Isatin-Schiff's base and chalcone hybrids as chemically apoptotic inducers and EGFR inhibitors; design, synthesis, anti-proliferative activities and in silico evaluation

Article abstract of DOI:10.1016/j.molstruc.2021.130159

Isatin derivatives have been found to possess anti-proliferative effects against different human cancer cell lines. A series of isatin-Schiff's base and chalcone were synthesized and screened for their anticancer activities against three human cell lines which are: MCF-7, HepG-2, and HCT-116. All the tested compounds exhibited moderate to high antitumor activity with IC₅₀ ranging from 0.68–35.60 μM compared to Imatinib as a reference standard. Compounds 2b, 5, 8b, 12, 13a and 13b were the most active, with IC₅₀ ranging from 0.68 to 5.85 μM for the three cell lines. The most active structures were selected for further investigations. Firstly, the IC₅₀ values on normal human cells (WI-38) have been studied to ensure the safety of our hits, which showed that our new compounds have exhibited (IC₅₀ >165.98 μM) as far as their cytotoxic effect is concerned. Moreover, up-regulation of BAX and Caspase-3 and down-regulation of Bcl-2 resulted in the induction of apoptosis from those active compounds. Further work has shown that the most potent derivative 8b, caused cell cycle arrest at the G2/M phase. Also, EGFR inhibitory activity for 8b showed IC₅₀ 0.014 μM versus wild EGFR^WT and 12.66 nM versus the mutant type, Lapatinib, and Erlotinib were used as reference standards with IC₅₀ values of 0.025, 0.0653 μM and 35.72, 59.56 nM versus both, respectively. Furthermore, the in-silico assessment showed that all-potent compounds were orally bioavailable without blood-brain barrier permeation. Finally, the molecular docking of 8b inside the active site of EGFR (1M17) showed a good binding through three hydrogen bonds and one arene-cation interaction.

Full text of DOI:10.1016/j.molstruc.2021.130159

Journal of Molecular Structure 1234 (2021) 130159
Contents lists available at ScienceDirect
Journal of Molecular Structure
journal homepage: www.elsevier.com/locate/molstr
Isatin-Schiff’s base and chalcone hybrids as chemically apoptotic
inducers and EGFR inhibitors; design, synthesis, anti-proliferative
activities and in silico evaluation
Eman A. Fayed a,∗, Rogy R. Ezz Eldinb, Ahmed B. M. Mehanyc, Ashraf H. Bayoumid,
Yousry A. Ammare,∗
a Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr City, Cairo, 11754, Egypt
b Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Port Said University, Egypt
c Zoology Department, Faculty of Science, Al-Azhar University, Nasr City, Cairo 11884, Egypt
d Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy (Boys), Al-Azhar University, Nasr City, Cairo, 11884, Egypt
e Chemistry Department, Faculty of Science (Boys), Al-Azhar University, Nasr City, Cairo, 11884, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Isatin derivatives have been found to possess anti-proliferative effects against different human cancer cell
lines. A series of isatin-Schiff’s base and chalcone were synthesized and screened for their anticancer ac-
tivities against three human cell lines which are: MCF-7, HepG-2, and HCT-116. All the tested compounds
exhibited moderate to high antitumor activity with IC50 ranging from 0.68–35.60 μM compared to Ima-
tinib as a reference standard. Compounds 2b, 5, 8b, 12, 13a and 13b were the most active, with IC50
ranging from 0.68 to 5.85 μM for the three cell lines. The most active structures were selected for fur-
ther investigations. Firstly, the IC50 values on normal human cells (WI-38) have been studied to ensure
the safety of our hits, which showed that our new compounds have exhibited (IC50 >165.98 μM) as far as
their cytotoxic effect is concerned. Moreover, up-regulation of BAX and Caspase-3 and down-regulation of
Bcl-2 resulted in the induction of apoptosis from those active compounds. Further work has shown that
the most potent derivative 8b, caused cell cycle arrest at the G2/M phase. Also, EGFR inhibitory activity
for 8b showed IC50 0.014 μM versus wild EGFRWT and 12.66 nM versus the mutant type, Lapatinib, and
Erlotinib were used as reference standards with IC50 values of 0.025, 0.0653 μM and 35.72, 59.56 nM
versus both, respectively. Furthermore, the in-silico assessment showed that all-potent compounds were
orally bioavailable without blood-brain barrier permeation. Finally, the molecular docking of 8b inside the
active site of EGFR (1M17) showed a good binding through three hydrogen bonds and one arene-cation
interaction.
Received 13 December 2020
Revised 10 February 2021
Accepted 14 February 2021
Available online 26 February 2021
Keyword:
Indole-2,3–dione schiff’s bases
Chalcone
Anti-proliferative activity
Apoptosis
EGFR
ADME properties and molecular docking
© 2021 Elsevier B.V. All rights reserved.
1. Introduction
In many developmental and physiological environments, apoptosis
plays a critical role in monitoring cell numbers. Apoptosis is de-
With the ecological changes and environmental deterioration,
cancer has gradually become a major threat to human health [1].
Cancer is one of the degenerative conditions that occur in the
age of rodents and humans due to age [2]. Chemotherapy remains
among all current therapeutic methods, the most common cancer
management options, either alone or in conjunction with surgery
and/or radiotherapy [3–5]. One of the best ways to treat cancer is
to avoid uncontrolled cancer cell development. The apoptotic ap-
proach is thus an exciting way to discover new anti-cancer agents.
creased in several human cancers, which is significantly responsi-
ble for transforming the normal cell into tumor cells by disrupting
apoptotic function. Apoptosis switching on and off is determined
by the relation of BAX proteins and Bcl-2 anti-apoptotic proteins
[6]. Two main signaling pathways have been specified for apop-
totic cell death, the first being the intrinsic mitochondrial apop-
totic pathway, which is primarily produced by cell stress, in which
mitochondrial permeability plays a crucial role. The second way is
the external cytoplasmic pathway, which is activated via the pro-
apoptotic ligands that bind to the death receptor on the cell’s sur-
face [7–11].
∗
Corresponding authors.
Caspase protease activity is vital for apoptosis; once active, cas-
pases cleave hundreds of different proteins leading to rapid cell
E-mail addresses: alfayed_e@azhar.edu.eg (E.A. Fayed), yossry@azhar.edu.eg (Y.A.
Ammar).
https://doi.org/10.1016/j.molstruc.2021.130159
0022-2860/© 2021 Elsevier B.V. All rights reserved.

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Fig. 1. Structures for some anticancer drugs derived from isatin and the newly synthesized compounds.
death with characteristic biochemical and morphological hallmarks
[12–15]. Caspase protease activity is essential to apoptosis; once
active, Caspases cluster hundreds of different proteins that result
in rapid cell death, with biochemical and morphological charac-
teristics [16,17]. Caspase activity can be initiated either through
apoptosis inherent in the pathway or through apoptosis extrinsic
pathways. Death of the cells also plays a significant role in cancer
therapy. The deregulation of apoptosis has been widely reported
as a feature of cancer during cancer disease. Accordingly, apopto-
sis initiation in tumor cells is an efficient strategy to counter var-
ious human malignancies during today’s medical age [18–21]. Fur-
thermore, a trans membranous glycope-protein tyrosine kinase re-
ceptor is the Epidermal Growth Factor (EGFR) receptor, which is
over-expressed by numerous cancer cells, such as (breast, ovar-
ian, human colon) [22]. The over-expression of the EGFR family
leads to autophosphorylation of multiple tyrosine residues within
the COOH-terminal waist receptor, contributing to a certain storm
causing cell proliferation, differentiation, and anti-apoptosis. There-
fore, the EGFR family’s inhibition has been found to play an im-
portant part in the development of targeted chemical-therapeutic
agents [23,24]. Currently, in clinical, the anticancer agents suffer
from several drawbacks correlated to drugs’ associated side ef-
fects and/or tumors’ multi-drug resistance [25–27]. However, with
the rapidly increasing multidrug-resistance, one of the strategies to
overcome this problem is to discover novel molecules with highly
potential anticancer activities.
It is well known that isatin is a natural product consisting of
many plants, which has also been found to be a common scaf-
fold in various drugs, agrochemicals, and dyes [28–30]. Isatin and
its derivatives display diverse pharmacological activities, including
antiviral, antitumor, anticonvulsants, antibacterial, and antifungal
[31–42]. Some isatin derivatives have been developed as commer-
cial anticancer drugs, such as Sunitinib and Semaxanib (Fig. 1),
which identify isatin moiety is an attractive pharmacophore in dis-
covering new drugs [43,44].
Some of the essential techniques for developing new drugs
are combining two or more drug-induced compounds in a single
molecule to achieve an effect of synergy or to produce new an-
titumor agents [45–49]. The moieties isatin-chalcone and isatin-
Schiff’s base have shown potent involvement in antitumor drugs,
in addition to the production of spiro-isatin that shows promising
antitumor properties. We employed a chalcone and Schiff’s base to
assemble some novel effective antiproliferative agents in this work.
The isatin group is set, and diversity was generated by adding the
other rings of various substituents. The target compounds were as-
sessed against cancer cell lines HepG-2, MCF-7and HCT-116, and
2

