatives, compounds
on the other hand, showed promising results against the cell lines used, with compound
being the most active. These results indicate that the presence of one methoxy group at
the 4-position, as in compound , is preferred for moderate antitumour activity against
5 (the monomethoxy) and 7 (the trimethoxy),
7
5
selected cell lines; however, the addition of a second methoxy group at the 3-position totally
abolished the activity. Surprisingly enough, the introduction of a third methoxy group
resulted in an enhancement of the activity, as observed in compound 7, which resulted in
the highest antitumour activity. These results indicate that the additional methoxy group is
essential for improving the activity.
It was previously confirmed that celecoxib (diaryl-substituted pyrazole structure) elec-
tively inhibits cyclo-oxygenase-2 activity (COX-2); COX-2 inhibition may result in apoptosis
and a reduction in tumours’ angiogenesis and metastasis [22]. Moreover, celecoxib was
found to inhibit the growth of hepatocellular carcinoma cells by activating retinoblastoma
protein (pRb) through its hypophosphorylation, thus repressing the DP1/E2F1 complex,
and inducing apoptosis through the activation of caspase-3 and caspase-9 [23].
Different molecular mechanisms were reported for cell death, including intrinsic
and extrinsic apoptosis, mitochondrial permeability transition (MPT)-driven necrosis,
necroptosis, pyroptosis, entotic cell death, lysosome-dependent cell death, autophagy-
dependent cell death, immunogenic cell death, cellular senescence, mitotic catastrophe,
and other different uncommon mechanisms [24,25].
In order to determine the mechanism of action of compounds
assays were conducted using an annexin flow cytometry assay. Contrary to previous
literature [26 27], our results show that there is no significant effect of the treatment of
5 and 7, apoptotic
,
experimental compounds on the viability of cells with regard to the apoptotic level against
A549 cells compared to the control untreated group. This suggests that the mechanism of
action of this anticancer compound is not mediated via apoptosis. These results were further
confirmed using the western blot technique. Our results show that there is no significant
change in the level of caspase-3 expression compared to the control untreated cells. On the
other hand, a new pyrazolopyrimidine analogue multiple tyrosine kinase inhibitor (CLM3)
performed both antiproliferative and proapoptotic properties via a mechanism mediated
by the suppression of phosphorylation of extracellular signal regulated kinases ERK1/2
and protein kinase B Akt, respectively [28].
Ki67 expression level was found to be decreased significantly following cells’ treatment
using compound
7. Ki67 is a nuclear protein that is associated with cellular proliferation.
It was reported before that Ki67 expression was decreased following chemotherapy. The

Molecules 2021, 26, 2961
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expression of Ki67 is strongly associated with tumour proliferation and growth. Therefore,
Ki67 is widely used as a biomarker for cancer proliferation. The Ki67 antigen encodes
two protein isoforms. Expression of Ki67 is low in the G1 phase and is increased during
the S and G2 phases. It reaches the maximum expression in the M phase. A significant
reduction in Ki67 expression levels occurs in the later phases of mitosis [29–31]. A prior
study indicated that pyrazolopyrimidine, PP2, diminished RET/PTC1-mediated mitogen-
activated protein (MAP) kinases signalling. Moreover, PP2 controlled the proliferation and
the invasive phenotype of human thyroid carcinoma cells [32].
Comparing the docking studies results on Ki67 (an indicator of cellular proliferation)
with those of caspase-3 (an indicator of apoptosis) revealed that a much better binding
of compound
H bonding, the compound formed two hydrogen bonds with Ki67. On the other hand,
compound showed only one arene–H bond with caspase-3. Reviewing the above, it was
found to be highly consistent with the experimentally obtained results, which indicates
7 was demonstrated towards Ki67, where, in addition to one weak arene–
7
that compound
than apoptosis.
7 inhibits cell growth through inhibition of cellular proliferation rather
4
. Materials and Methods
.1. Chemistry
The synthetic procedure of the target compounds was done according to reported
procedures [ 10 33], resulting in the new unreported compounds and 7, and is available
in the Supplementary Materials.
