A new CDK2 inhibitor with 3-hydrazonoindolin-2-one scaffold endowed with anti-breast cancer activity: Design, synthesis, biological evaluation, and in silico insights

Article abstract of DOI:10.3390/molecules26020412

Background: Cyclin-dependent kinases (CDKs) regulate mammalian cell cycle progression and RNA transcription. Based on the structural analysis of previously reported CDK2 inhibitors, a new compound with 3-hydrazonoindolin-2-one scaffold (HI 5) was well designed, synthesized, and biologically evaluated as a promising anti-breast cancer hit compound. Methods: The potential anti-cancerous effect of HI 5 was evaluated using cytotoxicity assay, flow cytometric analysis of apoptosis and cell cycle distribution, ELISA immunoassay, in vitro CDK2/cyclin A2 activity, and molecular operating environment (MOE) virtual docking studies. Results: The results revealed that HI 5 exhibits pronounced CDK2 inhibitory activity and cytotoxicity in human breast cancer MCF-7 cell line. The cytotoxicity of HI 5 was found to be intrinsically mediated apoptosis, which in turn, is associated with low Bcl-2 expression and high activation of caspase 3 and p53. Besides, HI 5 blocked the proliferation of the MCF-7 cell line and arrested the cell cycle at the G2/M phase. The docking studies did not confirm which one of geometric isomers (syn and anti) is responsible for binding affinity and intrinsic activity of HI 5. However, the molecular dynamic studies have confirmed that the syn-isomer has more favorable binding interaction and thus is responsible for CDK2 inhibitory activity. Discussion: These findings displayed a substantial basis of synthesizing further derivatives based on the 3-hydrazonoindolin-2-one scaffold for favorable targeting of breast cancer.

Full text of DOI:10.3390/molecules26020412

molecules
Communication
A New CDK2 Inhibitor with 3-Hydrazonoindolin-2-One
Scaffold Endowed with Anti-Breast Cancer Activity:
Design, Synthesis, Biological Evaluation, and In Silico Insights
1
, ,†
, Ahmad J. Obaidullah * , Mohamed E. Shaker , Garri Chilingaryan 5
,
2, ,†
3,4
Mohammad M. Al-Sanea *
Mohammed M. Alanazi 2 , Nawaf A. Alsaif , Hamad M. Alkahtani
2
2
, Sultan A. Alsubaie 2
and Mohamed A. Abdelgawad 1
,6
1
Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University,
Sakaka 72341, Aljouf Province, Saudi Arabia; mohamedabdelwahab976@yahoo.com
Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University,
Riyadh 11451, Saudi Arabia; mmalanazi@ksu.edu.sa (M.M.A.); nalsaif@ksu.edu.sa (N.A.A.);
ahamad@ksu.edu.sa (H.M.A.); 439106132@student.ksu.edu.sa (S.A.A.)
Department of Pharmacology, College of Pharmacy, Jouf University, Sakaka, Aljouf 72341, Saudi Arabia;
mshaker2222@yahoo.com
Department of Pharmacology and Toxicology, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
Institute of Biomedicine and Pharmacy, Russian-Armenian University, Yerevan 0051, Armenia;
garri.chilingaryan@rau.am
2
3
4
5
6
Department of Pharmaceutical Organic Chemistry, Beni-Suef University, Beni-Suef 62514, Egypt
Correspondence: mmalsanea@ju.edu.sa (M.M.A.-S.); aobaidullah@ksu.edu.sa (A.J.O.);
Tel.: +966-594076460 (M.M.A.-S.)
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
*
ꢀ
ꢁꢂꢃꢄꢅꢆ
†
These authors are equally contributed to this work.
Citation: Al-Sanea, M.M.;
Obaidullah, A.J.; Shaker, M.E.;
Chilingaryan, G.; Alanazi, M.M.;
Alsaif, N.A.; Alkahtani, H.M.;
Alsubaie, S.A.; Abdelgawad, M.A.
A New CDK2 Inhibitor with
Abstract: Background: Cyclin-dependent kinases (CDKs) regulate mammalian cell cycle progression
and RNA transcription. Based on the structural analysis of previously reported CDK2 inhibitors,
a new compound with 3-hydrazonoindolin-2-one scaffold (HI 5) was well designed, synthesized,
and biologically evaluated as a promising anti-breast cancer hit compound. Methods: The potential anti-
cancerous effect of HI 5 was evaluated using cytotoxicity assay, flow cytometric analysis of apoptosis
and cell cycle distribution, ELISA immunoassay, in vitro CDK2/cyclin A2 activity, and molecular
operating environment (MOE) virtual docking studies. Results: The results revealed that HI 5
exhibits pronounced CDK2 inhibitory activity and cytotoxicity in human breast cancer MCF-7
cell line. The cytotoxicity of HI 5 was found to be intrinsically mediated apoptosis, which in turn,
is associated with low Bcl-2 expression and high activation of caspase 3 and p53. Besides, HI 5 blocked
the proliferation of the MCF-7 cell line and arrested the cell cycle at the G2/M phase. The docking
studies did not confirm which one of geometric isomers (syn and anti) is responsible for binding
affinity and intrinsic activity of HI 5. However, the molecular dynamic studies have confirmed that
the syn-isomer has more favorable binding interaction and thus is responsible for CDK2 inhibitory
activity. Discussion: These findings displayed a substantial basis of synthesizing further derivatives
based on the 3-hydrazonoindolin-2-one scaffold for favorable targeting of breast cancer.
