Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles arrest cell cycle and induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological evaluation and in silico studies

Article abstract of DOI:10.1080/14756366.2020.1773814

As a continuation for our previous work, a novel set of N-alkylindole-isatin conjugates (7, 8a–c, 9 and 10a–e) is here designed and synthesised with the prime aim to develop more efficient isatin-based antitumor candidates. Utilising the SAR outputs from the previous study, our design here is based on appending four alkyl groups with different length (ethyl and n-propyl), bulkiness (iso-propyl) and unsaturation (allyl) on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to furnish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the growth inhibitory profile for the target conjugates in comparison to the reported lead VI. The best results were obtained with N-propylindole –5-methylisatin hybrid 8a which displayed broad spectrum anti-proliferative action with efficient sub-panel GI₅₀ (MG-MID) range from 1.33 to 4.23 μM, and promising full-panel GI₅₀ (MG-MID) equals 3.10 μM, at the NCI five-dose assay. Also, hybrid 8a was able to provoke cell cycle disturbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/PI assays. Furthermore, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2 protein (IC₅₀= 0.85 ± 0.03 and 0.46 ± 0.02 μM, respectively). Interestingly, molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising lead for further development and optimisation as an efficient antitumor drug.

Full text of DOI:10.1080/14756366.2020.1773814

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: 1475-6366 (Print) 1475-6374 (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel [(N-alkyl-3-
indolylmethylene)hydrazono]oxindoles arrest cell
cycle and induce cell apoptosis by inhibiting CDK2
and Bcl-2: synthesis, biological evaluation and in
silico studies
Tarfah Al-Warhi, Mahmoud F. Abo-Ashour, Hadia Almahli, Ohoud J. Alotaibi,
Mohammad M. Al-Sanea, Ghada H. Al-Ansary, Hanaa Y. Ahmed, Mahmoud
M. Elaasser, Wagdy M. Eldehna & Hatem A. Abdel-Aziz
To cite this article: Tarfah Al-Warhi, Mahmoud F. Abo-Ashour, Hadia Almahli, Ohoud J. Alotaibi,
Mohammad M. Al-Sanea, Ghada H. Al-Ansary, Hanaa Y. Ahmed, Mahmoud M. Elaasser, Wagdy
M. Eldehna & Hatem A. Abdel-Aziz (2020) Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles
arrest cell cycle and induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological
evaluation and inꢀsilico studies, Journal of Enzyme Inhibition and Medicinal Chemistry, 35:1,
1300-1309, DOI: 10.1080/14756366.2020.1773814
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JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
2020, VOL. 35, NO. 1, 1300–1309
https://doi.org/10.1080/14756366.2020.1773814
RESEARCH PAPER
Novel [(N-alkyl-3-indolylmethylene)hydrazono]oxindoles arrest cell cycle and
induce cell apoptosis by inhibiting CDK2 and Bcl-2: synthesis, biological
evaluation and in silico studies
Tarfah Al-Warhia, Mahmoud F. Abo-Ashourb, Hadia Almahlic, Ohoud J. Alotaibia, Mohammad M. Al-Sanead,
Ghada H. Al-Ansarye,f, Hanaa Y. Ahmedg, Mahmoud M. Elaasserg, Wagdy M. Eldehnah and Hatem A. Abdel-Azizi
b
aDepartment of Chemistry, College of Science, Princess Nourah bint Abdulrahman University, Riyadh, Saudi Arabia; Department of
c
Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, Cairo, Egypt; Department of Chemistry, Chemistry
d
Research Laboratory, University of Oxford, Oxford, UK; Department of Pharmaceutical Chemistry, College of Pharmacy, Jouf University, Sakaka,
e
Aljouf, Saudi Arabia; Department of Pharmaceutical Chemistry, Pharmacy Program, Batterejee Medical College, Jeddah, Saudi Arabia;
g
fDepartment of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; The Regional Center for Mycology and
h
Biotechnology, Al-Azhar University, Cairo, Egypt; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University,
i
Kafrelsheikh, Egypt; Department of Applied Organic Chemistry, National Research Center, Dokki, Giza, Egypt
ABSTRACT
ARTICLE HISTORY
Received 29 March 2020
Revised 3 May 2020
Accepted 17 May 2020
As a continuation for our previous work, a novel set of N-alkylindole-isatin conjugates (7, 8a–c, 9 and
10a–e) is here designed and synthesised with the prime aim to develop more efficient isatin-based antitu-
mor candidates. Utilising the SAR outputs from the previous study, our design here is based on appending
four alkyl groups with different length (ethyl and n-propyl), bulkiness (iso-propyl) and unsaturation (allyl)
on N-1 of indole motif, with subsequent conjugation with different N-unsubstituted isatin moieties to fur-
nish the target conjugates. As planned, the adopted strategy achieved a substantial improvement in the
growth inhibitory profile for the target conjugates in comparison to the reported lead VI. The best results
were obtained with N-propylindole –5-methylisatin hybrid 8a which displayed broad spectrum anti-prolif-
erative action with efficient sub-panel GI50 (MG-MID) range from 1.33 to 4.23 mM, and promising full-panel
GI50 (MG-MID) equals 3.10 mM, at the NCI five-dose assay. Also, hybrid 8a was able to provoke cell cycle dis-
turbance and apoptosis in breast T-47D cells as evidenced by the DNA flow cytometry and Annexin V-FITC/
PI assays. Furthermore, hybrid 8a exhibited good inhibitory action against cell cycle regulator CDK2 protein
kinase and the anti-apoptotic Bcl-2 protein (IC50¼ 0.85 0.03 and 0.46 0.02 mM, respectively). Interestingly,
molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites unveiled that N-propyl group is involved in
significant hydrophobic interactions. Taken together, the results suggested conjugate 8a as a promising
lead for further development and optimisation as an efficient antitumor drug.
KEYWORDS
Isatin; N-alkylindole;
hybridisation; anticancer;
Bcl-2 inhibitor;
CDK2 inhibitor
1. Introduction
cancer cell death, was found as an important consequence of
CDKs inhibition and can be assessed by cell cycle arrest at low
concentration or even mitochondrial damage at high concentra-
tion1. This fact was discovered from previous studies on CDKs
inhibitors as Roscovitine and Purvalanol and their effect on three
different prostate cancer cell lines2. In addition, the link between
CDKs and apoptosis was revealed in the investigational research
of the effect of Ibulocydine, a prodrug CDK inhibitor, on hepato-
cellular carcinoma3.
In the current medical era, cancer is considered as a major public
health problem worldwide and one of the most leading causes of
death throughout the world. So, development of more effective
new drugs for management of different human malignancies is a
major requirement. The expanded knowledge for the functional
relationship between the different molecules which constitute the
cell cycle and checkpoint pathways furnished novel promising
strategies for the management of tumours.