-(4-Methoxybenzylidene)-1-(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)hydrazine (
4
5,
,
5
2
5), White
◦
−1
crystals (65 mg, 86%), m.p: 183–185 C. IR (KBr, cm ): 3427 (NH), 2885, 3039, 3111 (Ar-
H), and 1675, 1597 and 1543 (C=N and Ar-C=C), 1253, 1171 (C-O). H-NMR (400 MHz,
1
DMSO-d6,
.37–7.49 (m, 4H, Ar-benzylidene-C3,5-H and pyrazolophenyl-C2,6-H), 7.55–7.58 (dist
t, 3H, pyrazolophenyl-C3,4,5-H), 8.03–8.05 (d, 2H, Ar-benzylidene-C2,6-H), 8.21 (s, 1H,
pyrazolopyrimidine-C3-H), 8.35 (s, 1H, pyrazolopyrimidine-C6-H), 12.56 (s, 1H, NH, D O
δ ppm): 4.05–4.07 (broad s, 3H, OCH ), 7.24 (singlet, 1H, benzyli dene-CH=N),
3
7
2
exchangeable). 13C-NMR (100 MHz,
δ_ppm): 56 (OCH ), 108, 114, 122, 123, 124, 127, 130,
3
1
32, 136, 138, 139, 149, 152, 157 (Ar-C). Anal. Calcd for C H N O (344.37): C, 66.27; H,
19 16 6
4
.68; N, 24.40. Found: C, 66.08; H, 4.56; N, 24.27.
-(3,4,5-Trimethoxybenzylidene)-1-(1-phenyl-1H-pyrazolo[3,4-d]pyrimidin4yl)hydrazine (7),
2
◦
−1
White crystals (80 mg, 90%), m.p: 196–198 C. IR (KBr, cm ): 3427 (NH), 2938, 2835
1
(
Ar-H), and 1665, 1599 (C=N and Ar-C=C), 1281, 1126 (C-O). H-NMR (400 MHz, DMSO-
d ,
6
δ ppm): 4.39 (broad s, 9H, OCH ), 7.26 (singlet, 1H, benzylidene-CH=N), 7.37–7.41
3
(t, J = 8 Hz, 1H, pyra zolophenyl-C4-H), 7.57–7.61 (t, J = 8 Hz, 2H, pyrazolophenyl-
C3,5-H), 7.69–7.73 and 7.94–7.98 (d, J = 16 Hz, 2H, Ar-benzylidene-C2,6-H), 8.21-8.26
(
1
d, J = 20 Hz, 2H, pyrazolophenyl-C2,6-H), 8.67 (s, 1H, pyrazolopyrimidine-C3-H), 8.92 (s,
13
H, pyrazolopyrimidine-C6-H), 11.01 (s, 1H, NH, D O exchangeable). C-NMR (100 MHz,
2
δ
ppm): 56.3, 56.6, 60 (3 “OCH ”), 106, 120, 121.3, 121.6, 127, 129, 130, 132, 134, 139, 140,
3
1
44.2, 144.3, 153.4, 153.6, 153.8, 154, 156 (Ar-C). Anal. Calcd for C H N O (404.42): C,
21 20 6 3
62.37; H, 4.98; N, 20.78. Found: C, 62.21; H, 4.96; N, 21.07.
4.2. In Vivo Toxicity Evaluation
Male albino rats (150 g average weight) were purchased from the animal house of the
Institute of Graduate Studies and Research, Alexandria University, Egypt. The animals
were housed under standardized environmental conditions, fed with a standard diet
and water, and left to acclimate to the environment for one week prior to inclusion in
the experiment. All of the animal experiments were conducted in accordance with the
guidelines for the care and use of laboratory animals of the National Institutes of Health
(
NIH 1985). The experimental procedures employed were ethically reviewed and approved
by the Institutional Animal Care and Use Committee (IACUC) of Alexandria University
Ethical approval number of the study: AU14-200922-1-8A) and “declaration of Helsinki”.
(

Molecules 2021, 26, 2961
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Four groups of rats were administered 20 mg/kg/day of compounds
5
,
6, and
7
orally twice
weekly for 6 weeks. Liver enzymes and other liver biomarkers together with creatinine
and urea were measured (Table 1). All of the results of the former parameters revealed that
compounds 5, 6, and 7 are safe compounds comparable to the healthy control group.
Assessment of Toxicity Parameters
The collected blood was allowed to clot, the serum was separated at 2000
×
g, and
the biochemical markers of hepatic damage, including serum ALT, AST [33], ALP [34],
albumin [35], total bilirubin [36], TG [37], urea [38], creatinine [39], and total choles-
terol [40], were estimated according to previously reported methods. These parameters
were determined using available commercial kits (ARE) following the instructions of
the manufacturer.
4.3. Docking Study
Computer-assisted simulation docking experiments were carried out under an MMFF94X
force field using Molecular Operating Environment (MOEDock 2015) software, Chemical
Computing Group, Montreal, QC, Canada.