3-Hydrazonoindolin-2-One Scaffold
Endowed with Anti-Breast Cancer
Activity: Design, Synthesis, Biological
Evaluation, and In Silico Insights.
Molecules 2021, 26, 412. https://
doi.org/10.3390/molecules26020412
Academic Editor: Pascal Marchand
Received: 7 December 2020
Accepted: 11 January 2021
Published: 14 January 2021
Publisher’s Note: MDPI stays neu-
tral with regard to jurisdictional clai-
ms in published maps and institutio-
nal affiliations.
Keywords: synthesis; CDK2; 3-hydrazonoindolin-2-one; MCF-7 breast cancer cell line; cytotoxicity;
apoptosis
Copyright: © 2021 by the authors. Li-
censee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and con-
ditions of the Creative Commons At-
tribution (CC BY) license (https://
creativecommons.org/licenses/by/
1
. Introduction
Cyclin-dependent kinases’ (CDKs) family members are protein kinases that have
an important role in regulating the sequential stages of the cell cycle [ ]. In particular,
CDK2 has a key role in cell cycle progression from the late G1-phase to the S-phase
alongside initiating DNA repair [ ]. Otherwise, CDK2-dependent cell cycle regulation
is critical for proliferation, proliferation, growth, and maintenance of different cancer
1
2
4
.0/).
Molecules 2021, 26, 412. https://doi.org/10.3390/molecules26020412
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 412
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tumors [
3,
4]. For instance, breast cancer falls under the most prevalent and lethal cancer
types affecting women worldwide [5]. Several studies have demonstrated that inhibition
of CDK2 could induce breast cancer cell apoptosis without damage to normal cells [6,7
].
Because of their substantial role in cell cycle regulation, CDKs have become attractive
therapeutic targets for limiting cancers in the breast and those occurring in the pancreas,
gastrointestinal tract, kidney, and other organs [1,4,8–10].
A vast number of studies has been performed to discover and develop new selective
CDK2 inhibitors for cancer therapy with minimal off-target toxicity. For instance, the flavone
alkaloid alvocidib (flavopiridol) was the first multi-CDK inhibitor (types 1, 2, 4, and 9) to be
studied in clinical trials but had limited efficacy and high toxicity [11
pyrazolo [1,5- ]pyrimidin-7-amine core structure is a potent and selective CDK2 activity
with an IC50 value of 1 nM under phase III clinical trial [13 14]. Milciclib is another CDK2
,12]. Dinaciclib with a
α
,
kinase inhibitor with a 4,5-dihydro-1H-pyrazolo[4,3-h]quinazolin-8-amine scaffold that
showed selectivity toward CDK2 (IC50 = 45 nM) at least three times more than other CDKs
1
2
, 2, 4, 5, and 7 [15
and 5 with suspending the proliferation of rapidly growing mammalian cancer cell
lines with an IC50 value of 16 M for CDK2 and induced growth arrest in G1-phase and
accumulation in G2-phase [17 19]. The 3-substituted indolinone compound SU9516 is
,16]. Besides, the small molecule roscovitine (seliciclib) inhibited CDKs
µ
–
another inhibitor of CDK2 that preferably binds to the ATP binding site of CDK2 with
an IC50 value of 22 nM than other CDKs (40 nM for CDK1 and 200 nM for CDK4) [20].
Lastly, NU6300 is the first irreversible CDK2 modulator that can bind covalently to the
cysteine residue in the ATP binding site [21
based compounds were shown to potently inhibit cyclin-dependent kinase 2 (CDK2)
activity as part of a G1 cell cycle arrest of human breast cancer cells [23 29]. A variety of
–23]. Different indole and indole isosteres-
–
oxindole scaffold-based compounds was synthesized as potential modulators for CDKs.
Sunitinib is an FDA approved anticancer agent with an oxindole core structure, and it
showed multikinase inhibitory activity towards CDK2, VEGF1-3, PDGFRb, RET, FLT3,
and CSF-1R [30,31].
