New hydrazonoindolin-2-ones: Synthesis, exploration of the possible anti-proliferative mechanism of action and encapsulation into PLGA microspheres

Article abstract of DOI:10.1371/journal.pone.0181241

The synthesis and molecular characterization of new isatin-based hydrazonoindolin-2-ones 4a-o and 7a-e are reported. The in vitro anti-proliferative potential of the synthesized compounds 4a-o and 7a-e was examined against HT-29 (colon), ZR-75 (breast) and A549 (lung) human cancer cell lines. Compounds 7b, 7d and 7e were the most active congeners against the tested human cancer cell lines with average IC₅₀ values of 4.77, 3.39 and 2.37 μM, respectively, as compared with the reference isatin-based drug, sunitinib, which exhibited an average IC₅₀ value of 8.11 μM. Compound 7e was selected for further pharmacological evaluation in order to gain insight into its possible mechanism of action. It increased caspase 3/7 activity by 2.4- and 1.85-fold between 4 and 8 h of treatment, respectively, at 10 μM and it caused a decrease in the percentage of cells in the G1 phase of the cell cycle with a corresponding increase in the S-phase. In addition, compound 7e increased phosphorylated tyrosine (p-Tyr) levels nearly two-fold with an apparent IC₅₀ value of 3.8 μM. The 7e-loaded PLGA microspheres were prepared using a modified emulsion-solvent diffusion method. The average encapsulation efficiency of the 7e-loaded PLGA microspheres was 85% ± 1.3. While, the in vitro release profile of the 7e-loaded microspheres was characterized by slow and continuous release of compound 7e during 21 days and the release curve was fitted to zero order kinetics. Incorporation of 7e into PLGA microspheres improved its in vitro anti-proliferative activity toward the human cancer cell line A549 after 120 h incubation period with an IC₅₀ value less than 0.8 μM.

Full text of DOI:10.1371/journal.pone.0181241

RESEARCH ARTICLE
New hydrazonoindolin-2-ones: Synthesis,
exploration of the possible anti-proliferative
mechanism of action and encapsulation into
PLGA microspheres
Mohamed I. Attia1,2*, Wagdy M. Eldehna3, Samar A. Afifi4,5, Adam B. Keeton6, Gary
A. Piazza6, Hatem A. Abdel-Aziz7*
1 Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi
Arabia, 2 Medicinal and Pharmaceutical Chemistry Department, Pharmaceutical and Drug Industries
Research Division, National Research Centre (ID: 60014618), Dokki, Giza, Egypt, 3 Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, Egypt, 4 Department
of Pharmaceutics, National Organization for Drug Control and Research, Giza, Egypt, 5 Department of
Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia, 6 Department of
Oncologic Sciences and Pharmacology, Drug Discovery Research Center, Mitchell Cancer Institute,
University of South Alabama, Mobile, AL, United States of America, 7 Department of Applied Organic
Chemistry, National Res earch Centre, (ID: 60014618), Dokki, Giza, Egypt
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* mattia@ksu.edu.sa (MIA); hatem_741@yahoo.com (HAA)
OPEN ACCESS
Citation: Attia MI, Eldehna WM, Afifi SA, Keeton
AB, Piazza GA, Abdel-Aziz HA (2017) New
hydrazonoindolin-2-ones: Synthesis, exploration of
the possible anti-proliferative mechanism of action
and encapsulation into PLGA microspheres. PLoS
ONE 12(7): e0181241. https://doi.org/10.1371/
journal.pone.0181241
Abstract
The synthesis and molecular characterization of new isatin-based hydrazonoindolin-2-ones
4a-o and 7a-e are reported. The in vitro anti-proliferative potential of the synthesized com-
pounds 4a-o and 7a-e was examined against HT-29 (colon), ZR-75 (breast) and A549 (lung)
human cancer cell lines. Compounds 7b, 7d and 7e were the most active congeners against
the tested human cancer cell lines with average IC50 values of 4.77, 3.39 and 2.37 μM, respec-
tively, as compared with the reference isatin-based drug, sunitinib, which exhibited an average
IC50 value of 8.11 μM. Compound 7e was selected for further pharmacological evaluation in
order to gain insight into its possible mechanism of action. It increased caspase 3/7 activity by
2.4- and 1.85-fold between 4 and 8 h of treatment, respectively, at 10 μM and it caused a
decrease in the percentage of cells in the G1 phase of the cell cycle with a corresponding
increase in the S-phase. In addition, compound 7e increased phosphorylated tyrosine (p-Tyr)
levels nearly two-fold with an apparent IC50 value of 3.8 μM. The 7e-loaded PLGA micro-
spheres were prepared using a modified emulsion-solvent diffusion method. The average
encapsulation efficiency of the 7e-loaded PLGA microspheres was 85% ± 1.3. While, the in
vitro release profile of the 7e-loaded microspheres was characterized by slow and continuous
release of compound 7e during 21 days and the release curve was fitted to zero order kinetics.
Incorporation of 7e into PLGA microspheres improved its in vitro anti-proliferative activity
toward the human cancer cell line A549 after 120 h incubation period with an IC50 value less
than 0.8 μM.
´
Editor: Stephanie Filleur, Texas Technical
University Health Sciences Center, UNITED
STATES
Received: March 12, 2017
Accepted: June 28, 2017
Published: July 25, 2017
Copyright: © 2017 Attia et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: This project was funded by the National
Plan for Science, Technology and Innovation
(MAARIFAH), King Abdulaziz City for Science and
Technology, Kingdom of Saudi Arabia, Award
Number 11-MED1924–02.
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Competing interests: The authors have declared
Introduction
that no competing interests exist.
Cancer is one of the most formidable health burdens with increasing annual frequency world-
wide [1]. In spite of precluding many of its causative risk factors, cancer still gives rise to about
550,000 deaths in the world every year and is the second leading cause of death next to heart dis-
ease [2]. The currently available cancer therapies usually suffer from poor selectivity between
normal and invaded cells, with serious side effects and the development of drug resistance [3].
Therefore, the continued search to identify novel antitumor compounds bestowed with low tox-
icity and minimum side effects remains critically important.
The 1H-Indole-2,3-dione (isatin) moiety constitutes the backbone of a number of agrochemi-
cals, dyes and bioactive molecules, owing to its appropriate size and unique electronic properties
[4–7]. Various 3-substituted isatins have been utilized as anticancer drugs or drug surrogates.
Example of this class of isatin derivatives is sunitinib (1, SU11248, SutentTM, Pfizer, Inc., Fig 1)
which was clinically approved in 2006 and is used now as the standard first-line treatment for
the management of gastrointestinal stromal cancers and renal cell carcinoma due to its multi-tar-
geting tyrosine kinase inhibiting activity [8]. Nintedanib (2, Fig 1) is another potential anticancer
agent of this class that possesses triple angiokinase inhibiting activity toward vascular endothelial
growth factor receptors (VEGFRs),platelet-derived growth factor receptors(PDGFRs) and fibro-
blast growth factor receptors (FGFRs)[9]. Semaxanib (3, Fig 1) was developed as a new antican-
cer candidate with multiple tyrosinekinase receptor inhibitor activity with GI50 values of less
than 10 nM against 46 out of 53 NCI cell lines, but it has been discontinued in clinical trials
owing to its dangerous side effects [10, 11].
Schiff bases of isatin are acknowledged to have a broad spectrum of biological activities
including anticonvulsant [12], antibacterial [13], antifungal [14], anti-HIV [15], antiviral [16]
and anticancer activity [17–19].
Indirubin (4, Fig 2) is a bis-isatin isomer of the widely used colorant indigo (5, Fig 2), and it
is the active ingredient of a traditional Chinese remedy (Danggui Longhui Wan) that is used
for the treatment of chronic myelogenous leukemia (CML) [20]. Moreover, indirubin (4)
inhibits both glycogen synthase kinase 3 beta (GSK-3β) and cyclin-dependent kinases (CDKs)
via binding to the ATP pocket with IC50 values of 0.190 and 0.075 mM, respectively [21, 22].
Azaindirubin (6, Fig 2) was developed to overcome the poor water solubility and hence low
bioavailability of indirubin (4) and it showed anti-proliferative activity toward ovarian ade-
noma cell lines [23]. In the same vein, the symmetric bis-isatin derivative (7, Fig 2) displayed
in vitro anti-proliferative activity against the HepG2 cell line with IC50 value about 4.23 mM
[21].
Microsphere carrier systems made from the naturally occurring biodegradable polymers
have attracted considerable attention for several years in the field of sustained drug delivery.
These carrier systems can precisely control the release rates and target drugs to a specific body
site with an enormous impact in formulation and development of novel drug delivery systems
[24]. Biodegradable polyesters such as copolymers of lactic and glycolic acid (PLGA) are
attractive biomaterials for the encapsulation of pharmaceuticals and bioactive compounds. In
this case, encapsulated drugs are not as readily available to biological systems as when they are
in solution because the release of the drug will occur only after the onset of polymeric degrada-
tion [25].
