Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design, synthesis, biological evaluation, and in silico studies

Article abstract of DOI:10.1080/14756366.2020.1862101

The serine/threonine protein kinases CDK2 and GSK-3β are key oncotargets in breast cancer cell lines, therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised as dual CDK2/GSK-3β inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N¹ -unsubstituted oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast cancer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC₅₀ of 3.41, 3.45 and 2.27 μM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 μM, respectively, on T-47D cell lines, in comparison to the used reference standard (staurosporine) IC₅₀ of 4.81 and 4.34 μM, respectively. On the other hand, the N¹ -substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activity on both breast cancer cell lines. CDK2 and GSK-3β enzyme inhibition assay of series 5 revealed that compounds 5d and 5f are showing potent dual CDK2/GSK-3β inhibitory activity with IC₅₀ of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3β. The most potent compounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of the CDK2/GSK-3β inhibition. Molecular docking studies showed that the newly synthesised N¹ -unsubstituted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3β. The oxindole ring is accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in GSK-3β. Whereas, in series 7 and 13, the N¹ -substitutions on the oxindole nucleus hinder the compounds from achieving these key interactions with hinge region amino acids what rationalises their moderate to low anti-proliferative activity.

Full text of DOI:10.1080/14756366.2020.1862101

Journal of Enzyme Inhibition and Medicinal Chemistry
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ienz20
Novel oxindole/benzofuran hybrids as potential
dual CDK2/GSK-3β inhibitors targeting breast
cancer: design, synthesis, biological evaluation,
and in silico studies
Wagdy M. Eldehna , Sara T. Al-Rashood , Tarfah Al-Warhi , Razan O.
Eskandrani , Amal Alharbi & Ahmed M. El Kerdawy
To cite this article: Wagdy M. Eldehna , Sara T. Al-Rashood , Tarfah Al-Warhi , Razan O.
Eskandrani , Amal Alharbi & Ahmed M. El Kerdawy (2021) Novel oxindole/benzofuran hybrids
as potential dual CDK2/GSK-3β inhibitors targeting breast cancer: design, synthesis, biological
evaluation, and inꢀsilico studies, Journal of Enzyme Inhibition and Medicinal Chemistry, 36:1,
270-285, DOI: 10.1080/14756366.2020.1862101
To link to this article: https://doi.org/10.1080/14756366.2020.1862101
©
2020 The Author(s). Published by Informa
View supplementary material
Submit your article to this journal
View Crossmark data
UK Limited, trading as Taylor & Francis
Group.
Published online: 16 Dec 2020.
View related articles
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
021, VOL. 36, NO. 1, 270–285
2
https://doi.org/10.1080/14756366.2020.1862101
ORIGINAL ARTICLE
Novel oxindole/benzofuran hybrids as potential dual CDK2/GSK-3b inhibitors
targeting breast cancer: design, synthesis, biological evaluation, and
in silico studies
a
b
c
b
b
Wagdy M. Eldehna , Sara T. Al-Rashood , Tarfah Al-Warhi , Razan O. Eskandrani , Amal Alharbi and
d,e
Ahmed M. El Kerdawy
a
b
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh, Egypt; Department of Pharmaceutical
c
Chemistry, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia; Department of Chemistry, College of Science, Princess Nourah Bint
d
Abdulrahman University, Riyadh, Saudi Arabia; Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Cairo, Egypt;
e
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, Cairo, Egypt
ABSTRACT
ARTICLE HISTORY
The serine/threonine protein kinases CDK2 and GSK-3b are key oncotargets in breast cancer cell lines,
therefore, in the present study three series of oxindole-benzofuran hybrids were designed and synthesised
as dual CDK2/GSK-3b inhibitors targeting breast cancer (5a–g, 7a–h, and 13a–b). The N -unsubstituted
Received 1 November 2020
Revised 28 November 2020
Accepted 1 December 2020
1
oxindole derivatives, series 5, showed moderate to potent activity on both MCF-7 and T-47D breast can-
cer cell lines. Compounds 5d–f showed the most potent cytotoxic activity with IC50 of 3.41, 3.45 and
KEYWORDS
Dual kinase inhibitors;
CDK2/GSK-3b inhibitors;
Benzofuran-2-carbohydra-
zide; Isatin;
2
.27 lM, respectively, on MCF-7 and of 3.82, 4.53 and 7.80 lM, respectively, on T-47D cell lines, in compari-
son to the used reference standard (staurosporine) IC50 of 4.81 and 4.34 lM, respectively. On the other
1
hand, the N -substituted oxindole derivatives, series 7 and 13, showed moderate to weak cytotoxic activ-
ity on both breast cancer cell lines. CDK2 and GSK-3b enzyme inhibition assay of series 5 revealed that
compounds 5d and 5f are showing potent dual CDK2/GSK-3b inhibitory activity with IC50 of 37.77 and
anticancer agents
5
2.75 nM, respectively, on CDK2 and 32.09 and 40.13 nM, respectively, on GSK-3b. The most potent com-
pounds 5d–f caused cell cycle arrest in the G2/M phase in MCF-7 cells inducing cell apoptosis because of
1
the CDK2/GSK-3b inhibition. Molecular docking studies showed that the newly synthesised N -unsubsti-
tuted oxindole hybrids have comparable binding patterns in both CDK2 and GSK-3b. The oxindole ring is
accommodated in the hinge region interacting through hydrogen bonding with the backbone CO and NH
of the key amino acids Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135, respectively, in
1
GSK-3b. Whereas, in series 7 and 13, the N -substitutions on the oxindole nucleus hinder the compounds
from achieving these key interactions with hinge region amino acids what rationalises their moderate to
low anti-proliferative activity.
1
. Introduction
dysregulation contributes to several diseases such as cancer, meta-
bolic disorders (such as diabetes), cardiovascular diseases, neuro-
degenerative diseases (such as Alzheimer’s disease), inflammatory
disorders and autoimmune diseases (such as rheumatoid arth-
Protein kinases (PKs) represent the fifth largest human protein
1–2
family comprising 518 proteins . They are key regulators of cell
functions through cellular signalling modulation and complex bio-
logical functions coordination such as cell growth, differentiation,
proliferation, metabolism, migration, and apoptosis (programmed
8
ritis) . The usage of PKs inhibitors is a promising strategy to man-
8–11
age their dysregulation in these diseases
. In cancer treatment,
1
,3–5
PK inhibitors represent a key class of targeted chemotherapy
which is devoid of the common side effects of conventional can-
cer chemotherapy because they target cancer cells’ signalling
pathways and microenvironment with minimum undesirable
cell death)
. PKs exert their functions through catalysing the
transfer of the gamma-phosphate group of an ATP molecule onto
a substrate protein hydroxyl group (substrate protein phosphoryl-
ation) accomplishing cellular signals transduction and amplifica-
1
2–15
1
effects on normal cells
.
tion . According to the phosphorylated residue in the substrate
Breast cancer is a very common cancer type all over the world,
and despite of its possible early detection by advanced diagnostic
techniques, breast cancer is still considered the leading cause of
protein, PKs are classified into tyrosine kinases (90 kinases) and
serine/threonine kinases (385 kinases), with a small group of dual
specificity kinases including MEK1 and MEK2 which could catalyse
1
6–17
the phosphorylation of both tyrosine and threonine on tar- death among women worldwide
. Thus, there is a serious con-
6–7
get proteins
.
tinuous demand for the discovery of more effective anti-breast
The prominent and critical importance of PKs dictates strict cancer agents. Several studies reported the overexpression of sev-
regulation of their cellular levels and activities, therefore, PKs eral PKs in primary as well as in metastatic breast cancers such as
CONTACT Wagdy M. Eldehna
3516, Egypt
Supplemental data for this article can be accessed here.
wagdy2000@gmail.com
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafr El-Sheikh,
3
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
271
1
8–20
28–29
vascular endothelial growth factor receptor (VEGFR)
, fibroblast correlates with poor prognosis in patients with breast cancer
,
20–21
growth factor receptor (FGFR)
, platelet-derived growth factor moreover, aberrant nuclear accumulation of GSK-3b in five human
, cyclin dependent kinases (CDKs) and their breast cancer cell lines and in 70% of human breast carcinomas
, and glycogen synthase kinase 3b (GSK- was reported , furthermore, GSK-3b inhibition suppressed the via-
and references therein.
bility of breast cancer cells in vitro, moreover, GSK-3b inhibition
CDKs, a family of serine/threonine protein kinases, are involved overcomes chemoresistance in human breast cancer .
2
0,22
receptor (PDGFR)
activating cyclins
2
3–27
29
2
8–30
3b)
5
2
in several cellular functions such as division, proliferation, apop-
tosis, and gene transcription. CDK1, CDK2, CDK4 and CDK6 sub- multitarget treatment strategies
The complex nature of cancer mandates the implementation of
5
3–55
. Moreover, in several cancer
types are responsible for the regulation of cell-cycle progression types especially solid tumours, such as breast cancer, more than
5
4,56
in its different phases, moreover, they play a pivotal role in cancer one PK is upregulated and contributes to carcinogenesis
cell continuous proliferation
.
31–33
. CDK2 subtype, specifically, Furthermore, drug resistance is commonly developed by target PK
received a great attention as a therapeutic target for cancer treat- mutation, target PK amplification/overexpression, or through upre-
5
4
ment due to its key role in several cellular processes upon com- gulation of alternative/downstream pathways . Thus, the use of
plexation with its activating cognate, cyclin A or E, in addition, multiple PK inhibitors is an emerging and appealing trend to over-
dysregulation of CDK2 or its cyclin partners was detected in vari- come cancer development, progression, and resistance. A possible
ous cancers such as ovarian, lung, pancreatic carcinomas, melan- drawback of this approach is the pharmacokinetics (ADME) inter-
23–27,34–37
oma as well as breast cancer
. The impact of CDK2 ference among the co-administered inhibitors, another concern is
inhibition on cancer cells confirmed the validity of CDK2 as a the potential toxicity of the used combination. To avoid these
promising anticancer drug target, therefore, many CDK2 inhibitors drawbacks, the design of small-molecules that are intentionally
3
4,35,38
have been progressed through clinical trials (Figure 1)
particular, CDK2 inhibition was found to effectively hinder breast considered
cancer cells proliferation including those resistant to hormonal ivity or multitarget selectivity (poly-specific inhibitors) might be
. In tailored to target more than one PK (multikinase inhibitors) was
5
3–55,57,58
. Multikinase inhibitors with favourable select-
3
9–41
therapy
GSK-3, another serine/threonine protein kinase and an essential toxicity
element of the WNT signalling pathway, contributes to several
Dual CDKs/GSK-3b inhibition is
physiological processes ranging from gene expression to glycogen approach to confront uncontrolled cancer cell prolifer-
.
more suitable for cancer treatment to balance efficacy and
5
3–55,57,58
.
a
promising therapeutic
4
2
35,36,51,59–66
metabolism . GSK-3 has two isozymes a and b encoded by two ation
. In particular, CDK2 and GSK-3b share a high
. GSK-3b is known as the “multi- homologous sequence (33% amino acid identity) and are used as
tasking kinase” due to its participation in several signalling path- examples for the homological CMGC protein kinase fam-
43–48
independent GSK-3 genes
49
59,64,67–75
ways . Dysregulated activity of GSK-3b is associated with several ily
diseases such as type 2 diabetes, heart disease, chronic inflamma- anticancer agents is an amenable and attractive strategy
tory diseases, neurodegenerative diseases, and cancer, therefore, 1H-indole-2,3-dione (isatin) is privileged scaffold that
GSK-3b inhibition is a potential approach for the treatment of emerged as a promising nucleus in medicinal chemistry demon-
. Therefore, designing dual CDK2/GSK-3b inhibitors as
6
5–66
.
a
3
0,50
these diseases
. GSK-3b is overexpressed in colon, pancreatic strating a broad range of pharmacological activities including anti-
as well as breast cancers as it plays a key role in cancer cell prolif- bacterial, anticonvulsant, antifungal, antiviral, as well as anticancer
28,29,51
76–78
eration and survival
.
The overexpression of GSK-3b activity
. Isatin derivatives exert their anticancer activity
Figure 1. CDKs inhibitors in the clinical trial phases.