Based on the structure analysis, most of the inhibitors mentioned above share an
amino-substituted heterocyclic scaffold, which plays a crucial role in developing bind-
ing affinity on CDK2. Despite the recent improvements in the discovery of CDK2 in-
hibitors, some restrictions that obstruct their applications at a clinical level, such as the
resistance to the drug, lack of specificity, and efficiency. Therefore, dire needs to develop
small molecule inhibitors that target CDK2 with potential and specificity to overcome
resistance among cancer cell populations. Stirred by these findings and in connection
with our current experimental work, it is thought beneficial to extend our investiga-
tions to examine a new 3-hydrazonoindolin-2-one scaffold-based compound (HI 5) as
potential anti-breast cancer and pro-apoptotic agent. Utilizing the virtual docking, the de-
sign here is based on attaching two pharmacologically interested hydrophobic moieties
(indole and isatin) via acetohydrazide linker (Figure 1). In this article, HI 5 was designed,
synthesized, and biologically evaluated as a potential anticancer agent. The target com-
pound was evaluated for its cytotoxicity toward two human breast cancer cell lines MCF-7
and MDA-MB-231 and the doxorubicin-resistant ovarian cell line NCI-ADR utilizing SRB
assay. Moreover, the growth inhibition mechanism in relation to cell cycle regulation and
apoptosis induction in breast MCF-7 cells cancer cells through the DNA flow cytometry
and Annexin V-FITC/PI assays were executed. Furthermore, inhibitory actions against
CDK2 kinase and the anti-apoptotic protein Bcl-2 were implemented. Lastly, virtual dock-
ing simulations were performed to explore the binding modes of the titled compound
within the CDK2 active site. The experimental results showed that the HI 5 is a novel
CDK2 inhibitor with promising antiproliferative activity against human breast MCF-7 and
resistant ovarian NCI-ADR cell lines. Additionally, HI 5 was able to block the cell cycle at
G2/M and induce marked apoptosis in MCF-7 cells.

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Figure 1. The structures of some potent CDK2 inhibitors.
2
2
. Results
.1. Chemistry
The new target indolohydrazid 5 (HI 5) was prepared through a four-steps reaction:
the first step is coupling dehydration of glycolic acid with indole to give indol-3yl-acetic
, which is esterified with ethanol in acidic medium to afford indole acetic acid
. The ester compound was reacted with hydrazine hydrate to afford hydrazide
to produce the target
HI 5 (Scheme 1) using previously described methods for reported compounds ( ) [32 33].
1
acid
ester
2
3
3
4.
5-methoxyindole 2,3-dione condensed in anhydrous condition with 4
2–
4
,
Scheme 1. Schematic representation for HI 5 synthesis: Reagent and conditions: (I) indole, glycolic acid;
+
KOH, HCl, H O; (II) ethanol, H , reflux for 10 h; (III) hydrazine hydrate, ethanol, reflux for 2 h;
2
(IV) acetic acid, 5-methoxyisatin, reflux for 4 h.

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The aliphatic NMR peaks, whether 13C-NMR or H-NMR, confirm the postulated
1
1
structure of HI 5. H-NMR peaks appeared at
δ 3.72–3.76 (m, 3H, OCH ), 3.88 (s, 1H, CH ),
3 2
13
4
.20 (s, 1H, CH ) are representing the aliphatic protons. Additionally, the C-NMR peaks,
2
which appeared at
δ
28.17, 32.36, 55.56, 55.66, confirmed the presence of aliphatic carbons.
2.2. Biological Screening
2.2.1. CDK2 Inhibitory Activity
Among the investigated protein kinases, HI 5 showed moderate potency against
CDK2 with single-digit micromolar EC50 value (6.32 µM) as shown in (Figure 2).
Figure 2. Effects of different concentrations of HI 5 and staurosporine on CDK2/E enzyme activity.
HI 5 showed high growth inhibitory activity CDK2/A2 (49%) and moderate (27%)
growth inhibitory effect against c-Met at a concentration of 10
ited certain selectivity toward CDK2/A2.
µM (Table 1). Hence, HI 5 exhib-
Table 1. Inhibitory effect of HI 5 on CDK2/A2 and c-Met kinases activity at a single dose of 10 µM.
Compound
% Enzyme Inhibitory Activity
CDK2/A2
c-Met
27
HI 5
49
Staurosporine
100
100
2.2.2. Annexin V-FITC Apoptosis Assay
The data showed that cytotoxicity of HI 5 is mainly apoptosis as indicated by an
increase in the percentage of cells in lower (early apoptotic) and upper (late apoptotic)
right quadrants (Figure 3 and Table 2).
Figure 3. Effect of HI 5 on the percentage of annexin V-FITC-positive staining in MCF-7 cells. The four
quadrants identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.

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Table 2. Distribution of apoptotic cells in the AnnexinV-FITC/PI dual staining assay in MCF-7 cells
after treatment with HI 5.
Early Apoptosis
Lower Right (LR) %)
Late Apoptosis
(Upper Right (UR) %)
Total
(LR % + UR %)
Comp.
(
HI 5
Control
5.39
1.03
10.32
0.29
15.71
1.32
2.2.3. Antiproliferative Activities toward Breast Cancer Cell Lines
The antiproliferative effect of the titled compound has shown four times more potency
than Dox in inhibiting the proliferation of the breast cancer cell line MCF-7, while it was
found to be equipotent with Dox in inhibiting the proliferation of the breast cancer cell
line MDA-MB-231 (Table 3). HI 5 has also shown antiproliferative potency on the ovarian
cancer cell line NCI-ADR (Table 3).
Table 3. In vitro antiproliferative activity of HI 5 against breast (MCF-7, MDA-MB-231) and ovarian
(NCI-ADR) cancer cell lines.
IC50 (µM) a
MDA-MB-231
Comp.