In this context, certain potential functionalized Schiff bases of isatin 4a-o were designed
and synthesized to evaluate their in vitro anti-proliferative activity against a panel of human
cancer cell lines. In addition, the non-symmetric bis-isatins 7a-e were also prepared and evalu-
ated as new anti-proliferative agents. The compound exhibiting the best pharmacological
activity was subjected to deeper pharmacological investigations in order to gain insight into its
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 1. Chemical structures of some clinically used isatine-based anticancer agents.
https://doi.org/10.1371/journal.pone.0181241.g001
pharmacological profile as well as its possible mechanism of action. Moreover, this compound
was encapsulated into PLGA microspheres in order to evaluate the influence of microencapsu-
lation on its in vitro anti-proliferative activity.
Experimental
General
Melting points were measured with a Stuart melting point apparatus and were uncorrected.
Infrared (IR) spectra were recorded as KBr disks using an FT-IR Spectrum BX apparatus(Per-
kin Elmer, CT, USA). NMR spectra were recorded on a Bruker NMR spectrometer (Bruker,
Reinstetten, Germany).1H spectra were run at 500 MHz and 13C spectra were run at 125 MHz
in deuterated dimethyl sulfoxide (DMSO-d6). Chemical shifts are expressed in values (ppm)
using the solvent peak as an internal standard. All coupling constant (J) values are given in Hz.
The abbreviations used are as follows: s, singlet; d, doublet; m, multiplet. Mass spectra were
recorded using an Agilent Quadrupole 6120 LC/MS with an ESI (electrospray ionization)
source (Agilent Technologies, Palo Alto, CA, USA). Elemental analyses were carried out at the
regional center for microbiology and biotechnology, Al-Azhar University, Cairo, Egypt. Ana-
lytical thin layer chromatography (TLC) on silica gel plates containing a UV indicator was
employed routinely to follow the course of reactions and to check the purity of the products.
All reagents and solvents were purified and dried by standard techniques. Compounds 4a [26],
Fig 2. Chemical structures of some bioactive bis-isatin compounds.
https://doi.org/10.1371/journal.pone.0181241.g002
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
4f [27], 4i [28] and 6a, b [29] have been previously reported. The microspheres were prepared
with poly (D, L-lactide co-glycolide) PLGA (50:50, mol. wt 30,000–60,000), which was pur-
chased from Sigma-Aldrich (St. Louis, USA). The emulsifier, low molecular weight polyvinyl
alcohol (PVA) was obtained from Alfa Aesar (Karlsruhe, Germany). Dichloromethane (DCM)
was purchased from Avonchem (United Kingdom). Dimethyl sulfoxide (DMSO) was obtained
from Loba Chemie (Mumbai, India). All ingredients used were of analytical grade. All cell
lines have been purchased from the American Type Culture Collection (ATCC).
Chemistry
3-Hydrazonoindolin-2-ones2a-c. To a stirred solution of indoline-2,3-diones 1a-c (10
mmol) in methanol (20 mL), 99% hydrazine hydrate (2.5 mL, 50 mmol) was added. The reac-
tion mixture was refluxed for one hour. The precipitate was filtered, washed with methanol,
dried and re-crystallized from glacial acetic acid to afford intermediates 2a-c [30].
General procedure for the synthesis of hydrazones 4a-o. A catalytic amount of glacial
acetic acid was added to equimolar quantities of the hydrazones 2a-c (1 mmol) and benzalde-
hydes 3a-e (1 mmol) in ethanol (10 mL).The reaction mixture was refluxed for four hours and
then cooled to room temperature. The obtained precipitate was collected by filtration, dried
and re-crystallized from ethanol/dioxane mixture (5:1) to afford compounds 4a-o with 67–
83% yields.
3-((4-Methoxybenzylidene)hydrazono)indolin-2-one (4a). Orange powder (yield 73%), m.
p. 277–279˚C; IR (KBr, ν cm-1): 3405 (NH), 1734 (C = O), 1607 (C = N); 1H NMR (DMSO-d6)
ppm: 3.87 (s, 3H, OCH3), 6.90 (d, 1H, H-7 isatin, J = 7.5 Hz), 7.05 (t, 1H, H-5 isatin, J = 7.5
Hz), 7.14 (d, 2H, H-3 and H-5 of 4-OCH3-C6H4, J = 8.0 Hz), 7.39 (t, 1H, H-6 isatin, J = 7.5
Hz), 7.96 (d, 2H, H-2 and H-6 of 4-OCH3-C6H4, J = 8.0 Hz), 8.04 (d, 1H, H-4 isatin, J = 7.5
Hz), 8.64 (s, 1H, -CH =), 10.84 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 56.0 (OCH3),
111.2, 112.4, 115.3, 117.1, 122.8, 126.6, 127.3, 129.4, 131.5, 133.9, 145.3, 162.4, 163.2 (Ar-CH
Ar-C and C = O); MS (ESI) m/z: 280.1 [M+1]+; Anal. Calcd. for C16H13N3O2: C, 68.81; H,
4.69; N, 15.05; Found: C, 69.03; H, 4.64; N, 14.94.
3-((3-Hydroxy-4-methoxybenzylidene)hydrazono)indolin-2-one (4b). Orange powder
(yield 71%), m.p. 273–275˚C; IR (KBr, ν cm-1): 3282 (NH), 1720 (C = O), 1615 (C = N); 1H
NMR (DMSO-d6) ppm: 3.87 (s, 3H, OCH3), 6.90–7.11 (m, 3H, Ar-H), 7.22–7.53 (m, 4H, Ar-
H), 8.52 (s, 1H, -CH =), 9.31 (s, 1H, OH), 11.00 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm:
56.1 (OCH3), 112.7, 113.8, 116.3, 119.4, 122.6, 123.0, 124.8, 127.6, 129.2, 133.3, 134.9, 145.1,
147.3, 162.4, 163.2 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 296.1 [M+1]+; Anal. Calcd. for
C16H13N3O3: C, 65.08; H, 4.44; N, 14.23; Found: C, 65.24; H, 4.39; N, 14.35.
3-((4-Hydroxy-3-methoxybenzylidene)hydrazono)indolin-2-one (4c). Red powder (yield
67%), m.p. 269–270˚C; IR (KBr, ν cm-1):3448 (NH), 1718 (C = O), 1618 (C = N); 1H NMR
(DMSO-d6) ppm: 3.88 (s, 3H, OCH3), 6.91 (d, 1H, H-7 isatin, J = 7.5 Hz), 6.96 (d, 1H, H-5 of
4-OH-3-OCH3-C6H3, J = 8.0 Hz), 7.02 (t, 1H, H-5 isatin, J = 7.5 Hz), 7.40 (t, 1H, H-6 isatin,
J = 7.5 Hz), 7.46 (d, 1H, H-6 of 4-OH-3-OCH3-C6H3, J = 8.5 Hz), 8.02 (d, 1H, H-4 isatin,
J = 8.0 Hz), 7.59 (s, 1H, H-2 of 4-OH-3-OCH3-C6H3), 8.70 (s, 1H, -CH =), 10.14 (s, 1H, OH),
10.98 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 56.1 (OCH3), 111.2, 112.1, 116.4, 117.2,
122.8, 124.7, 125.3, 127.5, 129.3, 133.8, 139.1, 145.2, 148.6, 151.0, 165.3 (C = O); MS (ESI) m/z:
296.1 [M+1]+; Anal. Calcd. for C16H13N3O3: C, 65.08; H, 4.44; N, 14.23; Found: C, 64.86; H,
4.39; N, 14.08.
3-((3,4-Dimethoxybenzylidene)hydrazono)indolin-2-one (4d). Orange powder (yield
79%), m.p. 276–278˚C; IR (KBr, ν cm-1): 3281 (NH), 1721 (C = O), 1616 (C = N); 1H NMR
(DMSO-d6) ppm: 3.87 (s, 3H, OCH3), 3.88 (s, 3H, OCH3), 6.90 (d, 1H, H-7 isatin, J = 7.5 Hz),
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
7.04 (t, 1H, H-5 isatin, J = 7.5 Hz), 7.16 (d, 1H, H-5 of 3,4-(OCH3)2-C6H3, J = 8.0 Hz), 7.39 (t,
1H, H-6 isatin, J = 7.5 Hz), 7.56–7.58 (m, 2H, H-2 and H-6 of 3,4-(OCH3)2-C6H3), 8.01 (d, 1H,
H-4 isatin, J = 8.0 Hz), 8.58 (s, 1H, -CH =), 10.84 (s, 1H, NHindolic); 13C NMR (DMSO-d6)
ppm: 55.9 (OCH3), 56.1 (OCH3), 109.6, 111.6, 111.9, 116.3, 123.0, 123.9, 127.2, 128.7, 134.9,
145.2, 145.7, 149.5, 152.1, 161.2, 163.9 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 310.1 [M+1]+;
Anal. Calcd. for C17H15N3O3: C, 66.01; H, 4.89; N, 13.58; Found: C, 65.79; H, 4.92; N, 13.72.