2
72
W. M. ELDEHNA ET AL.
through several mechanisms such as inhibition and/or modulation bioisosteric amido and uriedo moieties were used as linkers
of proteases, translation initiation, angiogenesis or tubulin poly- between the two nuclei, moreover, different substitution pattern
merisation, moreover, PK inhibition is one of the key anticancer and nature on the oxindole ring offering various electronic and
76–78
mechanisms of isatin derivatives
. Several oxindole-based mul- lipophilic environments were introduced to study their impact on
tikinase inhibitors (Figure 2) have been approved for cancer treat- the activity (Figure 4). On account of the hydrophobic nature of
ment such as sunitinib (2006) for gastrointestinal stromal tumour the CDK2 binding site, it was anticipated that the grafting a
79
and renal cell carcinoma as PDGFR and VEGFR inhibitor , and nin- hydrophobic substituent (such as Br) within the benzofuran motif
tedanib (2014) for idiopathic pulmonary fibrosis as FGFR, PDGFR could achieve a plenty of hydrophobic interactions.
8
0
and VEGFR inhibitor . Moreover, several oxindole derivatives have
The newly synthesised compounds were experimentally eval-
been designed and synthesised as inhibitors for diverse PKs such uated for their anti-proliferative activities against breast cancer
8
1
82–83
84
as FLT3 kinase , VEGFR
, polo-like kinase 4 (PLK4) , aurora B cell lines T-47D and MCF-7. Compounds showed promising anti-
8
5
86
kinase , p90 ribosomal S6 protein kinase 2 (RSK2) , microtubule proliferative activity were tested for their potential dual CDK2/
affinity-regulating kinase 4 (MARK4) as well as CDKs
87
78,88
, and GSK-3b inhibitory activity, moreover, compounds showed promin-
5
9
GSK-3b .
ent anti-proliferative activity and in vitro PK inhibitory activity
The naturally occurring oxindole derivative, indirubin, (Figure 2) were investigated further by studying their effect on cell cycle
has been identified as the main active ingredient of the traditional progression and cell apoptosis. Furthermore, molecular docking
Chinese medicinal remedy, Dang Gui Long Hui Wan which is used studies were also performed to study the interaction of the newly
64,66,89
to treat chronic myelocytic leukaemia (CML)
. Indirubin and synthesised hybrid compounds with CDK2 and GSK-3b kinase
its derivatives showed distinctive antiproliferative effect which is
attributed to their inhibition of CDKs and GSK-3 through compet-
domain hot spots (key amino acids) to rationalise their biological
activity and to reveal their probable binding mode.
74,90–93
ing with ATP at the kinase domain
.
Benzofuran is another privileged scaffold that demonstrated
several desirable biological activities among which are analgesic, 2. Results and discussion
anti-inflammatory, antipyretic, antibacterial, antifungal, antiviral,
antihyperglycemic, anti-hyperlipidemic, anti-oxidant, as well as
2.1. Chemistry
anticancer activities94–96. Benzofuran derivatives mediate their The synthetic strategies designed for the preparation of the target
anticancer activity through several mechanism such as farnesyl- final compounds (5a-g, 7a-h and 13a-b) were illustrated in
transferase, angiogenesis, oestrogen receptor, human peptide Schemes 1–3. In Scheme 1, bromobenzofuran-2-carbohydrazide 3
deformylase, tubulin polymerisation, and carbonic anhydrase was prepared via reaction of ethylbromacetate with 5-bromosali-
9
4,97
inhibition
and reference therein. Moreover, some benzofuran cylaldehyde 1 in acetonitrile to furnish ethyl 5-bromobenzofuran-
derivatives exert their anticancer activity through PK inhibition 2-carboxylate 2. Thereafter, the ester functionality was subjected
9
8
99–100
101
such as Pim-1 , mTOR signaling
, Src kinase , as well as to hydrazinolysis via refluxing hydrazine hydrate to afford the key
5
1
GSK-3b (Figure 3).
intermediate hydrazide 3, further; this key intermediate 3 was con-
Based on the reported PK inhibitory activity of isatin and ben- densed with different isatin derivatives 4a-g in absolute ethyl
zofuran nuclei, specially, on the homologous CDK2 and GSK-3b alcohol with catalytic drops of glacial acetic acid to get the final
kinases, a hybridisation strategy of these two privileged scaffolds novel compounds 5a-g.
was adapted to design dual CDK2/GSK-3b hybrid inhibitors which
In Scheme 2, isatin derivatives 4a and 4d were subjected to N-
could target breast cancer. For the hybridisation strategy, the alkylation in anhydrous DMF to furnish N-substituted isatin 6a-h,
Figure 2. Oxindole derivatives with multikinase activity.
Figure 3. Benzofuran derivatives with GSK-3b inhibitory activity.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
273
Figure 4. Designed oxindole/benzofuran dual CDK2/GSK-3b hybrid inhibitors.
2 3 2 2 2
Scheme 1. Synthesis of target compounds 5a–g; Reagents and conditions: (i) Ethyl bromoacetate/Acetonitrile/K CO /reflux 4 h, (ii) NH NH .H O/methanol/reflux 3 h,
(iii) Ethanol/glacial acetic acid (Cat.)/reflux 4–7 h.
2 2 3
Scheme 2. Synthesis of target compounds 7a-h; Reagents and conditions: (i) R -Br/anhydrous DMF/K CO /reflux 4 h, (ii) Ethanol/drops glacial acetic acid (Cat.)/
reflux 4–7 h.

2
74
W. M. ELDEHNA ET AL.
2 3
Scheme 3. Synthesis of target compounds 13a-b; Reagents and conditions: (i) HCl/reflux 6 h, (ii) (COCl) /anhydrous toluene/reflux 5 h, (iii) NaN /acetone/stirring r.t. 1 h,
(
iv) Dry toluene/reflux 1 h. (v) NH NH .H O/methanol/reflux 3 h, (vi) Anhydrous toluene/reflux 4 h.
2
2
2
which then condensed with the key intermediate 3 to afford tar- whereas, the rest of compounds showed weak cytotoxic activity
get compounds 7a-h.
with IC50 higher than 10 mM (Table 1).
In the last scheme, the ester moiety of 5-bromobenzofuran-2-
On T-47D cell line, the tested compounds showed an IC50
carboxylate 2 succumbed to acidic hydrolysis to afford the acid range of 1.27–43.27 mM, with compounds 5 b, 5d-e,7h and 13a
analogue 8, which was chlorinated with oxalyl chloride in toluene showing potent cytotoxic effect (IC50 of 1.27, 3.82, 4.53, 1.72 and
to get the acid chloride derivative 9 that reacted with NaN
acetone to produce 5-bromobenzofuran-2-carbonyl azide 10. The which showed an IC50 of 4.34 mM. Compounds 5a, 5c, 5f, 5 g and
in 3.22 mM, respectively) compared to the reference staurosporine
3
7
7
g showed moderate cytotoxic activity with IC50 of 9.67, 6.03,
.80, 6.65 and 9.67 mM, respectively, whereas, the rest of com-
pounds showed weak cytotoxic activity with IC50 higher than
azide derivative 10 was subjugated to Curtius Rearrangement
through heating in anhydrous toluene to furnish isocyanate ana-
logue 11. On the other hand, isatin derivatives 6c and 6e were
condensed with hydrazine hydrate to release their hydrazone ana-
logue 12a and 12 b, respectively, which reacted with isocyanate
analogue 11 to get the final target compounds 13a-b.
The structures of all the synthesised compounds were con-
firmed under the basis of spectral and elemental analyses which
were in full agreement with the proposed structures.
1
0 mM (Table 1).
These results indicate the superior cytotoxic activity of the N-
unsubstituted isatin derivatives 5a-g on both breast cancer cell
lines with IC50 range of 2.27–12.93 mM and 1.27–9.67 mM on MCF-7
and T-47D cell lines, respectively, which could be due to their bet-
ter binding to the target kinases. Compounds 5d–f with Br, CH
and OCH substituent on the isatin nucleus, respectively, showed
the best cytotoxic activity (Table 1).
3
3
2
.2. Biological evaluation
Series 7 showed moderate to weak cytotoxic activity on either
or both cell lines, except for the N-benzylisatin hybrid 7 h which
showed a potent cytotoxic activity on both cell lines with IC50 of
All the newly synthesised compounds were initially tested for their
anti-proliferative activity against breast cancer cell lines T-47D and
MCF-7 in MTT assay. Compounds showed promising anti-prolifera-
tive activity were tested for their PK inhibitory activity on the tar-
get kinases CDK-2 and GSK-3b. Furthermore, compounds showed
prominent activity at cellular level and on enzymes were further
evaluated for their effect on cell cycle progression and cell apop-
tosis in the breast cancer cell line MCF-7.
4
.32 and 1.72 mM on MCF-7 and T-47D, respectively. These results
indicate that the N-substitution at the isatin nucleus greatly affects
the cytotoxic activity in the newly synthesised compounds mostly
in a negative manner (Table 1).
Worthy of note, replacing the amido group in compound 7c
and 7e with the ureido moiety to afford 13a and 13 b greatly
enhanced the cytotoxic activity in both cell lines (37.04 and
8
.33 mM vs 18.21 and 5.70 mM, respectively, in MCF-7) and (43.27
and 11.80 mM vs 3.22 and 10.88 mM, respectively, in T-47D)
Table 1).
2
.2.1. In vitro anti-proliferative activity
(
All the newly synthesised benzofuran-oxindole hybrids were
screened for their in vitro anti-proliferative activity against the
breast cancer cell lines T-47D and MCF-7 using the MTT assay,
and the results were compared with the pan-kinase inhibitor
staurosporine as a reference standard
sented in Table 1.
2.2.2. CDK2 and GSK-3b inhibitory activities
The most potent hybrids on breast cancer cell lines (series 5)
were further evaluated biochemically for their kinase inhibitory
activity on CDK-2 and GSK-3b using their Kinase Assay Kits, and
were compared to the pan-kinase inhibitor staurosporine as a ref-
1
02
. The IC50 values are pre-
On MCF-7 cell line, the tested hybrids showed an IC50 range of
2
.27–37.04 mM, with compounds 5d-f, 7 b and 7 h showing potent erence standard. The results were presented in Table 2.
cytotoxic effect (3.41, 3.45, 2.27, 2.64 and 4.32 mM, respectively)
On CDK2, the tested compounds showed potent sub-micromo-
compared to the reference staurosporine which showed an IC50 of lar inhibitory activity with IC50 ranged from 37.80 to 177 nM in
.81 mM. Compounds 5a, 7d, 7e and 13 b showed moderate cyto- comparison to that of the used reference standard (IC50
toxic activity with IC50 of 5.47, 7.48, 8.33 and 5.70, respectively, 38.50 nM). The bromo isatin derivative 5d showed the most
4
¼