Isatin moiety served as a contributor in various CDKs inhibitors
having an apoptotic effect. For example, isatin dimers, such as
Indirubin-30-oxime (Figure 1), revealed a potent inhibition against
CDK1, CDK2, and CDK5 with IC50s ¼ 180 nM, 500 nM and 250 nM,
respectively4. In addition to its effect against CDKs, it possessed
an apoptotic effect by disruption of cell cycle phases through
Cyclin-dependent kinases (CDKs) are considered as crucial fac-
tors that affect diverse key transitions in cell cycle for the cancer
cell, in addition to regulation of apoptosis, transcription and exo-
cytosis, therefore therapeutic strategies based on inhibition of
CDKs stand out as a promising opportunity for anticancer drug
discovery and an efficient approach for management of different
human malignancies. On the other hand, apoptosis, an automatic arresting G2/M phase5. Indirubin-30-oxime is regarded as a lead
CONTACT Ghada H. Al-Ansary
6231, Jeddah, Saudi Arabia; Wagdy M. Eldehna
University, Kafrelsheikh, Egypt
ghada.yassin@bmc.edu.sa
Department of Pharmaceutical Chemistry, Pharmacy Program, Batterejee Medical College, P.O. Box
wagdy2000@gmail.com Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh
Supplemental data for this article can be accessed here.
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.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1301
Figure 1. Chemical structures for certain reported isatin-based anticancer agents (I–V), and lead compound VI.
compound. It is especially known in Chinese traditional medicine, different length (ethyl 7, n-propyl 8a–c), bulkiness (isopropyl 9)
and its other derivatives as 5-Bromoindirun and Indirubin-5-sulfon- and unsaturation (allyl 10a–e) on N-1 of indole motif, with subse-
ate proved to be potent CKD inhibitors against CDK2 with IC50s ¼
quent conjugation with different N-unsubstituted isatin moieties
1 and 0.5 mM, respectively. Moreover, they were co-crystallized
with CDK2 (2BHE and 1E9H, respectively) (Figure 1)6,7. This dimeric
scaffold is initiative for the design of other derivatives with a sig-
nificant activity against CDKs as reported8,9. Other isatin deriva-
tives decorated with 3-substitution expressed potent inhibition for
CDK2 as shown in SU9516 which inhibits CDK2 with IC50 ¼ 25 nM
(Figure 1)10. Moreover, compound (I) is another isatin derivative
with hydrazino linker at position 3 (CDK2; IC50 ¼ 60 nM) that was
co-crystallized with its target and submitted to protein data bank
(1VFT). Several modifications on compound (I) were performed to
get compound (II) which inhibits CDK2 in sub-nanomolar range
(IC50 ¼ 0.54 nM) (Figure 1)11.
to furnish the target conjugates.
All the synthesised conjugates were examined for their cyto-
toxicity towards a list of three different human cancer cell lines
HCT-116 (Colon), A-549 (NSCLC), and MDA-MB-231 (Breast) utilis-
ing SRB assay. Furthermore, seven hybrids (8a–c, 9, 10a, 10c and
10d) were screened for their possible in vitro anticancer action in
accordance with US-NCI protocol, then, 8a was furthermore
chosen for testing at the five-dose assay. Subsequently, we exam-
ined the growth inhibition mechanism of hybrid 8a in relation to
cell cycle regulation as well as apoptosis induction in breast T-47D
cancer cells through the DNA flow cytometry and Annexin V-FITC/
PI assays. Moreover, inhibitory actions of hybrid 8a against the
cell cycle regulator protein CDK2 kinase and the anti-apoptotic
protein Bcl-2 were explored. Finally, docking simulations were
conducted in order to explore the behaviour of 8a within the
active site of both CDK2 and Bcl-2, and to justify its activity.
In last few years, our research team has developed diverse
small molecules based on the isatin scaffold as efficient anticancer
agents (structures III–V12–14, Figure 1) with diverse cellular and
enzymatic targets; for example triggering of apoptosis in several
tumour cell lines15,16, inhibition of tumour-linked human carbonic
anhydrase isoform IX17,18, and inhibition of VEGFR-2 kinase19. In
2018, we have reported the conjugation between the isatin and
indole moieties via methylenehydrazono spacer (HC¼N–N¼) to
develop new three different series of [(3-indolylmethylene)hydra-
zono]indolinone derivatives as anticancer agents with promising
pro-apoptotic activity20. As concluded from SAR analysis for this
study20, hybridisation of N-propyl indole moiety with N-unsubsti-
tuted isatin moiety (compound VI, Figure 1) achieved the most
effective anticancer activity.
2. Results and discussion
2.1. Chemistry
Synthetic routes herein proposed in order to get the targeted
conjugates (7, 8a–c, 9 and 10a–e) have been illustrated in
Schemes 1 and 2. With regard to Scheme 1, preparation of 1H-
indole-3-carbaldehyde 2 was achieved through Vilsmeier formyla-
tion of indole 1 by the use of N, N-dimethylformamide (DMF) and
phosphorus oxychloride (POCl3), then aldehyde 2 was undergone
to N-alkylation using different alkyl halides 3a–d in DMF with the
aid of sodium hydride base to get N-substituted indole-3-carbalde-
hyde intermediates 4a–d. The different N-substituted indole-3-car-
baldehyde derivatives 4a–d were condensed with hydrazine
Inspired by these findings and in connection with our previous
research work, it is thought advantageous to broaden our investi-
gations to probe new N-alkylindole-isatin conjugates 7, 8a–c, 9
and 10a–e exerting promising anticancer and pro-apoptotic
actions. Utilising the SAR outputs from the previous study20, our
design here is based on appending four alkyl groups with hydrate via heating under reflux temperature in ethyl alcohol in

1302
T. AL-WARHI ET AL.
Scheme 1. Preparation of the key intermediates 5a–d; (i) DMF, POCl3, reflux 8 h.; (ii) DMF, NaH, stirring at R.T for 24 h.; (iii) Ethyl alcohol, NH2NH2ꢀH2O, reflux 2 h.
Scheme 2. Synthesis of target conjugates 7, 8a–c, 9 and 10a-e; (i) Ethyl alcohol, acetic acid, reflux 3 h.
order to produce the key intermediates N-substituted-3-(hydrazo- against three different cancer cell lines viz.; A-549, MDA-MB-231,
nomethyl)-1H-indoles 5a–d. In Scheme 2, the key intermediates
5a–d were reacted with different isatin derivatives 6a–g in abso-
lute ethyl alcohol with the aid of catalytic amount of glacial acetic
acid to furnish targeted [(3-indolylmethylene)hydrazono]indoli-
nones 7, 8a–c, 9 and 10a–e.
