Antitumor properties of certain spirooxindoles towards hepatocellular carcinoma endowed with antioxidant activity

Article abstract of DOI:10.1080/14756366.2020.1743281

In the current medical era, spirooxindole motif stands out as a privileged heterospirocyclic scaffold that represents the core for a wide range of bioactive naturally isolated products (such as Strychnofoline and spirotryprostatins A and B) and synthetic compounds. Interestingly, no much attention has been paid to develop spirooxindole derivatives with dual antioxidant and anticancer activities. In this context, a series of spirooxindoles 6a-p was examined for their anticancer effect towards HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell lines. Spirooxindole 6a was found to be an efficient anti-proliferative agent towards both HepG2 and PC-3 cells (IC50 = 6.9 and 11.8 μM, respectively). Afterwards, spirooxindole 6a was assessed for its apoptosis induction potential in HepG2 cells, where its pro-apoptotic impact was approved via the significant elevation in the Bax/Bcl-2 ratio and the expression levels of caspase-3,.

Full text of DOI:10.1080/14756366.2020.1743281

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Antitumor properties of certain spirooxindoles
towards hepatocellular carcinoma endowed with
antioxidant activity
Sara T. Al-Rashood, Ahmed R. Hamed, Ghada S. Hassan, Hamad M.
Alkahtani, Abdulrahman A. Almehizia, Amal Alharbi, Mohammad M. Al-
Sanea & Wagdy M. Eldehna
To cite this article: Sara T. Al-Rashood, Ahmed R. Hamed, Ghada S. Hassan, Hamad M.
Alkahtani, Abdulrahman A. Almehizia, Amal Alharbi, Mohammad M. Al-Sanea & Wagdy M. Eldehna
(2020) Antitumor properties of certain spirooxindoles towards hepatocellular carcinoma endowed
with antioxidant activity, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1, 831-839, DOI:
10.1080/14756366.2020.1743281
To link to this article: https://doi.org/10.1080/14756366.2020.1743281
© 2020 The Author(s). Published by Informa
UK Limited, trading as Taylor & Francis
Group.
Published online: 25 Mar 2020.
Article views: 37
Submit your article to this journal
View related articles
View Crossmark data
Full Terms & Conditions of access and use can be found at
https://www.tandfonline.com/action/journalInformation?journalCode=ienz20

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 831–839
https://doi.org/10.1080/14756366.2020.1743281
SHORT COMMUNICATION
Antitumor properties of certain spirooxindoles towards hepatocellular carcinoma
endowed with antioxidant activity
Sara T. Al-Rashooda, Ahmed R. Hamedb,c, Ghada S. Hassand, Hamad M. Alkahtania, Abdulrahman A. Almehiziaa
Amal Alharbia, Mohammad M. Al-Saneae and Wagdy M. Eldehnaf
,
b
aDepartment of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Chemistry of
c
Medicinal Plants, National Research Centre, Dokki, Egypt; Biology Unit, Central Laboratory of the Pharmaceutical & Drug Industries Research
d
Division, National Research Centre, Dokki, Egypt; Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura,
e
f
Egypt; Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka, Saudi Arabia; Department of Pharmaceutical
Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh, Egypt
ABSTRACT
ARTICLE HISTORY
Received 31 December 2019
Revised 4 March 2020
Accepted 10 March 2020
In the current medical era, spirooxindole motif stands out as a privileged heterospirocyclic scaffold that
represents the core for a wide range of bioactive naturally isolated products (such as Strychnofoline and
spirotryprostatins A and B) and synthetic compounds. Interestingly, no much attention has been paid to
develop spirooxindole derivatives with dual antioxidant and anticancer activities. In this context, a series
of spirooxindoles 6a-p was examined for their anticancer effect towards HepG2 hepatocellular carcinoma
and PC-3 prostate cancer cell lines. Spirooxindole 6a was found to be an efficient anti-proliferative agent
towards both HepG2 and PC-3 cells (IC50 ¼ 6.9 and 11.8 mM, respectively). Afterwards, spirooxindole 6a
was assessed for its apoptosis induction potential in HepG2 cells, where its pro-apoptotic impact was
approved via the significant elevation in the Bax/Bcl-2 ratio and the expression levels of caspase-3,
KEYWORDS
Anti-proliferative; antioxi-
dants; apoptosis;
spiroxindoles; synthesis
Introduction
produced by a range of bacteria, plants, and invertebrates10. Isatin
motif is a central privileged scaffold in a large number of bioactive
natural and synthetic products that possess a spectrum of bioac-
tivities such as antioxidant11 and anticancer activities12–14. On the
other hand, the natural spirooxindole alkaloids were first isolated
from plants of the Apocynaceae and Rubiacae families15.
Thereafter, spirooxindole motif stands out as a privileged hetero-
spirocyclic scaffold that represents the core for a wide range of
naturally isolated products such as alstonisine16, horsfiline17,
coerulescine18, and chitosenine19 with diverse bioactivity profiles.
Strychnofoline, spirotryprostatin A and spirotryprostatin B (Figure
1) are other examples for natural spirooxindoles possessing anti-
cancer activities20,21. Isolation and identification of such bioactive
spirooxindole alkaloids have inspired the researchers and paved
the way to design and synthesise various spirooxindole-based
small molecules with promising biological activities such as anti-
oxidant22 and anticancer23–25 activities.
Recently, we have reported a one-pot three-component syn-
thesis of novel spirooxindole derivatives 6a–p as efficient anti-pro-
liferative agents towards the aggressive triple-negative breast
cancer MDA-MB-231 cell line25. In the current study, the reported
promising anti-proliferative impact of spirooxindoles 6a–p spurred
us to extend our investigations around their different potential
biological activities, where they were evaluated for their potential
antioxidant activity via assessing their DPPH radical scavenging
capacities and Ferric reducing antioxidant power (FRAP). In add-
ition, 6a–p were examined for their anticancer effect towards
In the current medical era much attention has been paid to fight
the extreme assembly of reactive oxygen species (ROS), such as
hydrogen peroxide, hydroxyl radicals, or superoxide anion, that
aggressively interact with natural macromolecules in different tis-
sues by chain process resulting in an oxidative damage1,2
.