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
275
Table 1. In vitro anti-proliferative activity of hybrids 5a–g, 7a–h and 13a–b against breast T-47D and MCF-7 cancer cell lines.
a
IC50 (mM)
Comp.
R
R
1
R
2
MCF-7
T-47D
5
5
5
5
5
5
5
7
7
7
7
7
7
7
7
1
1
a
b
c
d
e
f
g
a
b
c
d
e
f
H
F
Cl
Br
CH
OCH
CH
H
H
H
H
H
H
Br
Br
–
–
–
H
–
–
–
–
–
–
–
CH
5.47 ± 0.16
12.93 ± 0.38
12.46 ± 0.37
3.41 ± 0.10
3.45 ± 0.10
2.27 ± 0.06
11.95 ± 0.35
10.43 ± 1.01
2.64 ± 0.07
37.04 ± 1.87
7.48 ± 0.22
8.33 ± 0.24
24.67 ± 0.72
24.82 ± 0.73
4.32 ± 0.12
18.21 ± 0.53
5.70 ± 0.16
4.81 ± 0.14
9.67 ± 0.31
1.27 ± 0.04
6.03 ± 0.19
3.82 ± 0.12
4.53 ± 0.14
7.80 ± 0.25
6.65 ± 0.21
11.79 ± 1.05
12.24 ± 0.40
43.27 ± 2.39
18.99 ± 0.62
11.80 ± 0.38
38.7 ± 1.26
9.67 ± 0.31
1.72 ± 0.04
3.22 ± 0.10
10.88 ± 0.35
4.34 ± 0.14
H
H
H
H
H
CH
–
–
–
–
–
–
–
–
–
–
–
3
3
3
3
3
CH
CH
CH
2
CH ¼ CH
CH(CH
COOC
2
2
3 2
)
5
2
2
H
CH
2
Ph
CH
2
2
CH Ph
CH(CH
g
h
3a
3b
CH
2
3
)
2
2
CH
2
Ph
CH
2
CH(CH
3
)
CH
2
Ph
Staurosporine
–
a
IC50 values are the mean ± SD of three separate experiments.
Table 2. Inhibitory activities of compounds 5a–g against CDK2 and GSK-3b.
a
IC50 (nM)
Comp.
CDK2
GSK-3b
5
5
5
5
5
5
5
a
b
c
d
e
f
140.6 ± 7.7
52.46 ± 2.9
85.36 ± 4.6
37.77 ± 2.1
176.5 ± 9.6
52.75 ± 2.9
104.8 ± 5.7
38.5 ± 2.1
136.9 ± 7.5
212.3 ± 12
102 ± 5.6
32.09 ± 1.7
80.75 ± 4.4
40.13 ± 2.2
75.54 ± 4.1
43.38 ± 2.4
g
Staurosporine
a
IC50 values are the mean ± SD of three separate experiments.
Figure 5. Effect of hybrids 5d, 5e and 5f on the phases of cell cycle of MCF-
cells.
7
potent inhibitory activity with IC50 of 37.80 nM comparable to that
of staurosporine. Compounds 5 b, 5c and 5f showed two-digit
nanomolar inhibitory activity with IC50 of 52.50, 85.40 and 52.80, 2.2.3. Cell cycle analysis
respectively (Table 2).
Cyclin E/CDK2 complexation plays an important role at G1 phase,
On GSK-3b, the tested compounds showed potent sub-micro- whereas, cyclin A/CDK2 complexation terminates S phase and
molar inhibitory activity as well with IC50 range of drives the cell cycle through normal G2/M phase progres-
2
5,37,103,104
3
2.09–212.30 nM in comparison to the used reference standard sion
. Furthermore, a significant percentage of CDK2-
(
IC50 ¼ 43.38 nM). The bromo isatin 5d and the methoxy isatin 5f deficient cells arrest in the G2/M phase, additionally, breast cancer
23,105
derivative showed more potent inhibitory activity than that of cells exposed to CDK2 inhibitors show G2/M phase arrest
. In
staurosporine (IC50 of 32.09 and 40.13 nM, respectively). the same vein, GSK-3b regulates the strength of the mitotic check-
Compounds 5e and 5 g showed two-digit nanomolar inhibitory point and connects the PI3K and WNT-signalling pathways to
activity with IC50 of 75.54 and 80.75 nM, respectively (Table 2).
mitosis, furthermore, GSK-3b inhibitors induce G2/M phase arrest
3
0,106,107
These results indicate the superiority of the bromo isatin 5d as well
.
and the methoxy isatin 5f derivatives on both kinases (Table 2).
Compounds showing promising antiproliferative as well as kin-
The methyl isatin derivative 5e shows a relatively less kinase ase inhibitory activity 5d, 5e and 5f were further investigated for
inhibitory activity than its bromo 5d and methoxy 5f congeners their influence on the cell cycle progression by flow cytometry
on both kinases despite of its obvious potent cellular cytotoxicity analysis using propidium iodide (PI) stain. Cell cycle parameters
which could be attributed to its probable further cytotoxic mecha- were compared for the breast cancer MCF-7 cells with DMSO as
nisms besides CDK2/GSK-3b dual inhibition.
control and after treatment with the compounds of interest and

2
76
W. M. ELDEHNA ET AL.
incubation for 24 h, and the results were presented in Figure 5 compounds 5d, 5e, and 5f (13.75, 19.74, and 26.10%, respectively)
and Table 3.
relative to control cells (0.82%) which is a significant indication of
From the obtained results Figure 5 and Table 3, it is noticeable the apoptotic effect of the compounds of interest. Compounds
that there is an increase in the percent of cell distribution in the 5d, 5e, and 5f produced an increase in the early apoptotic phase,
G2/M phase, from 11.30% in control to 30.26, 29.21 and 37.82% in from 0.63 to 3.61, 4.66, and 3.94, respectively, and an increase in
treated cells with 5d, 5e and, 5f, respectively, indicating a G2/M the late apoptotic phase, from 0.19 to 10.14, 15.08, and 22.16,
phase arrest which is the expected consequence from CDK2/GSK- respectively.
3b dual inhibition confirming the mechanism of action of the
designed compounds on the target kinases. Furthermore, an
increase in the percent of cells accumulated in the sub-G1 phase, 2.2.5. Cytotoxicity towards non-tumorigenic cells
from 1.29% in the control to 13.27, 17.73 and 22.02% in the In order to investigate their selectivity on cancer cells, hybrids 5d,
treated cells with 5d, 5e and, 5f, respectively, as a result of 5e and 5f, endowed with dual growth inhibitory action against
cell apoptosis.
MCF-7 and T-47D cells, were assesses for their cytotoxic action
The high efficacy of compound 5f in cell arrest at G2/M phase towards the non-tumorigenic breast cells (MCF-10A), via the MTT
and in cell apoptosis as indicated by its results in Table 3 relative assay. The obtained IC50s and the calculated mean tumour select-
to its congeners 5d and 5e aligns with its potent effect on MCF-7 ivity index (S.I.); IC50 for MCF-10A/IC50 average for (MCF-7 and
as indicated by its IC50 (2.27 lM) in comparison to the IC50 of com- T-47D) have been presented in Table 5. The examined hybrids
pounds 5d and 5e (3.41 and 3.45 lM, respectively).
exerted weak cytotoxic effect (IC50 ¼ 21.66 ± 1.05, 23.59 ± 0.86 and
9.95 ± 1.42 lM, respectively, with selectivity indexes equal 6.0, 5.9
and 7.9, respectively, Table 5.
3
2
.2.4. Apoptosis assay
To investigate further the effect of the promising compounds 5d,
e, and 5f on cell apoptosis, Annexin V-FITC/propidium iodide 3. Molecular docking study and structure activity
dual staining assay was performed according to the reported relationship
5
1
08
method . The morphological markers of apoptosis in the breast
cancer MCF-7 cell line were examined before and after treatment
with the compounds of interest. Apoptosis assay depends on the
translocation of the phosphatidylserine (PS) phospholipid to the
cell surface in cells undergoing apoptosis which can be easily
detected by staining with the fluorescent conjugate of annexin V
followed by flow cytometry analysis. Simultaneously, MCF-7 cells
were stained with propidium iodide (PI) which could enter cells
with damaged plasma membranes only. This enables the discrim-
ination between early apoptotic cells (positive for PS, but negative 5e
for PI) from late apoptotic and necrotic cells (positive for both PS 5f
and PI).
Figure 6 and Table 4 show that the percentage of the total
In the present molecular docking study, a pair of protein struc-
1
09
tures were used for CDK2 and GSK-3b, viz, PDB ID: 1FVT
and
Table 4. Distribution of apoptotic cells in the AnnexinV-FITC/PI dual staining
assay in MCF-7 cells upon treatment with compounds 5d, 5e and 5f.
Early
Late
Total
Apoptosis
Apoptosis
Comp.
(L.R % þ U.R %)
(Lower Right %)
(Upper Right %)
Necrosis
5d
13.75
19.74
26.10
0.82
3.61
4.66
3.94
0.63
10.14
15.08
22.16
0.19
1.56
1.81
2.14
0.49
Control
apoptotic cells in MCF-7 cell line increases after treatment with
Table 5. Cytotoxic action for hybrids 5d, 5e and 5f towards non-tumorigenic
breast cell line (MCF-10A), and mean tumour selectivity index (S.I.) (MCF-10A/
MCF-7 and T-47D).
Table 3. Effect of compounds 5d, 5e and 5f on the phases of cell cycle of MCF-
7
cells
IC50 (mM)
Comp.
%G0-G1
%S
%G2/M
%Sub-G1
Comp.
MCF-10A
MCF-7
T-47D
Mean tumour selectivity
5
5
5
d
e
f
33.97
29.85
22.28
53.59
22.5
30.26
29.21
37.82
11.3
13.27
17.73
22.02
1.29
23.21
17.88
33.82
5d
5e
5f
21.66 ± 1.05
23.59 ± 0.86
39.95 ± 1.42
3.41 ± 0.10
3.45 ± 0.10
2.27 ± 0.06
3.82 ± 0.12
4.53 ± 0.14
7.80 ± 0.25
6.0
5.9
7.9
Control
Figure 6. Effect of 5d, 5e and 5f on the percentage of annexin V-FITC-positive staining in MCF-7 cells. The experiments were done in triplicates. The four quadrants
identified as: LL, viable; LR, early apoptotic; UR, late apoptotic; UL, necrotic.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
277
Figure 7. (A) 2 D interaction diagram showing the oxindole derivative docking pose interactions with the key amino acids (hot spots) in the CDK2 active site.
Distances in Å) (B) 3 D representations of the superimposition of the docking pose (green) and the co-crystallized pose (red) of the oxindole derivative in the CDK2
(
active site, respectively, with RMSD of 0.894 Å.
Figure 8. (A) 2 D interaction diagram showing Indirubin-3’-monoxime docking pose interactions with the key amino acids (hot spots) in the GSK-3b active site.
(
Distances in Å) (B) 3 D representations of the superimposition of the docking pose (green) and the co-crystallized pose (red) of Indirubin-3’-monoxime in the GSK-3b
active site, respectively, with RMSD of 0.471 Å.
1
10
PDB ID: 1Q41 , respectively, which are co-crystallized with co-crystallized ligands accurately indicating the suitability of the used
potent CDK2 and GSK-3b oxindole-based inhibitors, respectively. docking setup for the planned simulations. This was demonstrated by
Molecular docking was carried out to investigate the interaction the small RMSD between the docked and the co-crystallized ligand
of the designed hybrid compounds with CDK2 and GSK-3b kinase poses in CDK2 (0.894 Å) and GSK-3b (0.471 Å), and by the ability of the
domain to rationalise their biological activity, to reveal their prob- attained docking poses to simulate the main interactions achieved by
able binding pattern and to elicit their SAR.
the co-crystallized ligands with the key amino acids in CDK2 and GSK-
Initially, self-docking of the co-crystallized ligands in CDK2 and GSK- 3b active sites (Figures 7 and 8, respectively).
3b active sites was performed to validate the used molecular docking
In the CDK2 active site the docking pose of the oxindole
protocol. The self-docking step stimulated the binding pattern of the derivative reproduced the key interactions of the co-crystalized