Postulated structures for the newly prepared key intermediates
and target compounds 7, 8a–c, 9 and 10a–e have been in full
consistent with the data of both spectral and elemental analyses.
and HCT-116, using the sulforhodamine B colorimetric (SRB)
assay21. Staurosporine, a clinically used antitumor drug, was co-
assayed as a reference agent for the experiment. The results have
been conveyed as IC50 values, and listed in Table 1.
Investigation of the obtained IC50 values (Table 1) hinted out
that the tested N-alkylindole-isatin derivatives (7, 8a–c, 9 and
10a–e) were more effective against colon HCT-116 cells than
NSCLC A-549 and TNBC MDA-MB-231 cancer cells, except com-
pound 8b that displayed enhanced anti-proliferative potency
against A-549 and MDA-MB-231 cell lines (IC50 ¼ 9.2 0.78 and
2.2. Biological evaluation
8.9 0.61 mM, correspondingly) than HCT-116 cells (IC50
¼
2.2.1. Antiproliferative activities towards A-549, MDA-MB-231 and
HCT-116 cancer cell lines
19.1 1.34 mM), and compound
enhanced antiproliferative action against breast MDA-MB-231
10.4 0.80 mM) than colon HCT-116 cells (IC50
were investigated for the prospective growth inhibitory actions ¼ 28.2 1.73 mM).
9
that exhibited slightly
All the newly prepared target conjugates (7, 8a–c, 9 and 10a–e) cells (IC50
¼

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1303
Table 1. Anti-proliferative activity of compounds 7, 8a–c, 9 and 10a–e against
human cancer A-549, MDA-MB-231 and HCT-116 cell lines.
2.2.2. Nci-USA cancer cell lines screening
Seven hybrids (8a–c, 9, 10a, 10c and 10d) have been selected
and screened by USA-National Cancer Institute (NCI-DTP; www.
dtp.nci.nih.gov) for their possible in vitro anti-proliferative actions
towards a panel of 59 human cancer cell lines representing breast,
ovarian, CNS, colon, NSCLC, leukaemia, melanoma, renal and pros-
tate cancers, according to the NCI, Bethesda, Drug Evaluation
Branch protocol22–24
.
2.2.2.1. Preliminary single (10 mM) dose screening. Antitumor activ-
ities of here reported conjugates 8a–c, 9, 10a, 10c and 10d were
first screened in an initial single dose (10 mM) screening, utilising
the SRB assay to estimate cells viability and growth21. The
obtained results were presented as percent growth inhibition (GI
%) for the examined conjugates towards the different treated
tumour cell lines, as presented in Table 2.
Exploring the attained GI% values (Table 2), disclosed that
hybrid 8a stood out as the most efficient anti-proliferative agent
in the herein reported NCI screening exhibiting mean GI % equals
to 59, with broad-spectrum action towards all human cancer cell
lines in all the herein examined cancer subpanels, except towards
Renal cancer TK-10 cell line.
Remarkably, conjugate 8a showed excellent growth inhibition
properties against Leukaemia (K-562 and SR), NSCLC (NCI-H522),
Colon cancer (HT29), Ovarian cancer (OVCAR-3), Prostate cancer
(PC-3), Renal cancer (RXF 393), and Breast cancer (MCF7) cell lines
with percentage inhibition of 83, 89, 86, 83, 98, 89, 82 and 91%,
respectively (Table 2, Figure 2). Furthermore, compound 8a
exerted good activity with growth inhibition % equal to or greater
than 60 towards NSCLC (HOP-92 and NCI-H460), Colon cancer
(SW-620, HCT-116 and HCT-15), CNS cancer (SF-539 and U251),
Melanoma (LOX IMVI, SK-MEL-2 and UACC-62), Ovarian cancer
(IGROV1) and Breast cancer (MDA-MB-231, T-47D and MDA-MB-
468) cell lines with inhibition percent of 63, 68, 60, 77, 64, 66, 60,
69, 65, 60, 61, 66, 61 and 75% respectivly (Table 2, Figure 2). On
the other hand, the remaining herein examined hybrids 8b, 8c, 9,
10a, 10c and 10d displayed moderate to weak antitumor activities
(GI % range: 4–11). It is worth stressing that hybrid 8a exerted a
cytotoxic impact with GI % >100 against some tumour cell lines.
Hybrid 8a was shown to be lethal towards Leukaemia (HL-60(TB),
CCRF-CEM, MOLT-4 and RPMI-8226) cells with GI % values equal
to 101, 123, 110 and 125, respectively, whereas, 8a exerted its
lethal action towards Colon cancer HCT-116 and Melanoma MDA-
MB-435 cells with GI % ¼ 116 and 126, respectively (Table 2).
IC50 (mM)a
Comp.
R
X
A-549
MDA-MB-231
HCT-116
7
8a
8b
8c
CH2CH3
5-Cl
5-CH3
5-OCF3
5-NO2
H
50.0 3.37
7.3 0.42
9.2 0.78
12.3 1.05
31.7 2.27
17.3 1.19
59.9 3.58
43.8 2.96
53.1 4.13
40.2 1.88
2.3 0.17
27.6 1.76
4.7 0.28
8.9 0.61
15.6 0.98
10.4 0.80
11.7 1.04
54.0 4.17
35.4 1.92
42.9 2.51
51.8 3.77
4.5 0.29
19.7 1.28
2.6 0.17
19.1 1.34
6.4 0.50
28.2 1.73
7.7 0.68
36.3 2.35
7.3 0.47
28.5 1.59
37.4 2.06
3.7 0.24
CH2CH2CH3
CH2CH2CH3
CH2CH2CH3
CH(CH3)2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
CH2CH¼CH2
-
9
10a
10b
10c
10d
10e
Dox.
H
5-F
7-F
5-Cl
5-OCF3
-
aIC50 values are the mean SE of 3 discrete experiments.
With respect to the cytotoxic activity against colon HCT-116
cell line, hybrid 8a stood out as the most potent analogue herein
reported with IC50 ¼ 2.6 0.17 mM, which is more effective than
reference drug doxorubicin (IC50 ¼ 3.7 0.24 mM). Also, conjugates
8c, 10a and 10c exhibited potent anti-proliferative activity (IC50
¼
6.4 0.50, 7.7 0.68 and 7.3 0.47 mM, respectively) with about 2-
fold decreased efficiency than doxorubicin. Moreover, compounds
7 and 8b possessed moderate potency with IC50 values equal
19.7 1.28 and 19.1 1.34 mM, respectively, whereas compounds 9,
10b, 10d and 10e elicited weak growth inhibitory activity towards
HCT-116 cells (IC50 ¼ 28.2 1.73, 36.3 2.35, 28.5 1.59 and
37.4 2.06 mM, respectively).