MCF-7
NCI-ADR
HI 5
Doxorubicin
1.15 ± 0.04
6.81 ± 0.22
10.54 ± 0.43
10.29 ± 0.72
9.17
ND
b
a
IC50 values are the mean ± S.D. of three separate experiments, b Not determined.
2.2.4. The Effect on the Apoptotic and Anti-Apoptotic Marker Levels
Interestingly, HI 5 lowered the cellular expression of Bcl-2 alongside raising Bax,
leading to a more than a 10-fold increase in the Bax/Bcl-2 ratio compared to the un-
treated MCF-7 control cells. Interestingly, HI 5-treated cells have shown 10-fold higher
expression of active Caspase 3 and 16 fold higher expression of p53 than untreated cells.
Thus, the markers of mitochondrial damage and apoptosis are elevated in cancer cells by
HI 5 pretreatment (Table 4).
Table 4. Effect of HI 5 on the expression levels of Bcl-2, Bax, active caspase-3, and p53 in MCF-7 cancer cells.
Bax
Bcl-2
Compound
Bax/Bcl-2
Caspase-3 (pg/mg)
p53 (pg/mg)
(
pg/mg of Total Protein)
(ng/mg of Total Protein)
HI 5
Control
297.4 ± 11.1
38.3 ± 2.2
2.56 ± 0.20
4.65 ± 0.23
116.2 ± 0.8
8.2 ± 0.7
387.5 ± 13.7
35.92 ± 1.8
673.2 ± 41.0
41.26 ± 2.7
2.2.5. Cell Cycle Assay
HI 5 treatment caused accumulation of MCF-7 cells in the G2/M phase of the cell
cycle, compared to the untreated control cells (Table 5). Thus, HI 5 affected the cell cycle
distribution of MCF-7 cells.
Table 5. Results of cell cycle analysis in MCF-7 expressed by (%) of cell in each phase when treated
with compound HI 5.
Comp.
%G0-G1
%S
%G2-M
%Sub-G1
HI 5
Control
41.54 ± 0.8
57.26 ± 0.2
22.17 ± 1.0
28.59 ± 0.5
36.29 ± 1.2
14.15 ± 1.0
17.34 ± 0.2
1.79 ± 0.8

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2.3. In Silico Studies
2.3.1. Virtual Docking of HI 5
To compare the two geometric isomers of HI 5, the two isomers were subjected to virtual
docking using MOE suite (Molecular Operating Environment (MOE), Chemical Computing
Group Inc.: Montreal, QC, Canada, 2016. The ATP binding site of CDK2 is positioned deep
at the joint of the C and N domains, including the pivotal catalytic amino acids with high
sensitivity and kinase hinge amino acids (Phe82, Glu81, and Leu83). Docking simulation
of both HI 5 isomers (E and Z) showed different binding orientations inside the CDK2
binding pocket (Figure 4).
Figure 4. Two-dimensional representation of docking poses of HI 5 geometric isomers (E and Z).
2.3.2. Molecular Dynamics Simulations
Simulations showed that syn conformation of the HI 5 interacts with the CDK2 ATP
binding site similar to the reference ligand fashion, while anti-isomer does not interact
with it. The ligand’s dynamic stability was studied by calculating the root mean square
deviation (RMSD) of the heavy atoms of the investigated compounds (Figure 5).
Figure 5. Root mean square deviation (RMSD) plot of HI 5 (syn and anti-isomers) and co-crystallized
ligand during simulations.

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The co-crystallized ligand binds to the site with the average binding energy of
−
106.7 kJ/mol. Syn-isomer of the HI 5 interacts with the ATP binding site with an average
binding energy of
−
91.6 kJ/mol. Van der Waals, electrostatic, polar solvation, solvent acces-
sible surface (SASA), and binding energies for the complex structures of HI 5 isomers and
CDK2 were calculated using the G_MMPBSA approach for the last 10 ns of the simulations
and presented in Table 6.
Table 6. HI 5 syn/anti-isomers and co-crystallized ligand energies (kJ/mol).
Compound
Van der Waals
Electrostatic
Polar Solvation
SASA
Binding Energy
Co-crystallized ligand 1
HI 5 (syn-isomer)
−178.546 ± 10.295
−186.753 ± 9.303
−102.060 ± 25.028
1
−20.344 ± 7.826
−53.024 ± 6.399
−28.865 ± 8.671
107.937 ± 11.584
165.947 ± 8.579
82.992 ± 56.836
−15.750 ± 0.720
−17.805 ± 0.788
−10.440 ± 2.166
−
−
−
106.703
±
3.411
12.345
47.381
91.636
58.374
±
HI 5 (anti-isomer)
±
4-(5-Bromo-2-oxo-2H-indol-3-ylazo)-benzenesulfonamide.
To distinguish the key amino acid residues that play an essential role in binding,
we calculated the energetic contributions of all amino acid residues to the ligand binding.
Comparison and differences in the values of total energies of the ATP binding site’s amino
acid residues with HI 5 syn-isomer and co-crystallized ligand are presented in Figure 6.