2
78
W. M. ELDEHNA ET AL.
Figure 9. 2 D diagram (A) and 3 D representation (B) of compound 5f showing its interaction with the CDK2 active site.
Figure 10. 2 D diagram (A) and 3 D representation (B) of compound 5f showing its interaction with the GSK-3b active site.
Table 6. Docking energy scores (S) in kcal/mol for the newly synthesised hybrid
ligand with the active site; it interacts in the hinge region through
hydrogen bonding with Glu81 backbone CO and Leu83 backbone
compounds and the co-crystalized compounds in CDK2 and GSK-3b kin-
ase domain
Energy score (S)
kcal/mol CDK2
Energy score (S) NH and CO. Furthermore, its sulfonamidophenylhydrazone group
kcal/mol GSK-3b projected outward towards the bulk solvent with the sulphona-
Compound
5
5
5
5
5
5
5
7
7
7
7
7
7
7
7
1
1
a
b
c
d
e
f
g
a
b
c
d
e
ꢀ9.53
ꢀ9.89
ꢀ9.99
ꢀ10.40
ꢀ10.59
ꢀ10.81
ꢀ10.53
ꢀ10.96
ꢀ10.60
ꢀ10.40
ꢀ10.24
ꢀ11.48
ꢀ11.76
ꢀ11.58
ꢀ10.68
ꢀ11.27
ꢀ11.46
ꢀ11.77
ꢀ11.92
ꢀ12.00
mide group interacting with Asp86 backbone NH and side-chain
þ
carboxyl group and Lys89 side chain NH
3
(Figure 7).
ꢀ10.07
ꢀ10.22
ꢀ10.05
ꢀ10.35
ꢀ10.39
ꢀ9.19
0
As for GSK-3b, the docking pose of indirubin-3 -monoxime
reproduced the key interactions of the co-crystalized ligand with
the active site; it interacts in the hinge region through hydrogen
bonding with Asp133 backbone CO and Val135 backbone NH and
CO (Figure 8). Furthermore, through a water mediated hydrogen
bonding network, it interacts with Gln185 and Thr138 by its oxime
group which is responsible for its selectivity towards GSK-3b
over CDK2.
ꢀ9.61
ꢀ10.03
ꢀ10.21
ꢀ9.98
1
f
ꢀ10.05
ꢀ10.56
ꢀ10.40
ꢀ13.60
ꢀ14.72
ꢀ10.63
In series 5, the newly synthesised N -unsubstituted oxindole
g
h
3a
3b
hybrids showed comparable binding patterns in both kinases
(Figures 9 and 10 and for further details, see supporting materi-
als). The oxindole ring is accommodated in the hinge region inter-
acting through hydrogen bonding by its NH and CO with the
backbone CO and NH of the key amino acids Glu81 and Leu83,
Co-crystalized ligand

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
279
respectively, in CDK2, and Asp133 and Val135, respectively, in MCF-7 and T-47D cell lines, respectively. CDK2 and GSK-3b inhib-
GSK-3b. ition testing of the potent series 5 indicated that compounds 5d
Moreover, through hydrophobic interaction the fused phenyl and 5f exhibit potent dual CDK2/GSK-3b inhibitory activity with
ring of the oxindole nucleus interacts with the hydrophobic side IC50 of 37.77 and 52.75 nM, respectively, on CDK2 and 32.09 and
chains of the surrounding amino acids; Val18, Ala31, Val64, Phe80, 40.13 nM, respectively, on GSK-3b. The most potent hybrids 5d-f
Leu134, and Ala144 in CDK2 and Val70, Ala83, Val110, Leu132, triggered cell cycle arrest in the G2/M phase as a consequence of
Leu188, and Cys199 in GSK-3b (Figures 9 and 10 and for further their dual CDK2/GSK-3b inhibition. Moreover, compounds 5d-f
details, see supporting materials). Therefore, increasing the sub- induced apoptosis in MCF-7 cells as indicated by the percent of
stituent hydrophobicity on the oxindole nucleus enhances the the total apoptosis of 13.75, 19.74, and 26.10%, respectively, in
binding affinity which is well reflected in the docking binding comparison to the untreated cells which showed 0.82% total
score and further in the biological activity (Br, 5d > Cl, 5c ꢁ CH , apoptosis. The molecular docking study showed that the newly
3
1
5
e > F, 5 b) (Tables 1, 2 and 6). The remarkable binding affinity of synthesised N -unsubstituted oxindole hybrids have comparable
the relatively polar methoxy group (OCH
3
, 5f) is attributed to the binding patterns in both CDK2 and GSK-3b. The oxindole ring is
fact that it is directed towards the bulk solvent and so decreased accommodated in the hinge region interacting through hydrogen
the solvation penalty during the binding scenario resulting in a bonding with the backbone CO and NH of the key amino acids
considerable increase in the binding affinity. On the other hand, Glu81 and Leu83, respectively, in CDK2 and Asp133 and Val135,
despite of its relatively higher hydrophobicity, the dimethyl substi- respectively, in GSK-3b. Moreover, through hydrophobic inter-
tuted hybrid 5 g does not show significant predicted binding action the fused phenyl ring of the oxindole nucleus interacts
score or experimental biological activity what could be attributed with the hydrophobic side chains of the surrounding amino acids;
7
to the C methyl substituent which sterically clashes with the bot- Val18, Ala31, Val64, Phe80, Leu134, and Ala144 in CDK2 and
tom of the hinge region hindering the key interaction of the NH Val70, Ala83, Val110, Leu132, Leu188, and Cys199 in GSK-3b. The
and CO of the oxindole ring with the key amino acids; Glu81 and hydrazono linker NH interacts through hydrogen bonding with
Leu83 in CDK2, respectively, and Asp133 and Val135 in GSK-3b, the backbone CO of Leu83 and Val135 in CDK2 and GSK-3b,
respectively (For further details, see supporting materials).
respectively, projecting the benzofuran moiety outward towards
The hydrazono linker NH interacts through hydrogen bonding the bulk solvent which is involved in a hydrophobic interaction
with the backbone CO of Leu83 and Val135 in CDK2 and GSK-3b, with the hydrophobic side chains of the surrounding amino acids
respectively, projecting the benzofuran moiety outward towards lining the gate of the hinge region; Ile10, Phe82, and Leu298 in
the bulk solvent which is involved in a hydrophobic interaction CDK2 and Ile62, Tyr134 and Pro136 in GSK-3b. In series 7 and 13,
1
with the hydrophobic side chains of the surrounding amino acids the N -substitutions on the oxindole nucleus hinder the com-
lining the gate of the hinge region; Ile10, Phe82, and Leu298 in pounds from achieving the key interactions with hinge region
CDK2 and Ile62, Tyr134 and Pro136 in GSK-3b (Figures 9 and 10 amino acids; Glu81 and Leu83 in CDK2, and Asp133 and Val135 in
and for further details, see supporting materials).
GSK-3b what rationalises their moderate to low antiprolifera-
1
In series 7 and 13, the N -substitutions on the oxindole tive activity.
nucleus hinder the compounds from achieving the key interac-
tions with hinge region amino acids; Glu81 and Leu83 in
5. Experimental
CDK2, and Asp133 and Val135 in GSK-3b what rationalises their
moderate to low antiproliferative activity (For further details,
see supporting materials). Molecular docking simulations show
their non-selective bindings and random binding patterns what
rationalise their low activity despite of their good docking binding
scores (Table 6).
5.1. Chemistry
5.1.1. General
Melting points were measured with a Stuart melting point appar-
atus and were uncorrected. Infra-red spectra were recorded on
Schimadzu FT-IR 8400S spectrophotometer. The NMR spectra were
1
These results point out the criticality of N being unsubstituted
1
3
1
recorded by Bruker spectrometer at 400 MHz. C NMR spectra
were run at 100 MHz in deuterated dimethylsulphoxide (DMSOd6).
Elemental analyses were carried out at the Regional Centre for
Microbiology and Biotechnology, Al-Azhar University, Cairo, Egypt.
Unless otherwise noted, all solvents and reagents were commer-
cially available and used without further purification. Compounds
for CDK2/GSK-3b kinase inhibition. The N -benzyloxindole deriva-
tive 7 h showing promising antiproliferative activity might be
exerting its effect through other mechanism than CDK2/GSK-3b
kinase inhibition.
9
7
111–112
113
114
4
. Conclusions
(2 and 3) , (6a–h)
, (8–9) , and (12 b)
were prepared
according to the reported methods.
In the present study three series of oxindole-benzofuran hybrids
were designed and synthesised as dual CDK2/GSK-3b inhibitors
targeting breast cancer (5a-g, 7a-h, and 13a-b). In MTT assay on
5
.1.1.2. Synthesis of 5-bromobenzofuran-2-carbohydrazide 3. To
hot stirred solution of ethyl 5-bromobenzofuran-2-carboxylate 2
1 g, 3.7 mmol) in 30 ml of methanol, hydrazine hydrate (0.25 ml,
.5 mmol) was added. The reaction solution was left for heating
1
the breast cancer cell lines MCF-7 and T-47D, the N -unsubstituted
(
7
oxindole derivatives, series 5, showed moderate to potent activity
on both breast cancer cell lines. Compounds 5d-f showed the
most potent cytotoxic activity with IC50 of 3.41, 3.45 and 2.27 lM,
respectively, on MCF-7 and IC50 of 3.82, 4.53 and 7.80 lM, respect-
ively, on T-47D cell lines, in comparison to the used reference
standard (staurosporine) IC50 of 4.81 and 4.34 lM, respectively. On
under reflux for 4 h, and then poured onto cold water. The formed
solid was collected by filtration, washed with diethyl ether and
recrystallized from EtOH/DMF mixture to furnish the key inter-
mediate 3.
1
the other hand, the N -substituted oxindole derivatives, series 7
and 13, showed moderate to weak cytotoxic activity on both 5.1.1.3. Synthesis of target compounds 5a–g and 7a–h. 5-
breast cancer cell lines except for compound 7 h which showed Bromobenzofuran-2-carbohydrazide 3 (0.3 g, 1.2 mmol) was added
potent antiproliferative activity with IC50 of 4.32 and 1.72 lM on to a hot solution of equivalent amount of the appropriate isatin