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
the most active compounds were docked within the active site of
EGFR to determine their potential mechanism of action anticancer
agents.
3-((4-(3-(4-Methoxyphenyl)acryloyl)phenyl)imino)indolin-2-
one (4)
To a solution of 2b (2.64 g, 0.01 mol) and p-anisaldehyde
(1.24 g, 0.01 mol) in ethanol (20 mL) NaOH (5%, 10 mL) was added.
The reaction mixture was stirred for 4 h and left overnight. The
precipitated solid was filtered and crystallized from ethanol.
Yellow powder, Yield: (70%); m.p.: 240–242 °C, IR:
υ/cm−1 = 3261 (NH), 3050 (CH-arom.), 2874 (CH-aliph.), 1742,
2. Experimental
2.1. Chemistry
1650 (2C O), 1598 (C = N); 1H NMR: δ/ppm= 3.82 (s, 3H, OCH3),
=
6.36 (d, 2H, J = 6 Hz, Ar-H), 6.71 (t, 1H, Ar-H), 6.89 (d, 2H,
J = 6 Hz, Ar-H), 7.01 (d, 2H, J = 6 Hz, Ar-H), 7.08, 7.87 (2d, 2H,
All melting points were taken on Electrothermal LA 9000 SERIS,
Digital Melting point Apparatus, and were uncorrected. IR Spectra
were determined using the KBr disk technique on Nikolet IR 200
FT IR Spectrophotometer at Pharmaceutical Analytical Unit, Faculty
of Pharmacy, Al-Azhar University, and values are represented in
cm−1. The NMR spectra were recorded on Gemini 300 MHz, and
Mercury 400 MHz NMR, 75 & 100 for 13C NMR Spectrometer at
the Main Chemical Warfare Laboratories, Chemical Warfare Depart-
ment, Ministry of Defense. DMSO-–d6 was used as a solvent; chem-
ical shifts were measured in δ ppm, relative to TMS as an inter-
nal standard. Mass Spectra were recorded at 70 ev on DI-50 unit
of Schimadzu GC/ MS-QP5050A Spectrometer at Regional Center
for Mycology and Biotechnology, Al-Azhar University. Microanalysis
was carried out at Regional Center for Mycology and Biotechnol-
ogy, Al-Azhar University. The anticancer screening was carried out
at Regional Center for Mycology and Biotechnology, Al-Azhar Uni-
versity. BAX, Bcl-2, and EGFR, both wide and mutant, were carried
out in VACSERA, Cairo, Egypt. The reaction’s progress was moni-
tored using TLC sheets pre-coated with UV fluorescent silica gel
Merck 60F254 plates and was visualized using a UV lamp. Solvent
for TLC: Hexane: Ethyl acetate in ratio 6:4 and 4:6.
=
J = 16 Hz, CH CH), 7.14 (d, 2H, J = 6 Hz, Ar-H), 7.33 (t, 1H,
Ar-H), 8.26 (d, 2H, J = 6 Hz, Ar-H), 10.98 (s, 1H, NH exchangeable
by D2O); Mass spectrum exhibited a molecular ion peak at m/z:
382.16 (M+, 88.3%) with a base peak at m/z: 237.11; Anal. Calc. for
C24H18 N2O3 (382.41): C, 75.38; H, 4.74; N, 7.33. Found: C, 75.52;
H, 4.81; N, 7.49.
3-(2-(4-Aminophenyl)−2-oxoethyl)−3-hydroxyindolin-2-one
(5) was prepared according to the reported method [52] as
A mixture of isatin (1) (1.47 g, 0.01 mol), p-aminoacetophenone
(1.35 g, 0.01 mol) and diethylamine (3 mL) in ethanol (30 mL), the
mixture was stirred for 5 h and left overnight, the obtained prod-
uct was collected by filtration and crystallized from ethanol.
Pale yellow powder, Yield: (83%); m.p.: 205–207 °C; IR:
υ/cm−1 = = 3437, 3317, 3233 (OH, NH & NH2), 3058 (CH-arom.),
2914 (CH-aliph.), 1705–1680 (2C= O); 1H NMR: δ/ppm = 3.43,
=
3.85 (2d, 2H, J = 18 Hz, CH2), 5.90 (s, 1H, OH exchangeable by
D2O), 6.03 (s, 2H, NH2 exchangeable by D2O), 6.51 (d, 2H, J = 6 Hz,
Ar-H), 6.77 (d, 1H, J = 9 Hz, Ar-H), 6.84 (t, 1H, Ar-H), 7.14 (t, 1H,
Ar-H), 7.21 (d, 1H, J = 9 Hz, Ar-H), 7.57 (d, 2H, J = 9 Hz, Ar-H),
10.12 (s,1H, NH exchangeable by D2O); 13C NMR; 56.46 (CH2), 73.57
(C3-indoline), 112.85, 121.37, 123.84, 124.48, 129.11, 130.72, 131.00,
3-((3-Acetylphenyl)imino)indolin-2-one
&
3-((4-Acetyl-
phenyl)imino)indolin-2-one (2 a,b): were prepared according the
reported method [50,51].
=
132.58, 143.45, 154.21 (Ar-Cs), 178.99, 193.68 (2C O); Mass spec-
trum exhibited a molecular ion peak at m/z: 282.72 (M+, 3.5%)
with a base peak at m/z: 83.11; Anal. Calc. for C16 H14 N2O3 (282.30):
C, 68.08; H, 5.00; N, 9.92. Found: C, 68.24; H, 4.98; N, 10.08.
3-Hydroxy-3-(2-(4-((2-hydroxybenzylidene)amino)phenyl)−2-
oxoethyl)indolin-2-one (6) was prepared according to the re-
ported method [52] as
3-((4-(1-(2-Phenylhydrazineylidene)ethyl)phenyl)imino)
indolin-2-one (3a)
To a solution of acetophenone derivative 2b (2.64 g, 0.01 mol)
in ethanol/acetic acid (1:1, 20 mL), phenylhydrazine (1.08 mol) was
added. The reaction mixture was heated under reflux for 3 h. The
solid formed was filtered and crystallized from ethanol.
Brownish powder; Yield: (75%); m.p.: 150–152 °C; IR:
υ/cm−1 = 3360, 3153 (2NH), 3052 (CH-arom.), 2825 (CH-aliph.),
1682 (C = O), 1600 (C=N); 1H NMR: δ/ppm= 2.37 (s, 3H, CH3),
6.53 (d, 2H, J = 9 Hz, Ar-H), 6.85 (d, 4H, J = 9 Hz, Ar-H), 7.05
(t, 1H, Ar-H), 7.24 (t, 2H, Ar-H), 7.47 (d, 1H, J = 6 Hz, Ar-H), 7.50
(d, 2H, J = 9 Hz, Ar-H), 7.64 (d, 1H, J= 6 Hz, Ar-H), 10.98, 12.74
(2 s, 2H, 2NH exchangeable by D2O); Mass spectrum exhibited a
molecular ion peak at m/z: 354.26 (M.+, 1.14%) with a base peak at
m/z: 65.07; Anal. Calc. for C22H18 N4O (354.41): C, 74.56; H, 5.12;
N, 15.81. Found: C, 74.69; H, 5.16; N, 15.93.
A
mixture of isatin (1) (1.47 g, 0.01 mol), 1-(4-((2-
hydroxybenzyldene)amino)phenyl)ethanone (0.01 mol) and di-
ethylamine (3 mL) in ethanol (30 mL), the reaction mixture was
stirred for 5 h, left for overnight, cooled and treated with ice-cold
water, the obtained product was filtered and crystallized from
benzene.
Orange powder; Yield: (83%); m.p.: 218–220 °C; IR:
υ/cm−1
=
3380, 3204 (NH & 2OH), 3059 (CH-Arom.), 2900,
2808 (CH-aliph.), 1695 (2C O), 1611 (C = N); 1H NMR: δ/ppm=
3.60, 4.09 (2d, 2H J = 18 Hz, CH2), 6.03 (s, 1H, OH exchangeable
by D2O), 6.79 (d, 1H J = 6 Hz, Ar-H), 6.85 (d, 1H J = 9 Hz, Ar-H),
6.96 (t, 2H, Ar-H), 7.13 (t, 2H, Ar-H), 7.26 (d, 2H J = 6 Hz, Ar-H),
7.45 (d, 1H J = 9 Hz, Ar-H), 7.68 (d, 1H J = 6 Hz, Ar-H),7.95 (d,
=
2-(1-(4-(2-Oxoindolin-3-ylidene)amino)phenyl)ethylidene)
hydrazine-1-carbothioamide (3b)
A solution of acetophenone derivative 2b (2.64 g, 0.01 mol)
in ethanol/acetic acid (1:1, 20 mL) was treated with thiosemicar-
bazide (0.01 mol). The reaction mixture was heated under reflux
for 4 h. The precipitated solid was filtered and crystallized from
ethanol.
=
2H J = 9 Hz, Ar-H), 8.97 (s, 1H, CH N), 10.23, 12.62 (2 s, 2H,
NH & OH exchangeable by D2O); 13C NMR; 46.18 (CH2), 73.49
(C3-isatin), 109.84, 117.15, 119.78, 121.56, 129.37, 129.98, 132.19,
133.05, 134.68, 134.66, 143.38, 152.99 (Ar-Cs), 160.75 (C = N),
–
=
165.35 (C OH), 178.77, 195.98 (2C O); Mass spectrum exhibited a
molecular ion peak at m/z: 386.12 (M.+, 1.21%) with a base peak
at m/z: 224.06; Anal. Calc. for C23H18 N2O4 (386.40): C, 71.49; H,
4.70; N, 7.25. Found: C, 71.66; H, 4.81; N, 7.38.
Brown powder, Yield: (71%); m.p.: 250–252 °C; IR:
υ/cm−1 = 3422, 3337, 3261, 3169 (NH2, 2NH), 3050 (CH-arom.),
2806 (CH-aliph.), 1680 (C = O), 1603 (C = N), 1208 (C = S); 1H
NMR: δ/ppm = 2.49 (s, 3H, CH3), 6.93 (d, 3H, J = 9 Hz, Ar-H),
7.10 (t, 1H, Ar-H), 7.37 (t, 1H, Ar-H), 7.66 (d, 3H, J = 9 Hz, Ar-H),
8.65, 9.01, 11.17 and 12.46 (4 s, 4H, 2NH and NH2 exchangeable
by D2O); Mass spectrum exhibited a molecular ion peak at m/z:
337.30 (M+, 0.82%) with a base peak at m/z: 40.17. Anal. Calc. for
C17 H15N5OS (337.40): C, 60.52; H, 4.48; N, 20.76. Found: C, 60.84;
H, 4.43; N, 20.92.
3-(2-(4-((−2-Hydroxybenzylidene)amino)phenyl)−2-
oxoethylidene)indolin-2-one (7) [52] was prepared by two
methods:
First method:
A mixture of 3–hydroxy-indolin-2-one derivative 5 (2.82 g,
0.01 mol), salicylaldehyde (0.01 mol) in ethanol (30 mL) and acetic
3