3-((3,4,5-Trimethoxybenzylidene)hydrazono)indolin-2-one (4e). Red powder (yield 80%), m.
p. 240–241˚C; IR (KBr, ν cm-1): 3280 (NH), 1721 (C = O), 1616 (C = N); 1H NMR (DMSO-d6)
ppm: 3.74 (s, 3H, OCH3), 3.85 (s, 6H, 2 OCH3), 6.92 (d, 1H, H-7 isatin, J = 7.5 Hz), 7.01 (t, 1H,
H-5 isatin, J = 7.5 Hz), 7.23 (s, 2H, H-2 and H-6 of 3,4,5-(OCH3)3-C6H2), 7.41 (t, 1H, H-6 isatin,
J = 7.5 Hz), 7.51 (d, 1H, H-4 isatin, J = 7.5 Hz), 8.67 (s, 1H, -CH =), 11.00 (s, 1H, NHindolic); 13
NMR (DMSO-d6) ppm: 56.4 (OCH3), 61.1 (OCH3), 106.0, 111.6, 116.2, 123.0, 128.7, 129.7,
C
134.9, 140.6, 145.2, 145.7, 153.6, 161.8, 163.9 (C = O); MS (ESI) m/z: 340.1 [M+1]+; Anal. Calcd.
for C18H17N3O4: C, 63.71; H, 5.05; N, 12.38; Found: C, 63.95; H, 5.01; N, 12.29.
5-Chloro-3-((4-methoxybenzylidene)hydrazono)indolin-2-one (4f). Orange powder (yield
75%), m.p. 282–284˚C; IR (KBr, ν cm-1): 3421 (NH), 1725 (C = O), 1618 (C = N); 1H NMR
(DMSO-d6) ppm: 3.88 (s, 3H, OCH3), 6.93 (d, 1H, H-6 isatin, J = 8.0 Hz), 7.17 (d, 2H, H-3 and
H-5 of 4-OCH3-C6H4, J = 8.5 Hz), 7.46 (d, 1H, H-7 isatin, J = 8.5 Hz), 7.94 (d, 2H, H-2 and H-
6 of 4-OCH3-C6H4, J = 8.5 Hz), 8.01 (s, 1H, H-4 isatin), 8.70 (s, 1H, -CH =), 10.98 (s, 1H,
NHindolic); 13C NMR (DMSO-d6) ppm: 56.1 (OCH3), 112.8, 115.4, 118.2, 126.3, 126.3, 128.5,
131.6, 133.3, 144.1, 150.5, 163.4, 163.9, 164.9 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 314.1
[M+1]+; Anal. Calcd. for C16H12ClN3O2: C, 61.25; H, 3.86; N, 13.39; Found: C, 61.08; H, 3.90;
N, 13.53.
5-Chloro-3-((3-hydroxy-4-methoxybenzylidene)hydrazono)indolin-2-one(4g). Orange pow-
der (yield 72%), m.p. 278–280˚C; IR (KBr, ν cm-1): 3364 (NH), 1725 (C = O), 1616 (C = N); 1H
NMR (DMSO-d6) ppm: 3.88 (s, 3H, OCH3), 6.93 (d, 1H, H-6 isatin, J = 8.0 Hz), 7.13 (d, 1H, H-5
of 3-OH-4-OCH3-C6H3, J = 8.5 Hz), 7.39 (d, 1H, H-7 isatin, J = 8.0 Hz), 7.46–7.48 (m, 2H, H-2
and H-6 of 3-OH-4-OCH3-C6H3), 8.02 (s, 1H, H-4 isatin), 8.61 (s, 1H, -CH =), 9.61 (s, 1H, OH),
10.97 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 56.2 (OCH3), 112.6, 112.8, 114.2, 118.3,
124.0, 126.3, 126.5, 128.4, 133.3, 144.0, 147.6, 150.3, 152.6, 164.4, 164.9 (Ar-CH, Ar-C and C = O);
MS (ESI) m/z: 330.1 [M+1]+; Anal. Calcd. for C16H12ClN3O3: C, 58.28; H, 3.67; N, 12.74; Found:
C, 58.41; H, 3.64; N, 12.67.
5-Chloro-3-((4-hydroxy-3-methoxybenzylidene)hydrazono)indolin-2-one (4h). Orange
powder (yield 70%), m.p. 269–271˚C; IR (KBr, ν cm-1): 3263 (NH), 1725 (C = O), 1615
(C = N); 1H NMR (DMSO-d6) ppm: 3.90 (s, 3H, OCH3), 6.92 (d, 1H, H-6 isatin, J = 8.0 Hz),
6.97 (d, 1H, H-5 of 4-OH-3-OCH3-C6H3, J = 8.5 Hz), 7.42–7.46 (m, 2H, H-6 of 4-OH-3-
OCH3-C6H3 and H-7 isatin), 7.59 (s, 1H, H-2 of 4-OH-3-OCH3-C6H3), 8.14 (s, 1H, H-4 isa-
tin), 8.66 (s, 1H, -CH =), 10.16 (s, 1H, OH), 10.96 (s, 1H, NHindolic); 13C NMR (DMSO-d6)
ppm: 55.8 (OCH3), 111.2, 112.8, 116.4, 118.4, 125.2, 125.7, 126.2, 128.8, 133.1, 143.9, 148.7,
150.7, 152.2, 164.8, 165.1 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 330.1 [M+1]+; Anal. Calcd.
for C16H12ClN3O3: C, 58.28; H, 3.67; N, 12.74; Found: C, 58.09; H, 3.70; N, 12.87.
5-Chloro-3-((3,4-dimethoxybenzylidene)hydrazono)indolin-2-one(4i). Red powder (yield
81%), m.p. 282–284˚C; IR (KBr, ν cm-1): 3420(NH), 1744 (C = O), 1818 (C = N); 1H NMR
(DMSO-d6) ppm: 3.88 (s, 6H, 2 OCH3), 6.93 (d, 1H, H-6 isatin, J = 8.0 Hz), 7.19 (d, 1H, H-5 of
3,4-(OCH3)2-C6H3, J = 8.5 Hz), 7.46 (d, 1H, H-7 isatin, J = 8.0 Hz), 7.54 (d, 1H, H-6 of 3,4-
(OCH3)2-C6H3, J = 8.0 Hz), 7.61 (s, 1H, H-2 of 3,4-(OCH3)2-C6H3), 8.01 (s, 1H, H-4 isatin),
8.69 (s, 1H, -CH =), 10.99 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 55.7 (OCH3), 56.3
(OCH3), 110.0, 112.3, 112.8, 118.3, 125.4, 126.2, 126.4, 128.8, 133.3, 144.0, 149.6, 150.9, 153.4,
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
164.0, 164.9 (C = O); MS (ESI) m/z: 343.9 [M+1]+; Anal. Calcd. for C17H14ClN3O3: C, 59.40;
H, 4.11; N, 12.22; Found: C, 59.61; H, 4.07; N, 12.34.
5-Chloro-3-((3,4,5-trimethoxybenzylidene)hydrazono)indolin-2-one (4j). Red powder
(yield 77%), m.p. 245–247˚C; IR (KBr, ν cm-1): 3315 (NH), 1734 (C = O), 1617 (C = N); 1H
NMR (DMSO-d6) ppm: 3.78 (s, 3H, OCH3), 3.90 (s, 6H, 2 OCH3), 6.93 (d, 1H, H-6 isatin,
J = 7.5 Hz), 7.34 (s, 2H, H-2 and H-6 of 3,4,5-(OCH3)3-C6H2), 7.46 (d, 1H, H-7 isatin, J = 7.0
Hz), 8.03 (s, 1H, H-4 isatin), 8.63 (s, 1H, -CH =), 11.01 (s, 1H, NHindolic); 13C NMR (DMSO-
d6) ppm: 56.4 (OCH3), 60.8 (OCH3), 106.7, 112.9, 118.2, 126.3, 128.9, 129.1, 133.4, 141.7, 144.2,
151.4, 153.8, 162.6, 164.8 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 373.2 [M+1]+; Anal. Calcd.
for C18H16ClN3O4: C, 57.84; H, 4.31; N, 11.24; Found: C, 58.07; H, 4.26; N, 11.31.
5-Bromo-3-((4-methoxybenzylidene)hydrazono)indolin-2-one (4k). Orange powder (yield
76%), m.p. 290–292˚C; IR (KBr, ν cm-1): 3422 (NH), 1744 (C = O), 1616 (C = N); 1H NMR
(DMSO-d6) ppm: 3.88 (s, 3H, OCH3), 6.88 (d, 1H, H-6 isatin, J = 8.0 Hz), 7.18 (d, 2H, H-3 and
H-5 of 4-OCH3-C6H4, J = 8.5 Hz), 7.59 (d, 1H, H-7 isatin, J = 8.0 Hz), 7.93 (d, 2H, H-2 and H-
6 of 4-OCH3-C6H4, J = 8.5 Hz), 8.16 (s, 1H, H-4 isatin), 8.69 (s, 1H, -CH =), 10.99 (s, 1H,
NHindolic); 13C NMR (DMSO-d6) ppm: 56.1 (OCH3), 113.3, 113.9, 115.4, 118.7, 126.3, 131.3,
131.6, 136.1, 144.2, 150.4, 163.4, 163.9, 164.8 (C = O); MS (ESI) m/z: 358.4 [M+1]+; Anal.
Calcd. for C16H12BrN3O2: C, 53.65; H, 3.38; N, 11.73; Found: C, 53.78; H, 3.36; N, 11.62.