Biological systems have the capability to defend themselves
against these ROS via enzymes (such as glutathione peroxidase,
superoxide dismutase, and catalase) or by non-enzymatic mecha-
nisms that involve organic antioxidants molecules (such as gluta-
thione, vitamin C, vitamin E). These antioxidants are capable of
direct scavenging the oxidant species through breaking of oxida-
tion propagation chain reactions or through the generation of
hydrogen atom in stoichiometric manner, hence preventing free
radical-associated damage1,2
.
Both depletion of antioxidant defences or abnormal ROS pro-
duction, result in the oxidative stress state that implicated in the
aetiology of a variety of disorders such as Alzheimer’s3, rheuma-
toid arthritis4, Huntington’s disease5, Parkinson’s disease6, coron-
ary heart disease7, and insulin resistance8. Furthermore, elevated
levels of ROS can promote tumorigenesis by initiation of mito-
chondrial or nuclear DNA mutations that promote neoplastic
transformation, promoting genomic instability, or activating pro-
oncogenic signalling pathways9. So, novel antioxidants featuring a
variety of chemical structures are needed for prevention of cancer
and to augment chemotherapies for different malignancies.
Being abundant in nature, isatins have been found in fluids
and tissues of mammals, in addition to natural products that are HepG2 hepatocellular carcinoma and PC-3 prostate cancer cell
CONTACT Wagdy M. Eldehna
wagdy.mohamad@pharm.kfs.edu.eg, wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Kafrelsheikh University, Kafr El-Sheikh, 33516, Egypt
ß 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

832
S. T. AL-RASHOOD ET AL.
Figure 1. Chemical structure of some naturally isolated anticancer spirooxindoles, and the synthetic spirooxindoles 6a–p.
lines. Furthermore, spirooxindole 6a was then assessed for its reported earlier30,31. The samples were analysed by a flow cytome-
ter (BectonDickinson FACSCalibur, BD, USA) and data were ana-
lysed using the CellQuest software (Becton Dickinson).
apoptosis induction potential in HepG2 hepatocellular carcinoma
cell line, so as to obtain some mechanistic insights for the anti-
proliferative impact of the herein reported spirooxindoles. Finally,
an in silico study was carried out by the use of the automated
SwissADME tool to afford clues about the physicochemical proper-
ties, ADME parameters, drug-like nature and pharmacokinetic
properties of spirooxindole 6a.
Antioxidant activity
DPPH radical scavenging activity. The antioxidant potential of spi-
rooxindoles 6a–p against DPPH_ was evaluated as per previously
described procedures by Brand-Williams et al.32. DPPH solution
was prepared by dissolving 0.024 g DPPH in methanol (100 mL). In
96-well plates, ꢁ lL sample and 200-x lL DPPH solution were
added. The plate was incubated for 30 min at room temperature
in the dark, and finally, absorbance was recorded at 517 nm wave-
length. Ascorbic acid was used as positive control. The percent
radical scavenging potential was calculated for each compound
via the following equation: Scavenging effect (%) ¼ f(Absorbance
of control ꢀ Absorbance of sample)/Absorbance of controlg ꢁ 100.
Materials and methods
Chemistry
Spirooxindoles 6a–p were prepared earlier by our group25.
Biological evaluation
Anti-proliferative activity
Ferric reducing antioxidant power (FRAP). The Benzie and Strain
method33 was adopted to assess the ferric reducing antioxidant
power (FRAP) for spirooxindoles 6a–p. Absorbance of mixtures
was recorded at 593 nm after incubation for 30 min at 37 ꢂC. FRAP
results were expressed as lM Fe2þ equivalents.
Anti-proliferative activity towards HepG2 and PC-3 cell lines.
Spirooxindoles 6a–p were evaluated for their anti-proliferative
potency towards HepG2 liver cancer and PC-3 prostate cancer cell
lines. Both cells lines were cultured as monolayers in DMEM sup-
plemented with 10% foetal bovine serum (FBS), L-glutamine
(2 mM), penicillin (100 U/mL) and streptomycin sulphate
(100 mg/mL). Then, cells were sub-cultured with trypsine/EDTA
solution, counted with haemocytometer and plated onto 96-well
plates (5000 cells/well) and left overnight so as to form a semi-
confluent monolayer. A modified method was employed utilising
the (3-[4,5-dimethylthiazol-2-yl] ꢀ 2,5-diphenyltetrazolium bromide
(MTT) dye, as reported earlier25–27. IC50 values were calculated
using non-linear regression curve fitting of the dose response
plots on GraphPad Prism V.6.0 software. Assessment of morpho-
logical changes of HepG2 cells after treatment with the active
compounds 6a, 6e and 6i were carried out by phase contrast
inverted microscope (Ziess, USA) and photomicrographs were
taken using digital camera.
Results and discussion
Chemistry
The synthetic pathway adopted for preparation of the desired spi-
rooxindoles is illustrated in Scheme 1. First, ethyl benzoates 2a–d
were reacted with acetonitrile under reflux temperature in dry
benzene and DMF, and in the presence of sodium hydride to fur-
nish
intermediates
3-oxo-3-phenylpropanenitriles
3a–d.