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
acid (3 mL) was heated under reflux for 2 h. The precipitated solid
was filtered and crystallized from ethanol.
A mixture of isatin (1) (1.47 g, 0.01 mol), the corresponding
thiourea derivative (2.70 g, 0.01 mol) and diethylamine (3 mL) in
ethanol (30 mL), the mixture was stirred for 5 h, left overnight,
cooled, and the obtained product was filtered and crystallized from
ethanol.
Second method:
Compound 6 (0.01 mol) in ethanol (30 mL), concentrated HCl
(2 mL) was added, the mixture was heated under reflux for
30 min., cooled, and the obtained product was filtered and crys-
tallized from ethanol.
Yellowish powder; Yield: (82%); m.p.: 185–187°C; IR:
υ/cm−1 = 3425, 3216 (OH, 3NH), 3031 (CH-arom.), 1705, 1666
(2C O), 1328 (C=S); 1H NMR: δ/ppm= 3.51, 4.00 (2d, 2H, J=12 Hz,
=
Dark orange powder; Yield: (78%); m.p.: 170–172 °C; IR:
υ/cm−1=3379, 3196 (OH & NH), 3050 (CH-arom.), 2950 (CH-aliph.),
1711 (C=O), 1601 (C = N); 1H NMR: δ/ppm= 6.60 (d, 2H J=6 Hz,
Ar-H), 6.73 (d, 2H J=6 Hz, Ar-H), 6.81 (t, 2H, Ar-H), 6.95 (m, 2H,
Ar-H), 7.05 (t, 2H, Ar-H), 7.45 (d, 1H J=6 Hz, Ar-H), 7.69 (d, 1H
CH2), 6.00 (s, 1H, OH exchangeable by D2O), 6.47 (d, 1H, J=8 Hz,
Ar-H), 6.52 (t, 1H, Ar-H), 6.73 (t, 1H, Ar-H), 6.78 (t, 1H, Ar-H),
7.17 (d, 1H, J=8 Hz, Ar-H), 7.42 (t, 2H, Ar-H), 7.62 (d, 2H, J=8 Hz,
Ar-H), 7.81 (d, 2H, J=8 Hz, Ar-H), 7.91 (d, 2H, J=8 Hz, Ar-H),
9.75, 10.09, 10.20 (3 s, 3H, 3NH exchangeable by D2O); Mass
spectrum exhibited a molecular ion peak at m/z: 417.31 (M+,
0.81%) with a base peak at m/z: 77.07; Anal. Calc. for C23H19 N3O3S
(417.48): C, 66.17; H, 4.59; N, 10.07. Found: C, 66.40; H, 4.63; N,
10.29.
=
J=6 Hz, Ar-H), 8.32, 8.51 (s, 2H, CH N), 10.24, 10.67 (2 s, 2H,
NH & OH exchangeable by D2O); 13C NMR: 110.56, 117.67, 119.83,
119.92, 120.70, 122.56, 122.73, 125.13, 126.83, 128.89, 129.59,
131.84, 132.35, 133.03, 136.85, 144.64 (Ar-Cs), 155.22 (C = C), 161.17
–
=
(C = N), 168.81 (C OH), 188.39, 192.11 (2C O); Mass spectrum ex-
hibited a molecular ion peak at m/z: 369.12 (M +1, 10.19%), 368.10
(M.+, 32.27%) with a base peak at m/z: 224.06; Anal. Calc. for
C23H16 N2O3 (368.38): C, 74.99; H, 4.38; N, 7.60. Found: C, 75.12;
H, 4.42; N, 7.69.
N-(4-(2-(3-Hydroxy-2-oxoindolin-3-yl)acetyl)phenyl)−4-
methylbenzenesulfonamide (10)
A mixture of isatin (1) (1.47 g, 0.01 mol), the corresponding
acetyl sulfonamide derivative (2.89 g, 0.01 mol) and diethylamine
(3 mL) in ethanol (30 mL), the mixture was stirred for 5 h, left
overnight, cooled, and the obtained product was filtered and crys-
tallized from ethanol.
5‘-(4-((2Hydroxybenzylidene)amino)phenyl)−4‘-hydrospiro
(indoline-3,3‘-pyrazol)−2-one (8a,b):
A mixture of isatin derivative 7 (3.69 g, 0.01 mol) and hy-
drazine hydrate or phenylhydrazine (0.012 mol) in ethanol (20 mL)
containing acetic acid (5 mL) was heated under reflux for 7 h,
cooled, and the obtained product was filtered and crystallized from
ethanol.
Yellowish powder; Yield: (78%); m.p.: 165–167°C; IR:
υ/cm−1 = 3454, 3218 (br. OH & 2NH), 3050 (CH-arom.), 2938
(CH-aliph.), 1720, 1668 (2C O); 1H NMR: δ/ppm= 2.31 (s, 3H,
=
CH3), 3.39, 3.98 (2d, 2H, J=12 Hz, CH2), 6.00 (s, 1H, OH exchange-
able by D2O), 6.73 (d, 1H, J=8 Hz, Ar-H), 6.78 (t, 1H, Ar-H), 7.12 (d,
1H, J=8 Hz, Ar-H), 7.16 (d, 2H, J=8 Hz, Ar-H), 7.33 (d, 2H, J=8 Hz,
Ar-H), 7.66 (t, 1H, Ar-H), 7.71 (d, 2H, J=8 Hz, Ar-H), 7.79 (d, 2H,
J=8 Hz, Ar-H), 10.19, 10.73 (2 s, 2H, 2NH exchangeable by D2O);
13C NMR: 26.55 (CH3), 56.50 (CH2), 80.00 (C3-indolinone), 106.03,
116.52, 116.59, 117.42, 117.67, 118.52, 119.52, 121.60, 123.77, 128.33,
5‘-(4-((2Hydroxybenzylidene)amino)phenyl)−2‘,4‘-dihy-
drospiro(indoline-3,3‘-pyrazol)−2-one (8a)
Brown powder, Yield: (83%); m.p.: 220–222 °C; IR:
υ/cm−1 =3337, 3262, 3169 (OH & 2 NH), 3025 (CH-arom.), 2996
(CH-aliph.), 1668 (C = O), 1598 (C = N); 1H NMR: δ/ppm = 3.60,
3.85 (2d, 2H, J=18 Hz, CH2), 5.62, (1 s, 1H, 1NH exchangeable by
D2O), 6.72 (d, 1H J = 6 Hz, Ar-H), 6.78 (d, 2H J = 6 Hz, Ar-H),
6.84 (m, 1H, Ar-H), 6.87 (t, 2H, Ar-H), 7.11 (t, 2H, Ar-H), 7.28 (d,
2H, Ar-H), 7.52 (d, 1H J = 6 Hz, Ar-H), 7.71 (d, 1H J = 6 Hz, Ar-H),
=
128.40, 129.48, 142.37, 145.09 (Ar-Cs), 162.01, 162.10 (2C O); Mass
spectrum exhibited a molecular ion peak at m/z: 436.30 (M.+
,
2.24%) with a base peak at m/z: 91.08; Anal. Calc. for C23H20N2O5S
(436.11): C, 63.29; H, 4.62; N, 6.42. Found: C, 63.42; H, 4.68; N,
6.65.
=
8.36 (s, 1H, CH N), 11.10, 12.49 (2 s, 2H, OH & 1NH exchangeable
by D2O); 13C NMR (101 MHz, DMSO) δ 43.80 (CH2), 67.95 (C-
spiro), 111.42, 115.76, 119.48, 121.15, 122.30, 123.50, 124.67, 125.87,
127.54, 129.98, 131.95, 133.93, 135.59, 140.46, 143.50, 151.60,
3-(2-Oxo-2-(2-oxo-2H-chromen-3-yl)ethylidene)indolin-2-one
(11) [53,54]
–
154.59, 157.76, 161.57 (2 C=N), 165.28 (C OH), 168.61(C=O); Mass
A mixture of isatin (1) (1.47 g, 0.01 mol), acetyl coumarin
(1.90 g, 0.01 mol) and diethylamine (3 mL) in ethanol (30 mL),
the mixture was stirred for 5 h, left overnight, cooled, and the ob-
tained product was filtered and crystallized from ethanol.
Pale yellow powder; Yield: (73%); m.p.: 250–252°C; IR:
spectrum exhibited a molecular ion peak at m/z: 382.24 (M.+
,
2.18%) with a base peak at m/z: 120.09; Anal. Calc. for C23H18 N4O2
(382.41): C, 72.24; H, 4.74; N, 14.65. Found: C, 72.39; H, 4.79; N,
14.78.
5ꢀ-(4-((2-Hydroxybenzylidene)amino)phenyl)−2ꢀ-phenyl-2ꢀ,4ꢀ-
υ/cm−1 = 3428 (NH), 3112 (CH-arom.), 1717–1645 (3C O); 1H
=
dihydrospiro[indoline-3,3ꢀ-pyraz-ol]−2-one (8b)
NMR: δ/ppm= 7.47 (s, 1H, CH-ethylinic), 7.52 (d, 1H, J=6 Hz, Ar-H),
7.59 (d, 1H, J=6 Hz, Ar-H), 7.67 (d, 2H, J=6 Hz, Ar-H), 7.84 (t, 2H,
Ar-H), 8.22 (t, 2H, Ar-H), 8.39 (s, 1H, CH-coumarin), 11.09 (1 s, 1H,
NH exchangeable by D2O); 13C NMR: 113.45, 121.94, 125.55, 126.08,
127.53, 128.30, 128.39, 128.53, 129.41, 130.61, 134.55, 135.65, 141.13
Deep orange powder; Yield: (71%); m.p.: 120–122°C; IR:
υ/cm−1 = 3370, 3310 (OH & NH), 3038 (CH-arom.), 2822 (CH-
aliph.), 1680 (C=O), 1597 (C=N); 1H NMR: δ/ppm= 3.24, 3.35
(2d, 2H,
J
=
18 Hz, CH2), 6.73 (t, 2H, Ar-H), 6.85 (d, 2H,
–
J=9 Hz, Ar-H), 6.96 (d, 1H, J=6 Hz, Ar-H), 7.13 (d, 2H, J=6 Hz,
(Ar-Cs), 146.06, 155.37 (C=C), 161.55 (C O), 161.69, 163.74, 172.36
=
Ar-H), 7.20 (t, 2H, Ar-H), 7.36–743 (m, 6H, Ar-H), 7.53 (d, 2H,
(3C O); Mass spectrum exhibited a molecular ion peak at m/z:
J=9 Hz, Ar-H), 8.23 (s, 1H, CH N), 10.37, 10.55 (2 s, 2H, OH
317.12 (M.+, 1.57%) with a base peak at m/z: 156.21; Anal. Calc. for
C19 H11 NO4 (317.30): C, 71.92; H, 3.49; N, 4.41, Found: C, 71.75; H,
3.69; N, 4.18.
=
&
NH exchangeable by D2O); 13C NMR (101 MHz, DMSO) δ
45.06 (CH2), 66.42 (C-spiro), 111.52, 114.67, 116.32, 118.79, 121.22,
123.26, 124.84, 125.90, 128.20, 130.00, 132.02, 133.75, 134.92,
136.59, 138.09, 141.00, 142.13, 143.68, 147.57, 149.15, 151.79, 155.70,
Spiro[chroman-2,3‘-indoline]−2‘,4–dione (12)
A solution of isatin)1((1.47 g, 0.01 mol), 2-hydroxyacetophenone
(1.38 g, 0.01 mol) in ethanol (30 mL), was treated with diethy-
lamine (3 mL), the mixture was stirred for 5 h, left overnight,
cooled and the obtained product was filtered and crystallized from
ethanol.
–
160.92 (2 C=N), 165.94 (C OH), 169.46 (C=O); Mass spectrum
exhibited
with
a , 0.88%)
molecular ion peak at m/z: 458.13 (M.+
a
base peak at m/z: 44.03; Anal. Calc. for C29H22N4O2
(458.52): C, 75.97; H, 4.84; N, 12.22. Found: C, 76.12; H, 4.91; N,
12.34.
Orange powder; Yield: (83%); m.p.: 240–242°C; IR:
υ/cm−1 = 3418 (NH), 3050 (CH-arom.), 2992, 2805 (CH-aliph.),
1-(4-(2-(3-Hydroxy-2-oxoindolin-3-yl)acetyl)phenyl)−3-
1655 (br 2C O); 1H NMR: δ/ppm= 3.45, 3.97 (2d, 2H, J=8 Hz,
=
phenylthiourea (9)
4