5-Bromo-3-((3-hydroxy-4-methoxybenzylidene)hydrazono)indolin-2-one (4l). Orange
powder (yield 74%), m.p. 278–280˚C; IR (KBr, ν cm-1): 3361 (NH), 1725 (C = O), 1611 (C = N);
1H NMR (DMSO-d6) ppm: 3.88 (s, 3H, OCH3), 6.89 (d, 1H, H-6 isatin, J = 8.0 Hz), 7.14 (d, 1H,
H-5 of 3-OH-4-OCH3-C6H3, J = 8.5 Hz), 7.51 (d, 1H, H-7 isatin, J = 8.0 Hz), 7.45–7.47 (m, 2H,
H-2 and H-6 of 3-OH-4-OCH3-C6H3), 8.25 (s, 1H, H-4 isatin), 8.69 (s, 1H, -CH =), 9.59 (s, 1H,
OH), 10.99 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 56.3 (OCH3), 112.6, 113.3, 113.9,
114.4, 118.7, 123.9, 126.5, 131.2, 136.1, 144.4, 147.6, 150.2, 152.6, 164.2, 164.8 (Ar-CH, Ar-C and
C = O); MS (ESI) m/z: 374.1 [M+1]+; Anal. Calcd. for C16H12BrN3O3: C, 51.36; H, 3.23; N,
11.23; Found: C, 51.52; H, 3.26; N, 11.08.
5-Bromo-3-((4-hydroxy-3-methoxybenzylidene)hydrazono)indolin-2-one (4m). Red pow-
der (yield 72%), m.p. 285–287˚C; IR (KBr, ν cm-1): 3420 (NH), 1745 (C = O), 1609 (C = N); 1H
NMR (DMSO-d6) ppm: 3.92 (s, 3H, OCH3), 6.87 (d, 1H, H-6 isatin, J = 8.0 Hz), 6.97 (d, 1H,
H-5 of 4-OH-3-OCH3-C6H3, J = 8.0 Hz), 7.41 (d, 1H, H-7 isatin, J = 8.0 Hz), 7.57 (d, 1H, H-6
of 4-OH-3-OCH3-C6H3, J = 8.5 Hz), 7.60 (s, 1H, H-2 of 4-OH-3-OCH3-C6H3), 8.31 (s, 1H, H-
4 isatin), 8.66 (s, 1H, -CH =), 10.17 (s, 1H, OH), 10.97 (s, 1H, NHindolic); 13C NMR (DMSO-d6)
ppm: 55.8 (OCH3), 110.8, 113.2, 113.9, 116.3, 118.9, 125.3, 125.9, 131.7, 135.8, 144.3, 148.7,
150.7, 152.2, 164.8, 164.9 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 374.1 [M+1]+; Anal. Calcd.
for C16H12BrN3O3: C, 51.36; H, 3.23; N, 11.23; Found: C, 51.09; H, 3.29; N, 11.35.
5-Bromo-3-((3,4-dimethoxybenzylidene)hydrazono)indolin-2-one (4n). Red powder (yield
83%), m.p. 280–282˚C; IR (KBr, ν cm-1): 3421 (NH), 1744 (C = O), 1618 (C = N); 1H NMR
(DMSO-d6) ppm: 3.88 (s, 6H, 2 OCH3), 6.88 (d, 1H, H-6 isatin, J = 8.5 Hz), 7.18 (d, 1H, H-5 of
3,4-(OCH3)2-C6H3, J = 8.5 Hz), 7.52 (d, 1H, H-7 isatin, J = 8.5 Hz), 7.58 (d, 1H, H-6 of 3,4-
(OCH3)2-C6H3, J = 8.5 Hz), 7.60 (s, 1H, H-2 of 3,4-(OCH3)2-C6H3), 8.27 (s, 1H, H-4 isatin),
8.69 (s, 1H, -CH =), 10.99 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 55.8 (OCH3), 56.3
(OCH3), 111.4, 113.3, 119.3,123.6, 126.5, 129.4, 132.3, 136.9, 141.4, 144.3, 147.6, 149.7, 151.1,
162.3, 164.0(Ar-CH, Ar-C and C = O); MS (ESI) m/z: 388.2[M+1]+; Anal. Calcd. for
C17H14BrN3O3: C, 52.60; H, 3.64; N, 10.82; Found: C, 52.80; H, 3.61; N, 10.71.
5-Bromo-3-((3,4,5-trimethoxybenzylidene)hydrazono)indolin-2-one (4o). Red powder
(yield 81%), m.p. 261–263˚C; IR (KBr, ν cm-1): 3413 (NH), 1737 (C = O), 1612 (C = N); 1H
NMR (DMSO-d6) ppm: 3.78 (s, 3H, OCH3), 3.90 (s, 6H, 2 OCH3), 6.88 (d, 1H, H-6 isatin,
J = 8.0 Hz), 7.34 (s, 2H, H-2 and H-6 of 3,4,5-(OCH3)3-C6H2), 7.58 (d, 1H, H-7 isatin, J = 8.0
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Hz), 8.21 (s, 1H, H-4 isatin), 8.64 (s, 1H, -CH =), 11.01 (s, 1H, NHindolic); 13C NMR (DMSO-
d6) ppm: 56.4 (OCH3), 60.8 (OCH3), 106.7, 113.36, 113.9, 118.7, 129.1, 131.9, 136.2, 141.7,
144.5, 151.1, 153.8, 162.8, 164.7 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 418.2 [M+1]+; Anal.
Calcd. for C18H16BrN3O4: C, 51.69; H, 3.86; N, 10.05; Found: C, 51.84; H, 3.82; N, 9.91.
General procedure for the synthesis of target indolin-2-ones7a-e. A mixture of interme-
diates 6a, b (1 mmol) and the appropriate 3-hydrazonoindolin-2-one2a-d (1 mmol) in ethyl
alcohol and catalytic amount of glacial acetic acid was heated under reflux for six hours, fil-
tered while hot and the precipitate was washed with ethanol. The solid product was collected
and re-crystallized from an ethanol/DMF mixture (3:1) to furnish compounds 7a-e with 67–
79% yield.
5-Fluoro-3-((1-methyl-2-oxoindolin-3-ylidene)hydrazono)indolin-2-one (7a). Red powder
(yield 72%), m.p. 295–297˚C; IR (KBr, ν cm-1): 3409 (NH), 1737, 1702 (2 C = O), 1611
(C = N); 1H NMR (DMSO-d6) ppm: 3.22 (s, 3H, CH3), 6.92–6.94 (m, 1H, Ar-H), 7.10–7.14 (m,
2H, Ar-H), 7.31–7.33 (m, 2H, Ar-H), 7.52–7.55 (m, 2H, Ar-H), 11.03 (s, 1H, NHindolic); 13
C
NMR (DMSO-d6) ppm: 27.2 (CH3), 110.3, 112.6, 115.7, 116.6, 121.3, 121.4, 123.5, 128.6, 134.9,
142.1, 145.3, 146.9, 157.4, 158.7, 162.6 (C = O), 163.9 (C = O); MS m/z: 323.1 [M+1]+; Anal.
Calcd. for C17H11FN4O2: C, 63.35; H, 3.44; N, 17.38; Found: C, 63.53; H, 3.40; N, 17.51.
5-Chloro-3-((methyl-2-oxoindolin-3-ylidene)hydrazono)indolin-2-one (7b). Red powder
(yield 74%), m.p. 269–271˚C; IR (KBr, ν cm-1): 3409 (NH), 1735, 1707 (2C = O),1638 (C = N);
1H NMR (DMSO-d6) ppm: 3.22 (s, 3H, CH3), 6.94 (d, 1H, Ar-H, J = 8.5 Hz), 7.08–7.14 (m, 2H,
Ar-H), 7.48–7.56 (m, 4H, Ar-H), 11.14 (s, 1H, NHindolic);); 13C NMR (DMSO-d6) ppm: 26.7
(CH3), 110.3, 113.1, 115.7, 117.5, 123.5, 125.3, 128.1, 128.7, 134.3, 135.0, 137.6, 144.5, 145.3,
147.5, 162.4, 163.7 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 339.0 [M+1]+; Anal. Calcd. for
C17H11ClN4O2: C, 60.28; H, 3.27; N, 16.54; Found: C, 60.49; H, 3.31; N, 16.41.
1-Benzyl-3-((oxoindolin-3-ylidene)hydrazono)indolin-2-one (7c). Red powder (yield
70%), m.p. 255–257˚C; IR (KBr, ν cm-1): 3422 (NH), 1718 (C = O), 1608 (C = N); 1H NMR
(DMSO-d6) ppm: 5.01 (s, 2H, CH2), 6.93 (d, 1H, Ar-H, J = 8.0 Hz), 6.97–7.12 (m, 3H, Ar-H),
7.28–7.49 (m, 9H, Ar-H), 11.02 (s, 1H, NHindolic); 13C NMR (DMSO-d6) ppm: 43.5 (CH2),
110.9, 111.6, 115.9, 116.2, 123.1, 123.7, 127.8, 128.1, 128.9,129.2, 134.7, 134.8, 135.1, 136.3,
144.6, 144.8, 145.4, 145.8, 162.8 (C = O), 164.0 (C = O); MS (ESI) m/z: 381.1[M+1]+; Anal.