2
80
W. M. ELDEHNA ET AL.
derivative (4a–g or 6a–h) in ethanol (15 ml) with catalytic amount 3320, 3341 (2NH) and 1721, 1700 (2 C ¼ O); Analysis calculated for
of ethanoic acid. The reaction mixture was heated under reflux for C H BrN O : C, 54.29; H, 3.04; N, 10.55; found C, 54.57; H, 3.01;
1
8
12
3 3
4–7 h with TLC monitoring, once the reaction completed, the reac- N, 10.62.
tion mixture was left for cooling then was filtered-off. The pro-
duced solid was washed with water, diethyl ether and
recrystallized from dioxane/propanol mixture to produce target
compounds 5a–g and 7a–h, respectively.
5
2
.1.1.3.6. 5-Bromo-N’-(5-methoxy-2-oxoindolin-3-ylidene)benzofuran-
ꢂ
1
-carbohydrazide 5f. Red powder (yield 82%), m.p. > 300 C;
H
NMR ppm: 3.80, 3.83 (2 s, 3H, OCH ), 6.85–6.91 (m, 1H, Ar-H),
3
6
.98–7.07 (m, 1H, Ar-H), 7.18 (s, 1H, Ar-H), 7.68–7.76 (m, 2H, Ar-H),
5
.1.1.3.1. 5-Bromo-N’-(2-oxoindolin-3-ylidene)benzofuran-2-carbohy- 7.82, 8.01 (2 s, 1H, Ar-H), 8.08–8.13 (m, 1H, Ar-H), 10.72,11.20 (2 s,
drazide 5a. Yellow powder (yield 75%), m.p. > 300 C; H NMR 1H, NH indolin-2-one), 11.97, 14.10 (2 s, 1H, NH); IR (KBr, ꢀ cm
ꢂ
1
ꢀ1
)
ppm: 6.94–7.00 (m, 1H, Ar-H), 7.11 (t, 1H, Ar-H, J ¼ 8.0 Hz), 3324, 3301 (2NH) and 1718, 1710 (2 C ¼ O); MS m/z [%]: 416
þ
þ
7
.41–7.47 (m, 1H, Ar-H), 7.64–7.84 (m, 3H, Ar-H), 7.99 (t, 1H, Ar-H, [M þ2, 75.42], 414 [M , 77.51], 402 [100]; Analysis calculated for
J ¼ 8.0 Hz), 8.09, 8.11 (2 s, 1H, Ar-H), 10.91, 11.39 (2 s, 1H, NH indo-
C
18
H
12BrN
O
3 4
: C, 52.19; H, 2.92; N, 10.14; found C, 52.01; H, 2.95;
13
lin-2-one), 11.89, 14.06 (2 s, 1H, NH);
C NMR d ppm: 111.32, N, 10.25.
1
1
14.73, 116.72, 120.10, 121.76, 122.45, 123.30, 126.13, 127.23,
29.61, 131.18, 132.70, 133.70, 143.29, 153.87, 163.35 (C ¼ O indo-
5
.1.1.3.7.
5-Bromo-N’-(5,7-dimethyl-2-oxoindolin-3-ylidene)benzo-
ꢀ
1
lin-2-one), 165.04 (C ¼ O hydrazide); IR (KBr, ꢀ cm ) 3380, 3345
ꢂ
furan-2-carbohydrazide 5 g. Red powder (yield 76%), m.p. > 300 C;
þ
(
3
5
2NH) and 1711, 1701 (2 C ¼ O); MS m/z [%]: 384 [M þ2, 50.94],
1
H NMR ppm: 2.20, 20.22 (2 s, 3H, CH
.29, 2.32 (2 s, 3H, CH of C-5 of indolin-2-one), 7.06, 7.11 (2 s, 1H,
Ar-H), 7.30 (s, 0.5H, Ar-H), 7.69–7.71 (m, 1.5H, Ar-H), 7.75–7.79 (m,
H, Ar-H), 7.82, 8.00 (2 s, 1H, Ar-H), 8.09, 8.11 (2 s, 1H, Ar-H),
.1.1.3.2. 5-Bromo-N’-(5-fluoro-2-oxoindolin-3-ylidene)benzofuran-2- 10.82,11.29 (2 s, 1H, NH indolin-2-one), 11.78, 14.06 (2 s, 1H, NH);
3
of C-7 of indolin-2-one),
þ
82 [M , 48.60], 160 [100]; Analysis calculated for C H BrN O : C,
17
10
3 3
2
3
3.15; H, 2.62; N, 10.94; found C, 53.33; H, 2.64; N, 10.82.
1
5
ꢂ
1
13
carbohydrazide 5b. Red powder (yield 77%), m.p. > 300 C;
NMR ppm: 6.92–7.00 (m, 1H, Ar-H), 7.24–7.33 (m, 1H, Ar-H), 7.49, of indolin-2-one), 114.67, 116.88, 119.47, 120.97, 123.31, 126.13,
.86 (2br s, 1H, Ar-H), 7.67–7.71 (m, 1H, Ar-H), 7.74 (t, 1H, Ar-H, 129.61, 131.11, 132.34, 134.53, 135.97, 139.65, 141.47, 142.47,
J ¼ 8.0 Hz), 7.96 (d, 0.6H, Ar-H, J ¼ 8.0 Hz), 8.04–8.13 (m, 1.4H, Ar- 153.84, 163.82 (C ¼ O indolin-2-one), 165.16 (C ¼ O hydrazide); IR
H
3 3
C NMR d ppm: 16.31 (CH C-7 of indolin-2-one), 20.88 (CH C-5
7
ꢀ
þ
1
H), 10.92,11.42 (2 s, 1H, NH indolin-2-one), 12.02, 14.02 (2 s, 1H, (KBr, ꢀ cm ) 3350, 3315 (2NH) and 1723, 1701 (2 C ¼ O); MS m/z
ꢀ
1
þ
NH); IR (KBr, ꢀ cm ) 3280, 3245 (2NH) and 1723, 1705 (2 C ¼ O); [%]: 414 [M þ2, 17.45], 412 [M , 14.19], 160 [100]; Analysis calcu-
Analysis calculated for C H BrFN O : C, 50.77; H, 2.26; N, 10.45; lated for C H BrN O : C, 55.36; H, 3.42; N, 10.19; found C, 55.53;
17
9
3
3
19 14
3 3
found C, 50.90; H, 2.24; N, 10.57.
H, 3.38; N, 10.27.
5
.1.1.3.3. 5-Bromo-N’-(5-chloro-2-oxoindolin-3-ylidene)benzofuran-2- 5.1.1.3.8. 5-Bromo-N’-(1-methyl-2-oxoindolin-3-ylidene)benzofuran-2-
ꢂ
1
ꢂ
1
carbohydrazide 5c. Orange powder (yield 83%), m.p. > 300 C; H carbohydrazide 7a. Yellow powder (yield 81%), m.p. 260–262 C; H
NMR ppm: 6.94–7.01 (m, 1H, Ar-H), 7.45–7.50 (m, 1H, Ar-H), NMR ppm: 3.27 (s, 3H, N-CH ), 7.19 (t, 2H, Ar-H, J ¼ 8.0 Hz), 7.50 (t,
3
7
1
.64–7.76 (m, 2.3H, Ar-H), 8.04, 8.10 (2 s, 1H, Ar-H), 8.14–8.17 (m, 1H, Ar-H, J ¼ 8.0 Hz), 7.68–7.71 (m, 2H, Ar-H), 7.76 (d, 1H, Ar-H,
.7H, Ar-H), 11.03,11.50 (2 s, 1H, NH indolin-2-one), 12.08, 13.96 J ¼ 8.0 Hz), 7.85 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 11.97, 14.02 (2 s,
1
3
13
(2 s, 1H, NH);
C NMR d ppm: 112.66, 114.60, 116.94, 118.92, 1H, NH); C NMR d ppm: 26.26 (N-CH ), 110.59, 114.68, 116.94,
3
1
1
21.28, 124.34, 126.29, 126.77, 131.04, 132.93, 137.31, 141.43, 119.40, 121.41, 123.84, 126.16, 129.60, 130.33, 131.24, 132.62,
43.41, 153.82, 163.17 (C ¼ O indolin-2-one), 164.88 (C ¼ O hydra- 138.18, 144.51, 151.44, 153.87, 161.58 (C ¼ O indolin-2-one), 165.10
ꢀ
1
ꢀ1
zide); IR (KBr, ꢀ cm ) 3300, 3240 (2NH) and 1735, 1702 (2 C ¼ O); (C ¼ O hydrazide); IR (KBr, ꢀ cm ) 3345 (NH) and 1720, 1695
þ
þ
Analysis calculated for C H BrClN O : C, 48.77; H, 2.17; N, 10.04; (2 C ¼ O); MS m/z [%]: 400 [M þ2, 92.41], 398 [M , 94.85], 91
17
9
3 3
found C, 48.82; H, 2.16; N, 9.93.
[100]; Analysis calculated for C H BrN O : C, 54.29; H, 3.04; N,
18 12 3 3
1
0.55; found C, 54.48; H, 3.02; N, 10.64.
5
.1.1.3.4. 5-Bromo-N’-(5-bromo-2-oxoindolin-3-ylidene)benzofuran-2-
ꢂ
1
carbohydrazide 5d. Orange powder (yield 87%), m.p. > 300 C; H 5.1.1.3.9. N’-(1-Allyl-2-oxoindolin-3-ylidene)-5-bromobenzofuran-2-car-
ꢂ
1
NMR ppm: 6.91–6.97 (m, 1H, Ar-H), 7.60 (d, 1H, Ar-H, J ¼ 8.0 Hz), bohydrazide 7 b. Orange powder (yield 73%), m.p. 207–209 C; H
7
1
.71–7.76 (m, 2H, Ar-H), 8.03–8.15 (m, 2H, Ar-H), 8.31 (s, 1H, Ar-H), NMR ppm: 4.46 (s, 2H, N-CH ), 5.22–5.31 (m, 2H, ¼CH ), 5.88–5.97
2
2
1.05,11.52 (2 s, 1H, NH indolin-2-one), 12.09, 13.99 (2 s, 1H, NH); (m, 1H, N-CH
2
-CH), 7.13 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.19 (t, 1H, Ar-H,
ꢀ
1
IR (KBr, ꢀ cm ) 3295, 3271 (2NH) and 1732, 1701 (2 C ¼ O); J ¼ 8.0 Hz), 7.47 (t, 1H, Ar-H, J ¼ 8.0 Hz), 7.68–7.72 (m, 2H, Ar-H),
Analysis calculated for C H Br N O : C, 44.09; H, 1.96; N, 9.07; 7.76 (d, 1H, Ar-H, J ¼ 8.0 Hz),) , 7.85 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H),
17
9
2 3 3
ꢀ
1
found C, 43.81; H, 1.98; N, 9.16.
11.99, 13.97 (2 s, 1H, NH); IR (KBr, ꢀ cm ) 3304 (NH) and 1715,
1
3
701 (2 C ¼ O); Analysis calculated for C20
3 3
H14BrN O : C, 56.62; H,
.33; N, 9.90; found C, 56.77; H, 3.31; N, 10.01.
5
.1.1.3.5. 5-Bromo-N’-(5-methyl-2-oxoindolin-3-ylidene)benzofuran-2-
ꢂ
1
carbohydrazide 5e. Brown powder (yield 84%), m.p. > 300 C;
H
NMR ppm: 2.23, 2.35 (2 s, 3H, CH ), 6.84–6.88 (m, 1H, Ar-H), 5.1.1.3.10. 5-Bromo-N’-(1-isobutyl-2-oxoindolin-3-ylidene)benzofuran-
3
ꢂ
7
.22–7.27 (m, 1H, Ar-H), 7.47 (s, 0.6H, Ar-H), 7.68–7.71 (m, 1H, Ar- 2-carbohydrazide 7c. Yellow powder (yield 65%), m.p. 216–218 C;
H), 7.74–7.79 (m, 1H, Ar-H), 7.82–7.88 (m, 1H, Ar-H), 8.00 (s, 0.4H,
1
H NMR ppm: 0.94 (d, 6H, -CH-(CH
)
3 2
, J ¼ 8.0 Hz), 2.09–2.16 (m, 1H,
Ar-H), 8.09–8.14 (m, 1H, Ar-H), 10.80, 11.28 (2 s, 1H, NH indolin-2- N-CH
2
-CH), 3.36 (d, 2H, N-CH
2
, J ¼ 8.0 Hz), 7.17 (t, 1H, Ar-H,
1
3
one), 11.83, 14.05 (2 s, 1H, NH) ; C NMR d ppm: 20.97 (CH3), J ¼ 8.0 Hz), 7.24 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.47 (t, 1H, Ar-H,
1
1
(
11.57, 114.65, 116.89, 120.09, 122.08, 126.14, 127.54, 129.60, J ¼ 8.0 Hz), 7.67–7.70 (m, 2H, Ar-H), 7.77 (d, 1H, Ar-H, J ¼ 8.0 Hz),
30.92, 131.15, 132.44, 133.12, 141.02, 142.44, 153.84, 163.41 7.84 (s, 1H, Ar-H), 8.08 (s, 1H, Ar-H), 13.99 (s, 1H, NH); IR (KBr, ꢀ
C ¼ O indolin-2-one), 165.09 (C ¼ O hydrazide); IR (KBr, ꢀ cmꢀ
1
)
cm ) 3312 (NH) and 1720, 1711 (2 C ¼ O); Analysis calculated for
ꢀ1