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
CH2), 6.73 (d, 1H, J=6 Hz, Ar-H), 6.82 (t, 2H, Ar-H), 6.90 (d, 1H,
J=6 Hz, Ar-H), 7.47 (t, 2H, Ar-H), 7.60 (d, 2H, J=9 Hz, Ar-H), 10.39
(s, 1H, NH exchangeable by D2O); 13C NMR: 56.05 (CH2), 113.46
(C3-indoline), 114.50, 122.37, 125.59, 126.12, 127.52, 128.31, 130.64,
confluence, washed with PBS, and then treated with different con-
centrations of compounds in fresh maintenance medium from 50
to 1.56 μg and incubated at 37 °C for 24 h. Control of the un-
treated cells was made in the absence of the test compound. The
untreated cells were used as negative control. Serial two-fold di-
lutions of the tested compounds were added into a 96-well tissue
culture plate using a multichannel pipette (Eppendorf, Germany).
After treatment (24 h), the culture supernatant was replaced by
fresh medium. Then, the cells in each well were incubated at 37°C
with 100 μL of MTT solution (5 mg/mL) for 4 h. After incubation,
the MTT solution was removed, and then 100 μL of DMSO was
added to each well. The absorbance was detected at 570 nm using
a microplate reader (Sun Rise TECAN, Inc, USA). The absorbance
of untreated cells was considered as 100%. The results were deter-
mined by three independent experiments [55].
–
131.39, 141.16, 142.93, 146.05 (Ar-Cs), 155.62 (C O), 163.70, 187.84
=
(2C O); Mass spectrum exhibited a molecular ion peak at m/z:
265.16 (M.+, 2.72%) with a base peak at m/z: 76.07; Anal. Calc. for
C16 H11 NO3 (265.26): C, 72.45; H, 4.18; N, 5.28. Found: C, 72.68; H,
4.22; N, 5.43.
N-(Aryl)−2-imino-2H-furo[2,3-b]indole-3-carboxamides (13a,
b):
To a solution of isatin (1) (1.47 g, 0.01 mol) in ethanol (30 mL),
cyanoacetranilide derivatives (0.01 mol) and piperidine (0.5 mL)
were added. The reaction mixture was heated under reflux for 3 h,
left overnight, cooled, and the obtained product was filtered and
crystallized from ethanol.
N-(2,4-Dimethoxyphenyl)−2-imino-2H-furo[2,3-b]indole-3-
carboxamide (13a):
2.2.2.1. Data analysis. The percentage of cell viability was calcu-
lated as follows = [1−(ODt/ODc)] × 100%, where ODt is the mean
optical density of wells treated with the tested compound and ODc
is the mean optical density of untreated cells. The tested com-
pounds were compared using the IC50 value, i.e., the concentra-
tion of an individual compound leading to 50% cell death estimated
from graphical plots of surviving cells verses compound concentra-
tions.
Reddish brown powder; Yield: (66%); m.p.: 165–167 °C; IR:
υ/cm−1= 3423, 3365 (br 2NH), 3050 (CH-arom.), 2942, 2842 (CH-
aliph.), 1660 (C=O), 1613 (2C N); 1H NMR: δ/ppm= 3.60, 3.67 (2 s,
=
6H, 2OCH3), 6.48 (d, 1H, J=6 Hz, Ar-H), 6.70 (s, 1H, Ar-H), 6.93 (t,
1H, Ar-H), 6.82 (d, 1H, J=9 Hz, Ar-H), 7.30 (t, 1H, Ar-H), 8.22 (d,
1H, J=6 Hz, Ar-H), 9.04 (d, 1H, J=6 Hz, Ar-H), 10.98, 11.52 (2 s, 2H,
2NH exchangeable by D2O); Mass spectrum exhibited a molecu-
lar ion peak at m/z: 349.62 (M.+, 4.30%) with a base peak at m/z:
138.12; Anal. Calc. for C19 H15N3O4 (349.35): C, 65.32; H, 4.33; N,
12.03. Found: C, 65.12; H, 4.94; N, 12.21.
2.2.3. Apoptosis detection studies
2.2.3.1. Determination of the active caspase-3. The manufacturer ’s
protocol calculated the active level of Caspase-3 with an active
Quantikine-Human Caspase-3 Immunoassay (R&D Systems, Inc.
Minneapolis, USA). After cell washing with PBS, the cells were col-
lected and lysed by adding them to the protease inhibitor extrac-
tion buffer (1 mL per 1 × 107 cell). The lysate was diluted imme-
diately before the test. Finally, a microplate reader set at 450 nm
calculated the optical density of each well in 30 min [46].
N-(4-Fluorophenyl)−2-imino-2H-furo[2,3-b]indole-3-
carboxamide (13b):
Dark red powder; Yield: (67%); m.p.: 300–302 °C; IR:
υ/cm−1 = 3224 (2NH), 3050 (CH-arom.), 2815 (CH-aliph.), 1665
(C=O), 1612 (2C N); 1H NMR: δ/ppm= 6.93 (d, 2H, J=6 Hz, Ar-H),
=
7.12 (t, 1H, Ar-H), 7.58 (d, 1H, J=6 Hz, Ar-H), 7.45 (t, 1H, indolin-
5), 7.61 (d, 1H, J=6 Hz, Ar-H), 7.93 (d, 2H, J=9 Hz, Ar-H), 10.88,
10.99 (2 s, 2H, 2NH exchangeable by D2O); 13C NMR: 108.76, 111.57,
115.07, 116.21, 119.45, 122.36, 123.03, 124.26, 125.58, 134.39, 140.81
2.2.3.2. Determination of mitochondrial apoptosis pathway proteins
BAX and Bcl-2. Cells were extracted from the American Culture
Set, cells were grown in RPMI 1640 containing 10% fetal serum
from bovine animals at 37°C, the compounds were stimulated for
the BAX or Bcl-2 test, and the cell extraction buffer was lysed.
This lysate was diluted across the test range and tested for hu-
man active BAX or Bcl-2 content in Regular Diluent Buffer. (Cells
are Plated in the density of 1.2 – 1.8 × 10,000 cells/well in a vol-
ume of 100 μL complete growth medium + 100 uL of the tested
compound per well in a 96-well plate for 24 h before measuring
for human active BAX or Bcl-2) [6].
=
=
(Ar-Cs), 144.85, 145.40 (2C C), 158.21, 158.54 (2C N), 158.83 (C-
F), 165.51 (C=O); Mass spectrum exhibited a molecular ion peak
at m/z: 307.15 (M.+, 2.03%) with a base peak at m/z: 111.10; Anal.
Calc. for C17 H10FN3O2 (307.28): C, 66.45; H, 3.28; N, 13.67. Found:
C, 66.67; H, 3.26; N, 13.
2.2. Biological activities
2.2.1. Cell lines
Human breast (MCF-7), hepatocellular (Hep-G2), and colon
(HCT-116) carcinoma cells were obtained from the American Type
Culture Collection (ATCC, Rockville, MD, USA). The cells grown in
RPMI-1640 medium, supplemented with 10% inactivated fetal calf
serum and 50 μg/mL gentamycin. The cells were maintained at
37°C in a humidified atmosphere with 5% CO2.
2.2.4. Cell cycle analysis
The MCF-7 cancer cells were seeded into a 6-well plate and
incubated for 24 h at a concentration of 1 × 105 cells per well.
Cells have been treated with a vehicle (0.1 percent DMSO) or with
a compound 8b for 24 h. Afterward, cells were harvested with ice-
cold 70% ethanol at 4 °C and fixed for 12 h. Ethanol and cold PBS
are extracted from the cells and incubated in 0.5 mL P BS contain-
ing 1 mg /mL Rnase for 30 min. at a temperature of 37°C. The cells
were stained with propidium iodide in the dark for about 30 min.
The DNA contents were then measured by the flow cytometer [56].
2.2.2. Evaluation of the antitumor activity by MTT assay
Viability of control and treated cells were evaluated using
the MTT assay in triplicate. The MTT assay is a laboratory test
and
a standard colorimetric assay (an assay which measures
color changes) for measuring cellular growth, Yellow MTT (3-(4,5-
dimethylthiazol-2-yl)−2,5-diphenyl tetrazolium bromide, a tetra-
zole) was reduced to purple formazan in the mitochondria of liv-
ing cells. A solubilization solution (dimethyl sulfoxide) was added
to dissolve the insoluble purple formazan product into a colored
solution. Briefly, three tumor cell lines were seeded in 96-well
plates containing 100 μL of the growth medium at a density of
1 × 104 cells/well. Cells were permitted to adhere for 24 h till
2.2.5. Annexin v-FITC assay
The MCF-7 cancer cells have been plated in a 6-well plate, incu-
bated for 24 h, then treated in a vehicle (0.1% DMSO) or compound
8b for 24 h. The binding buffer (10 mM HEPES, 140 mM NaCl, and
2.5 mM CaCl2 at pH 7.4) was used to harvest cells, wash them with
pBS and spray them in the dark with Annexin V-FITC and propid-
ium iodide (PI). The flow cytometer was then analyzed [57–59].
5