Calcd. for C23H16N4O2: C, 72.62; H, 4.24; N, 14.73; Found: C, 72.39; H, 4.30; N, 14.87.
1-Benzyl-3-((5-chloro-2-oxoindolin-3-ylidene)hydrazono)indolin-2-one (7d). Red powder
(yield 75%), m.p. 258–260˚C; IR (KBr, ν cm-1): 3411 (NH), 1735 (C = O), 1603 (C = N); 1H
NMR (DMSO-d6) ppm: 5.01 (s, 2H, CH2), 6.95 (d, 1H, Ar-H, J = 8.5 Hz), 7.01 (d, 1H, Ar-H,
J = 8.0 Hz), 7.08 (t, 1H, Ar-H, J = 8.0 Hz), 7.29–7.60 (m, 9H, Ar-H), 11.15 (s, 1H, NHindolic);
13C NMR (DMSO-d6) ppm: 43.4 (CH2), 111.5, 111.9, 117.4, 119.5, 123.8, 124.9, 125.2, 126.8,
127.7, 127.8, 128.1, 129.1, 129.2, 129.3, 136.0, 138.4, 151.6, 157.4, 163.1 (C = O), 165.8 (C = O);
MS m/z: 415.1[M+1]+; Anal. Calcd. for C23H15ClN4O2: C, 66.59; H, 3.64; N, 13.51; Found: C,
66.72; H, 3.61; N, 13.62.
1-Benzyl-3-((5-bromo-2-oxoindolin-3-ylidene)hydrazono)indolin-2-one (7e). Red powder
(yield 79%), m.p. 265–267˚C; IR (KBr, ν cm-1): 3421 (NH), 1734 (C = O), 1602 (C = N); 1H
NMR (DMSO-d6) ppm: 5.01 (s, 2H, CH2), 6.89 (d, 1H, Ar-H, J = 8.0 Hz), 7.00 (d, 1H, Ar-H,
J = 8.0 Hz), 7.08 (t, 1H, Ar-H, J = 7.5 Hz), 7.27–7.45 (m, 6H, Ar-H), 7.59–7.70 (m, 3H, Ar-H),
11.15 (s, 1H, NHindolic)[31]; 13C NMR (DMSO-d6) ppm: 43.5 (CH2), 110.9, 113.6, 114.2, 115.9,
117.9, 123.7, 127.8, 128.1, 128.9, 129.2, 130.9, 134.9, 136.3, 137.2, 144.9, 145.6, 145.9, 146.3,
162.8, 163.6 (Ar-CH, Ar-C and C = O); MS (ESI) m/z: 459.0[M+1]+; Anal. Calcd. for C23H15Br
N4O2: C, 60.15; H, 3.29; N, 12.20; Found: C, 59.89; H, 3.33; N, 12.28.
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Pharmacological investigations
The detailed experimental procedures for pharmacological evaluation of the synthesized com-
pounds were provided as supplementary materials.
Microspheres
Preparation of 7e -loaded PLGA microspheres. PLGA microspheres were prepared by a
modified emulsion-solvent diffusion method as described previously [32]. Briefly, the DCM (3
mL) phase containing PLGA was poured into the DMSO (4 mL) phase containing compound
7e to obtain a 7e-polymer ratio of 1:10. The resulting organic phase was added dropwise with
the help of a syringe into an aqueous phase (25 mL) containing 1% (w/v) PVA using magnetic
stirrer at 300 rpm and then homogenized at 13,500 rpm at room temperature for 3 min. The
resulting emulsion was then transferred into water (25 mL) at room temperature and stirred at
200 rpm with a magnetic stirrer overnight until the solvent was evaporated. The resulting
PLGA microspheres were recovered by centrifugation at 15000 rpm using a 3–30 K centrifuge
(Sigma, Germany) for 15 min at 4˚C, washed three times with distilled water (50 mL) to
remove the residual PVA, re-suspended in distilled water (15 mL) and then lyophilized in a
Telstar freeze-dryer (Terrassa, Spain) at -50˚C with a pressure below 1 mbar for 24 h. Plain
microspheres were prepared in a similar manner except for the absence of compound 7e in the
DMSO phase.
Microsphere characterization. Microsphere yield: The percentage yield of microspheres
was calculated using the weight in mg of the final product after drying by lyophilization with
respect to the initial total weight in mg of the drug and the polymer used for the preparation in
the emulsifying process according to Eq (1) [32, 33]. The equation below shows the percentage
yield calculation.
Weightoftheobtsainedmicrospheres
Weightofthepolymer þ weightofthedrug
% Yield ¼
 100
ð1Þ
Encapsulation efficiency and compound 7eloading: 10 mg of quantitatively weighed micro-
spheres were dissolved in DMSO (2 mL) and then sonicated for 15 min, then 0.1N HCl (5 mL)
was added to precipitate the polymer. The solution was centrifuged at 10,000 rpm for 10 min.
then the supernatant was diluted appropriately and analyzed for the content of 7e spectropho-
tometrically (Biochrom Libra S22, UK) at λ max of 318 nm to determine the 7e content. The
polymer did not interfere with the absorbance of7e at the specified wavelength. Theoretical 7e-
loading was determined by the entire 7e present in the polymer solution in the microspheres.
The 7e-loading and entrapment efficiency were determined by formulas (2) and (3), respec-
tively.
Amountofthedruginthemicrospheres
% Drug loading ¼
 100
ð2Þ
ð3Þ
Amountofthemicrospheres
Actualdrugloadinginmicrospheres
Theoriticaldrugloading
Entrapmentefficiency ¼
 100
All of the measurements were conducted in triplicate.
Determination of the particle size of microspheres: Particle size of the prepared microspheres
was measured using a dynamic light scattering particle size analyzer, model Zetasizer Nano-
ZS, ZEN 3500, Malvern Instruments (Worcestershire, UK) at 25.0 0.1˚C[34, 35]. Lyophilized
microspheres (1 mg) were re-suspended in distilled water (100 mL) and the particle size was
recorded in triplicate.
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Morphological characterization of 7e-loaded microspheres: To investigate the morphological
characterization of the prepared microspheres, they were mounted onto a double-sided adhe-
sive tape attaching to an aluminum stub, then coated with gold and examined by scanning
electron microscope (SEM, JEOL JSM-7001F, Japan) at 20 kV acceleration voltage with a sec-
ondary detector. The magnification was adjusted until a clear image of the surface of the
microspheres appeared and the picture was then recorded [36].
In vitro7e-release: Studies on the in vitro release of 7e from microspheres were carried out
using the vial method as reported by Dubey et al. [34]. Compound 7e-PLGA-microspheres
containing 5 mg equivalent of compound 7e were suspended in a vial containing phosphate
buffer (10 mL, pH 7.4) with 0.3% Tween 80 to improve the solubility of 7e. The vial was incu-
bated in a water bath at 37 0.5˚C and vibrated horizontally at speed of 100 rpm. In vitro 7e
release was assessed by intermittently sampling the vial (2 mL) at predetermined time intervals
(1, 2, 3, 4, 5, 6, 10, 12, 15, 18, 20 and 21 days), then replacing the volume with 2 mL of fresh
phosphate buffer.
Cell viability assay: The cell viability assay was performed as described in the section of anti-
proliferative activity in the supplementary materials.
Results and discussion
Chemistry
The synthetic strategy adopted for the preparation of the target derivatives is depicted in S3
and S4 Files. Indoline-2,3-diones 1a-c were refluxed with 99% hydrazine hydrate in methanol
to obtain the corresponding hydrazone derivatives 2a-c.The reaction of hydrazones 2a-c with
different substituted benzaldehydes 3a-e in ethanol in the presence of a catalytic amount of
glacial acetic acid furnished the target derivatives 4a-o with 67–83% yields (S3 File).
The IR spectra of compounds 4a-o displayed absorption bands around 3400 cm-1 for the
indolic NH group in addition to the absorption bands of carbonyl groups near 1720 cm-1 which
is consistent with the previously reported ones for compounds 4a [26] and 4f [27]. Also, their 1H
NMR spectra revealed a D2O-exchangeable signals in the region δ 10.84–11.01 ppm attributable
to the NH protons of the indolin-2-one moieties, in addition to the signal of the methine protons
(-CH = N-) in the region δ 8.55–8.70 ppm which is in accordance with those previously reported
for compounds 4a [26], 4f [27] and 4i [28]. Moreover, the 13C NMR spectra of compounds 4a-o
showed signals resonating around δ 163 ppm, characteristic of C = O carbons.
Direct methylation and benzylation of indoline-2,3-dione 1a with dimethyl sulfate and ben-
zyl bromide were carried out in the presence of sodium hydroxide or potassium carbonate to
furnish intermediates 6a and 6b, respectively. The second group of the target indolin-2-ones
7a-e was obtained in good yields (67–79%) through the reaction of the two intermediates 6a
and 6b with the appropriate 3-hydrazonoindolin-2-one 2a-d in refluxing ethyl alcohol using
catalytic amount of glacial acetic acid (S4 File).