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
281
C
21
H
18BrN
3
O
3
: C, 57.29; H, 4.12; N, 9.54; found C, 57.08; H, 4.17; 5.1.1.4. Synthesis of 5-bromo-2-isocyanatobenzofuran 11. 5-
Bromobenzofuran-2-carbonyl chloride 9 (1 g, 3.8 mmol) was dis-
N, 9.47.
solved in dry acetone (30 ml), then added drop wisely to aqueous
ꢂ
solution of equivalent amount of NaN
3
at 0 C over 30 min. The
5
2
2
CH
7
.1.1.3.11. Ethyl-2–(3-(2–(5-bromobenzofuran-2-carbonyl)hydrazono)-
formed precipitate was filtrated under vacuum and washed with
pet. ether, then heated without purification in dry toluene for 1 h
to produce the intermediate 11.
-oxoindolin-1-yl)acetate 7d. Orange powder (yield 69%), m.p.
ꢂ
1
15–216 C; H NMR ppm: 1.23 (t, 3H, CH
, J ¼ 8.0 Hz), 4.76 (s, 2H, N-CH ), 7.22 (t, 1H, Ar-H, J ¼ 8.0 Hz),
.50 (t, 1H, Ar-H, J ¼ 8.0 Hz), 7.96–7.78 (m, 2H, Ar-H), 7.87 (s, 1H,
3
, J ¼ 8.0 Hz), 4.18 (q, 2H,
2
2
13
5
.1.1.5. Synthesis of hydrazones 12a-b. To hot stirred solution of
Ar-H), 8.10 (s, 1H, Ar-H), 13.81 (s, 1H, NH); C NMR d ppm: 14.49
the appropriate indolin-2-one 6c and 6e (6 mmol) in isopropyl
alcohol (22 ml), hydrazine hydrate (0.50 ml, 15 mmol) was added.
The reaction solution was heated under reflux for 3 h, then cold
to room temperature. The formed solid was collected via filtration,
washed with n-hexane and recrystallized from acetonitrile to
afford the key intermediates hydrazones 12a-b in a good
yield (87–92%).
(
1
1
CH ), 41.47 (-CH -CH ), 61.97 (N-CH ), 110.93, 113.40, 114.68,
16.96, 119.28, 121.62, 124.20, 124.67, 126.19, 129.59, 131.33,
3 2 3 2
32.68, 143.48, 150.87, 153.88, 161.55 (C ¼ O indolin-2-one), 165.22
ꢀ
1
(
C ¼ O hydrazide), 167.89 (C ¼ O ester); IR (KBr, ꢀ cm ) 3324 (NH)
and 1745, 1715, 1698 (3 C ¼ O); Analysis calculated for
16BrN : C, 53.63; H, 3.43; N, 8.94; found C, 53.80; H, 3.42;
N, 8.88.
C
21
H
3 5
O
5
.1.1.5.1. 3-Hydrazono-1-isobutylindolin-2-one 12a. Yellow powder
5
.1.1.3.12. N’-(1-Benzyl-2-oxoindolin-3-ylidene)-5-bromobenzofuran-2-
ꢂ
(yield 65%), m.p. 91–93 C; H NMR ppm: 0.98 (d, 6H, -CH-(CH ) ,
3 2
1
ꢂ
1
carbohydrazide 7e. Yellow powder (yield 80%), m.p. 248–250 C; H
J ¼ 6.8 Hz), 2.02–2.12 (m, 1H, N-CH -CH), 3.55 (d, 2H, N-CH ,
2
2
NMR ppm: 5.01, 5.05 (2 s, 2H, N-CH ), 7.06 (t, 1H, Ar-H, J ¼ 8.0 Hz),
2
J ¼ 7.6 Hz), 7.02 (t, 1H, Ar-H, J ¼ 7.6 Hz), 7.08 (d, 1H, Ar-H,
J ¼ 8.0 Hz), 7.21 (t, 1H, Ar-H, J ¼ 8.0 Hz), 7.41 (d, 1H, Ar-H,
J ¼ 7.6 Hz), 9.67 (d, 1H, NH , D O exchangeable, J ¼ 14.8 Hz), 10.54
7
2
7
.17 (t, 1H, Ar-H, J ¼ 8.0 Hz), 7.29– 7.32 (m, 4H, Ar-H), 7.42–7.48 (m,
H, Ar-H), 7.69–7.73 (m, 1.5H, Ar-H), 7.77 (t, 1H, Ar-H, J ¼ 8.0 Hz),
.87, 8.02 (2 s, 1H, Ar-H), 8.09–8.12 (m, 1.5H, Ar-H), 12.01, 13.99
2
2
1
3
ꢀ
1
(d, 1H, NH
20.45 (–CH–(CH
17.73, 121.83, 122.23, 125.66, 127.45, 140.07, 161.49 (C ¼ O indo-
lin-2-one); Analysis calculated for C H N O: C, 66.34; H, 6.96; N,
2
, D
2
O exchangeable, J ¼ 14.8 Hz);
C NMR d ppm:
(
2 s, 1H, NH); IR (KBr, ꢀ cm ) 3309 (NH) and 1712, 1701 (2 C ¼ O);
þ þ
3 2 2 2
) ), 27.39 (N–CH –CH), 46.40 (N–CH ), 109.59,
MS m/z [%]: 476 [M þ2, 16.41], 474 [M , 21.15], 475 [100];
1
Analysis calculated for C H BrN O : C, 60.77; H, 3.40; N, 8.86;
24
16
3 3
1
2 15 3
found C, 60.98; H, 3.38; N, 8.75.
1
9.34; found C, 66.53; H, 6.91; N, 19.39.
5
.1.1.3.13.
5-Bromo-N’-(2-oxo-1-phenethylindolin-3-ylidene)benzo-
5
.1.1.6. Synthesis of target compounds 13a-b. To the previously
furan-2-carbohydrazide 7f. Yellow powder (yield 78%), m.p.
2
2
7
1
1
prepared solution of isocyanate 11, equimolar amount of hydra-
zone 12a–b was added. The reaction mixture was heated for 4 h,
the formed precipitate was filtrated while hot, washed with
diethyl ether and recrystallized from DMF/EtOH mixture to pro-
duce targeted compounds 13a–b, respectively.
ꢂ
1
39–241 C; H NMR ppm: 2.99 (br s, 2H, N-CH
2 2
-CH ), 4.03 (br s,
H, N-CH -CH ), 7.16–7.22 (m, 3H, Ar-H), 7.28–7.32 (m, 4H, Ar-H),
2
2
.45 (t, 1H, Ar-H, J ¼ 8.0 Hz), 7.46 (t, 2H, Ar-H, J ¼ 8.0 Hz), 7.78 (d,
H, Ar-H, J ¼ 8.0 Hz), 7.85 (s, 1H, Ar-H), 8.09 (s, 1H, Ar-H), 11.97,
1
3
3.94 (2 s, 1H, NH); C NMR d ppm: 33.43 (N-CH -CH ), 41.36 (N-
2 2
2 2
CH -CH ), 110.86, 114.71, 116.95, 119.34, 120.21, 121.53, 123.75,
5
.1.1.6.1. N-(5-Bromobenzofuran-2-yl)-2–(1-isobutyl-2-oxoindolin-3-yli-
1
1
26.15, 127.02, 127.92, 128.93, 129.34, 129.59, 130.55, 131.24,
dene)hydrazine-1-carboxamide 13a. Yellow powder (yield 73%),
32.58, 135.38, 138.58, 141.28, 143.61, 153.87, 161.36 (C ¼ O indo-
ꢂ
1
ꢀ
1
m.p. 208–210 C; H NMR ppm: 0.94 (d, 6H, –CH–(CH
.06–2.13 (m, 1H, N–CH –CH), 3.57 (d, 2H, N-CH
3
)
2
, J ¼ 8.0 Hz),
lin-2-one), 165.06 (C ¼ O hydrazide); IR (KBr, ꢀ cm ) 3356 (NH)
and 1721, 1703 (2 C ¼ O); Analysis calculated for C25H18BrN3O3:
C, 61.49; H, 3.72; N, 8.60; found C, 61.63; H, 3.70; N, 8.66.
2
2
2
, J ¼ 7.6 Hz), 6.55
(s, 1H, Ar-H), 6.63 (s, 0.5H, Ar-H), 7.15–7.21 (m, 1H, Ar-H), 7.28–7.32
(m, 1.5H, Ar-H), 7.41–7.47 (m, 2H, Ar-H), 7.70–7.74 (m, 2H, Ar-H),
1
3
1
2
1
1
0.12 (s, 1H, NH urea), 11.33 (s, 1H, NH urea); C NMR d ppm:
5
.1.1.3.14.
5-Bromo-N’-(5-bromo-1-isobutyl-2-oxoindolin-3-ylidene)-
0.44 (–CH–(CH ), 27.28 (N–CH –CH), 66.82 (N–CH ), 110.60,
)
3 2
2
2
benzofuran-2-carbohydrazide 7 g. Orange powder (yield 74%), m.p.
12.52, 116.02, 119.90, 121.04, 122.52, 123.30, 125.07, 131.39,
32.27, 133.49, 143.34, 148.57, 148.99, 150.31, 151.01, 161.27
ꢂ
1
2
2
6
2
44–246 C; H NMR ppm: 0.94 (d, 6H, –CH–(CH
3
)
2
, J ¼ 7.6 Hz),
ꢀ
1
.11–2.17 (m, 1H, N-CH -CH), 3.51 (d, 2H, N-CH
.94–7.12 (m, 1H, Ar-H), 7.70 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.73–7.80 (m,
H, Ar-H), 8.10–8.18 (m, 2H, Ar-H), 8.35 (s, 1H, Ar-H), 12.12, 13.98
2
2
, J ¼ 8.0 Hz),
(
3
C ¼ O indolin-2-one), 165.09 (C ¼ O hydrazide); IR (KBr, ꢀ cm
)
345, 3240 (2NH) and 1730, 1711 (2 C ¼ O); MS m/z [%]: 457
þ þ
[
C
M þ2, 27.45], 455 [M , 29.72], 414 [100]; Analysis calculated for
ꢀ
1
(
2 s, 1H, NH); IR (KBr, ꢀ cm ) 3358 (NH) and 1724, 1700 (2 C ¼ O);
21
H
19BrN : C, 55.40; H, 4.21; N, 12.31; found C, 55.59; H, 4.16;
4 3
O
Analysis calculated for C21H17Br2N3O3: C, 48.58; H, 3.30; N, 8.09; N, 12.42.
found C, 48.73; H, 3.27; N, 8.14.
5
2
.1.1.6.2. 2–(1-Benzyl-2-oxoindolin-3-ylidene)-N-(5-bromobenzofuran-
-yl)hydrazine-1-carboxamide 13 b. Yellow powder (yield 68%), m.p.
5
.1.1.3.15. N’-(1-Benzyl-5-bromo-2-oxoindolin-3-ylidene)-5-bromoben-
ꢂ
1
zofuran-2-carbohydrazide 7 h. Yellow powder (yield 81%), m.p. 217–219 C; H NMR ppm: 4.98 (s, 2H, N-CH ), 6.53 (s, 1H, Ar-H),
2
ꢂ
1
2
36–237 C; H NMR ppm: 5.00, 5.04 (2 s, 2H, N-CH ), 7.03 (d, 1H, 6.61 (s, 1H, Ar-H), 7.02 (d, 1H, Ar-H, J ¼ 8.0 Hz), 7.11 (t, 1H, Ar-H,
2
Ar-H, J ¼ 8.0 Hz), 7.29–7.32 (m, 1H, Ar-H), 7.35–7.39 (m, 2H, Ar-H), J ¼ 8.0 Hz), 7.25–7.30 (m, 2H, Ar-H), 7.31–7.38 (m, 3H, Ar-H),
7
1
1
.42 (d, 2H, Ar-H, J ¼ 8.0 Hz), 7.51–7.61 (m, 1H, Ar-H), 7.66–7.70 (m, 7.41–7.45 (m, 2H, Ar-H), 7.68–7.71 (m, 2H, Ar-H), 10.10 (s, 1H, NH
13
H, Ar-H), 7.73–7.77 (m, 2H, Ar-H), 7.87, 8.13 (2 s, 1H, Ar-H), 8.07 (s, urea), 11.38 (s, 1H, NH urea); C NMR d ppm: 42.51 (CH ), 115.57,
2
ꢀ
1
H, Ar-H), 12.19, 13.87 (2 s, 1H, NH); IR (KBr, ꢀ cm ) 3335 (NH) 119.67, 120.62, 122.17, 123.04, 124.56, 124.73, 127.43, 127.63,
and 1731, 1708 (2 C ¼ O); Analysis calculated for C24
H
15Br
2
N
3 3
O
: C, 128.74, 130.81, 131.68, 132.92, 135.84, 142.18, 148.06, 148.21,
5
2.11; H, 2.73; N, 7.60; found C, 51.86; H, 2.74; N, 7.69.
148.48, 149.75, 150.56, 150.69, 160.57 (C ¼ O indolin-2-one), 165.06