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
2.2.6. EGFR assay
α and 7.87 ppm for H-β with coupling constant (J=15 Hz), which
indicated the trans isomer.
Inhibitory behavior of the most promising compound 8b on
the MCF-7 cells was carried with the same instructions and the
On the other hand, aldol condensation of p-aminoacetophenone
with isatin in absolute ethanol in the presence of few drops of N,N-
diethylamine (DEA) as a catalyst, the final product separated and
identified as 3-(2-(4-aminophenyl)−2-oxoethyl)−3-hydroxyindolin-
2-one (5). The structure of product 5 was ascertained from their
spectral features. The IR spectrum of compound 5 showed ab-
sorption bands at ν 3437, 3317, and 3233 cm−1 assignable to OH,
NH2, and NH groups, and a band at ν 1705–1680 cm−1 due to
two carbonyl functions. Its 1H NMR spectrum showed two dou-
blets singlet at δ 3.43, 3.85 ppm corresponding for CH2 protons.
Three singlet signals were observed at δ 5.9, 6.03, and 10.12 ppm
for OH, NH2, and NH, respectively. The phenyl protons appeared
in addition to the indoline doublets and triplet signals between δ
6.51–7.57 ppm for eight protons. Typical 13C NMR spectrum dis-
played signals for CH2 and C3-indoline carbon at 56.45 and 73.57
besides aromatic carbon in the region of δ 112.85–154.21 ppm,
and carbonyl carbons appeared at δ 178.99 and 193.68 ppm. Con-
densation of compound 5 with salicylaldehyde afforded a prod-
uct that was formulated as the corresponding Schiff’s base 7,
and dehydration occurred. This compound was confirmed chem-
ically by another way where the reaction of isatin with 1-(4-
(2-hydroxybenzyldene)amino)phenyl)ethanone to furnish the 3–
hydroxy-3-substituted-2-oxoindole derivative 6 which exposed to
dehydration with ethanolic hydrochloric. Unfortunately, both ele-
mental analysis and spectral data of the 3–hydroxyl 2-oxo-indole
derivatives 6 and Schiff’s base 7 were on assignment with the pos-
sible structures. The structure of product 6 was confirmed from
their spectral features. The IR revealed two hydroxy (OH) and NH
groups as a broad band around at ν 3380, 3204 cm−1 and two car-
recorded method against EGFRwt and EGFR
from the pro-
L858R-TK
tocol producer [23,24].
2.2.7. In silico physicochemical and ADME properties prediction
Using Chemdraw 12.0, the molecular structures have been con-
verted to SMILES. Such SMILS were then introduced into the Swiss
ADME website for the purposes of measuring physicochemical de-
scriptors, lipophilicity, pharmacokinetics, ADME parameters, and
medicinal chemistry friendliness [60].
2.2.8. Molecular docking
Molecular modeling was carried out using Molecular Operat-
ing Environment software 10.2008 (MOE), Chemical Computing
Group Inc., Montreal, Quebec, Canada, as the computational soft-
ware and docking process performed as described in previous work
[23]. The enzyme (1M17) [24] was downloaded from the pro-
tein data bank, water molecule removed, all hydrogen atom was
added, then refined, and energy minimized were performed with
MOE. The validation process was performed by redocking the Er-
lotinib into the EGFR binding site using the MOE software de-
fault set to confirm that the protein is ready for docking. The
target compound was drawn in chem draw then transferred to
MOE where protonated 3D, render hidden hydrogen and finally,
energy minimized and saved as mdb for docking inside active
site. Then, the docking process was performed using the default
protocol.
bonyl groups at ν 1695 cm−1 1H NMR spectrum (DMSO–d6) ex-
.
3. Results and discussion
hibited two doublets at δ 3.60, 4.09 ppm assignable to CH2 pro-
tons, other three singlet signals at δ 6.03, 10.23, 12.62 ppm due to
two hydroxy and NH protons, the aromatic protons were observed
as multiplet signals at δ 6.79–7.95 ppm. Also, 13C NMR exhibited
three signals at δ 46.18, 73.49 ppm due to CH2 and C3-indoline
besides Ar-Cs at δ 109.84–152.99 ppm and two signals a δ 160.75,
3.1. Chemistry
In continuation of our work to synthesize some heterocyclic
compounds in the field of medicinal chemistry [61–66], it seemed
of interest to design and synthesize a novel series of isatin deriva-
tives bearing biologically active units to evaluate their anti-cancer
activity. Preparation of the isatin derivatives is outlined in schemes
1&2. The behavior of isatin (1) with aminoacetophenone deriva-
tives was discussed. The aminoacetophenone has two nucleophilic
centers; thus, its reaction with isatin dependent on the reaction
conditions. Condensation of p-aminoacetophenone with isatin in
acetic acid afforded Schiff’s base product, which was identified as
3-((3 or 4-acetylphenyl)imino)indolin-2-one (2a,b), where the con-
densation occurred through the amino group that prepared ac-
cording to reported methods [50,51]. The resulting Schiff base 2b
has acetyl function, which was subjected to react with phenyl-
hydrazine, thiosemicarbazide, or anisaldehyde to afford a single
product in each case. The product formed was formulated as
the phenylhydrazone, thiosemicarbazone, and unsaturated ketone
derivatives 3–4, respectively. The structure of these products 3a,
b, and 4 was confirmed based on their correct elemental analyses
and spectral data. The IR spectra of 3a showed two NH stretching
bands at ν 3360, 3153 cm−1, the carbonyl function was observed
at 1682. The 1H NMR spectra of 3b displayed a singlet signal at
δ 2.49 ppm due to CH3, with four singlet signals related to NH &
NH2 protons of the thiosemicarbazone moiety and NH indole at δ
8.65, 9.01, 11.17, and 12.46 ppm, which all exchanged by D2O. The
aromatic protons were observed as a multiplet at δ 6.93–7.66 ppm
integrated for (8H) protons. 1H NMR spectral data of unsaturated
ketone with 2-oxo-indole derivative 4 showed a singlet signal at δ
3.82 ppm due to methoxy group, and a singlet at δ 10.98 ppm indi-
cating the presence of NH. Besides, the protons of α,β-unsaturated
system that observed as two doublets around δ 7.08, ppm for H-
–
165.35 ppm for (C=N) and (C OH) respectively, and two C=O at
δ 178.77 and 195.98 ppm. The mass spectrum showed a molecu-
lar ion peak at m/z = 386, corresponding to a molecular formula
C23H18 N2O4. The structure of compound 7 was confirmed on the
basis of its spectral data; its IR spectrum showed absorption bands
at ν 3379, 3196 cm−1 assignable to OH and NH groups, and a band
at ν 1711 cm−1 due to C=O function in addition to the band at
ν 1601 cm−1 corresponding for C=N. 1H NMR spectrum showed
two singlet signals were observed at δ 10.24, 10.67 ppm for OH,
and NH respectively, the aromatic protons have appeared at range
δ 6.60–7.91 ppm. 13C NMR spectrum displayed signals for aromatic
carbons & C=C in the region of δ 110.56–155.22 ppm, and carbonyl
carbons appeared at δ 188.39 and 192.11 ppm. Cyclo-condensation
of α, β-unsaturated ketone 7 with hydrazine or phenylhydrazine
was carried out in ethanol containing acetic acid and afforded
spiropyrazoline derivatives 8a,b (Scheme 1). Compound 8a,b was
suggested for the reaction product based on both elemental and
spectral analyses. The IR spectrum of 8a as an example showed
intense peaks at ν 3337, 3262, 3169 cm–1 for OH and NH groups,
respectively, in addition to absorption peak at ν 1668 cm–1 for car-
bonyl (C=O) and ν 1598 cm−1 for (C=N) stretching. Its 1H NMR
spectrum showed two doublet signal at δ 3.60, 3.85 ppm due to
CH2, a multiplet at δ 6.72–7.71 ppm was observed for the aro-
matic twelve protons, a singlet was observed at δ 8.36 ppm due
=
to CH N. Besides, three D2O-exchangeable signals at δ 5.62, 11.10,
12.49 ppm due to two NH and phenolic hydroxyl groups. 13C NMR
showed signal at δ 43.80 ppm due to CH2, 67.95 for sipro carbon
(C3-indoline), and two signals at δ 157.76 and 161.57 ppm for two
6