The IR spectra of the target compounds 7a-e revealed the presence of the characteristic
absorption bands due to the NH and carbonyl groups of indolin-2-one moieties. Moreover,
the 1H NMR spectra of 7a-e revealed the presence of the singlet indolic NH protons at a δ
11.02–11.15 ppm. Also, the signals of the aliphatic protons (N-CH3) of compounds 7a, b were
observed as singlet signals around δ 3.22 ppm, while the benzylic protons (-CH2) of 7c-e
appeared as singlet signals close to δ 5.01 ppm.
Pharmacological investigations
Anti-proliferative activity. Twenty compounds, i.e. 4a-o and 7a-e, were analyzed for can-
cer cell growth inhibitory activity. These studies were carried out using cells derived from
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Table 1. Anti-proliferative (cell growth inhibitory activity at 30 μM concentration) activity of the target compounds 4a-o, 7a-e and sunitinib against
HT-29, ZR-75 and A-549 cell lines.
Compound No.
HT-29
ZR-75
A-549
Average Growth Inhibition %
4a
4b
4c
4d
4e
4f
19.5 ± 12.4
81.9 ± 3.0
74.9 ± 5.3
81.7 ± 5.2
81.9 ± 5.4
6.1 ± 8.6
34.8 ± 23.7
59.3 ± 11.1
55.4 ± 23.4
58.8 ± 4.3
86.1 ± 3.8
10.9 ± 14.0
46.0 ± 6.4
27.1 ± 20.3
66.3 ± 5.7
54.2 ± 5.0
82.5 ± 6.9
93.6 ± 3.6
8.4 ± 10.0
44.2 ± 16.5
27.2
69.2
61.5
74.3
87.2
8.4
4g
4h
4i*
4j
33.5 ± 11.3
41.2
36.6
11.4
8.9
41.6 ± 10.4
30.9 ± 9.5
37.3 ± 8.1
-3.6 ± 9.7
6.9 ± 4.2
33.0 ± 13.0
18.1 ± 21.2
-5.3 ± 18.1
44.3 ± 18.4
58.7 ± 16.0
12.5 ± 21.9
3.1 ± 8.2
4.9 ± 3.3
1.7 ± 17.5
-6.9 ± 9.4
46.6 ± 9.5
83.7 ± 10.3
0.1 ± 5.1
4k
4l
-0.6 ± 7.2
45.9 ± 9.8
42.1 ± 1.8
-2.0 ± 10.2
-1.1 ± 9.0
84.8 ± 3.6
95.5 ± 3.4
73.8 ± 4.4
97.0 ± 1.8
95.7 ± 4.2
59.5 ± 2.3
-4.3
45.6
61.5
3.5
4m
4n*
4o
7a
7b
7c
7d
7e
-3.4 ± 6.6
91.6 ± 4.8
95.2 ± 2.8
86.6 ± 9.9
98.4 ± 0.6
97.1 ± 3.3
85.7 ± 2.7
-0.5
85.0
95.4
84.2
97.7
96.3
78.7
78.6 ± 9.9
95.5 ± 6.0
92.1 ± 3.8
97.8 ± 1.8
96.1 ± 4.6
90.7 ± 4.5
Sunitinib
https://doi.org/10.1371/journal.pone.0181241.t001
human lung, colon and breast tumors (A549, HT-29 and ZR-75 cells, respectively). This initial
assessment of activity tested each compound in quadruplicate at a single concentration of
30 μM, if solubility permitted.
As indicated in Table 1, the test compounds 4a-j, 4l-n and 7a-e exhibited anti-proliferative
activity against A549, HT-29 and ZR-75 cells with an average growth inhibition in the range
from 3.5 to 97.7%, whereas compounds 4k and 4o stimulated the growth of the tested cell
lines.
Compounds 7b, 7d and 7e were the most active compounds, showing an average growth
inhibition from 95.4 to 97.7%. Therefore, they were subjected to quantitative inhibitory concen-
tration 50% (IC50) determination for their cell growth inhibitory activity towards the A549, HT-
29 and ZR-75 cancer cell lines. The results are presented in Table 2 and Fig 3. Extra sum-of-
Table 2. Inhibitory concentration 50% (IC50) of anti-proliferative activity of the selected compounds 7b, 7d, 7e and sunitinib against HT-29, ZR-75
and A-549 cell lines.
Compound No.
IC50 (μM)
ZR-75
3.75a
2.42a
1.79a
Average IC50 (μM)
HT-29
5.38a
3.62a
2.49a
10.14a
A-549
5.19b
4.13b
2.83b
5.87b
7b
7d
4.77
3.39
2.37
8.11
7e
Sunitinib
8.31a
adenotes those in which the P< 0.01
bdenotes those in which the P< 0.05
https://doi.org/10.1371/journal.pone.0181241.t002
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 3. IC50 of the anti-proliferative activity of compounds 7b, 7d, 7e and sunitinib against HT-29, ZR-75 and A-549 cell lines.
https://doi.org/10.1371/journal.pone.0181241.g003
squares F test was performed on dose response curves to determine if the IC50 values obtained
in a cell line were significantly different for each compound tested.
Compound 7e exhibited the best average IC50 value (2.37 μM), being nearly three-fold
more potent than the positive control, sunitinib (average IC50 value = 8.11 μM). Therefore,
compound 7e was subjected to deeper pharmacological investigations in order to obtain
insight into its pharmacological profile.
Apoptosis and caspase 3/7 activity. Compound 7e was analyzed for apoptosis inducing
activity in cancer cells. These studies were carried out using cells derived from human lung
(A549). This follow-up assessment of activity tested compounds in quadruplicate at concentra-
tions equivalent to the IC50 value to inhibit growth and a concentration three-fold above the
IC50 concentrations over a time course ranging from 2 to 48 h. As indicated in Fig 4, com-
pound 7e at 10 μM increased caspase activity by 2.4- and 1.85-fold between 4 and 8 h of treat-
ment, respectively, while the lower tested concentration had no effect on caspase 3/7 activity.
Cell cycle effects. Compound 7e was analyzed for effects on various aspects of cell cycle
progression in human cancer cells. These studies were carried out using cells derived from
lung adenocarcinoma (A549). This follow-up assessment of activity tested compounds used
immunofluorescent imaging of phosphorylated Rb protein and the total DNA content of each
cell to determine the cell cycle phase. The ability of the test compounds to affect the cell cycle
and Rb phosphorylation was tested over a range of concentrations from less than 100 nM to
50 μM.
As indicated in Fig 5, compound 7e had dose-dependent effects on the tested parameters. It
caused a significant reduction in the total cell number after 24 h of treatment with an IC50
value = 2.67 μM and with an IC50 value = 2.36 μM after 48 h (Table 3), reflective of the dose-
dependent growth inhibition observed in the homogenous assay.
In addition, compound 7e caused a decrease in the percentage of cells in the G1 phase of
the cell cycle with a corresponding increase in the S-phase. This suggests that part of the com-
pound effects on growth may be attributable to a decreased rate of progression through the cell
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 4. A-549 human lung cancer cells were treated with 3.3 and 10 μM 7e over a time course from 2–48
h. Caspase 3/7 activity was measured by a luminescence-based homogenous assay.
https://doi.org/10.1371/journal.pone.0181241.g004
cycle and corresponding decreases in proliferation, similar to the positive control, sunitinib.
Arrest in G2 may represent a checkpoint blockade, whereas mitotic arrest may, in some cases,
lead to mitotic catastrophe and subsequent programmed death of cells with multiple or aber-
rant nuclei. The increase in S-phase cells without a concomitant G2/M phase fraction may
indicate a very rapid onset of apoptosis in the case of compound 7e.
As with other cell cycle parameters, levels of phosphorylated Rb protein were substantially
reduced in a dose-dependent manner by the control and the test compound 7e. After 24 h of
treatment, the IC50 value was lower than the IC50 value for reductions in the cell number
caused by the test compound 7e (Table 3). This may support the hypothesis that the inhibition
of cyclin dependent kinases by isatin compounds plays a role in their growth inhibitory activ-
ity. However, the correlation was less apparent at the 48 h time point.
Furthermore, compound 7e was analyzed for its effect on total cellular levels of phos-
phorylated tyrosine (p-Tyr) residues in human cancer cells. These studies were carried out
using cells derived from lung adenocarcinoma (A549) and immunofluorescent imaging.
The ability of the test compound 7e to affect acute serum stimulation of tyrosine phosphor-
ylation was tested over a range of concentrations from less than 100 nM to 50 μM. Com-
pound 7e increased p-Tyr levels nearly two-fold with an apparent IC50 value of 3.8 μM,
which is consistent with the growth inhibitory IC50 value of this compound. In contrast, the
positive control compound sunitinib caused a modest dose-dependent decrease in the level
of cellular tyrosine phosphorylation activity with an IC50 value of 0.9 μM, well below its
growth inhibitory IC50value (Fig 6).
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 5. Dose-dependent changes in cell cycle. Distribution of cells within the cell cycle as well as total cell numbers
were quantitated by fluorescent staining of nuclear DNA with DAPI. Phosphorylated Rb protein was detected by
indirect immunofluorescence followed by automated image acquisition and analysis.
https://doi.org/10.1371/journal.pone.0181241.g005
Selectivity. As an indicator of selectivity for tumor cells, compound 7e was analyzed for
cell growth inhibitory activity in three non-tumorigenic cell lines.IEC-6 cells derived from rat
intestine exhibit morphologic and karyotypic features of normal intestinal epithelial cells [37].