Figure 6. Comparison of interacting amino acid residues of the ATP binding site of CDK2 (PDB ID 1FVT)
with HI 5 syn-isomer (Syn) and co-crystallized ligand (Crystal).
Thirteen amino acids with a difference in energy values more than ~2 times were care-
fully compared to gain insights into four energetic components (
and Gtotal). Figure 7 depicts the contribution of these four energetic components to the
overall binding affinity.
∆EMM, ∆Gpolar, ∆Gapolar,
∆
Figure 7. Energetic components’ values of the selected amino acid residues with HI 5 syn-isomer
Syn) and co-crystallized ligand (Crystal).
(

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3. Discussion
3
.1. Chemistry
1H-NMR and 13C-NMR data were effectively used to elucidate the chemical structures
1
of HI 5. The shielded H-NMR peaks at
protons OCH and CH . The three NH groups are represented by six peaks in H-NMR
at
at
δ
3.72, 3.76, 3.88, and 4.20 represent the aliphatic
1
3
2
13
δ
range of 10.89 to 13.01 ppm. C-NMR spectrum showed four aliphatic carbons
28.17, 32.36, 55.56, 55.66, and the total 38 peaks confirm the presence of the two
δ
geometric isomers. The NMR spectra of HI 5 showed characteristic double peaks in
equal percentage, which indicated that the two isomers (Syn/Anti) were presented in an
equal percentage [34,35].
3.2. Biological Screening
3.2.1. CDK2 Inhibitory Activity
Protein kinases constitute a major class of enzymes that have importance in cell
division and signaling [36]. Pharmacological inhibitors of certain protein kinase subtypes
such as serine/threonine and tyrosine kinases represent the targeted cancer therapy class
members. Accordingly, we determined whether cytotoxicity of HI 5 is related to inhibition
of certain types of serine/threonine and/or tyrosine kinases. Of the investigated protein
kinases, HI 5 showed marked inhibition of the activity of the serine/threonine kinase CDK2
acting on cyclin E with an IC50 of 6.32µM as shown in (Figure 2). Besides, HI 5 exhibited high
inhibitory activity (49%) against CDK2 acting on cyclin A and moderate inhibitory effect
against the tyrosine kinase c-Met at a concentration of 10 µM (Table 1).
3.2.2. Annexin V-FITC Apoptosis Assay
Induction of apoptosis is a well-known therapeutic strategy for mediating tumor
remission. The ongoing research on chemotherapeutics works on developing novel small
molecules that augment the extrinsic and intrinsic pathways of apoptosis. These apoptotic
pathways are commonly impaired by the cancer cells’ resistance mechanisms to maintain
their progression and evasion from the host immune surveillance. To test whether HI 5
has potential cytotoxicity, flow cytometric analysis was performed using annexin V-FITC
and PI stainings to detect apoptosis and necrosis of MCF-7 cells, respectively (Figure 3 and
Table 2).
3.2.3. Antiproliferative Activities toward Breast Cancer Cell Lines
Next, the antiproliferative potency of HI 5 was compared with the classical chemother-
apeutic agent doxorubicin (Dox) by determining the IC50 on breast MCF-7 and MDA-
MB-231 cancer cell lines (Table 3). Besides, the antiproliferative effect of HI 5 was further
confirmed on resistant ovarian cancer cell line NCI-ADR (Table 3).
3.2.4. The Effect on the Apoptotic and Anti-Apoptotic Marker Levels
The extrinsic pathway is mediated by exogenous stimulation of death receptors by
their ligands, while the intrinsic apoptotic cell death pathway is initiated by endogenous
signals arising from DNA damage and oxidative stress. These signals induce the mito-
chondrial permeability transition pore opening by inactivating the anti-apoptotic proteins
such as Bcl-2. Thus, it is not surprising that cancer cells dampen these damaging signals by
overexpressing Bcl-2. The overexpression of Bcl-2 also acts as an inhibitor for apoptotic
proteins such as Bax to mediate the mitochondrial permeability transition pore opening.
Therefore, the influence of HI 5 on Bcl-2 and Bax expression levels in MCF-7 cancer cells
was quantified to ascertain whether HI 5 induces cytotoxicity via the mitochondrial per-
meability transition pore opening (Table 4). The data indicated that HI 5 induced the
mitochondrial permeability transition pore opening as evidenced by the higher Bax/Bcl-2
ratio, compared to control cells.
Following the mitochondrial permeability transition pore opening, cytochrome C is re-
leased from the mitochondria and activates caspase 9 via the apoptosome assembly.

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Subsequently, the activated caspase-9 cleaves and activates other executioner Caspases, such as
0
caspase-3 and caspase-7, followed by DNA damage and apoptosis. Accordingly, HI 5 s effect
on active caspase 3 and p53 expressions in MCF-7 cancer cells was evaluated as indicators
of DNA damage and cell death (Table 4). Based on these results, HI 5-induced cytotoxicity
is mediated by the mitochondrial intrinsic pathway of apoptosis.