Exploring anti-proliferative activities towards NSCLC A-549 cells
revealed that hybrid 8a was the most efficient counterpart (IC50
¼
2.2.2.2. In vitro full NCI panel five dose assay. The obtained data
from the preliminary single dose (10 mM) screening disclosed that
hybrid 8a (NSC: D-771612/1) is the most efficient anticancer agent
here in this study, displaying promising effectiveness against
numerous cancer cell lines from different subpanels (Table 2,
Figure 2). Subsequently, hybrid 8a was chosen to carry out further
biological screening at a (0.01–100 mM) five-dose assay. Three
response parameters (GI50, TGI and LC50) were evaluated for
hybrid 8a towards each herein examined cancer cell line, and
listed in Table 3. GI50 values reflect the level for growth inhibitory
effect, whereas TGI represents cytostatic impact. In addition, LC50
parameter is considered to be the cytotoxicity parameter for the
examined hybrid. Moreover, both full panel and subpanel mean
graph midpoints (MG-MID), for full panel and individual subpanels
cell lines, were calculated on the level of GI50 parameter, provid-
ing an average potency parameter for the tested hybrid 8a
(Table 4).
7.3 0.42 mM), with about 3-fold dwindled efficiency compared to
the reference compound doxorubicin (IC50 ¼ 2.3 0.17 mM) against
A-549 cells. Moreover, compounds 8b, 8c and 10a exhibited effi-
cient anti-proliferative action towards A-549 cells with IC50 values
equal 9.2 0.78, 12.3 1.05 and 17.3 1.19 mM, respectively. With
regards to the cytotoxicity against the breast MDA-MB-231 cell
line, the data shown in Table 1 ascribed to the examined hybrids
excellent to weak efficacy in inhibiting the proliferation of MDA-
MB-231 cells with IC50 values ranging between 4.7 0.28 and
42.9 2.51 mM, except compounds 10b and 10e that failed to
inhibit the growth of breast MDA-MB-231 cells up to 50 mM (IC50
¼ 54.0 4.17, and 51.8 3.77 mM, respectively). Superiorly, com-
pound 8a emerged as the most effective derivative in the current
study towards MDA-MB-231 cells with IC50 value equals
4.7 0.28 mM. Besides, compounds 8b, 8c, 9 and 10a displayed
good anti-proliferative activity against MDA-MB-231 cells (IC50
8.9 0.61, 15.6 0.98, 10.4 0.80 and 11.7 1.04 mM, respectively).
¼

1304
T. AL-WARHI ET AL.
Table 2. In vitro GI % for compounds (8a–c, 9 and 10a, c, d), at 10 lM concen-
% Sub-G1
%G2/M
%S
100
50
0
tration, towards the subpanel cancer cell lines
Compounda
Subpanel / Cell line
8a
8b
52
12
33
50
30
–
8c
9
10a
10c
10d
%G0-G1
Leukaemia
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
101
123
83
110
125
89
–
10
18
–
–
19
–
18
–
13
–
11
–
22
30
35
13
50
17
–
20
19
–
–
–
–
14
–
33
58
l
o
8a
r
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
nt
Co
36
50
25
63
116
52
53
68
86
28
40
21
–
–
12
–
13
–
–
11
–
–
–
19
–
–
38
–
14
16
–
–
–
10
–
–
–
–
15
–
–
56
–
–
–
34
–
33
–
–
41
–
–
–
17
Figure 2. Impact of hybrid 8a on the cell cycle progression in breast cancer
T-47D cells.
–
13
12
–
–
31
As shown in Table 3, hybrid 8a displayed potent anti-prolifera-
tive action at a single-digit micromolar concentration against all
herein examined human cancer cell lines with GI50 values range:
1.17–8.41 mM, except for Melanoma (UACC-257), Renal (TK-10) and
Breast (HS 578 T) cell lines which possessed GI50 values equal
12.30, 11.10 and 11.30 mM, respectively (Table 3). Interestingly,
hybrid 8a showed superior sub-micromolar activity towards leu-
kaemia (MOLT-4 and SR), Ovarian cancer (OVCAR-4) and Breast
11
19
116
53
60
77
83
40
64
–
–
21
23
25
–
–
–
20
–
–
11
–
–
–
–
–
–
–
–
30
–
24
15
10
–
–
–
28
22
–
–
–
–
10
–
–
HT29
KM12
SW-620
–
–
cancer (T-47D) cells (GI50
respectively).
¼
0.68, 0.52, 0.93 and 0.41 mM,
–
–
–
CNS cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
54
29
66
37
15
60
–
–
–
–
–
–
–
10
22
–
–
–
–
–
33
–
–
–
–
–
18
10
–
–
–
–
–
–
–
–
–
–
–
–
In addition, hybrid 8a showed potent cytostatic activity at sin-
gle-digit micromolar concentration (TGI range: 2.96–8.03 mM)
against 22 cancer cell lines belonging to all herein examined can-
cer subpanels, except CNS cancer subpanel (Table 3). While hybrid
8a had no cytostatic impact (TGI > 100 mM) against leukaemia
(CCRF-CEM), NSCLC (EKVX and NCI-H322M), colon cancer (HT29),
CNS cancer (SNB-19), renal cancer (ACHN and TK-10), ovarian can-
cer (OVCAR-8), and breast cancer (HS 578 T) cells, it exhibited
good to weak cytostatic activity towards the remaining cancer cell
lines with TGI spanning in the interval: 10.6–96.6 mM. On the other
hand, compound 8a emerged as a non-lethal agent that possesses
LC50 values more than 100 mM for the most of cancer cell lines
herein examined, except for Colon cancer (COLO 205, HCC-2998
and HCT-116), NSCLC (NCI-H460), Prostate cancer (PC-3),
Melanoma (LOX IMVI, M14, MDA-MB-435 and SK-MEL-5), and
Breast cancer (MDA-MB-468) cell lines (LC50 ¼ 75.7, 7.46, 19.0,
9.96, 7.41, 17.2, 5.85, 39.0, 47.40 and 79.40 mM, respectively
(Table 3).
13.32
28
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
69
49
44
127
65
41
51
41
60
–
–
–
–
–
–
–
–
11
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
26
12
–
–
–
–
–
25
–
–
–
–
–
–
–
–
15
–
–
–
–
–
–
–
–
19
61
98
58
16
53
24
12
–
–
–
–
–
–
–
29
–
–
–
–
–
–
–
–
28
21
27
–
15
–
31
13
22
–
11
–
23
–
–
–
–
–
15
15
–
–
–
With regards to the sensitivity for diverse tumour cell lines,
hybrid 8a possessed relatively homologous growth inhibitory
action for the whole of NCI panel, with efficient sub-panel GI50
(MG-MID) range of 1.33–4.23 mM, and promising full-panel GI50
(MG-MID) ¼ 3.10 mM. leukaemia, prostate cancer, colon cancer,
and breast cancer subpanels were the most vulnerable herein
examined cancer subpanels to the impact of hybrid 8a [GI50 (MG-
MID) ¼ 1.33, 2.47, 2.85 and 3.09 mM, respectively] (Table 4).