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Scheme 1. synthesis of compounds 2–8.
=
2C N as well as two significant signals at δ 165.28, 168.61 ppm
for carbon attached for hydroxy and carbonyl groups, besides the
aromatic-carbons.
ture of compound 10 was deduced based on their spectral data
and elemental analyses. The IR spectrum of compound 10 revealed
an absorption band at ν 3454, 3218 cm−1 due to OH and two NH
groups, and absorption bands at ν 1668, 1720, and 1597 attributed
to two C=O and C=N respectively. Its 1H NMR spectrum showed a
singlet at δ 2.31 ppm assigned to the CH3 protons and the pres-
ence of the CH2 protons at δ 3.44, 3.98 ppm that confirmed the
structure. Also, a multiplet in the region of δ 6.73–7.79 ppm due
to aromatic protons besides two D2O exchangeable signals at δ
10.09, 10.73 ppm assigned to 2 NH. 13C NMR showed a signal at
26.55 ppm attributed to CH3, methylene carbon at δ 56.50 ppm,
and two signals at δ 162.01 and 162.10 ppm due to two carbonyl
carbons besides the aromatic carbons.
In continuation of this investigation, another type of acetyl
derivative was used as reagents to react with isatin in the hope
of obtaining more pharmacologically active compounds. Thus,
reaction of isatin (1) with 1-(4-acetylphenyl)−3-phenylthiourea
furnished
a product was formulated as 1-(4-(2-(3–hydroxy-2-
oxoindolin-3-yl)acetyl)-phenyl)−3-phenylthiourea (9). The struc-
ture of the compound 9 was elucidated based on their spec-
tral data and elemental analyses. Its IR spectra revealed absorp-
tion bands at ν 3425, 3216 cm−1 assignable to OH and three
NH groups and absorption bands at 1705 and 1666 due to C=O
groups, 1328 for C=S. The 1H NMR spectrum of compound 9
exhibited two doublet signals at δ 3.51 and 4.00 ppm rep-
resenting the protons of methylene group (CH2) and four ex-
changeable D2O signals at δ 6.00, 9.75, 10.09 and 10.20 ppm
for OH and three NH groups, besides aromatic protons which
observed in the region δ 6.47–7.93 ppm. Its mass spectrum
showed a peak corresponding to the molecular ion at m/z 417
(0.81%) with a base peak at m/z 77. Also, the interaction of isatin
(1) with N-(4-acetylphenyl)−4-methylbenzenesulfonamide in the
presence of diethylamine, the 3–hydroxyl 2-oxindole derivative
was obtained and identified as N-(4-(2-(3–hydroxy-2-oxoindolin-
3-yl)acetyl)phenyl)−4-methylbenzene-sulfonamide (10). The struc-
On
the
other
hand,
3-(2-oxo-2-(2-oxo-2H-chromen-3-
yl)ethylidene)indolin-2-one (11) was obtained upon treatment
isatin (1) with 3-acetyl-2H-chromen-2-one in the presence of
diethylamine. The reaction proceeded via acetyl group’s addition
to afford the intermediate, which was subjected to dehydration
under the reaction condition. The structure of compound 11 was
deduced on the basis of their elemental analyses and spectral data.
Its IR spectrum of the compound revealed an absorption band at
ν 3428 cm−1 due to NH and three carbonyl groups observed at ν
1645–1717 cm−1. Its 1H NMR spectrum exhibited signals around
δ 7.47–8.39 ppm attributed aromatic protons, CH=ethylinic, and
4-H coumarin. Its 13C NMR displayed three carbonyl carbons at δ
7

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Scheme 2. synthesis of compounds 9–13.
161.69, 163.74, and δ 172.36 ppm besides the aromatic carbons.
The mass spectrum showed a molecular ion peak at m/z 317
(1.57%) with a base peak at m/z 156.
imino-2H-furo[2,3-b]indole-3-carboxamides (13a, b) based on the
elemental analyses and the spectral data. The formation of com-
pound 13a,b is assumed to proceed via the nucleophilic addition
of the active methylene in the cyano acetanilide derivatives to the
carbonyl group of isatin in position three followed by dehydra-
tion to afford the arylidine intermediate D, which underwent nu-
cleophilic attack of the hydroxyl group to the nitrile function to
give the final product. The IR spectrum of 13a revealed the lack of
nitrile band and the presence of absorption bands at ν 3423 and
1660 cm−1 due to 2 NH, and carbonyl groups, respectively. 1H NMR
spectrum of 13a displayed two singlet signals at δ 3.60, 3.67 ppm
due to two methoxy groups and the seven aromatic protons, be-
sides the D2O exchangeable protons were observed as two singlet
signals at δ 10.98, 11.52 ppm. Its mass spectrum showed a molec-
ular ion peak at m/z 349, which is characteristic of the molecular
formula C19 H15N3O4 together with base peak at m/z 111. 13C NMR
spectra of compound 13b exhibited two C=N and C=O carbons at
δ 158.54, 158.83 and 165.51 ppm besides the aromatic protons.
Furthermore, spiro[chroman-2,3‘-indoline]−2‘,4dione (12) was
prepared in one step on the reaction of isatin 1 with 2hydroxy
acetophenone in the presence of diethylamine as a catalyst. The
structure of 12 was established based on elemental and spectral
data. Its IR spectrum revealed the presence of an absorption band
at ν 3418 cm−1 due to NH, and two carbonyl groups observed at
1653 cm−1. The 1H NMR spectrum revealed singlet signal at δ 3.45
attributable to CH2 in addition to the aromatic protons and D2O
exchangeable proton at δ 10.39 ppm related to NH of isatin deriva-
tives. Also, 13C NMR revealed a signal at δ 56.05 ppm due to CH2
and three signals at δ 113.46, 163.70 and 187.84 ppm assigned to
spirocarbon (C3-indoline) and two carbonyl carbons besides the
aromatic carbons that ranged between δ 114.50 to 146.05 ppm.
The formation of compound 12 is assumed to proceed via the for-
mation of unsaturated ketone (intermediate B subjected to hydra-
tion). Then, the hydroxyl group present in the ortho position of
acetophenone acts as nucleophilic addition on alkene double bond
through intramolecular cyclization (intermediate C) to afford the
final product.
3.2. Biological activity
3.2.1. Anti-proliferative activity and structure activity relationship
study
Finally, isatin was subjected to react with cyano acetanilide
derivatives as an active methylene compound, and the actual struc-
ture of the product was assigned as N-(substitutedphenyl)−2-
All the newly synthesized compounds were screened for their
anticancer activity against three cell lines, which are human breast
8