2
82
W. M. ELDEHNA ET AL.
1
(
1
3
C ¼ O hydrazide); IR (KBr, ꢀ cmꢀ ) 3334, 3270 (2NH) and 1728, References
707 (2 C ¼ O); Analysis calculated for C24
4 3
H17BrN O : C, 58.91; H,
1.
2.
3.
Manning G, Whyte DB, Martinez R, et al. The protein kinase
complement of the human genome. Science 2002;298:
.50; N, 11.45; found C, 59.22; H, 3.53; N, 11.52.
1
912–34.
Parang K, Sun G. Protein kinase inhibitors drug discovery.
In: Gad SC, ed. Drug discovery handbook. Hoboken, New
Jersey: John Wiley & Sons, Inc; 2005:1191–1257.
Roskoski R. Jr. Classification of small molecule protein kin-
ase inhibitors based upon the structures of their drug-
enzyme complexes. Pharmacol Res 2016;103:26–48.
5
.2. Biological evaluations
All experimental procedures utilised in the biological assays herein
conducted were performed as reported earlier; anti-prolifer-
, Annexin V-FITC Apoptosis
assays, and have been provided in the
Supplementary Materials.
115
116–117
118
ation , cell cycle
kinase
and CDK2
1
19
4. Allam HA, Aly EE, Farouk A, et al. Design and synthesis of
some new 2,4,6-trisubstituted quinazoline EGFR inhibitors
as targeted anticancer agents. Bioorg Chem 2020;98:
103726.
5
.3. Molecular docking study
5
.
Al-Sanea MM, Elkamhawy A, Paik S, et al. Sulfonamide-
based 4-anilinoquinoline derivatives as novel dual Aurora
kinase (AURKA/B) inhibitors: synthesis, biological evaluation
and in silico insights. Bioorg Med Chem 2020;28:115525.
Alonso A, Sasin J, Bottini N, et al. Protein tyrosine phospha-
tases in the human genome. Cell 2004;117:699–711.
All the molecular modelling simulations were performed using
Molecular Operating Environment (MOE, 2010.10) software. All
minimizations were carried out with MOE until an RMSD gradient
ꢀ
1
ꢀ1
of 0.05 kcalꢃmol
Å
with MMFF94ꢄ force field and the partial
6
.
.
charges were automatically calculated. The X-ray crystallographic
structure of CDK2 co-crystallized with an oxindole derivative (IC50
7
Bononi A, Agnoletto C, De Marchi E, et al. Protein kinases
and phosphatases in the control of cell fate. Enzyme Res
1
09
¼
60 nM) as inhibitor (PDB ID: 1FVT)
lized with the oxindole derivative Indirubin-3 -monoxime (IC50
and of GSK-3b co-crystal-
0
¼
2
011;2011:329098.
1
10
2
2 nM) as inhibitor (PDB ID: 1Q41)
were downloaded from the
8
.
.
Zhang J, Yang PL, Gray NS. Targeting cancer with small
molecule kinase inhibitors. Nat Rev Cancer 2009;9:28–39.
Fabbro D, Ruetz S, Buchdunger E, et al. Protein kinases as
targets for anticancer agents: from inhibitors to useful
drugs. Pharmacol Ther 2002;93:79–98.
1
20
protein data bank . The selection of these two protein structures
specifically attributed to their co-crystallization with potent CDK2
and GSK-3b oxindole-based inhibitors, respectively.
9
For the CDK2 protein structure (PDB ID: 1FVT); water molecules
were first removed, as for the GSK-3b protein structure (PDB ID:
1
0. Pearson M, Garc ꢀı a-Echeverr ꢀı a C, Fabbro D, Protein tyrosine
kinases as targets for cancer and other indications. In:
Fabbro D, Mc Cormick F, eds. Protein tyrosine kinases -
from inhibitors to useful drugs. Totowa (NJ): Humana Press
Inc.; 2006:1–29.
1
Q41); Chain B and water molecules which are not involved in
binding were first removed, whereas, two conservative water mol-
ecules near chain A Thr138 that serve a functional role in the co-
110
crystalized inhibitor binding were kept . Then, the proteins were
prepared for the docking study using LigX protocol in MOE with
default options. The co-crystalized ligands were used to define the
active site for docking. Triangle Matcher placement method and
London dG scoring function were used for docking. Self-docking
of the co-crystallized ligands in the active site of the kinase
domains was first performed to validate the used docking proto-
col giving a docking pose with an energy score (S) ¼ ꢀ10.63 kcal/
mol and an RMSD of 0.894 Å in CDK2 (PDB ID: 1FVT) and energy
score (S) ¼ ꢀ12.00 kcal/mol and an RMSD of 0.471 Å in GSK-3b
1
1. Sedlacek HH. Kinase inhibitors in cancer therapy: a look
ahead. Drugs 2000;59:435–76.
12. Weinstein IB, Joe AK. Mechanisms of disease: oncogene
addiction-a rationale for molecular targeting in cancer ther-
apy. Nat Clin Pract Oncol 2006;3:448–57.
13. Padma VV. An overview of targeted cancer therapy.
BioMedicine 2015;5:19.
14. Aggarwal S. Targeted cancer therapies. Nat Rev Drug
Discov 2010;9:427–8.
15. Topcul M, Cetin I. Endpoint of cancer treatment: targeted
therapies. Asian Pac J Cancer Prev 2014;15:4395–403.
(PDB ID: 1Q41), Figure 7 and Table 5.
The validated docking protocols were then used to study the
1
6. Bray F, Ferlay J, Soerjomataram I, et al. Global cancer statis-
tics 2018: GLOBOCAN estimates of incidence and mortality
worldwide for 36 cancers in 185 countries. CA Cancer J
Clin 2018;68:394–424.
ligand-protein interactions of the newly synthesised compounds
in the active site of the target kinases to predict their binding
mode and establish their structure activity relationship (SAR) to
rationalise their binding affinity.
1
7. American Cancer Society. Cancer Facts & Figures 2019.
Available from: https://www.cancer.org/content/dam/can-
cer-org/research/cancer-facts-and-statistics/annual-cancer-
facts-and-figures/2019/cancer-facts-and-figures-2019.pdf.
8. Yan JD, Liu Y, Zhang ZY, et al. Expression and prognostic
significance of VEGFR-2 in breast cancer. Pathol Res Pract
Disclosure statement
No potential conflict of interest was reported by the author(s).
1
1
2
015;211:539–43.
Funding
9. Guo S, Colbert LS, Fuller M, et al. Vascular endothelial
growth factor receptor-2 in breast cancer. Biochim Biophys
Acta 2010;1806:108–21.
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-1439–065. Also, this research was funded by the Deanship of
Scientific Research at Princess Nourah Bint Abdulrahman
University through the Fast-track Research Funding Program.
20. Abdel-Mohsen HT, Abd El-Meguid EA, El Kerdawy AM, et al.
Design, synthesis, and molecular docking of novel 2-aryl-
benzothiazole multiangiokinase inhibitors targeting breast
cancer. Arch Pharm (Weinheim) 2020;353:e1900340.