Furthermore, dividing the full-panel MG-MID (mM) for the
examined derivative by its individual subpanel MG-MID (mM) pro-
vides an index which is regarded as a measure for its selectivity. A
value between three and six refers to moderate selectivity against
the corresponding cancer cell line, ratio more than six indicates
high selectivity, whereas if the compound does not meet any of
such criteria is considered as non-selective25. In this regards,
hybrid 8a was found to be a non-selective anticancer agent that
exhibits broad-spectrum potency towards all cancer subpanels
herein examined at the GI50 level, with selectivity ratios spanning
in the range from 0.73 to 2.33 (Table 4).
–
–
–
Renal cancer
786-0
A498
ACHN
RXF 393
SN12C
TK-10
UO-31
Prostate
PC-3
36
43
45
89
51
–
–
–
15
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
14
–
–
–
11
–
–
–
–
11
–
–
–
–
–
–
–
–
–
23
40
19
33
29
82
34
91
32
13
29
–
–
15
–
–
10
22
–
33
10
–
31
–
–
18
DU-145
MCF7
Breast cancer
MDA-MB-231
HS 578T
T-47D
MDA-MB-468
BT-549
66
26
61
75
37
58
–
–
35
11
–
–
–
–
17
–
14
11
–
13
–
–
12
–
–
56
20
–
–
–
50
54
–
–
–
30
36
–
Sensitive cell lines no.
aOnly GI % higher than 10% are shown.
26
27
20
12

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1305
Table 3. Results of NCI-USA in vitro five-dose testing for conjugate 8a (NSC: D- Table 4. Median GI50 (mM) values for subpanel cancer cell lines for conju-
795311/1).
gate 8a.
Compound
8a
8a
Subpanel tumour cell line
MG-MID
Selectivity index
Leukaemia
1.33
3.22
2.47
4.08
3.45
3.24
4.23
2.85
3.09
3.10
2.33
0.96
1.25
0.75
0.89
0.95
0.73
1.08
1.00
Subpanel/tumour cell lines
GI50 (mM)
TGI (mM)
LC50 (mM)
NSCL Cancer
Colon Cancer
CNS Cancer
Leukaemia
CCRF-CEM
HL60(TB)
K-562
MOLT-4
RPMI-8226
SR
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
HOP-92
1.94
1.31
2.22
0.68
1.33
0.52
>100
4.80
20.5
4.54
3.96
8.72
>100
>100
>100
>100
>100
>100
Melanoma
Ovarian Cancer
Renal Cancer
Prostate Cancer
Breast Cancer
Full panel MG-MID
7.18
2.36
4.17
1.87
2.95
3.13
3.42
2.17
1.81
39.6
>100
25.0
6.57
20.6
38.2
>100
10.6
6.59
>100
>100
>100
>100
>100
>100
>100
75.7
2.2.3. Cell cycle analysis
The superior sub-micromolar anti-proliferative activity of conjugate
8a on breast cancer T-47D cells (GI50 ¼ 0.41 mM, Table 3)
prompted us to further investigate about the growth inhibitory
mechanism of the target conjugates. Both regulation of cell cycle
progression and apoptosis induction have been considered as sig-
nificant strategies to control the proliferation of different cancer
cells, accordingly, we primarily examined the growth inhibition
mechanism of hybrid 8 in relation to cell cycle progression and
regulation in human breast T-47D cancer cells.
The impact on cell cycle distribution was assessed by a DNA
flow cytometry analysis, upon incubation of T-47D cells with con-
jugate 8a at its GI50 concentration (0.41 mM) for 24 h (Figure 3).
From the obtained results it was found that T-47D cells exposed
to hybrid 8 significantly arrested at the G2/M phase of the cell
cycle with an escalation in G2/M phase fraction from 19.19% (in
control cells) to 34.05% (in 8a-treated T-47D cells). Furthermore,
the cell population in sub-G1 phase was drastically augmented
from 1.25% (in control cells) to 17.73% (in 8a-treated T-47D cells).
Generally the upsurge of populations in the sub-G1 phase indi-
cates the induction of apoptotic cell death. So, a subsequent
study will be conducted to reveal whether the G2/M phase cell
cycle arrest afforded by conjugate 8a was accompanied
by apoptosis.
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
CNS cancer
SF-268
>100
1.25
2.09
1.75
2.63
3.4
3.22
2.97
3.06
5.14
4.17
42.2
>100
19.7
35.3
7.46
19.0
9.96
>100
>100
>100
>100
3.33
8.41
2.69
4.24
2.31
3.51
28.0
96.6
17.1
>100
46.0
17.3
>100
>100
>100
>100
>100
>100
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
RXF 393
SN12C
TK-10
UO-31
1.26
1.88
1.92
1.50
2.42
4.70
2.86
12.30
2.24
3.06
6.03
4.70
2.96
8.03
7.41
100
17.2
5.85
>100
>100
39.0
>100
>100
36.9
13.50
66.60
12.10
3.38
1.36
0.93
4.04
5.41
6.39
1.17
63.10
5.11
25.60
25.8
>100
89.90
67.10
>100
>100
>100
>100
>100
>100
>100
2.2.4. Apoptosis assay
In an attempt to further investigate whether the antiproliferative
activity for conjugate 8a is harmonious with the apoptosis induc-
tion within T-47D cells pointed out by the increased cell popula-
tion in sub-G1 phase in 8a-treated T-47D cells (Figure 3), Annexin
V-FITC/PI dual staining analysis was used for the apoptosis assay
(Figure 4).
The outcomes of the Annexin V-FITC/PI assay suggested that
treatment of T-47D cells with conjugate 8a led to an early and
late cellular apoptosis, which proved through the significant
increase for the apoptotic cells percentage in both the early apop-
totic phase (from 0.26% to 4.82%) and the late apoptotic phase
(from 0.47% to 14.62%) that signifies about 26-fold increase in
total apoptosis, when compared to the untreated control
(Figure 4).
5.94
1.37
3.56
2.17
3.20
11.10
2.28
71.20
7.50
>100
7.94
75.90
>100
6.18
>100
>100
>100
>100
>100
>100
>100
Prostate cancer
PC-3
DU-145
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
MDA-MB-468
1.83
3.88
5.17
40.60
47.40
>100
1.54
1.81
11.3
1.79
0.41
1.69
15.50
7.40
>100
6.33
18.40
5.78
>100
>100
>100
>100
>100
79.40
2.2.5. Cdk2 and Bcl-2 inhibitory activity
The promising anti-proliferative impact of conjugate 8a, in add-
ition to its cell cycle disruption and pro-apoptotic effects, pro-
voked a further exploration for their possible inhibitory activities
against the cell cycle regulator CDK2 protein kinase and the anti-
NT: not tested.