3.2.5. Cell Cycle Assay
The cell division cycle comprises G1 phase of cellular growth and preparation for DNA
synthesis, S phase of DNA synthesis, G2 phase of preparation for mitosis, and M phase
of mitosis. We investigated the effect of HI 5 on the cell cycle. According to the results,
we can determine whether HI 5 is a cell cycle-specific agent that acts selectively on cancer
cells when they are traversing, or not. (Table 5). HI 5 treatment raised the percentage
of MCF-7 cells in the G2/M phase of the cell cycle in comparison to the control cells
(
36% vs. 14%). Thus, HI 5 seems to impair the phases of DNA replication and mitosis of
cancer cells, leading to arrest of division.
3.3. In Silico Studies
3.3.1. Virtual Docking of HI 5
The molecular docking simulations for HI 5 in its two geometric isomers (E and Z)
showed three important hydrogen bond interactions with Leu83, Glu81, and Leu83 via
acetohydrazide NH and 2-oxoindolin N and 2-oxoindolin O, respectively, which represent
key interactions with kinase active domain (Figure 4). Besides, the docking poses of HI 5
showed that 2-oxoindolin moiety made anchoring hydrophobic interactions mixed with
VDW interactions with Val18 and Val64 and Ala144 and Ala31 from both sides, as revealed
in Figure 4.
3.3.2. Molecular Dynamics Simulations
Results of molecular dynamics simulation of HI 5 syn-isomer at the ATP binding site
of the CDK2 showed highly stable (RMSD < 0.2 nm) binding affinity with the maintenance
of initial binding pose during 150 ns simulation. However, the anti-isomer leaves the ATP
binding site after ~36 ns then keeps localized close to binding site until ~70 ns followed
by complete left until the end of the simulation (Figure 5). According to the performed
molecular dynamics simulation results, the HI 5 anti-isomer does not interact properly
with the ATP binding site of the CDK2.
HI 5syn-isomer and reference ligand interact with the ATP binding site of CDK2 with
the close average binding energy (Table 6). Total interaction energies of the following amino
acids: Ala31, Val64, Glu81, His84, Lys89, and Leu134 are similar for the two compounds.
Glu81 and Asp86 have relatively high positive values of total binding energy with both
compounds, while Asp145 has high total positive binding energy only with HI 5syn-
isomer. Gln85 interacts only with co-crystallized ligand with high negative binding energy.
Asn132, in contrast to Gln85, interacts only with syn-isomer of the HI 5. Ile10, Phe80, Phe82,
and Leu83 interact with co-crystallized ligand with relatively higher binding affinity than
syn-isomer. At the same time, Gly11, Val18, Lys33, Gln131, Leu134, and Ala144 interact
with higher binding affinity with the syn-isomer of HI 5.
Asp145 interacts with syn-isomer of the HI 5 through relatively strong VdW and elec-
trostatic energy ( 12.1 kJ/mol), but overwhelmingly, high polar energy (33 kJ/mol) overall
−
results in the repelling effect between HI 5 syn-isomer and Asp145, while co-crystallized
ligand does not interact with this residue. High polar energies in cases of Asp86 and Glu81
also result in repelling effects.
Both VdW and electrostatic energies control most of the analyzed interactions between
amino acid residues of the ATP binding site of CDK2 and studied compounds. The similar
binding affinity of the HI 5 syn and co-crystallized ligand and the difference in the binding
site’s interacting amino acid residues pattern characterize syn-isomer of the HI 5, a potential
competitive inhibitor of the CDK2. The obtained results have valuable information that

Molecules 2021, 26, 412
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0
can be used for future design and modifications of the HI 5 s syn-isomer, in particular,
to neutralize high polar energy values of the mentioned binding site’s amino acid residues.
4. Materials and Methods
4.1. Chemistry
Compound 1 was commercially available, and it was obtained from Sigma Aldrich
Company, St. Louis, MO, USA. Compounds
the reported methods [37].
2–4 were chemically synthesized according to
Synthesis of (syn/anti) 1H-indol-3-yl)-acetic acid (5-methoxy-2-oxo-1,2-dihydro-indol-
-ylidine)-hydrazide (HI 5). A mixture of (1H-indol-3-yl)-acetic acid hydrazide (0.01 mol,
3
1
.89 gm) and 5-methoxy-1H-indole 2,3-dione (0.01 mol, 1.77 gm) was dissolved in absolute
ethanol (30 mL) containing glacial acetic acid (0.01 mL) [38 40]. The reaction mixture was
–
heated under reflux for 6h, and the reaction progress was followed by TLC. The reaction
mixture was cooled, and the formed precipitate was filtered and crystallized from ethanol
◦
with yield of 62%. Yellow solid, m.p.: 232.5 234 C, H-NMR (DMSO-d , 400 MHz)
6
1
−
δ
ppm; 3.72–3.76(m, 3H, OCH ), 3.88(s, 1H, CH ), 4.20(s, 1H, CH ), 6.78–6.891(m, 2H, ArH),
3 2 2
6
.96–7.07(m, 2.5 H, ArH), 7.18 (s,0.5H, ArH), 7.30–7.36(m, 2H, ArH), 7.51–7.60(m, 1H, ArH),
1
1
1
1
0.89–11.08(m, 2H, 2NH), 12.54(s, 0.5H, NH), 13.01(s, 0.5H, NH). 13C-NMR (DMSO-d6,
00 MHz) ppm: 28.17, 32.36, 55.56, 55.66, 105.49, 105.60, 105.74, 106.35, 107.05, 111.44,
δ
11.50, 111.55, 111.89, 117.38, 117.82, 118.25, 118.46, 118.60, 118.65, 120.63, 121.04, 121.20,
21.32, 124.39, 124.83, 126.99, 127.10, 127.27, 134.02, 135.95, 136.20, 136.32, 137.02, 137.20,
+
+
155.34, 162.60, 168.42, 173.40. MS m/z [%]: 348.7 [M , 13.44], 190.5 [M , 100]; elemental analysis
for C H N O ; Calc. C; 65.51, H; 4.63, N; 16.08, found C; 65.22, H; 4.69, N; 15.98.