Docking Protocol
The coordinates from the X-ray crystal structure of Ki67, caspase-3, P53, Bax, Bcl-2,
and P21 used in this simulation were obtained from the Protein Data Bank (PDB ID: 2aff,
6
bdv, 5mct, 6eb6, 5whh, and 6nzv, respectively), where the active site is bound to the
appropriate ligand. The ligand molecules were constructed using the builder molecule
and were energy minimized. The active sites of the proteins were generated using the
MOE-Alpha Site Finder, and then ligands were docked within this active site using the
MOE Dock. The lowest energy conformation was selected, and the ligand interactions
(
hydrogen bonding, arene–H, arene–arene interactions together with other hydrophobic
interactions) with the proteins were recorded.
4.4. Cell Culture
Human fibrosarcoma (HT1080), cervical cancer (Hela), colorectal cancer (Caco-2), and
human lung cancer cell lines (A549) were purchased from American Type Culture Collec-
tion (ECACC, Salisbury, UK). Cervical cancer cells (Hela) and fibrosarcoma cells (HT-1080)
were maintained in a RPMI medium supplemented with 10% foetal bovine serum (FBS)
◦
and were expanded at 37 C and 5% CO . Caco-2 and A549 cells were grown in Dulbecco’s
2
modified eagle medium (DMEM)-high glucose (biowest, Riverside, MO, USA) enriched
with (10% v/v) (foetal bovine serum) FBS (biowest, Riverside, MO, USA) and antibiotics
(
100 U/mL penicillin, 100
µ
g mL streptomycin) (Lonza, Basel, Switzerland). Cells were
◦
maintained at 37 C in an incubator with 5% CO atmosphere. Part of the experiments
2
were carried out in CERRMA (Center of Excellence for Research in Regenerative Medicine
and its Applications), Faculty of Medicine, Alexandria University.
4.5. Experimental Design
Hela, Caco-2, HT1080, and A549 cancer cell lines were screened for their integrity
and to ensure that they were free from contamination before starting the study and then
cultured as previously mentioned [41–44].
4.6. Cell Viability Assays
The cell viability of A549 and Caco-2 cells was measured using MTT assays. Cells
were incubated for 48 h in triplicates with the compounds of interest. The MTT assay shows
the effect of the drug on the viability and proliferation of different cancer cell lines. Cells
3
were seeded in 96-well plates at a density of 5
×
10 cells/well and were maintained in
complete media. After adherence for 48 h, cells were treated with different concentrations
of newly synthesized compounds ranging from 10 to 500 M. The plates were incubated at
µ

Molecules 2021, 26, 2961
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◦
% CO and 37 C. After 48 h, the medium was discarded, and cells were incubated with
2
5
1
3
00 µL MTT (Serva, Heidelberg, Germany) solution (0.5 mg/mL) for 3 h at 5% CO and
7 C. Finally, the reagent was discarded and replaced with 100 µL DMSO. The optical
2
◦
density was measured at a wavelength of 570 nm (Tecan, Infinite F50, Magellan reader
control and data analysis software, Mennedov, Switzerland). The percentage cell viability
data were obtained by normalization of the optical density (OD) values for all groups to
the OD of the control group.
4
Cervical Hela and HT-1080 cells (2
culture plates in 100
×
10 per well) were seeded in 96-well tissue
µL cell culture medium. Cells were incubated for 48 h in triplicates
with the compounds of interest. The cytotoxic effect was assessed by crystal violet staining
and measured at 595 nm. To normalize cell viability values each plate included a triplicate
of cells treated with DMSO, the vehicle of the compound to calculate 100% viable cells. At
the same time, a triplicate of cells was simulated with a cytotoxic combination (200 ng/mL
TNF, 200 ng/mL CD95L, 200 ng/mL TRAIL, 25 µg/mL CHX, 1% (w/v) sodium azide)
to elucidate maximal cell death, and thus 0% viability. All other viability values were
normalized depending on the averages of these triplicates and evaluated by the Graph Pad
Prism 5 software (La Jolla, CA, USA).
4.7. Apoptosis Assays
For evaluating cell apoptosis, cells were plated in 6-well plates at a density of
2
× 104 cells per well. After incubation for 24 h, 70% confluency is reached. Cells were
then labelled using the FITC Annexin V apoptosis detection kit I (BD Pharminogen, San
Diego, CA, USA) according to manufacturer’s instructions (Cell Signaling Technology;
Flow Cytometry, Methanol Permeabilization Protocol) and analysed by using BD FACS
Calibur flow cytometry and Cell Quest™ software.