1306
T. AL-WARHI ET AL.
Figure 3. Effect of conjugate 8a over the AV-FITC-positive staining percentages in breast T-47D cells.
Figure 4. (a) Docking pose of hybrid 8a within the active site of CDK2 (pdb code 2BHE) showing hydrogen bond with Leu83 (green dots) and hydrophobic interaction
(light purple dots). (b) Hydrophobic surface of active site of CDK2 surround compound 8a.
molecules8–11. In the docking simulations 8a has taken the planar
Table 5. Inhibitory activities of compound 8a against CDK2 and Bcl-2.
IC50 (lM)a
orientation, although it has a certain flexibility, with some little
deviation at the indole moiety to fill the part of the corresponding
hydrophobic pocket by hydrophobic contact with Lys89 as shown
in (Figure 4(b)), whereas the N-propyl group interacted with the
part of the terminal hydrophobic pocket by alkyl hydrophobic
contact with Lys20. Moreover, the Molecular docking simulations
showed an important hydrogen bond interaction with Leu83, via
C¼O of isatin, which is a key interaction with distance 2.5 Å. Also
the docking poses of hybrid 8a showed that 5-methyl group, dec-
orated on the benzenoid part of the isatin moiety, made an
anchoring hydrophobic mixed with p interactions with Val18 and
Compound
CDK2
Bcl-2
8a
0.85 0.03
0.1 0.01
–
0.46 0.02
–
0.09 0.01
Roscovitine
Venetoclax
aIC50 values are the mean SE of three separate experiments.
apoptotic Bcl-2 protein. The results expressed as IC50 values are
presented in Table 5.
Results in Table 5 showed that the tested hybrid 8a exhibited
good inhibitory action against CDK2 and Bcl-2 with IC50 values Leu134 from both sides as shown in Figure 4(a).
On the other hand, the molecular docking studies explored the
equal 0.85 0.03 and 0.46 0.02 mM, respectively.
binding pattern of hybrid 8a within the active site of Bcl-2 to jus-
tify its apoptotic effect. As reported, the pro-apoptotic effect of
the small molecules could be done at the molecular level by mim-
icking the BH3 a-helix and then binding to the hydrophobic
groove of anti-apoptotic Bcl-2 proteins preventing their heterodi-
merization, which eventually leads to apoptosis27. The attitude of
a-helical BH3 domains complexed with Bcl-2 family besides the
docking poses of other small molecules as Bcl-2 inhibitors were
3. Molecular docking studies
The docking simulation studies were conducted to investigate the
behaviour of compound 8a in the active site of both CDK2 and
Bcl-2 (pdb code 2BHE7 and 426). From several X-ray structures for
CDK2 enzyme, as mentioned before in the introduction, it was
observed that the active site should be filled by planar discussed as a guide for our docking study28–31
.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1307
Figure 5. (a) Compound 8a docked into the groove of Bcl-2 (4AQ3) showing the different interaction from both sides to fill this groove. (b) Compound 8a embedded
inside the active site of Bcl-2. The macromolecule was surrounded by hydrophobic surface to focus on the hydrophobic contacts.
Molecular docking of compound 8a, as a small molecule inhibi- several times, dried and recrystallized from isopropyl alcohol to
tor, inside the hydrophobic groove of Bcl-2 showed significant furnish N-substituted-3-(hydrazonomethyl)-1H-indole derivatives
5a–d, respectively.
interactions at the front of this groove (Figure 5(a)). The (NH) func-
tionality of isatin moiety can donate a hydrogen bond (2.00 Å) to
the key amino acid Asn102 at the margin of the hydrophobic
groove. This anchoring hydrogen bond was supported by the
p-stack of isatin ring with Arg105 (Figure 5(a)). On the other side
of the groove, N-propyl group attached to indole ring was
embedded in the hydrophobic pocket formed of three amino
acids; Phe112, Phe71 and Met74. The p-stack interaction of indole
ring with Leu96 and Ala108 would act as a middle support for the
whole compound inside the opening of the hydrophobic groove
(Figure 5(b)).
4.1.3. General procedures for preparation of target compounds
7, 8a–c, 9 and 10a–e
To a hot stirred solution of isatin derivatives 6a–g (1 mmol) in
absolute ethyl alcohol (5 ml) and catalytic drops of acetic acid, the
appropriate N-substituted-3-(hydrazonomethyl)-1H-indole deriva-
tive 5a–d (1 mmol) was added. The resulting mixture was stirred
at reflux for 3 h, and then filtrated. The collected precipitate was
dried and then recrystallized from isopropyl alcohol to afford tar-
get hybrids 7, 8a–c, 9 and 10a–e, respectively.
Full characterisation for the key intermediates (5a, 5c and 5d)
as well as the target conjugates (7, 8a–c, 9 and 10a–e) have been
presented in the Supporting Information.
4. Experimental
4.1. Chemistry
4.1.1. General
4.2. Biological evaluation
Melting points were measured with a Stuart melting point appar-
atus, and are uncorrected. Nuclear magnetic resonance; 1H NMR
and 13C NMR spectra were recorded using a Bruker NMR spec-
trometer (400/100 MHz), in deuterated dimethylsulphoxide (DMSO-
d6). Chemical shifts (dH) were reported relative to TMS as an
internal standard. The coupling constant values (J) were given in
hertz. Chemical shifts (dC) were reported as follows: s, singlet; d,
doublet; m, multiplet. HRMS spectra were performed on a Bruker
MicroTOF spectrometer. Infra-red (IR) Spectra are obtained by the
use of Schimadzu FT-IR 8400S spectrophotometer as KBr discs and
expressed in wave number (cmꢁ1). Compounds 2 and 4a–d were
prepared as reported previously32.
Experimental procedures for biological evaluations for the herein
reported conjugates (7, 8a-c, 9 and 10a-e) were provided in the
Supplementary material (Supplementary materials can be found at
www.mdpi.com/xxx/s1).
4.2.1. Antiproliferative action against A-549, MDAMB-231 and
HCT-116 cancer cell lines
The three examined cancer cell lines (non-small cell lung A-549,
Breast MDA-MB-231 and colon HCT-116 cells) were obtained from
American Type Culture Collection (ATCC). Examination of the cyto-
toxic activity for the target conjugates was carried out in accord-
ance with the SRB colorimetric assay protocol21, as described
previously33.