Furthermore, synthesis of 5-aminopyrazole
5
was achieved
through the heterocyclocondensation of intermediate 4 with phe-
nylhydrazine in ethyl alcohol under reflux temperature. Finally, the
target spirooxindoles 6a–p were prepared through a one-pot
three-component reaction of equimolar amounts of isatins 1a–d,
Apoptosis study. The levels of the apoptotic markers caspase-3, 3-oxo-3-phenylpropanenitriles 3a–d, and 1-phenyl-3-(thiophen-2-
caspase-9, p53 and Bax, as well as the anti-apoptotic marker Bcl-2 yl)-1H-pyrazol-5-amine 5 in HOAc/H2O mixture (1:1 v/v) at 70 ꢂC
(Scheme 1).
were evaluated by the use of ELISA colorimetric kits per the man-
ufacturer’s instructions, as reported earlier28,29. Furthermore, the
pro-apoptotic potential of spirooxindole 6a towards HepG2 cells
was assessed using FITC Annexin V Apoptosis Detection Kit by
flow cytometry, according to the manufacturer’s protocol and
Biological evaluation
Antitumor activity
referring to the reported procedures30,31
.
Anti-proliferative activity towards HepG2 and PC-3 cell lines.
Spirooxindoles 6a–p were tested for their in vitro anti-HCC activity
against HepG2 cell line, and for their anti-prostate cancer activity
against PC-3 cell line by the use of MTT reduction assay27. The
Cell cycle analysis. Cell cycle distributions in HepG2 cells were
examined through PI staining and analysed by flow cytometry
after treatment with spirooxindole 6a at its IC50 (6.9 mM), as anticancer drug Adriamycin was co-assayed as the positive

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
833
Scheme 1. Synthesis of target compounds 6a–p; Reagents and conditions: (i) CH3CN, DMF, NaH, benzene, reflux 4 h; (ii) Ethanol, phenylhydrazine, reflux 1 h; (iii) HOAc/
H2O (1:1 v/v), heating at 120 ꢂC, 8–11h.
Table 1. Anti-proliferative activities of spirooxindoles 6a–p against HepG2 hepa-
respectively) than HepG2 cells (IC50 ¼ 32.7, 30.6 and >50.0 mM,
tocellular carcinoma and PC-3 prostate cancer cell lines.
respectively).
Regarding the activity towards HepG2 cells, the data displayed
in Table 1 ascribed to the tested spirooxindoles excellent to weak
efficacy in inhibiting the growth of the HCC HepG2 cells (IC50 val-
ues ranging between 6.3 and 49.1 mM, Table 1), except com-
pounds 6 g and 6n–6p which failed to inhibit the growth of
HepG2 cells up to 50 lM. In particular, spirooxindoles 6a and 6i
were found to be the most potent derivatives in this study against
HepG2 cells with IC50 values equal to 6.9 and 6.3 lM, respectively.
In addition, spirooxindoles 6e and 6j exhibited good activity
towards HepG2 cells (IC50 ¼ 8.4 and 9.9 mM, respectively), whereas,
IC50 (mM)
Compound
R
Ar
C6H5––
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
C6H5–
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
C6H5–
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
C6H5–
HepG2
PC-3
compounds 6 b, 6c, 6 h, 6k and 6 l elicited moderate potency
against HepG2 cells with IC50 range: 12.7 ꢀ 21.0 mM. Morphological
assessment revealed the toxic effects of spirooxinodoles 6a, 6e
and 6i as presented in Figure 2 including dramatic cellular effects
including cell rounding, shrinkage and monolayer disruption.
On the other hand, it was apparent from the obtained results
that most of the prepared spirooxindoles possessed moderate to
modest growth inhibitory activity towards prostate cancer PC-3
cells with IC50 in the range of 11.8 ꢀ 43.7 mM. Compounds 6a and
6e were found to be the most potent spirooxindoles towards PC-
3 cells with IC50 values equal to 11.8 and 13.5 lM, respectively.
Moreover, spirooxindoles 6i, 6j and 6 m were moderately active
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
6.9
21.0
19.1
49.1
8.4
32.7
>50.0
19.9
6.3
11.8
34.2
43.7
>50.0
13.5
29.3
>50.0
>50.0
17.9
26.6
39.1
>50.0
24.3
42.8
>50.0
>50.0
0.62
Cl
Cl
Cl
Cl
Br
Br
Br
Br
OCH3
OCH3
OCH3
OCH3
9.9
13.2
12.7
30.6
>50.0
>50.0
>50.0
0.12
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
against PC-3 cells with (IC50
respectively).
¼
17.9, 26.6 and 24.3 mM,
Adriamycin
Observing the aforementioned results for the anti-proliferative
activity MTT assay against HepG2 cells, certain structure activity
relationships could be concluded. Firstly, we examined the effect
of C-5 substitution of the indoline moiety. 5-Br substituted spi-
rooxindoles 6i–6l displayed more enhanced anti-proliferative
activity (IC50 ¼ 6.3, 9.9, 13.2 and 12.7 mM, respectively) than their
corresponding unsubstituted spirooxindoles 6a–6d (IC50 ¼ 6.9,
21.0, 19.1 and 49.1 mM, respectively), 5-Cl substituted spirooxin-
doles 6e–6h (IC50 ¼ 8.4, 32.7, >50.0 and 19.9 mM, respectively),
and 5-OCH3 substituted spirooxindoles 6 m–6p (IC50 ¼ 30.6,
>50.0, >50.0 and >50.0 mM, respectively), which highlighted that
control. The anti-proliferative activities of tested spirooxindoles
against the two cancer cell lines were measured as IC50 (in lM)
value (the dose that affords a 50% inhibition of cell growth after
48 h of incubation) and displayed in Table 1.
Investigation of the obtained results in Table 1 revealed that
the tested spirooxindoles were more effective towards HCC
HepG2 cells rather than prostate cancer PC-3 cells, except com-
pounds 6f, 6 m and 6n which caused slightly enhanced growth
inhibitory activity against PC-3 (IC50 ¼ 29.3, 24.3 and 42.8 mM, C-5 substitution of the indoline moiety with the large lipophilic Br

834
S. T. AL-RASHOOD ET AL.
25 µM
50 µM
3.125 µM
6.25 µM
12.5 µM
Conc.