4.8. Cell Proliferation Assay
For evaluating the proliferation status of cells, cells were plated in 6-well plates at a
4
density of 2
7
×
10 cells per well for the cell proliferation assay. After incubation for 24 h,
0% confluency is reached. Cells treated with IC50 concentration of the tested compound
for 48 h were labelled using the Ki67 Proliferation Kit (D3B5, Rabbit mAb Alexa Fluor
88 Conjugate, Cell Signaling Technology) according to manufacturer’s instructions (Cell
®
4
Signaling Technology; Flow Cytometry, Methanol Permeabilization Protocol) and analysed
by using BD FACS Calibur flow cytometry and Cell Quest™ software.
4.9. Investigation of Caspase-3 Protein Expression Levels Involved in the Anticancer Effect
A western blot assay was carried out to examine cleaved caspase-3 protein expression
levels following the different treatments with different concentrations of the compound that
5
were employed in the current study. Cells (5
×
10 cells/well) were seeded in 12-well plates
and were induced the next day with the compounds of interest for 48 h. The cells were
collected and centrifuged (2 min, 10,000 g). The cells were washed two times with ice-cold
PBS, and the cell pellet was suspended in 4 Laemmli sample buffer (8% (w/v) SDS, 0.1 M
dithiothreitol, 40% (v/v) glycerol, 0.2 M Tris, pH 8.0) supplemented with a phosphatase
inhibitor cocktail II (Sigma) by sonification (20 pulses) and heating for 5 min at 95 C. After
×
×
◦
removal of the remaining insoluble debris by centrifugation (2 min, 10,000
the lysate were applied to SDS–PAGE. The isolated proteins were blotted from the gel to
a nitrocellulose. The proteins of interest were detected with a suitable primary antibody
×
g) 8 µL of
(
Anti-Caspase-3, 17,19 kD, Rabbit polyclonal = 9661, from Cell Signaling Technology,
6
Beverly, MA, USA) and horseradish peroxidase conjugated secondary antibodies (Dako,
Glostrup, Denmark and Cell Signaling Technology, Beverly, MA, USA).
Statistics
The in vitro data are representative of at least three independent experiments and
shown as mean
±
standard deviation (SD). Statistical significance among multiple groups

Molecules 2021, 26, 2961
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was tested with the Anova post hoc tukey test. A p value of less than 0.05 is considered to
be statistically significant for all results.
5. Conclusions
The target of the present study was to synthesize and evaluate the antineoplastic
activities of some new pyrazolo[3,4-d]pyrimidines. The obtained results clearly revealed
that compounds 5 and 7 are promising antineoplastic agents against four cancer cell lines
investigated in the current study and thus represent a fruitful matrix that deserves further
investigation and derivatization.
Supplementary Materials: The following are available online, a description of all reagents, solvents
and instrumentation used in the chemistry part is supplied together with the general procedure for
the synthesis of the target compounds. In addition the yield and m.p. of the reported compound (
are given.
6)
Author Contributions: Conceptualization, E.M.O., T.D., and A.W.; methodology, H.M.R., A.A.B.,
and M.A.A.; software, H.M.R. and A.W.; validation, E.M.O., A.A.B., T.D. and M.A.A.; investigation,
A.W., H.M.R., and E.M.O.; resources, A.A.B. and M.A.A.; writing—original draft preparation, E.M.O.,
A.W., and H.M.R.; writing—review and editing, E.M.O., T.D., A.W. and A.A.B.; visualization, H.M.R.
and M.A.A.; supervision, A.W. and E.M.O.; project administration, A.W. and H.M.R. All authors
have read and agreed to the published version of the manuscript.
Funding: We thank Land Bavaria (contribution of DFG-project 324392634/TRR 221 to T.D. and E.M.O.).
Institutional Review Board Statement: No human subjects involved, for the animal experiment.
Informed Consent Statement: No human subjects involved, Not applicable.
Data Availability Statement: All results and data are given in the manuscript and its supplements.
Further details are available from the corresponding author. The data presented in this study are
available on request from the corresponding author.
Acknowledgments: We acknowledge Harald Wajant, Division of Molecular Internal Medicine,
Department of Internal Medicine II, University Hospital Wuerzburg, Wuerzburg, Germany, for his
input in the study.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Samples are limited, but we have protocols and can advise how to gener-
ate them.
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