19
16
4
3
4.2. Antiproliferative Activities Toward Breast and Resistant Ovarian Cancer Cell Lines
Breast (MCF-7 and MDA-MB-231) and doxorubicin-resistant ovarian (NCI-ADR)
cancer cell lines were obtained from Nawah Scientific Inc., (Mokatam, Cairo, Egypt).
In Dulbecco’s Modified Eagle’s media, cells were maintained and supplemented with
1
00 mg/mL of streptomycin, 100 units/mL of penicillin, and 10% heat-inactivated fetal
◦
bovine serum in humidified, 5% (v/v) CO atmosphere at 37 C.
2
4.3. Cytotoxicity Assay
Cell viability was assessed by SRB assay according to the reported procedure. Aliquots
3
of 100
µ
L cell suspension (5
×
10 cells) were taken in 96-well plates and incubated for 24 h.
Cells were treated with another aliquot of 100 µL media containing the vehicle DMSO or
the HI5 at various concentrations. After 72 h of drug exposure, cells were fixed by replacing
◦
media with 150
µ
L of 10% TCA and incubated at 4 C for 1 h. The cell wells were then
washed five times with distilled water, followed by the addition of 70
0.4% w/v) and incubation in the dark at room temperature for 10 min. Plates were washed
three times with 1% acetic acid and allowed to air-dry overnight. Then, 150 L of TRIS
10 mM) was added to dissolve the protein-bound SRB stain; the absorbance was measured
at 540 nm.
µL of SRB solution
(
µ
(
4.4. Flow Cytometric Analysis of Apoptosis and Cell Cycle Distribution
MCF-7 cells were treated by HI 5 at IC50 concentration or the vehicle DMSO for 24 h.
After that, the cells were collected for flow cytometry detection of exposure of internal
membrane phosphatidylserine occurring during apoptosis using staining with Annexin-V-
FITC (BD Pharmingen, San Diego, CA, USA) and propidium iodide (PI, Sigma-Aldrich,
St. Louis, MO, USA) using FACS Calibur and CellQuest software (Becton Dickinson,
Totowa, NJ, USA). Flow cytometry was also performed for MCF-7 cells after PBS wash,
ethanol fixation, ribonuclease, and PI additions when treated or not treated by HI 5 at
IC50 concentration for 24 h to analyze the distribution of cell cycle phases as previously
described [38] (See supplementary file).

Molecules 2021, 26, 412
11 of 14
4.5. ELISA Immunoassay
The concentrations of Bax, Bcl-2, cleaved caspase-3, p53, and phosphorylated c-Met
were determined using ELISA kits (Abcam, Boston, MA, USA). In brief, MCF-7 cells were
treated by HI 5 at IC50 concentration or the vehicle DMSO for 24 h. After that, the cells were
◦
rinsed with PBS and lysed in RIPA lysis buffer at 4 C for 30 min, followed by centrifugation
◦
at 10,000
×
g for 20 min. Supernatants were then collected and stored at
−
80 C for later
protein determination using Bradford’s protein assay. Then, equal protein concentrations
of cell lysates were incubated in pre-coated ELISA kits with primary antibodies specific to
Bax, Bcl-2, and cleaved caspase-3. After one hour, the appropriate secondary biotin-linked
antibody was added, followed by streptavidin-HRP and TMB/H O . A stop solution
2
2
was finally added to the plate wells when the developed color was optimal, followed by
measuring the optical absorbance at 450 nm by a microplate reader (BMG LABTECH-FLUO
star Omega, Ortenberg, Germany) (See supplementary file).
4.6. In Vitro CDK2/Cyclin A2 and c-Met Activity
The CDK2/cyclin A2 and c-Met protein kinase assays were performed using Kinase-
Glo Plus luminescence kinase assay kit (Promega, Madison, WI, USA), as illustrated
earlier [19 24 41]. In brief, the kinase activity was assessed by measuring the unreacted
,
,
ATP remaining in solution after the kinase reaction. The percentage inhibition of the HI 5
against CDK2/cyclin A2 and c-Met protein kinases were compared to that of staurosporine
at a single concentration of 10 µM (See supplementary file).