Cultures derived from human fibrocystic mammary tissue (MCF-10A) are non-tumorigenic
and exhibit features of primary cultures of breast tissue, including dome formation [38].Fibro-
blasts derived from embryonic tissue from mice (Swiss 3t3 fibroblasts) are both non-tumori-
genic and contact inhibited [39].For comparison, the A549 human non-small cell lung cancer
(NSCLC) cell line was included. This assessment of the growth inhibitory activity of the
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Table 3. IC50 for reductions in the total cell number and cell cycle effects of compound 7e and
sunitinib.
Compound No.
IC50 (μM) for IC50(μM)for
reductions in the reduction in Rb
Cell cycle effects
total cell number phosphorylation
24 h
2.67a
12.54a
48 h
2.36
3.48
24 h
2.55b
3.16b
48 h
7e
2.47
G1 decreased, S-phase increased
Sunitinib
14.01 G1 decreased and G2/M-phases increased
adenotes those in which the P< 0.01
bdenotes those in which the P< 0.05
https://doi.org/10.1371/journal.pone.0181241.t003
compounds was tested in quadruplicate at a maximum concentration of 25 μM, followed by 10
serially diluted concentrations.
As indicated in Fig 7 and Table 4, compound 7e inhibited growth in both normal and
tumor cell lines >50%. Compound 7e displayed a selectivity value of 1.7 while the positive
control sunitinib showed a selectivity value of 1.4 (the difference between the mean IC50 in
non-tumor cell lines versus the NSCLC cells).
Multidrug resistant lung cancer cell line. Compound 7e was analyzed for cancer cell
growth inhibitory activity in a sensitive NSCLC cell line (A549) and a multidrug resistant lung
cancer cell line (NCI-H69AR) which expresses the ABCC1 efflux pump protein. This assess-
ment of activity tested compounds in quadruplicate at a maximum concentration of 25 μM,
followed by 10 serially diluted concentrations.
As indicated in Fig 8 and Table 5, compound 7e inhibited growth in sensitive A549 and
resistant NCI-H69AR cell lines with IC50 values of1.6 and 12.7μM, respectively. The resistant
NCI-H69AR cell line was nearly eight-fold less sensitive, indicating that this compound may
be subjected to efflux by ABCC1. The positive control sunitinib showed a lesser degree of resis-
tance, i.e. the resistant NCI-H69AR cell line was 1.9-fold less sensitive.
Evaluation of 7e-loaded microspheres
Encapsulation efficiency and 7e loading capacity. The prepared microspheres had high
yield of 82% w/w recovery with respect to the initial amounts of polymer and 7e used in the
microsphere formulation. The average encapsulated efficiency of 7e in PLGA microspheres
was 85% 1.3 using a 1:10 7e:polymer ratio. The high encapsulation efficiency may be due to
the presence of a high amount of the polymer, which can reduce 7e loss during the fabrication
process. The percentage 7e-loading capacity was 9.84 0.15.
Fourier transform infrared spectroscopic (FT-IR) analysis. The FT-IR spectra of pure
7e, PLGA, their physical mixture, plain microspheres and 7e-loaded microspheres were
obtained to verify the chemical interaction between 7e and the polymer. The functional group
bands of 7e remained the same in the spectra of both pure 7e as well as in the physical mixture
and the formulation. This indicated that no interaction took place between 7e and the poly-
mer. In the FT-IR spectrum of 7e-loaded microspheres, it was found that there was no signifi-
cant spectral shift or disappearance of the bands of 7ein any spectrum of 7e with the polymer,
as shown in Fig 9, indicating compatibility between 7e and the PLGA polymer.
Particle size determination. The particle size of drug-loaded microspheres is considered
an important characteristic as it could affect drug release properties. The particle size determi-
nation revealed that the produced microspheres were uniform with a size range of 1–3μm. The
small polydispersity index (PdI) suggested that the size distribution of the particles was fairly
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 6. Dose-dependent changes in total levels of tyrosine phosphorylation. Cells were stimulated for 10 min with 10% fetal bovine
serum. Phosphotyrosine levels were determined by indirect immunofluorescence followed by automated image acquisition and analysis.
https://doi.org/10.1371/journal.pone.0181241.g006
monomodal (Fig 10). The loading of 7edidnot alter the particle size, as the average particle size
of the plain microspheres and the loaded microspheres was 2.26 0.2 and 2.287 0.17 μm,
respectively (mean SD, n = 3). Fig 10 shows the particle size distribution of the 7e-loaded
microspheres around 3 μm. A narrow particle size distribution is an important aspect in pas-
sive targeting of microspheres as well as for stability issues. The results demonstrate that the
particle size of the prepared microspheres was uniform.
Morphological analysis of 7e-loaded microspheres. Scanning electron microscope
(SEM) images revealed that the microspheres were intact spheres showing the formation of
spherical and smooth surface microspheres with almost no pores or cavities, which may pro-
long the release of the encapsulated 7e over an extended period of time. No crystals of 7e were
detected, and there was no clogging or deformation and few fragments of polymer adhering,
as shown in Fig 11. No difference was observed in the morphological properties of micro-
spheres due to the presence of compound 7e. The particle size observed by SEM was consistent
with that obtained from the Zetasizer particle size analyzer. These results indicate the efficiency
of the preparation method and optimization of the preparation parameters such as the concen-
tration and viscosity of the polymer solution. The obtained results are consistent with the pre-
viously reported studies for different drugs [40–42].
Drug release evaluation. Drug release from biodegradable polymeric particles occurs
through a combination of several mechanisms. Generally, it occurs through desorption of sur-
face-bound drug, diffusion of the drug through the polymeric matrix and erosion of the poly-
mer particles.
Fig 7. Tumor cell selectivity. Dose-dependent growth inhibition by 7e or sunitinib in a NSCLC cell line (A-549) compared with
three non-tumorigenic cell lines: Sw-3t3 (murine fibroblasts), IEC-6 (murine inestinal epithelial cells), or MCF-10A (human
mammary epithelial cells).
https://doi.org/10.1371/journal.pone.0181241.g007
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Table 4. Selectivity for compound 7e and sunitinib toward tumor and non-tumorigenic cell lines.
Compound No.
IC50 (μM)
Breast MCF-10A
Mean
tumor selectivity
Intestine IEC-6
Fibroblast Swiss 3t3
NSCLC
A-549
7e
3.72a
4.56a
1.75
4.43a
2.56 a
4.07b
1.56
3.06
1.7
1.4
Sunitinib
adenotes those in which the P< 0.01
bdenotes those in which the P< 0.05
https://doi.org/10.1371/journal.pone.0181241.t004
In vitro 7e release was assessed in phosphate buffer (pH 7.4) containing a surfactant to simu-
late the physiological milieu and maintain sink conditions. Samples were incubated at 37˚C,
and the amount of 7e was determined using a UV spectrophotometer. Fig 12 shows the release
profile of raw 7e powder and the prepared 7e-PLGA microspheres. Linear prolonged 7e release
rates from the microspheres were observed for 21 days without an initial burst release (Fig 12).
On the other hand, the release of compound 7e from the raw powder was faster than its release
from the microspheres, as 65% of 7e was released after 5 days. This uncontrolled burst release of
compound 7e from raw powder could result in too high local concentration which could lead to
severe local tissue injure and hamper appropriate delivery to the target.
It can be seen that 7e-loaded microspheres exhibited biphasic drug release kinetics [34, 43]
with an initial release up to 6% after 48 h and 27% after six days followed by cumulative release
of 90% after 20 days. No initial burst release was observed, suggesting low 7e density at the sur-
face of the microspheres and hence the homogenous distribution of 7e within the micro-
spheres. The high polymer concentration led to the formation of a dense polymer matrix
structure in the microspheres and no pores as observed in the SEM images, which resulted in a
significant decrease in the initial burst release from the microspheres. The kinetics of 7e release
from microspheres was close to the zero order model. The correlation coefficient (r2) was
0.9884 and the release curve was fitted to zero order kinetics. This release pattern was due to
diffusion and degradation of the microspheres.
Fig 12 shows the initial slow release of 7e from microspheres, which could be attributed to
the hydrophobicity of 7e. Moreover, the interaction of the hydrophobic 7e molecule with the
polymer matrix via hydrophobic binding forces might cause a trapping of the drug inside the
particles and, therefore, slower release. The rate of 7e release from the loaded microspheres
gradually increased, as 10% release was observed after three days (Fig 12).This was probably
due to the diffusion of the drug, which was present at the surface of the microspheres. Thereaf-
ter, constant release was observed, which may have been due to 7e diffusion and matrix
Fig 8. Tumor cell selectivity. Dose-dependent growth inhibition by 7e or Sunitinib in a NSCLC cell line (A-549) compared with
three non-tumorigenic cell lines: Sw-3t3 (murine fibroblasts), IEC-6 (murine inestinal epithelial cells), or MCF-10A (human
mammary epithelial cells).
https://doi.org/10.1371/journal.pone.0181241.g008
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Table 5. Cancer cell growth inhibitory activity of compound 7e and sunitinib toward sensitive (A-549) and resistant (NCI-H69AR) cell lines.