6a
6e
0.1% DMSO
(Vehicle)
6i
Figure 2. Morphological changes following 48 h exposure of HepG2 cells to indicated concentrations of 6a, 6e and 6i. Signs of toxicity indicated with arrows represent
cell rounding, shrinkage and/or loss of monolayer integrity. Total magnification ¼ 300.
Table 2. Cytotoxic action of spirooxindoles 6a–c, 6e, 6f, 6i–k and 6 m towards
non-tumorigenic human MCF-10A cell line, and selectivity index (S. I.) for tumour
cells (MCF-10A/HepG2).
IC50 (mM)
Compound
MCF-10A
HepG2
Tumour S. I.
6a
6b
6c
6e
6f
6i
6j
94.56
6.9
13.7
5.8
7.4
8.3
4.9
12.2
14.8
10.3
6.1
121.61
141.30
69.74
158.79
76.94
146.41
136.27
185.96
21.0
19.1
8.4
32.7
6.3
9.9
13.2
30.6
6k
6m
Figure 3. Effect of spirooxindole 6a on the phases of cell cycle of HepG2 cells.
spirooxindoles S. I. lied in the range: 4.9–14.8. Superiorly, spiroox-
indoles 6a and 6j displayed excellent safety profile with S. I. equal
13.7 and 14.8, respectively, Table 2.
group is more advantageous for the growth inhibitory activity
against HepG2 cells than substitution with smaller lipophilic (as Cl
and OCH3) groups and unsubstitution.
We then explored the impact of substitution of the pendant
phenyl group at C-6 of pyrazolo[3,4-b]pyridine moiety on the
Flow cytometric analysis and apoptotic studies in HepG2 cells.
activity against HepG2 cells. Spirooxindoles 6a, 6e, 6i and 6 m Spirooxindole 6a was found to be an efficient anti-proliferative
that were entailed with an unsubstituted phenyl group showed agent towards both HepG2 and PC-3 cells (IC50 ¼ 6.9 and 11.8 mM,
respectively, Table 1), in addition to its promising antioxidant
better activity (IC50 ¼ 6.9, 8.4, 6.3 and 30.6 mM, respectively) than
activities (Tables 5 and 6). Accordingly, spirooxindole 6a was
selected for further evaluations to acquire mechanistic insights
into its anti-proliferative activity against HepG2 cells.
their corresponding 4-substitutedphenyl counterparts 6c–d (IC50
range: 213 ꢀ 49.1 mM), 6f–h (IC50 range: 19.9 – > 50 mM), 6j–l (IC50
range: 9.9 ꢀ 13.2 mM) and 6n–p (IC50 > 50 mM), suggesting that
incorporation of an unsubstituted phenyl group at C-6 of pyra-
zolo[3,4-b]pyridine moiety is indispensable for the anti-proliferative
activity towards HCC HepG2 cells.
Cell cycle analysis. The impact of spirooxindole 6a on the normal
cell cycle progression was investigated using flow cytometric ana-
lysis of the DNA ploidy in HepG2 cells. Exposure of HepG2 cells to
spirooxindole 6a at its IC50 (6.9 mM) for 24 h revealed the ability of
6a to disrupt the normal cell cycle by decreasing both G0-G1
and S phases by approximately 0.53- and 0.37-folds with respect
to the control (Figure 3). Furthermore, both Pre-G and G2/M
phases of the treated HepG2 cell were significantly increased
by 11.9- and 2.1-folds related to the control (Figure 3).
Alteration of the Pre-G phase and arrest of G2-M phase may imply
On the other hand, spirooxindoles 6a–c, 6e, 6f, 6i-k and 6 m,
with dual activity against both here examined HepG2 and PC-3
cancer cell lines, were tested for their potential cytotoxic impact
towards the non-tumorigenic human breast MCF-10A cell line in
order to explore the safety profile of the target spirooxindole scaf-
fold towards the normal cells, Table 2.
The tested spirooxindoles 6a–c, 6e, 6f, 6i-k and 6 m exerted
non-significant cytotoxicity towards the non-tumorigenic MCF-10A
cells with IC50 values spanning between 69.74 and 185.96 mM,
apoptosis as
a potential mechanism for 6a-induced cancer
respectively. In addition, the calculated for the tested cell death.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
835
Effect of 6a on the levels of the apoptotic markers (Bax, caspase- staining assay was carried out at IC50 of 6a (6.9 mM). Examining
the results of this flow cytometric analysis (Figure 4), revealed that
HepG2 cells treated with spirooxindole 6a exhibited a significant
increase in the percentage of annexin-V positive cells indicating
an early (lower right quadrant) and late (upper right quadrant)
3, caspase-9 and p53), and the anti-apoptotic marker Bcl-2. To
investigate the ability of spirooxindole 6a to provoke apoptosis,
herein we assessed the expression levels of the hallmark parame-
ters of apoptosis in HepG2 cells upon treatment with 6a at its IC50
(6.9 mM) (Tables 3 and 4). In this study, exposure of HepG2 cells to apoptosis from 0.95% to 6.76%, and from 0.33% to 14.14%,
spirooxindole 6a resulted in a significant 5.7-fold increase in the
expression of the pro-apoptotic protein Bax, with concurrent sig-
nificant down-regulation of the expression levels of the anti-apop-
totic protein Bcl-2 by ꢃ 42% compared to the control (Table 3).
Analysing these results declared that spirooxindole 6a boosted the
Bax/Bcl-2 ratio nine folds in comparison to the control. The Bax/Bcl-
2 ratio is considered as a decisive value that gives a real insight to
the overall pro-apoptotic impact of the examined compound.
The elevated Bax/Bcl-2 ratio aroused the assessment of the
respectively, which comprises about 16.3-folds total increase with
respect to control (Figure 4).