4.1.2. Synthesis of N-substituted-3-(hydrazonomethyl)-1H-indole
derivatives 5a–d
To a solution of N-substituted-1H-indole-3-carbaldehyde deriva-
tives 4a–d (4 mmol) in ethyl alcohol (25 ml), an excess of 99% 4.2.2. Nci-60 cancer cell lines screening
hydrazine hydrate (1.25 ml, 25 mmol) was added. The reaction mix- The NCI-USA anticancer screening was carried out following the
ture was stirred at reflux temperature for 2 h, and then filtrated NCI, Bethesda, Drug Evaluation Branch protocol22–24, utilising the
upon cooling. The obtained precipitate was washed with water SRB protein assay21, as reported earlier34,35
.

1308
T. AL-WARHI ET AL.
4.2.3. Cell cycle analysis
Funding
Flow cytometric analysis (FACS) was carried out to examine the
cell cycle distributions in breast T-47D cancer cells upon treatment
with conjugate 8a, by BD FACS Calibur flow cytometer, as
described earlier36.
The authors extend their appreciation to the Deanship of
Scientific Research at Princess Nourah bint Abdulrahman
University for funding this work through the Research Groups
Programme [Grant No. RGP-1440–0025].
4.2.4. Apoptosis assay
Phosphatidylserine externalisation effect of conjugate 8a over
breast T-47D cancer cell line was investigated using Annexin-V-
FITC Apoptosis Detection Kit by flow cytometry, as
described earlier37.
References
1. Schwartz GK. CDK inhibitors: cell cycle arrest versus apop-
tosis. Cell Cycle 2002;1:113–4.
2. Damla Arisan E, Obakan P, Coker-Gurkan A, et al. CDK inhibi-
tors induce mitochondria-mediated apoptosis through the
activation of polyamine catabolic pathway in LNCaP, DU145
and PC3 prostate cancer cells. Curr Pharm Des 2014;20:
180–8.
4.2.5. Cdk2 and bcl-2 inhibitory activity
These assays were carried out as reported earlier38,39
.
3. Cho S-J, Kim Y-J, Surh Y-J, et al. Ibulocydine is a novel pro-
drug Cdk inhibitor that effectively induces apoptosis in hep-
atocellular carcinoma cells. J Biol Chem 2011;286:19662–71.
4. Damiens E, Baratte B, Marie D, et al. Anti-mitotic properties
of indirubin-3’-monoxime, a CDK/GSK-3 inhibitor: induction
of endoreplication following prophase arrest. Oncogene
2001;20:3786–97.
4.3. Molecular docking studies
The target proteins, CDK2 (2BHE) and BCl-2 (4AQ3), were down-
loaded from protein databank and prepared by the default proto-
col in Discovery Studio 4. All other steps including preparation of
small molecules, determination of the active site and docking
protocol were followed as reported40.
0
€
5. Perabo FGE, Frossler C, Landwehrs G, et al. Indirubin-3 -mon-
oxime, a CDK inhibitor induces growth inhibition and apop-
tosis-independent up-regulation of survivin in transitional
cell cancer. Anticancer Res 2006;26:2129–35.
5. Conclusions
To conclude, a novel series of N-alkylindole-isatin conjugates (7,
8a–c, 9 and 10a-e) was designed and synthesised, utilising the
SAR outputs from the previous study. The design is based on
appending four alkyl groups with different length (ethyl, n-propyl),
bulkiness (isopropyl) and unsaturation (allyl) on N-1 of indole
motif, with subsequent conjugation with different N-unsubstituted
isatin moieties to furnish the target conjugates. As planned, the
adopted strategy achieved a substantial improvement in the
growth inhibitory profile for the target conjugates in comparison
to the reported lead VI. All conjugates were screened for their
potential cytotoxicity towards a panel of three different human
cancer cell lines HCT-116 (Colon), A-549 (NSCLC), and MDA-MB-
231 (Breast) utilising SRB assay. Conjugate 8a, superiorly, displayed
the best anti-proliferative activity against the three examined cell
lines (IC50 ¼ 7.3 0.42, 4.7 0.28 and 2.6 0.17 mM, respectively).
Furthermore, 8a displayed broad spectrum anti-proliferative action
with efficient subpanel GI50 (MG-MID) range of 1.33–4.23 mM, and
promising full panel GI50 (MG-MID) ¼ 3.10 mM, at the NCI five-
dose assay. In particular, hybrid 8a showed superior sub-micromo-
lar activity towards leukaemia (MOLT-4 and SR), ovarian cancer
(OVCAR-4) and breast cancer (T-47D) cell lines (GI50 ¼ 0.68, 0.52,
0.93 and 0.41 mM, respectively). Conjugate 8a was able to provoke
cell cycle disturbance and apoptosis in breast T-47D cells as evi-
denced by the DNA flow cytometry and Annexin V-FITC/PI assays.
Moreover, hybrid 8a exhibited good inhibitory action against cell
cycle regulator CDK2 protein kinase and the anti-apoptotic Bcl-2
protein (IC50 ¼ 0.85 0.03 and 0.46 0.02 mM, respectively). The
molecular docking for hybrid 8a in CDK2 and Bcl-2 active sites
unveiled that N-propyl group is involved in significant hydropho-
bic interactions. Taken together, the results suggested conjugate
8a as a promising lead for further development and optimisation
as an efficient antitumor drug.
6. Davies TG, Tunnah P, Meijer L, et al. Inhibitor binding to
active and inactive CDK2: the crystal structure of CDK2-cyc-
lin A/indirubin-5-sulphonate. Structure 2001;9:389–97.
€
7. Jautelat R, Brumby T, Schafer M, et al. From the insoluble
dye indirubin towards highly active, soluble CDK2-inhibitors.
ChemBioChem 2005;6:531–40.
8. Yan L, Lai F, Chen X, Xiao Z. Discovery of novel indirubin-3’-
monoxime derivatives as potent inhibitors against CDK2 and
CDK9 . Bioorg Med Chem Lett 2015;25:2447–51.
9. Ahn M-Y, Kim T-H, Kwon S-M, et al. 5-Nitro-5’-hydroxy-indir-
ubin-3’-oxime (AGM130), an indirubin-3’-oxime derivative,
inhibits tumor growth by inducing apoptosis against non-
small cell lung cancer in vitro and in vivo. Eur J Pharm Sci
2015;79:122–31.
10. Wood DJ, Korolchuk S, Tatum NJ, et al. Differences in the
conformational energy landscape of CDK1 and CDK2 sug-
gest a mechanism for achieving selective CDK inhibition.
Cell Chem Biol 2019;26:121–30.
11. Bramson HN, Corona J, Davis ST, et al. Oxindole-based inhib-
itors of cyclin-dependent kinase 2 (CDK2): design, synthesis,
enzymatic activities, and X-ray crystallographic analysis.
J Med Chem 2001;44:4339–58.
12. Attia MI, Eldehna WM, Afifi SA, et al. New hydrazonoindolin-
2-ones: Synthesis, exploration of the possible anti-prolifera-
tive mechanism of action and encapsulation into PLGA
microspheres. PLoS One 2017;12:e0181241.