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
cancer cell line (MCF-7), human liver carcinoma (HepG-2), and
colon cancer cell line (HCT-116). The entire tested compound
showed moderate to high anticancer activity against MCF-7 cells
with IC50 ranging from 0.68 to 35.60 μM (Table 1). Compounds
2b, 5, 8b, 12 and 13a,b elicited the highest antitumor activity
against the MCF-7 cell lines with IC50 0.76, 2.81, 0.68, 1.28, 2.47
and 2.87 μM respectively compared with the standard Imatinib,
which has IC50 6.06 μM. For the HepG-2 cell line, it found that the
same compounds 2a, 2b, 5, 6, 8b, 12, 13a and 13b were showing
the highest activity with IC50 4.73, 2.44, 2.88, 5.33, 2.80.95, 1.30,
3.02, and 2.95 μM, respectively, in comparison to Imatinib which
has IC50 5.50 μM for this cell line.
Also, compounds 2b, 8b, and 12 represent the highest activ-
ity against HCT-116 cell line in which the IC50 was 2.48, 0.74, and
0.76 μM, respectively. Other compounds showed moderate activity
against the three cell lines. This series’s most active compounds
were 8b containing spiropyrazole moiety with IC50 0.74, 0.95, and
0.68 μM compared with the standard Imatinib with IC50 6.06 5.50,
and 4.70 μM against the tested cell lines. Also, compound 2b,
which have IC50 0.76, 2.44, and 2.48 μM, indicates that the free
acetyl group’s presence showed good activity against the cell lines.
Upon reaction of compound 2 with hydrazine hydrate or thiosemi-
carbazide to afford 3a and 3b, the activity was diminished, and this
may be due to the absence of carbonyl in acetyl group and formed
hydrazone derivatives.
Finally, furo[2,3-b]indol-3-carboxamide derivatives 13a,b dis-
played the least Caspase-3 activator level by 8.01 and 8.27 folds,
respectively. The presence of 4-florophenyl derivative is favored
and induced apoptosis by activate caspase 3 proteins with slightly
higher than 2,4-dimethyl phenyl derivative at carboxamide group.
3.2.2.2. Study on mitochondrial apoptosis pathway BAX and Bcl-
2 proteins. For further evaluation, BAX and Bcl-2 are proteins
present in the mitochondria-mediated pathway and play an im-
portant role in tumor resistance to chemotherapy, so an increase
in BAX expression and decrease in Bcl-2 can induce the apop-
totic process and eliminate cancer cells [70,71]. In general, de-
pending on obtained data from Table 2, it found that treatment
of MCF-7 cells with the most promising derivatives 2b, 5, 8b,
12, 13a, and 13b displayed an upregulation of pro-apoptotic pro-
tein (BAX) and a down-regulation of anti-apoptotic, (Bcl-2) pro-
tein. Furthermore, compounds 8b, 12, 2b induced pro-apoptosis
with BAX protein values (461.92, 439.15 and 419.71 pg/mL) by
(9.76, 9.28 and 8.87 folds), respectively, in comparison to control
value (47.32 pg/mL). Additionally, design polynuclear structure as
furo[2,3-b]indol-3-carboxamide derivatives 13a, 13b demonstrated
BAX values (328.58 and 365.41 pg/mL) related to substituent on
carboxamide moiety. By the same way, all the tested compounds
2b, 5, 8b, 12, 13a and 13b suppress apoptosis with Bcl-2 values
(1.95, 2.12, 1.54, 1.75, 2.58 and 2.31pg/mL) respectively, and less
than untreated MCF-7 cells 6.16 pg/mL.
Chalcone 4, which formed through the reaction of 2b with
anisaldehyde, have moderate activity against the three cell lines
with IC50 8.91, 11.30, and 24.90 μM against MCF-7, HepG-2, and
HCT-116 cell lines respectively, this may be due to the presence
of methoxy group which has + M effect. On the other hand, 3-
hydroxyindolin-2-one derivative 5 showed higher activity against
the three cell lines with IC50 2.81, 2.88, and 3.22 μM, which means
that the free amino group’s presence increases activity. Moreover,
when this amino group blocked as in compound 7 through salicy-
laldehyde reaction, the antitumor activity decreased. Furthermore,
in Scheme 2, the most active compounds were 12, 13a and 13b
with IC50 (1.28, 1.30 and 0.76 μM), (2.47, 3.02, and 5.85 μM) and
(2.87, 2.95, and 4.76 μM) respectively and this may be due to the
formation of spiro-chromane moiety in compound 12. The increase
of anticancer activity for compounds 13a and 13b may be due to
the presence of furo[2,3-b]indole and carboxamide moieties in ad-
dition to isatin moiety.
Finally, it was reported that the increased ratio of up-regulating
BAX expression to down-regulating Bcl-2 expression plays a signif-
icant role in the collapse of mitochondrial membrane, then lead to
Cyt c release and cell death [72]. By applying BAX/ Bcl-2 ratio to
consideration value of the MCF-7 cell, the most active compound
8b showed ratio nearly to 39 folds as compared to the control, at
the same time the other compounds 2b, 5, 12, 13a and 13b es-
tablished to have ratio 27.95, 23.09, 32.60, 16.59 and 20.62 folds
respectively.
3.2.3. Flow cytometric analysis and apoptotic studies
One of the most promising cancer therapy strategies is cell cy-
cle targeting for cell line [73]. In this study compound, 8b was cho-
sen among the most active compounds for further studies and in-
vestigated its molecular mechanism of action.
3.2.3.1. Cell cycle analysis. Compound 8b that have spiroindolie-
3,3ꞌ-pyrazole derivative on its structure was evaluated cell cycle
distribution by flow cytometric analysis in MCF-7 cells, upon treat-
ing the cell with ten μM to study its effect on the cell cycle phases.
As shown in Fig. 3 and Table 3, the obtained result observed that
spiroindolie-3,3ꞌ-pyrazole derivative 8b arrested in G2/M phase by
47.95% and reduced in S phase (29.21%) in comparison to control
as well as induced apoptosis at priG1 and G0-G1 phases by 17.36
and 22.84%, respectively.
Finally, it found that the most active compounds contained
free acetyl or amino groups in p- position, spiropyrazole, spiro-
chromane, and furo[2,3-b]indole carboxamide moieties; this is be-
side the isatin structure as shown in Table 1.
3.2.2. Apoptosis detection studies
3.2.2.1. Effect on the active caspase-3 level. Caspase-3 is one of the
central ‘executioners’ of apoptosis that stimulates apoptosis by at-
tacking many profitable proteins required by the cell and there-
fore induced programmed cell death. Caspase-3 consists of two
subunits, with 12 and 17-kDa, and each subunit contains three
and five thiol functions, respectively [67–69]. Caspase-3 activation
for the most promising compounds depending on the IC50 results
was evaluated to explain the anti-proliferative activity mechanism,
and the obtained data are represented in (Fig. 2 and Table 2).
Spiro pyrazole derivatives 8b reveled the best Caspase-3 activator
with concentration 537.29 pg/mL with 10.81 folds than un-treated
3.2.3.2. Annexin-V FTIC apoptotic study. The apoptotic activity of
spiroindoline-3,3ꞌ-pyrazole derivative 8b was measured via flow
cytometry detection using Annexin V-FITC and propidium iodide
(PI) double staining. As shown in Fig. 5 and Table 4, upon treat-
ing the MCF-7 cells by compound 8b, the results demonstrate a
significant increase in the total apoptosis (17.36%) when compared
with the control (3.17%). Also, compound 8b induced apoptosis in
the late-stage higher than early stage 8.15, 6.46%. These data rep-
resented in Figs. 4 and 5 and Table 4 suggest that compound 8b is
a good apoptotic agent.
ꢀ
MCF-7 cell 49.71 pg/mL. Further, both spiro-choroman-2,3 indolin-
2-one derivatives 12 and Schiff’s base of 4-aminoacetophenone
with isatin 2b causes overexpression to Caspase-3 level (514.63 and
509.47 pg/mL) by 10.35 and 10.25 folds, respectively. Moreover, 3–
hydroxy-2-substituted-2-oxindole derivatives 5 demonstrated the
level of Caspase-3 by 8.62 folds.
3.2.4. In vitro EGFR inhibition assay
One of the important biomarkers for several different tumor
types and an attractive drug target in cancer therapy is the Epider-
mal Growth Factor Receptor. EGFR (also known as HER1 or erbB-1)
9

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Table 1
In vitroa cytotoxic activity of the synthesized compounds and Imatinib against MCF-7, HepG-2, and HCT-116
.
Cpd.
No.
IC50 μM/mL/SIb
MCF-7
R
HepG-2
HCT-116
WI-38
2a
2b
11.40 0.89
4.73 0.34
5.80 0.45
ND
0. 0.03
(250.7)
2. 0.11
(78)
2.48 0.12
(78.8)
190.5 1.87
3a
3b
35.60 1.6
18.60 1.25
21.70 1.4
ND
ND
50<
50<
50<
4
5
8.
0.73
11.30 0.90
24.90 1.5
ND
2.81 0.15
(67)
2.88 0.16
(65.5)
3.22 0.21
(58.6)
188.76 1.65
6
12.10 0.94
5.33 0.45
4.50 0.32
ND
7
9.82 0.84
11.10 0.92
24.30 1.5
ND
ND
8a
50<
50<
50<
8b
0.68 0.01
(300.5)
0.95 0.03
(215)
0.74 0.02
(276)
204.36 2.78
9
6. 0.51
7.16 0.70
5.21 0.35
ND
(continued on next page)
10

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Table 1 (continued)
Cpd.
No.
R
IC50 μM/mL/SIb
MCF-7
HepG-2
HCT-116
WI-38
10
11
8.76 0.76
10.18 0.84
13.10 0.95
19.20 1.2
ND
21.20 1.3
22.40 1.4
ND
12
202.08 2.11
1.28 0.06
(157.9)
1.30 0.07
(155)
0.76 0.02
(265)
13a
2.47 0.17
(67)
3.02 0.2
(55)
5.85 0.39
(28)
165.98 0.98
13b
2.87 0.14
(65)
2.95 0.18
(63)
4.76 0.35
(39)
186.55 1.34
Imatinib
6. 0.51
5.50
0.44
4.70 0.37
ND
ND: Not determined.
a
Data represent the mean values of three independent determinations.
b
SI: Selectivity index = (IC50 of WI-38)/ (IC50 of cancer cell line).
Fig. 2. A diagram indicating the effect of the most active compounds (2b, 5, 8b, 12, 13a, and 13b) on apoptotic induction of Caspase-3.
that can be defined as an intracellular signal translation receptor
that regulates pathway of proliferation, appointment, angiogene-
sis, adhesion, and motility of intracellular signal inside the cell and
therefore many drug therapy was approved by FDA that use this
receptor as a specific target [74–77].
ing MCF-7 cells Table (5). Compound 8b showed inhibitory con-
centration for EGFRWT with IC50= 0.014 μM in comparison to Lap-
atinib (IC50= 0.025 μM) and Erlotinib (IC20= 0.065 μM) with nearly
two and six folds higher than the positive control, respectively.
While in the case of mutant EGFRL858R-TK compound 8b displayed
IC50 value 12.66 nM with 2.82, 4.72 folds greater than reference
drugs, Lapatinib and Erlotinib, respectively Table 5.
The most promising spiroindoline-3,3ꞌ-pyrazole derivative 8b
was evaluated for two types of EGFR enzymatic activity assays us-
11

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Fig. 3. Cell cycle analysis and apoptosis effect of the most potent compound 8b on MCF-7 cell line.
Fig. 4. Cell cycle and flow cytometer analysis: i- 8b/ MCF-7 and ii- control MCF-7.
12