4.7. In Silico Studies
4.7.1. Virtual Docking of HI 5
All molecular docking studies were performed using molecular operating environ-
ment (MOE) software. The crystal structure of CDK2 was downloaded from the RCSB
Protein Data Bank (PDB ID: 1FVT). The protein crystal structure was then prepared,
and the docking protocol was validated and performed according to the reported method-
ologies [28,42].
4.7.2. Molecular DYNAMICS simulations
Missing atoms of the Leu25, Arg36, and missing amino acid residues (Leu37, Asp38,
Thr39, Glu40, Thr41, Glu42, Gly43, Val154, Pro155, Val156, Arg157, Thr158, Tyr159, Thr160,
His161, Glu162) of the CDK2 structure (PDB ID:1FVT) were modeled using CHARMM-
GUI [43]. Three molecular dynamics simulations: syn/anti-isomers of the HI 5 and co-
crystallized ligand with CDK2, were performed, every 150 ns. Docking binding poses
of syn and anti-isomers of HI 5 were used as starting points for the corresponding simu-
lations; co-crystallized ligand (106) of the 1FVT was used as a reference compound [44].
Molecular dynamics simulations were performed using GROMACS 2019.25 molecular
dynamics package. AMBER99SB-ILDN force field was used for simulations [45,46]. As the
force field does not contain parameters for the ligand, all ligands were parameterized
using ANTECHAMBER to generate consistent parameters with the General Amber Force
Field (GAFF) [41]. The AM1-BCC method was used to assign charges. ACPYPE was
used for conversion of ligands topologies to format compatible with GROMACS [45–47].
All simulations were performed in an explicit water environment, using the TIP3P model.
+
Complexes were solvated in a dodecahedron box system and were neutralized with Na
−
and Cl ions. Steepest descent was used for depreciation, and Fmax was set of no greater
−
1
−1
than 1000 kJ mol nm . Systems were equilibrated using NVT and NPT ensembles
for 200 ps duration each. The temperature was sustained at 300 K using the V-rescale
algorithm [48]. For the regulation of systems pressure, the Parrinello–Rahman barostat
was used. The linear constraint solver (LINCS) algorithm was used for the bond’s length
constraints [49]. The particle mesh Ewald (PME) method was used for long-range electro-
statics calculations. For all simulations, time step was set to 2 fs. Van der Waals’s cut-off
distance was set to 1 nm. Binding-free energies were calculated using the G_MMPBSA

Molecules 2021, 26, 412
12 of 14
program [50
,51], with the MM-PBSA method adapted for GROMACS. The binding energy
consists of three energetic terms, potential energy in a vacuum, polar-solvation energy,
and non-polar solvation energy. The SASA model was used to calculate the non-polar
solvation energy. For the calculations of all interaction energies, the last ten ns (every frame
with 100 ps interval) of the trajectories were used.
5
. Conclusions
In conclusion, we have successfully designed and synthesized a novel molecule with
-hydrazonoindolin-2-one scaffold (HI 5). This compound has shown marked cytotoxic
3
activity on certain human breast cancer cell lines through intrinsic apoptosis. The an-
tiproliferative effects against MCF7 cell line showed an IC50 of 1.15 M. The cell cycle
µ
assay showed growth arrest of MCF7 cell lines at G2/M phase. Compound HI 5 showed
selectivity toward CDK2/A2 with high (49%) growth inhibitory activity against CDK2/A2.
The molecular docking studies of HI 5 in the CDK2 active site disclosed that the indole
and isatine groups could be exchanged with possible other bioisosteric equivalents with
different Es, π, and σ values. Moreover, the syn- and anti-isomers of HI 5 showed almost
the same binding types, strength, and energy score. However, the molecular dynamic
simulations confirm the responsibility of syn-isomer (rather than the anti-isomer) for the
CDK2 antagonistic activity of HI 5. Further development of 3-hydrazonoindolin-2-one
scaffold will be applied to synthesize a new series of bioisosteric analogs with more potent
and selective CDK2 inhibitory activities.
1
Supplementary Materials: The following are available online, Figure S1: H-NMR chart for com-
1
13
pound HI 5, Figure S2: H-NMR expanded chart for compound HI 5, Figure S3: C-NMR chart for
compound HI 5.
Author Contributions: Conceptualization, M.M.A.-S. and A.J.O.; methodology, M.E.S.; software,
G.C.; validation, G.C., M.E.S., and M.M.A.; formal analysis, N.A.A.; investigation, H.M.A.; re-sources,
M.M.A.-S.; data curation, M.E.S.; writing—original draft preparation, S.A.A.; writ-ing—review and
editing, M.A.A.; visualization, M.A.A.; supervision, A.J.O.; project administra-tion, A.J.O.; funding
acquisition, A.J.O. and M.M.A. All authors have read and agreed to the published version of the
manuscript.
Funding: The authors extend their appreciation to the Deanship of Scientific Research at King Saud
University for funding this work through research group no. (RG-1441-398).
Data Availability Statement: The data presented in this study are available on request from the
corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.
Sample Availability: Sample of the compound HI 5 is available from the authors.
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