Antioxidant activity
DPPH radical scavenging activity. The DPPH (2,2-diphenyl-1-picryl-
hydrazyl) radical scavenging activity assay is considered as one of
the most common and simple methods for measuring the antioxi-
dant ability to trap free radicals. DPPH is a commercially available
expression levels of the pro-apoptotic active caspase-3, caspase-9 stable free radical that possesses the ability to accept an electron
and p53 tumour suppressor protein. Treatment of HepG2 cells to produce a stable molecule. Substances that have the potential
with spirooxindole 6a resulted in a significant elevation in the
expression levels of caspase-3, caspase-9 and p53 proteins by
about 8.88, 12.27 and 10.03 folds, respectively, compared to con-
trol (Table 4).
The simultaneous up-regulation of the downstream caspase 3,
the hallmark key player and the key executor of apoptosis, along-
side to p53 highlighted that a cascade of apoptotic markers was
activated as a consequence of Bax/Bcl2 elevation that eventually
resulted in apoptosis.
to act as electron donors result in reduction of this DPPH radical
to the corresponding DPPH2 disclosing their antioxidant potential.
The principle of the method relies upon measuring the absorb-
ance of the odd electrons of DPPH at 517 nm. The absorbance
decreases, in the presence of free radical scavengers, proportional
to the decrease of the DPPH radical concentration34. In the cur-
rent study, the scavenging effect of compounds 6a–p was eval-
uated via the DPPH radical scavenging assay, and compared with
the standard antioxidant; ascorbic acid. The results, summarised in
Table 5, are expressed in terms of IC50 values that produce 50%
inhibition of the DPPH radical; where the lower is this value, the
more active is the examined compound.
Annexin V-FITC apoptosis assay. To confirm the apoptotic effect
of spirooxindole 6a in HepG2 cells, Annexin-V FTIC/PI dual
From the obtained results, it was obvious that most of the pre-
pared spirooxindoles 6a–p exhibited excellent to moderate DPPH
radical scavenging capacities with IC50 spanning in the range:
28.07 ꢀ 113.45 mg/mL. Superiorly, both spirooxindoles 6a and 6 m
emerged as the most efficient DPPH radical scavengers with IC50
values of 29.76 1.82 and 28.07 1.63 mg/mL, respectively, which
are 1.6-fold more potent than the standard antioxidant Ascorbic
acid (IC50 ¼ 45.89 2.39 mg/mL). Also, compounds 6c–6f, 6i, 6j
and 6 l showed higher DPPH radical scavenging capacities (IC50
range: 33.43 1.24 ꢀ 43.42 3.04 mg/mL) than Ascorbic acid.
Furthermore, spirooxindoles 6 h, 6k and 6n–6p displayed moder-
ate antiradical activity with IC50 values of 46.29 2.18,
69.25 4.25, 46.29 2.52, 58.82 3.14 and 55.05 3.41 mg/mL,
respectively. Contrariwise, compounds 6 b and 6 g elicited weak
DPPH_ scavenging activity with IC50 values of 113.45 5.59 and
97.47 5.62 mg/mL, respectively.
Table 3. Effect of compound 6a on the expression levels of Bax and Bcl-2 in
HepG2 cells treated with the compound at its IC50
.
Bax
ng/mL
Bcl-2
ng/mL
4.34
2.51 (0.57)
Comp.
Bax/Bcl-2 ratio
Control
6a
40.63
232.2 (5.7)
9.4
92.5
ꢄ
ꢄ
ꢄ
Numbers given between parentheses are the numbers of folds of control.
Table 4. Effect of compound 6a on the expression levels of active caspases-3
and -9, and p53 in HepG2 cells treated with the compound at its IC50
.
Caspase-3
pg/mL
Caspase-9
ng/mL
p53
pg/mL
Comp.
Control
6a
53.57
475.9 (8.88)
2.19
26.92 (12.27)
77.15
774.5 (10.03)
ꢄ
ꢄ
ꢄ
ꢄ
Numbers given between parentheses are the numbers of folds of control.
Figure 4. Effect of spirooxindole 6a on the percentage of annexin V-FITC-positive staining in HepG2 cells. The experiments were done in triplicates. The four quadrants
identified as: LL: viable; LR: early apoptotic; UR: late apoptotic; UL: necrotic.

836
S. T. AL-RASHOOD ET AL.
Table 5. The in vitro antioxidant activity of spirooxindoles (6a–p) in DPPH_ scav-
enging assay.
6c–6f, 6 h–6j, 6 l and 6n–6p possessed good reducing capacity in
this assay with FRAP range: 10.12 ꢀ 11.90 mM.
Physicochemical, ADME and pharmacokinetic
properties prediction
For prediction of the pharmacokinetic properties, ADME parame-
ters and drug-like nature of spirooxindole 6a, the automated
SwissADME online web tool36 was employed. Articulating such
properties is of great significance in the context of pharmaceutical
chemistry to develop efficient clinical candidates.
Compound
R
Ar
IC50 (mg/mL)a
Figure 5(A) displays the BOILED-Egg graph of the WLOGP vs.
TPSA (topological polar surface area) for spirooxindole 6a37, where
it is located in the region of human intestinal absorption (HIA)
with no BBB permeability so it is anticipated to display low occur-
rence for CNS side effects. Also, this graph revealed that spirooxin-
dole 6a is not a P-glycoprotein substrate (PGPꢀ), therefore it is
not susceptible to the efflux mechanism carried out by this trans-
porter that utilised by many cancer cell lines as a drug-resistance
mechanism38,39. Figure 5(B) represents the bioavailability radar
chart for spirooxindole 6a, which consists of six axes for six key
properties for oral bioavailability; polarity (POLAR), solubility
(INSOLU), lipophilicity (LIPO), flexibility (FLEX), saturation (INSATU)
and size (SIZE) 39,40. The range for optimal property values is dis-
played as a pink area, whereas the red line represents the pre-
dicted properties for the examined molecule.