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Fig. 5. Diagram illustrates the effect of the compound 8b on cell death percentage.
3.2.5. In silico computational analysis studies
quirements in the screening process according to Veber ’s rules.
Both compounds have rotary bonds from 2 to 10 that demonstrate
molecular stability against their bio-targets, except compound 12,
which has zero rotary bonds.
3.2.5.1. In silico evaluation of physicochemical and ADME properties.
Computational testing was performed to evaluate physical and
ADME properties of the most biologically active synthesized com-
pounds (2b, 5, 8b, 12, 13a, and 13b, in addition to Imatinib and
Erlotinib). Both synthesized compounds are in no way in viola-
tion of the Lipinski rule for oral drugs for their physical-chemical
characteristics (Table 6). Therefore, all hits follow the drug-like re-
The total topological polar area (TPSA) (Table 6) is higher than
55.40 °A2. The absorption (percent of ABS) was further estimated
by using the equation percentage ABS %= 109 – (0.345 x TPSA)
[78] and found that the calculated percent ABS of all these hits var-
ied between 76.92 and 89.89 %, which showed that the cell mem-
brane permeability and bioavailability of such synthesized deriva-
tives were required.
Table 2
Effect of most promising compounds (2b, 5, 8b, 12, 13a, and 13b) on the gene’s
In relation to parameters of pharmacokinetic and some medic-
inal chemistry parameters for the newly synthesized compounds,
it was observed that all tested compounds with good gastrointesti-
nal absorption and no blood-brain barrier permeation, except 2b,
12, and 13b, which can pass the blood-brain barrier and can cause
CNS side effects. (Table 7)
expression and apoptosis markers.
Cpd.
No.
Caspase-3
BAX
Bcl-2
BAX/Bcl-2
ratio
Pg/mL (Fold)
Pg/mL(Fold)
Pg/mL (Fold)
2b
509.47 (10.25)
428.72 (8.62)
537.29 (10.81)
514.63 (10.35)
398.43 (8.01)
411.55 (8.28)
49.71
419.71 (8.87)
375.64 (7.94)
461.92 (9.76)
439.15 (9.28)
328.58 (6.94)
365.41 (7.72)
47.32
1.95 (3.15)
2.12 (2.86)
1.54 (3.99)
1.75 (3.51)
2.58 (2.39)
2.31 (2.67)
6.16
27.95
23.09
38.98
32.60
16.59
20.62
1
5
8b
Another significant factor throughout the preclinical research
review was the examination of a non-substrate P-glycoprotein (P-
gp) candidature. P-gp acts as an effluent transport mechanism
that pumps medicines and other compounds out of the cell and
its substrate is one of the factors behind its resistance to can-
cer chemotherapy. P-gap was found to be a vital carrier of the
medicament, thus resisting drugs such as Imatinib [79]. Thus, our
compounds were analyzed on the website of SwissADME (http:
//swissadme.ch/index.php), and most of the hits are not P-gp pro-
tein substrates, as seen in Table 7. It indicates that 2b, 8b and 13a,
have very little chance of effluxing out of the cell, leading to full
impact.
12
13a
13b
MCF-7
Table 3
Results of cell cycle analysis in MCF-7 expressed by (%) of cells in each phase
when treated with 8b.
Cpd.
No.
%G0-G1
%S
%G2/M
%Pre-G1
8b
22.84
50.49
29.21
34.09
47.95
15.42
17.36
3.17
MCF-7
Bioavailability is a plasma quantity index and is seen as the
most important factor affecting absorption. It is important to note
the high bioavailability levels of 0.55 of all active synthesized
derivatives. SwissADME [47,60] has shown zero hit warnings for
pan-assay interference compounds (PAINS). PAINS, while impor-
tant features must be taken into consideration when designing
medicines for the avoidance of false-positive effects, will lead only
to the exclusion of promising hits based on fantastic PAINS [80].
All analogs were found to have synthetic accessibility between 2.58
and 4.72 values, suggesting that they can be easily synthesized on
a large basis (Table 7).
Table 4
Different Percent of cell death induced by compounds 8b on MCF-7 cells.
Cpd.
No.
Apoptosis
Total
Necrosis
Early
Late
8b
17.36
3.17
6.46
1.47
8.15
1.25
2.75
0.45
MCF-7
Table 5
IC50 of the representative anticancer active compound
8b on EGFR in MCF-7 cells.
Cpd.
No.
Enzyme inhibitory activity IC50
3.2.5.2. Molecular docking studies. In recent years, the computer
docking technique plays a major role in drug design by placing
a molecule into the binding site of the target enzyme in a non-
covalent fashion, and therefore these models provide a more accu-
rate picture of the biologically active molecules at the atomic level
EGFRWT (μM)
EGFRL858R-TK(nM)
8b
0.014
0.025
0.0653
12.66
35.72
59.72
Lapatinib
Erlotinib
13

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Fig. 6a. Superimposition 3D of the co-crystallized (red) and the docking pose (blue) of Erlotinib with RMSD of 1.10 °A in the active site of 1M17. Fig. 6b: 3D interactions of
Erlotinib in the active site of 1M17. Fig. 6c: 2D & 3D interaction maps of compound 8b inside 1M17 active site.
14

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
Table 6
Physicochemical properties based on Lipinskis´ rule of five, Vebers´ rule, TPSA, and% ABS.
Cpd.
no.
HBD
HBA
MlogP
MW
TPSA∗
ABS
%
Lipinskis´
Vebers´
˂ 5
˂ 10
˂ 4.15
˂ 500
RT∗
°A²
Violations
violations
2b
1
3
2
1
2
2
2
1
3
3
4
3
6
5
6
6
1.5
264.28
282.29
458.51
265.26
349.34
307.28
493.60
393.44
2
58.53
92.42
77.29
55.40
93.00
74.54
86.28
74.73
88.81
77.12
82.33
89.89
76.92
83.28
79.23
83.22
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
5
0.75
3.60
1.58
1.46
2.45
2.15
1.62
3
8b
4
12
0
13a
5
13b
3
Imatinib
Erlotinib
8
10
∗RT= rotatable bond, TPSA =topological polar surface area,.
Table 7
Pharmacokinetic properties and medicinal chemistry parameters.
Cpd. no.
GI Absorption
BBB Permeation
Pgp substrate
Bioavailability Score
PAINS Alerts
Synthetic Accessibility
2b
High
High
High
High
High
High
High
High
Yes
No
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
No
Yes
Yes
No
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0.55
0
0
0
0
0
0
0
0
2.59
2.58
4.72
3.38
3.65
3.38
3.78
3.19
5
8b
12
13a
13b
Imatinib
Erlotinib
[81]. In order to have an insight into the molecular interaction of
most potent compound 8b with epidermal growth factor receptor
kinase domain (EGFR), the docking study was performed to explore
the mode of interactions of compound 8b with amino acids in the
active site of the enzyme (PDB ID 1M17) [24], that was down-
loaded from protein data bank (www.rcsb.org) as crystal structure
and co-crystalized with Erlotinib inhibitor (4-anilinoquinazoline).
Redocking process of co-crystallized ligand observed docking score
S = −17.84 kcal/mol, and RMSD of 1.10 °A with only one hydrogen
bond with Met 769 and nitrogen of quinazoline with bond length
(3.04 °A) (Fig. 6a,b). Molecular modeling was carried out using
Molecular Operating Environment software 10.2008 (MOE), Chem-
ical Computing Group Inc., Montreal, Quebec, Canada, the docking
results were achieved, and the best confirmation model with the
high docking score was investigated for analysis.
different interaction as hydrogen bond (three bonds), arene-cation,
and hydrophobic interactions.
4. Conclusions
A new series of isatin-linked chalcones and Schiffʼs base in this
work were synthesized and evaluated as novel apoptotic agents.
The target compounds were examined for their cytotoxic activity
against three human cell lines, which are breast cancer cell lines
(MCF-7), liver hepatocellular carcinoma (HepG-2), and colon car-
cinoma cell lines (HCT-116). Compounds 2b, 5, 8b, 12, 13a, and
13b displayed to be the most potent in this study compared to
Imatinib as the reference standard drug. Compound 8b was found
to be the most potent against the three cell lines. Furthermore,
apoptotic studies conducted by these active compounds reveal-
ing that apoptosis was accomplished by up-regulation of BAX and
Caspase-3 and down-regulation of Bcl-2. Moreover, 8b, the most
potent compound tested for their activity on the cell cycle MCF-
7, revealed that they arrest cells at G2/M phase in the cell cycle.
Also, the inhibitory activity for compound 8b against wild EGFRWT
and mutant EGFRL858R-TK were done. It showed IC50= 0.014 μM
Compound 8b admitted
a
lower docking score energy
(−18.76 kcal/mol), with two hydrogen bonds acceptor, one
hydrogen bond bonded between Cys773 with the carbonyl of
isatin with bond length (2.99 °A) with a strength of 11 %, and
another one between Lys692 and hydroxy group of salicylaldehyde
derivative with bond length (2.63 °A) and strength of 64 %. Also,
there is one H-bond sidechain donor between NH of isatin deriva-
tive and Asp776 with bond length 2.46 °A and strength (54%) and
an arene-cation interaction observed between Lys 704 and phenyl
group of salicylaldehyde derivative (Figure 6c).
in comparison to Lapatinib (IC50= 0.025 μM) and Erlotinib (IC20
=
0.065 μM), while in the case of mutant EGFRL858R-TK 8b displayed
IC50 value 12.66 nM with 2.82, 4.72 folds greater than reference
drugs, Lapatinib and Erlotinib, respectively. Physicochemical and
ADME properties are also done for the most active compounds as
well as docking simulation for compound 8b. Finally, depending
on the above studies, we can conclude that among the most ac-
tive compounds, only one compound (spiroindoline-3,3ꞌ-pyrazole
derivative) 8b was selected for further studies to elucidate the
mechanism of cell death and therefore, the advanced studies dis-
played that compound 8b capable of inducing apoptosis by multi-
ple mechanisms, which helps us in the future work for this struc-
ture.
Our synthetic strategy in this research involves three chiral
compounds as 8a, 8b, and 12. However, only one derivative 8b
showed good anti-proliferative activity with lower IC50 values
against EGFR expression; for the previous reason, further docking
study for both derivatives 8b and 12 was performed to illustrate
this conflict. Docking of Compound 8a displayed binding energy
S = −14.52 kcal/mol, with only one hydrogen bond between the
nitrogen of pyrazole and Cys773 with bond length 3.15 °A and
strength 13%. Furthermore, Compound 12 exhibited binding energy
S = −12.72 kcal/mol, with two hydrogen bonds between the car-
bonyl of chromone and isatin with the residues Lys721 and Thr766
with bond length 3.10°A 2.67°A respectively (See supplementary
material). Finally, it can be concluded that spiro indoline-3,3ꞌ-
pyrazole derivative 8b could enhance anti-proliferative activity
with EGFR inhibition by lower binding energy (−18.76) and
Declaration of Competing Interest
The authors declare that they have no known competing finan-
cial interests or personal relationships that could have appeared to
influence the work reported in this paper. The authors declare the
15

E.A. Fayed, R.R.E. Eldin, A.B. M. Mehany et al.
Journal of Molecular Structure 1234 (2021) 130159
following financial interests/personal relationships which may be
considered as potential competing interests.
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CRediT authorship contribution statement
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A.B.M. Mehany, Design, synthesis, antiproliferative activity, molecular dock-
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doi:10.1016/j.ejmech.2018.06.061.
Eman A. Fayed: Conceptualization, Methodology, Validation,
Formal analysis, Investigation, Resources, Writing - original draft,
Writing
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Yousry A. Ammar: Conceptualization, Validation, Formal analysis,
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