Compound No.
IC50 (μM)
Fold resistant
Sensitive A-549
Resistant NCI-H69AR
7e
1.6
12.7 a
5.8a
8.1
1.9
Sunitinib
3.1
adenotes those in which the P< 0.01
https://doi.org/10.1371/journal.pone.0181241.t005
erosion mechanisms of the biodegradable PLGA polymer. Slow drug release from micro-
spheres maybe due high encapsulation of the drug with low swelling of polymer in the release
medium, leading to slow diffusion of the drug particles from polymeric matrices. Furthermore,
the degradation of PLGA 50:50 is slow. Therefore, the release of 7e from microspheres may
depend on drug diffusion, the PLGA surface and bulk erosion or swelling [34].Conclusively,
compound 7e was slowly and continuously released from the PLGA microspheres with no
obvious burst release. This behavior is consistent with a previous study using PLGA micro-
spheres for sustained drug release [44] and with several anticancer drugs incorporated into
microspheres [33, 40, 45–47]. This formula was used for further studies on the human lung
cancer cell line A549.
Cell viability assay. The human lung cancer A549 cell line was incubated with various
concentrations (0.8, 1.6, 3.13, 6.25, 12.5, 25 and 50 μM) of both free 7e and 7e-loaded micro-
spheres to evaluate the anti-proliferative activity by assessing their effect on cell viability.
Unloaded PLGA microspheres (lacking compound 7e) were coincubated with the same cell
line to show that tumor growth was not inhibited due to the PLGA microspheres alone.
In Fig 13A, the cytotoxicity of free 7e was evaluated at0.8, 1.6, 3.13 and 6.25 μM. After 24 h
of incubation time, no cytotoxicity was observed at low concentrations (0.8 and 1.6 μM), while
after 72 h, only a 30% reduction in cell viability could be seen at 1.6 μM. Moreover, at 3.1 μM,
20 and 70% reductions in the cell viability were noted after 24 h and 72 h of incubation,
Fig 9. FT-IR spectrum of a) PLGA, b) Pure 7e, c) Physical mixture of the PLGA + 7e, d) Plain microspheres, and e) 7e-
loaded microspheres.
https://doi.org/10.1371/journal.pone.0181241.g009
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 10. Particle size distribution of 7e-loaded PLGA microspheres.
https://doi.org/10.1371/journal.pone.0181241.g010
respectively, and almost total inhibition was observed after 120 h of incubation. A remarkable
reduction in cell viability was observed at 6.25 μM = after 24 h of incubation and was main-
tained for 120 h of incubation. Therefore, the cytotoxicity effect of free 7e needs higher con-
centrations to obtain minimal to no growth of the human lung cancer cell lineA549.
Fig 11. Scanning electron micrographs of (A) Free microspheres and (B) 7e-Loaded PLGA microspheres.
https://doi.org/10.1371/journal.pone.0181241.g011
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 12. Cumulative in vitro release of 7e from PLGA microspheres. Each point represents the mean ± SD
obtained from triplicate samples.
https://doi.org/10.1371/journal.pone.0181241.g012
Fig 13B illustrates the obtained results when 7e-loaded microspheres were incubated with
the human lung cancer cell lineA549for incubation periods and concentrations similar to that
of the free 7e. There was no cytotoxicity observed at 0.8 μM after 24 h of incubation, but at 1.6,
3.13 and 6.25 μM, 30, 40 and 50% reductions in cell viability were observed, respectively. After
72 h of incubation, cell viability was decreased to 10, 7 and 5%, respectively, for the same con-
centrations. This assay demonstrated that entrapped 7e was more effective in arresting cell
growth as compared with free 7e. The enhanced cytotoxicity of 7e-loaded microspheres was
observed with increasing incubation time in a concentration dependent manner. Therefore,
the decrease in percentage cell viability was not immediate but rather gradual and continuous
due to the polymer that allowed for slow release of 7e from microspheres and hence its gradual
cytotoxic effect. The extended activity of 7e from 7e-loaded microspheres might be explained
by the fact that they can be adsorbed onto the cell membrane, generating a drug concentration
Fig 13. Cell viability of A549 cells incubated with free 7e (A) and 7e-loaded microspheres (B) at 0.8, 1.6, 3.13, and 6.25 μM drug
concentrations after 24, 72, and 120 h incubation time.
https://doi.org/10.1371/journal.pone.0181241.g013
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
Fig 14. The IC50 values of the free 7e and 7e-PLGA loaded microspheres.
https://doi.org/10.1371/journal.pone.0181241.g014
gradient near the cell surface that could favor 7e to penetrate into the cell. Furthermore, the
cell can phagocytize 7e-loaded microspheres, allowing 7e to be released inside the cytoplasm,
thus contributing to a sustained 7e concentration [48].
Quantitative in vitro cytotoxicity of 7e-loaded microspheres was investigated via the deter-
mination of theIC50 value toward A549cells as compared with that of free 7e. The IC50 value
was almost the same for both the free 7e (IC50 value = 6.51 μM) and the 7e-loaded micro-
spheres (IC50 value = 6.50 μM) after 24 h of incubation (Fig 14). Increasing the incubation
time of 7e-loaded microspheres to 72 and 120 h resulted in a significant reduction in its IC50
value by approximately 85 and 90%, respectively. On the other hand, the IC50 value of 7e-
loaded microspheres was reduced to approximately 60 and 70% after 72 and 120 h, respec-
tively, as compared with the IC50 values of free 7e. Statistical analysis revealed that these values
were significantly different (P< 0.05) from the IC50 values of free 7e.
The enhanced7e activity mediated by its incorporation into microspheres can be attributed
to its sustained release, as shown in Fig 12. Microspheres can act as a reservoir for 7e, protecting
the drug from hydrolysis and provide not only sustained release of the drug, but also contribute
to the maintenance of drug activity. Several reported studies have emphasized that incorpo-
ration of anticancer drugs in such delivery systems improves their cytotoxic effects[49, 50].
Conclusions
The synthesis and molecular characterization using different spectroscopic tools of certain isatin-
based hybrids 4a-o and 7a-e are described. Compounds 4a-o and 7a-e were subjected to in vitro
anti-proliferative assessment against HT-29 (colon), ZR-75 (breast) and A549 (lung) human can-
cer cell lines. The most active congeners in the preliminary in vitro anti-proliferative screening,
compounds 7b, 7d and 7e, showed average IC50 values of 4.77, 3.39 and 2.37 μM, respectively, as
compared with the reference drug sunitinib which exhibited an average IC50 value of 8.11 μM
towards the tested human cancer cell lines. Compound 7e was selected for deeper pharmacologi-
cal testing to gain insight into its possible in vitro anti-proliferative mechanism of action. It
increased caspase activity by 2.4- and 1.85-fold between 4 and 8 h of treatment, respectively, at
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Synthesis and anticancer activity of certain hydrazonoindolin-2-ones
10 μM. Moreover, compound 7e decreased the percentage of cells in the G1 phase of the cell cycle
with a corresponding increase in the S-phase. The increase in S-phase cells without a concomitant
G2/M phase fraction may indicate a very rapid onset of apoptosis. In addition, compound 7ein-
creased p-Tyr levels by nearly two-fold with an apparent IC50 value of 3.8 μM, which is consistent
with its growth inhibitory IC50 value. Compound 7e was incorporated into biodegradable PLGA
microspheres to improve its in vitro anti-proliferative activity. The results of the in vitro anti-pro-
liferative assay indicated that 7e-loaded PLGA microspheres exhibited better cytotoxicity as com-
pared with free 7e toward the human lung cancer cell line A549 after 3 and 5 days of incubation.
Supporting information
S1 File. Experimental protocols of the pharmacological evaluation of the synthesized tar-
get compounds.
(DOCX)
S2 File. Representative examples of the NMR (1H and 13C) spectra of the synthesized target
compounds.
(DOC)
S3 File. Synthetic pathway to achieve the target compounds 4a-o.
(DOCX)
S4 File. Synthesis of the target compounds 7a-e.
(DOCX)
Author Contributions
Conceptualization: Mohamed I. Attia.
Data curation: Mohamed I. Attia, Samar A. Afifi, Adam B. Keeton, Hatem A. Abdel-Aziz.
Formal analysis: Samar A. Afifi, Gary A. Piazza, Hatem A. Abdel-Aziz.
Investigation: Wagdy M. Eldehna, Samar A. Afifi, Adam B. Keeton, Hatem A. Abdel-Aziz.
Methodology: Wagdy M. Eldehna, Samar A. Afifi, Adam B. Keeton, Hatem A. Abdel-Aziz.
Resources: Mohamed I. Attia.
Supervision: Mohamed I. Attia, Gary A. Piazza, Hatem A. Abdel-Aziz.
Writing – original draft: Mohamed I. Attia, Wagdy M. Eldehna, Samar A. Afifi, Adam B. Kee-
ton, Gary A. Piazza, Hatem A. Abdel-Aziz.
Writing – review & editing: Mohamed I. Attia, Wagdy M. Eldehna, Samar A. Afifi, Adam B.
Keeton, Gary A. Piazza, Hatem A. Abdel-Aziz.
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