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
H
H
H
H
C6H5–
29.76 1.82
113.45 5.59
43.04 2.39
33.43 1.24
36.03 1.37
40.31 2.61
97.47 5.62
46.29 2.18
36.38 2.71
43.42 3.04
69.25 4.25
41.36 2.17
28.07 1.63
46.29 2.52
58.82 3.14
55.05 3.41
45.89 2.39
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
C6H5–
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
C6H5–
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
C6H5–
4–CH3–C6H4–
4–OCH3–C6H4–
4–Cl–C6H4–
–
Cl
Cl
Cl
Cl
Br
Br
Br
Br
OCH3
OCH3
OCH3
OCH3
–
Ascorbic acid
aIC50 values are the mean SD of three separate experiments.
Furthermore, as shown in Table 7, spirooxindole 6a showed
high predicted level of gastrointestinal absorption, and predicted
to be inhibitor of three of CYP isoforms (CYP1A2, CYP2C9 and
CYP2C19) whereas predicted to be non-inhibitor of CYP3A4 and
CYP2D6 isoforms.
Table 6. The in vitro antioxidant activity of spirooxindoles (6a–p) in FRAP assay.
Compound
mM (Fe2þ Eq.)
Moreover, SwissADME tool showed that spirooxindole 6a fulfils
the drug-likeness characteristics as defined by the major pharma-
ceutical companies (Table 8) and passed their filters; Lipinski’s
(Pfizer)41, Veber’s (GSK)42 and Egan’s (Pharmacia)43 filters, whereas
it failed to pass Ghose’s (Amgen) filter due to two violations;
molecular weight > 480, and molar refractivity >13044. However,
it was not classified as lead-like because its molecular weight is
higher than 350 and its predicted XLOGP345 exceeded 3.5.
In summary, this in silico study for predicting the physicochemical
and pharmacokinetic properties of spirooxindole 6a revealed that it
is not only with promising antioxidant and anti-proliferative bio-
logical activities, but also with promising pharmacokinetic properties.
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
11.98
6.933
10.32
10.99
10.12
11.36
7.79
11.35
11.88
10.34
8.499
11.59
12.13
11.58
11.90
11.18
13.42
Ascorbic acid
Conclusions
In summary, a series of spirooxindole-based derivatives (6a–p)
was examined for its anticancer effect towards HepG2 hepatocel-
lular carcinoma and PC-3 prostate cancer cell lines. Spirooxindoles
6a–p were more effective towards HCC HepG2 cells rather than
prostate cancer PC-3 cells, except compounds 6f, 6 m and 6n. In
particular, spirooxindoles 6a and 6i were found to be the most
potent derivatives in this study against HepG2 cells with IC50 val-
ues equal to 6.9 and 6.3 lM, respectively. The SAR outcomes high-
lighted that C-5 substitution of the indoline moiety with a large
lipophilic Br group, and incorporation of unsubstituted phenyl
group at C-6 of pyrazolo[3,4-b]pyridine moiety are indispensable
factors for the growth inhibitory activity against HepG2 cells.
Furthermore, spirooxindole 6a was assessed for its apoptosis
induction potential in HepG2 cells, where its pro-apoptotic impact
was approved via the significant elevation in the Bax/Bcl-2 ratio
and the expression levels of caspase-3, caspase-9 and p53 proteins
Ferric reducing antioxidant power (FRAP). FRAP assay is a widely
used antioxidant assay that uses antioxidant substances as reduc-
ing agents in a redox-linked colorimetric reaction, wherein Fe3þ is
reduced to Fe2þ at acidic pH that results in a formation of a col-
oured ferrous-probe complex from a colourless ferric-probe com-
plex35. In this assay, the electron donation capacity (reflecting the
electron transfer ability) of the examined compounds is evaluated.
In this study, all spirooxindoles 6a–p herein reported were
evaluated for their potential FRAP. The results were expressed as
mM Fe2þ equivalents and presented in Table 6, where the higher
mM Fe2þ equivalents value means higher FRAP and subsequently
higher antioxidant capacity. As in the above DPPH assay, both spi-
rooxindoles 6a and 6 m exhibited the best FRAP activity (11.98
and 12.13 mM, respectively) that is comparable to the activity of
the reference Ascorbic acid (13.42 mM). Moreover, compounds by about 9, 8.8, 12.2 and 10 folds, respectively, and via the

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
837
Figure 5. (A) Predicted Boiled-Egg plot from swissADME online web tool for spirooxindole 6a; (B) Bioavailability radar chart for spirooxindole 6a; The pink area repre-
sents the range of the optimal property values for oral bioavailability and the red line is spirooxindole 6a predicted properties.
Table 7. In silico predictions of the pharmacokinetics properties for spirooxindole 6a.
Cpd.
GIA
BBB
No
P-gp substrate
No
CYP1A2 inhibitor
Yes
CYP2C9 inhibitor
Yes
CYP2C19 inhibitor
Yes
CYP3A4 inhibitor
No
CYP2D6 inhibitor
No
6a
High
GIA: gastrointestinal absorption; BBB: blood-brain barrier permeation; P-gp: permeability glycoprotein; CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 are the
five major isoforms of cytochromes P450.
Table 8. In silico predictions of the drug-likeness properties for spirooxindole 6a.
Cpd.
Lipinski #violations
0
Ghose #violations
2
Veber #violations
0
Egan #violations
0
PAINS #alerts
0
Brenk #alerts
0
Bioavailability Score
0.55
6a
All calculations were performed using SwissADME [36].
significant increase in the annexin V-FITC positive apoptotic cells
percent. Besides, 6a–p were screened for their potential antioxidant
activity via assessing their DPPH radical scavenging capacities and
Ferric reducing antioxidant power (FRAP). The obtained results indi-
cated that spirooxindoles 6a and 6 m are the most efficient DPPH
radical scavengers (IC50 ¼ 29.76 1.82 and 28.07 1.63 mg/mL,
respectively), and exhibited the best FRAP activity (11.98 and
12.13 mM, respectively). Finally, an in silico study was carried out to
predict the physicochemical and pharmacokinetic properties of spi-
rooxindole 6a, which revealed that 6a is not only with promising
antioxidant and anti-proliferative activities, but also with proper
drug-likeness and pharmacokinetic properties.