13. 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 Enzyme
Inhib Med Chem 2018;33:867–78.
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
Devel Ther 2017;11:2333–46.
Disclosure statement
No potential conflict of interest was reported by the author(s).

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
1309
15. Eldehna WM, Almahli H, Al-Ansary GH, et al. Synthesis and 28. Czabotar PE, Lee EF, van Delft MF, et al. Structural insights
into the degradation of Mcl-1 induced by BH3 domains.
Proc. Natl. Acad. Sci. U.S.A 2007;104:6217–22.
29. Porter J, Payne A, de Candole B, et al. Whitcombe, tetrahy-
droisoquinoline amide substituted phenyl pyrazoles as
selective Bcl-2 inhibitors. Bioorg Med Chem Lett 2009;19:
230–3.
30. Enyedy IJ, Ling Y, Nacro K, et al. Discovery of small-molecule
inhibitors of Bcl-2 through structure-based computer screen-
ing. J Med Chem 2001;44:4313–24.
in vitro antiproliferative activity of some novel isatins conju-
gated with quinazoline/phthalazine hydrazines against tri-
ple-negative breast cancer MDA-MB-231 cells as apoptosis-
inducing agents. J Enzym Inhib Med Chem 2017;32:600–13.
16. 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.
31. Oltersdorf T, Elmore SW, Shoemaker AR, et al. An inhibitor
of Bcl-2 family proteins induces regression of solid tumours.
Nature 2005;435:677–81.
32. Abo-Ashour MF, Eldehna WM, George RF, et al. Novel
indole-thiazolidinone conjugates: design, synthesis and
whole-cell phenotypic evaluation as a novel class of anti-
microbial agents. Eur J Med Chem 2018;160:49–60.
33. Eldehna WM, El Kerdawy AM, Al-Ansary GHA, et al. Type
IIA–Type IIB protein tyrosine kinase inhibitors hybridization
as an efficient approach for potent multikinase inhibitor
development: Design, synthesis, anti-proliferative activity,
multikinase inhibitory activity and molecular modeling of
novel indolinone-based ureides and amides. Eur J Med
Chem 2019;163:37–53.
34. Abou-Seri SM, Eldehna WM, Ali MM, El Ella DA. 1-
Piperazinylphthalazines as potential VEGFR-2 inhibitors and
anticancer agents: Synthesis and in vitro biological evalu-
ation. Eur J Med Chem 2016;107:165–79.
35. Eldehna WM, Hassan GS, Al-Rashood ST, et al. Synthesis and
in vitro anticancer activity of certain novel 1-(2-methyl-6-
arylpyridin-3-yl)-3-phenylureas as apoptosis-inducing agents.
J Enzyme Inhib Med Chem 2019;34:322–32.
36. Sabt A, Abdelhafez OM, El-Haggar RS, et al. Novel coumarin-
6-sulfonamides as apoptotic anti-proliferative agents:
Synthesis, in vitro biological evaluation, and QSAR studies. J
Enzyme Inhib Med Chem 2018;33:1095–107.
37. 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 MDA-
MB-231 cells. J Enzyme Inhib Med Chem 2018;33:309–18.
38. Said MA, Eldehna WM, Nocentini A, et al. Sulfonamide-based
ring-fused analogues for CAN508 as novel carbonic anhy-
drase inhibitors endowed with antitumor activity: design,
synthesis, and in vitro biological evaluation. Eur J Med
Chem 2020;189:112019.
39. Abo-Ashour MF, Eldehna WM, Nocentini A, et al. 3-
Hydrazinoisatin-based benzenesulfonamides as novel car-
bonic anhydrase inhibitors endowed with anticancer activity:
Synthesis, in vitro biological evaluation and in silico insights.
Eur J Med Chem 2019;184:111768.
17. Eldehna WM, Nocentini A, Al-Rashood ST, et al. Tumor-asso-
ciated carbonic anhydrase isoform IX and XII inhibitory
properties of certain isatin-bearing sulfonamides endowed
with in vitro anticancer activity towards colon cancer. Bioorg
Med Chem 2018;81:425–32.
18. Eldehna WM, Abo-Ashour MF, Nocentini A, et al.
Enhancement of the tail hydrophobic interactions within the
carbonic anhydrase IX active site via structural extension:
design and synthesis of novel N-substituted isatins-SLC-0111
hybrids as carbonic anhydrase inhibitors and antitumor
agents. Eur J Med Chem 2019;162:147–60.
19. Eldehna WM, Fares M, Ibrahim HS, et al. Indoline ureas as
potential anti-hepatocellular carcinoma agents targeting
VEGFR-2: synthesis, in vitro biological evaluation and
molecular docking. Eur J Med Chem 2015;100:89–97.
20. Eldehna WM, Abo-Ashour MF, Ibrahim HS, et al. Novel [(3-
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.
21. Skehan P, Storeng R, Scudiero D, et al. New colorimetric
cytotoxicity assay for anticancer-drug screening.
J Natl
Cancer Inst 1990;82:1107–12.
22. Monks A, Scudiero D, Skehan P, et al. Feasibility of a high-
flux anticancer drug screen using a diverse panel of cultured
human tumor cell lines. J Natl Cancer Inst 1991;83:757–66.
23. Boyd MR. Cancer. In: Teicher BA, ed. Drug discovery and
development: anticancer drug development guide: preclin-
ical screening, clinical trials and approval, 2nd ed. Totowa,
NJ: Humana Press; 2014:41–62.
24. Boyd MR, Paull KD. Some practical considerations and appli-
cations of the National Cancer Institute in vitro anticancer
drug discovery screen. Drug Dev Res 1995;34:91–109.
25. Ibrahim HS, Eldehna WM, Fallacara AL, et al. One-pot synthe-
sis of spiro(indoline-3,4’-pyrazolo[3,4-b]pyridine)-5’-carboni-
triles as p53-MDM2 interaction inhibitors. Future Med Chem
2018;10:2771–89.
26. Perez HL, Banfi P, Bertrand J, et al. Schmidt, Identification of
a phenylacylsulfonamide series of dual Bcl-2/Bcl-xL antago-
nists. Bioorg Med Chem Lett 2012;22:3946–50.
27. Delgado-Soler L, Pinto M, Tanaka-Gil K, Rubio-Martinez J.
Molecular determinants of Bim(BH3) peptide binding to pro-
survival proteins. J Chem Inf Model 2012;52:2107–18.
40. Ibrahim HS, Albakri ME, Mahmoud WR, et al. Synthesis and
biological evaluation of some novel thiobenzimidazole
derivatives as anti-renal cancer agents through inhibition of
c-MET kinase. Bioorg Chem 2019;85:337–48.