References
1. (a) de Torre MP, Cavero RY, Calvo MI, Vizmanos JL, A simple
and a reliable method to quantify antioxidant activity
in vivo. Antioxidants 2019;8:142.(b) Kundu T, Bhattacharjee
B, Hazra S, et al. Synthesis and biological assessment of pyr-
rolobenzoxazine scaffold as a potent antioxidant. J Med
Chem 2019;62:6315–29.
ꢀ
2. Habrant D, Poigny S, Segur-Derai M, et al. Evaluation of anti-
oxidant properties of monoaromatic derivatives of pulvinic
acids. J Med Chem 2009;52:2454–64.
3. (a) Yanovsky I, Finkin-Groner E, Zaikin A, et al. Carbamate
derivatives of indolines as cholinesterase inhibitors and anti-
oxidants for the treatment of Alzheimer’s disease. J Med
Chem 2012;55:10700–15.(b) Zhang C, Zhou Q, Wu XN, et al.
Discovery of novel PDE9A inhibitors with antioxidant activ-
ities for treatment of Alzheimer’s disease. J Enzym Inhib
Med Chem 2019;34:260–70.
Disclosure statement
No potential conflict of interest was reported by the author(s).
Funding
The authors would like to extend their sincere appreciation to the
Deanship of Scientific Research at King Saud University for its
funding of this research through the Research Group Project no.
RG-1439–65.
4. Hassan SZ, Gheita TA, Kenawy SA, et al. Oxidative stress in
systemic lupus erythematosus and rheumatoid arthritis
patients: relationship to disease manifestations and activity.
Int J Rheum Dis 2011;14:325–31.
5. Segovia J, Perez-Severiano F. Oxidative damage in
Huntington’s disease. Methods Mol Biol 2004;277:321–34.
6. Secci D, Carradori S, Petzer A, et al. 4-(3-Nitrophenyl) thia-
zol-2-ylhydrazone derivatives as antioxidants and selective
hMAO-B inhibitors: synthesis, biological activity and compu-
tational analysis. J Enzym Inhib Med Chem 2019;34:597–612.
ORCID
Abdulrahman A. Almehizia
8711-3873
http://orcid.org/0000-0001-

838
S. T. AL-RASHOOD ET AL.
7. Signorelli SS, Scuto S, Marino E, et al. Oxidative stress in
Peripheral Arterial Disease (PAD) mechanism and bio-
markers. Antioxidants 2019;8:367.
8. Hao X, Qi G, Ma H, et al. Novel 2-phenoxypyrido [3, 2-b] pyr-
azin-3 (4 H)-one derivatives as potent and selective aldose
reductase inhibitors with antioxidant activity. J Enzym Inhib
Med Chem 2019;34:1368–72.
9. Franco R, Schoneveld O, Georgakilas AG, Panayiotidis MI.
Oxidative stress, DNA methylation and carcinogenesis.
Cancer Lett 2008;266:6–11.
carbonitriles as p53-MDM2 interaction inhibitors. Future
Med Chem 2018;10:2771–89.
25. Eldehna WM, El-Naggar DH, Hamed AR, et al. One-pot three-
component synthesis of novel spirooxindoles with potential
cytotoxic activity against triple-negative breast cancer
MDAMB-231 cells. J Enzym Inhib Med Chem 2018;33:309–18.
26. Denizot F, Lang R. Rapid colorimetric assay for cell growth
and survival: modifications to the tetrazolium dye procedure
giving improved sensitivity and reliability.
J Immunol
Methods 1986;89:271–7.
10. Kakkar R. Isatin and its derivatives: a survey of recent syn- 27. Mosmann T. Rapid colorimetric assay for cellular growth and
theses, reactions, and applications. MedChemComm 2019;
survival: application to proliferation and cytotoxicity assays.
10:351–68.
J Immunol Methods 1983;65:55–63.
11. Thanh ND, Giang NTK, Quyen TH, et al. Synthesis and evalu- 28. Eldehna WM, Hassan GS, Al-Rashood ST, et al. Synthesis and
ation of in vivo antioxidant, in vitro antibacterial, MRSA and
antifungal activity of novel substituted isatin N-(2, 3, 4, 6-
tetra-O-acetyl-b-d-glucopyranosyl) thiosemicarbazones. Eur J
Med Chem 2016;123:532–43.
in vitro anticancer activity of certain novel 1-(2-methyl-6-
arylpyridin-3-yl)- 3-phenylureas as apoptosis-inducing
agents. J Enzym Inhib Med Chem 2019;34:322–32.
29. El-Naggar M, Eldehna WM, Almahli H, et al. Novel thiazolidi-
none/thiazolo[3,2-a] benzimidazolone-isatin conjugates as
apoptotic anti-proliferative agents towards breast cancer:
one-pot synthesis and in vitro biological evaluation.
Molecules 2018;23:1420.
12. Eldehna WM, Al-Wabli RI, Almutairi MS, et al. Synthesis and
biological evaluation of certain hydrazonoindolin-2-one
derivatives as new potent anti-proliferative agents. J Enzym
Inhib Med Chem 2018;33:867–78.
13. Eldehna WM, El Kerdawy AM, Al-Ansary GH, et al. Type IIA- 30. Eldehna WM, Abo-Ashour MF, Ibrahim HS, et al. Novel [(3-
Type IIB protein tyrosine kinase inhibitors hybridization as
an efficient approach for potent multikinase inhibitor devel-
opment: Design, synthesis, anti-proliferative activity, multiki-
nase inhibitory activity and molecular modeling of novel
indolinone-based ureides and amides. Eur J Med Chem
2019;163:37–53.
indolylmethylene)hydrazono]indolin-2-ones as apoptotic
anti-proliferative agents: design, synthesis and in vitro bio-
logical evaluation.
J Enzym Inhib Med Chem 2018;33:
686–700.
31. Sabt A, Abdelhafez OM, El-Haggar RS, et al. Novel coumarin-
6-sulfonamides as apoptotic anti-proliferative agents: syn-
thesis, in vitro biological evaluation, and QSAR studies. J
Enzym Inhib Med Chem 2018;33:1095–107.
32. Brand-Williams W, Cuvelier ME, Berset C. Use of a free rad-
ical method to evaluate antioxidant activity. LWT-Food Sci
Technol 1995;28:25–30.
33. Benzie IFF, Strain JJ. The ferric reducing ability of plasma
(FRAP) as a measure of “antioxidant power”: The FRAP assay.
Anal Biochem 1996;239:70–6.
14. Abdel-Aziz HA, Eldehna WM, Keeton AB, et al. Isatin-benzoa-
zine molecular hybrids as potential antiproliferative agents:
synthesis and in vitro pharmacological profiling. Drug Des
Dev Ther 2017;11:2333–46.
15. Bindra JS. Oxindole alkaloids. In: Manske RHF, ed. The alka-
loids. Vol 14. New York: Academic Press; 1973:84–121.
16. (a) Elderfield RC, Gilman RE. Alkaloids of Alstonia muelleriana.
Phytochemistry 1972;11:339–43.(b) Ghedira K, Zeches-Hanrot
M, Richard B, et al. Alkaloids of Alstonia angustifolia.
Phytochemistry 1988;27:3955–62.
17. Jossang A, Jossang P, Hadi HA, et al. Horsfiline, an oxindole
alkaloid from Horsfieldia superba. J Org Chem 1991;56:
6527–30.
18. Anderton N, Cockrum PA, Colegate SM, et al. Oxindoles
from Phalaris coerulescens. Phytochemistry 1998;48:437–9.
19. Sakai S, Aimi N, Yamaguchi K, et al. Gardneria alkaloids-IX
ꢀ
34. Sordon
S,
Popłonski
J,
Milczarek
M,
et
al.
Structure–antioxidant–antiproliferative activity relationships
of natural C7 and C7–C8 hydroxylated flavones and flava-
nones. Antioxidants 2019;8:210.
35. Stana A, Vodnar DC, Marc G, et al. Antioxidant activity and
antibacterial evaluation of new thiazolin-4-one derivatives as
potential tryptophanyl-tRNA synthetase inhibitors. J Enzym
Inhib Med Chem 2019;34:898–908.
structures of chitosenine and three other minor bases: from 36. Swiss Institute of Bioinformatics. http://www.swissadme.ch/
Gardneria multiflora makino. Tetrahedron Lett 1975;16:
715–8.
20. M Heravi M, Zadsirjan V, Malmir M. Application of the asym-
metric pictet–spengler reaction in the total synthesis of nat-
ural products and relevant biologically active compounds.
Molecules 2018;23:943.
index.php.
37. Daina A, Zoete V. A boiled-egg to predict gastrointestinal
absorption and brain penetration of small molecules. Chem
Med Chem 2016;11:1117–21.
38. Szakacs G, et al. The role of ABC transporters in drug
absorption, distribution, metabolism, excretion and toxicity
(ADME-Tox). Drug Discov Today 2008;13:379–93.
39. Daina A, Michielin O, Zoete V. SwissADME: a free web tool
to evaluate pharmacokinetics, drug-likeness and medicinal
chemistry friendliness of small molecules. Sci Rep 2017;7:
42717.
21. Cui C-B, Kakeya H, Osada H. Novel mammalian cell cycle
inhibitors, spirotryprostatins
A
and B, produced by
Aspergillus fumigatus, which inhibit mammalian cell cycle at
G2/M phase. Tetrahedron 1996;52:12651–66.
22. Kaur M, Singh B, Singh B, Arjuna A. Synthesis and evaluation
of novel spiro [oxindole-isoxazolidine] derivatives as potent
antioxidants. J Heterocycl Chem 2017;54:1348–54.
23. Yu B, Yu D-Q, Liu H-M. Spirooxindoles: promising scaffolds
for anticancer agents. Eur J Med Chem 2015;97:673–98.
40. Daina A, Michielin O, Zoete V. iLOGP: a simple, robust, and
efficient description of n-octanol/water partition coefficient
for drug design using the GB/SA approach. J Chem Inf
Model 2014;54:3284–301.
24. Ibrahim HS, Eldehna WM, Fallacara AL, et al. One-pot synthe- 41. Lipinski CA, Lombardo F, Dominy BW, Feeney PJ.
sis of spiro (indoline-3, 40-pyrazolo [3, 4-b] pyridine)-50-
Experimental and computational approaches to estimate

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
839
solubility and permeability in drug discovery and develop- 44. Ghose AK, Viswanadhan VN, Wendoloski JJ. A knowledge-
ment settings. Adv Drug Deliv Rev 2001;46:3–26.
42. Veber DF, Johnson SR, Cheng HY, et al. Molecular properties
that influence the oral bioavailability of drug candidates. J
Med Chem 2002;45:2615–2623.
based approach in designing combinatorial or medicinal
chemistry libraries for drug discovery. 1. A qualitative and
quantitative characterization of known drug databases. J
Comb Chem 1999;1:55–68.
43. Egan WJ, Merz KM, Baldwin JJ. Prediction of drug absorption 45. Cheng T, Zhao Y, Li X, et al. Computation of octanol ꢀ water
using multivariate statistics. J Med Chem 2000;43:3867–77.
partition coefficients by guiding an additive model with
knowledge. J Chem Inf Model 2007;47:2140–8.