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
283
2
1. Elbauomy Elsheikh S, Green AR, Lambros MBK, et al. FGFR1
amplification in breast carcinomas: a chromogenic in situ
hybridisation analysis. Breast Cancer Research 2007;9:R23.
2. Vrekoussis T, Stathopoulos EN, Kafousi M, et al. Expression
of endothelial PDGF receptors alpha and beta in breast
cancer: up-regulation of endothelial PDGF receptor beta.
Oncol Rep 2007;17:1115–9.
40. W eR sierska-G a˛ dek J, Gritsch D, Zulehner N, et al.
Roscovitine, a selective CDK inhibitor, reduces the basal
and estrogen-induced phosphorylation of ER-a in human
ER-positive breast cancer cells. J Cell Biochem 2011;112:
761–72.
41. Johnson N, Bentley J, Wang LZ, et al. Pre-clinical evaluation
of cyclin-dependent kinase 2 and 1 inhibition in anti-estro-
gen-sensitive and resistant breast cancer cells. Br J Cancer
2
2
2
3. He X, Xiang H, Zong X, et al. CDK2-AP1 inhibits growth of
breast cancer cells by regulating cell cycle and increasing
docetaxel sensitivity in vivo and in vitro. Cancer Cell Int
2
010;102:342–50.
4
2. Welsh GI, Proud CG. Glycogen synthase kinase-3 is rapidly
inactivated in response to insulin and phosphorylates
eukaryotic initiation factor eIF-2B. Biochem J 1993;294:
2014;14:130.
4. Alexander A, Karakas C, Chen X, et al. Cyclin E overexpres-
sion as a biomarker for combination treatment strategies
in inflammatory breast cancer. Oncotarget 2017;8:
6
25–9.
4
3. Frame S, Cohen P, Biondi RM. A common phosphate bind-
ing site explains the unique substrate specificity of GSK3
and its inactivation by phosphorylation. Mol Cell 2001;7:
14897–911.
2
2
2
2
2
3
5. Ding L, Cao J, Lin W, et al. The roles of cyclin-dependent
kinases in cell-cycle progression and therapeutic strategies
in human breast cancer. Int J Mol Sci 2020;21:1960.
6. Harwell RM, Porter DC, Danes C, Keyomarsi K. Processing of
cyclin E differs between normal and tumor breast cells.
Cancer Res 2000;60:481–9.
7. Cooley A, Zelivianski S, Jeruss JS. Impact of cyclin E overex-
pression on Smad3 activity in breast cancer cell lines. Cell
Cycle 2010;9:4900–7.
8. Quintayo MA, Munro AF, Thomas J, et al. GSK3b and cyclin
D1 expression predicts outcome in early breast cancer
patients. Breast Cancer Res Treat 2012;136:161–8.
9. Walz A, Ugolkov A, Chandra S, et al. Molecular pathways:
revisiting glycogen synthase Kinase-3b as a target for the
treatment of cancer. Clin Cancer Res 2017;23:1891–7.
0. Duda P, Akula SM, Abrams SL, et al. Targeting GSK3 and
associated signaling pathways involved in cancer. Cells
1
321–7.
4
4. Hooper C, Killick R, Lovestone S. The GSK3 hypothesis of
Alzheimer’s disease. J Neurochem 2008;104:1433–9.
4
5. Bhat RV, Budd Haeberlein SL, Avila J. Glycogen synthase
kinase 3: a drug target for CNS therapies. J Neurochem
2
004;89:1313–7.
4
4
6. Hughes K, Nikolakaki E, Plyte SE, et al. Modulation of the
glycogen synthase kinase-3 family by tyrosine phosphoryl-
ation. Embo J 1993;12:803–8.
7. Martinez A, Castro A, Dorronsoro I, Alonso M. Glycogen
synthase kinase 3 (GSK-3) inhibitors as new promising
drugs for diabetes, neurodegeneration, cancer, and inflam-
mation. Med Res Rev 2002;22:373–84.
4
8. Avila J, Leon-Espinosa G, Garcia E, et al. Tau phosphoryl-
ation by GSK3 in different conditions. Int J Alzheimers Dis
2
012;2012:578373.
2020;9:1110.
4
5
9. Doble BW, Woodgett JR. GSK-3: tricks of the trade for a
multi-tasking kinase. J Cell Sci 2003;116:1175–86.
0. El Kerdawy AM, Osman AA, Zaater MA. Receptor-based
pharmacophore modeling, virtual screening, and molecular
docking studies for the discovery of novel GSK-3b inhibi-
tors. J Mol Model 2019;25:171.
3
3
3
3
1. Malumbres M. Physiological relevance of cell cycle kinases.
Physiol Rev 2011;91:973–1007.
2. Malumbres M, Barbacid M. Mammalian cyclin-dependent
kinases. Trends Biochem Sci 2005;30: 630–41.
3. Lapenna S, Giordano A. Cell cycle kinases as therapeutic
targets for cancer. Nat Rev Drug Discov 2009;8:547–66.
4. Luk KC, Simcox ME, Schutt A, et al. A new series of potent
oxindole inhibitors of CDK2. Bioorg Med Chem Lett 2004;
5
1. Gaisina IN, Gallier F, Ougolkov AV, et al. From a natural
product lead to the identification of potent and selective
benzofuran-3-yl-(indol-3-yl)maleimides as glycogen syn-
thase kinase 3beta inhibitors that suppress proliferation
and survival of pancreatic cancer cells. J Med Chem 2009;
14:913–7.
3
3
3
5. Chohan TA, Qayyum A, Rehman K, et al. An insight into
the emerging role of cyclin-dependent kinase inhibitors as
potential therapeutic agents for the treatment of advanced
cancers. Biomed Pharmacother 2018;107:1326–41.
6. Chohan TA, Qian H, Pan Y, Chen JZ. Cyclin-dependent kin-
ase-2 as a target for cancer therapy: progress in the devel-
opment of CDK2 inhibitors as anti-cancer agents. Curr Med
Chem 2015;22:237–63.
7. Abd El-Karim SS, Syam YM, El Kerdawy AM, Abdelghany
TM. New thiazol-hydrazono-coumarin hybrids targeting
human cervical cancer cells: synthesis, CDK2 inhibition,
QSAR and molecular docking studies. Bioorg Chem 2019;
5
2:1853–63.
5
2. Ugolkov A, Gaisina I, Zhang JS, et al. GSK-3 inhibition over-
comes chemoresistance in human breast cancer. Cancer
Lett 2016;380:384–92.
5
5
3. Branca MA. Multi-kinase inhibitors create buzz at ASCO.
Nat Biotechnol 2005;23:639.
4. Broekman F, Giovannetti E, Peters GJ. Tyrosine kinase
inhibitors: multi-targeted or single-targeted? World J Clin
Oncol 2011;2:80–93.
55. Krug M, Hilgeroth A. Recent advances in the development
of multi-kinase inhibitors. Mini Rev Med Chem 2008;8:
1312–27.
56. Muller-Tidow C, Diederichs S, Bulk E, et al. Identification of
metastasis-associated receptor tyrosine kinases in non-
small cell lung cancer. Cancer Res 2005;65:1778–82.
57. Garuti L, Roberti M, Bottegoni G. Multi-kinase inhibitors.
Curr Med Chem 2015;22:695–712.
86:80–96.
3
8. Horiuchi D, Huskey NE, Kusdra L, et al. Chemical-genetic
analysis of cyclin dependent kinase 2 function reveals an
important role in cellular transformation by multiple onco-
genic pathways. Proc Natl Acad Sci U S A 2012;109:
E1019–27.
3
9. Ali S, Heathcote DA, Kroll SH, et al. The development of a
selective cyclin-dependent kinase inhibitor that shows anti-
tumor activity. Cancer Res 2009;69:6208–15.
58. Abdel-Mohsen HT, Omar MA, El Kerdawy AM, et al. Novel
potent substituted 4-amino-2-thiopyrimidines as dual

2
84
W. M. ELDEHNA ET AL.
VEGFR-2 and BRAF kinase inhibitors. Eur J Med Chem 2019;
Alzheimer’s disease. A property common to most cyclin-
dependent kinase inhibitors? J Biol Chem 2001;276:251–60.
75. Bhat R, Xue Y, Berg S, et al. Structural insights and bio-
logical effects of glycogen synthase kinase 3-specific inhibi-
tor AR-A014418. J Biol Chem 2003;278:45937–45.
76. Vine KL, Matesic L, Locke JM, et al. Cytotoxic and anti-
cancer activities of isatin and its derivatives: a comprehen-
sive review from 2000–2008. Anticancer Agents Med Chem
179:707–22.
5
6
6
6
6
9. Lozinskaya NA, Babkov DA, Zaryanova EV, et al. Synthesis
and biological evaluation of 3-substituted 2-oxindole deriv-
atives as new glycogen synthase kinase 3b inhibitors.
Bioorg Med Chem 2019;27:1804–17.
0. Ouach A, Boulahjar R, Vala C, et al. Novel optimization of
valmerins (tetrahydropyrido[1,2-a]isoindolones) as potent
dual CDK5/GSK3 inhibitors. Eur J Med Chem 2016;115:
2
009;9:397–414.
3
11–25.
1. Xu M, Wang SL, Zhu L, et al. Structure-activity relationship
SAR) studies of synthetic glycogen synthase kinase-3b
inhibitors: a critical review. Eur J Med Chem 2019;164:
48–70.
7
7
7
7. Abdel-Aziz HA, Eldehna WM, Keeton AB, et al. Isatin-
benzoazine molecular hybrids as potential antiproliferative
agents: synthesis and in vitro pharmacological profiling.
Drug Des Devel Ther 2017;11:2333–46.
8. Al-Warhi T, El Kerdawy AM, Aljaeed N, et al. Synthesis, bio-
logical evaluation and in silico studies of certain oxindole-
indole conjugates as anticancer CDK inhibitors. Molecules
(
4
2. Boulahjar R, Ouach A, Bourg S, et al. Advances in tetrahy-
dropyrido[1,2-a]isoindolone (valmerins) series: potent glyco-
gen synthase kinase 3 and cyclin dependent kinase 5
inhibitors. Eur J Med Chem 2015;101:274–87.
2
020;25:2031.
9. Goodman VL, Rock EP, Dagher R, et al. Approval summary:
sunitinib for the treatment of imatinib refractory or intoler-
ant gastrointestinal stromal tumors and advanced renal cell
carcinoma. Clin Cancer Res 2007;13:1367–73.
3. Boulahjar R, Ouach A, Matteo C, et al. Novel tetrahydropyr-
ido[1,2-a]isoindolone derivatives (valmerins): potent cyclin-
dependent kinase/glycogen synthase kinase 3 inhibitors
with antiproliferative activities and antitumor effects in
human tumor xenografts. J Med Chem 2012;55:9589–606.
4. Polychronopoulos P, Magiatis P, Skaltsounis AL, et al.
Structural basis for the synthesis of indirubins as potent
and selective inhibitors of glycogen synthase kinase-3 and
cyclin-dependent kinases. J Med Chem 2004;47:935–46.
5. Mettey Y, Gompel M, Thomas V, et al. Aloisines, a new fam-
ily of CDK/GSK-3 inhibitors. SAR study, crystal structure in
complex with CDK2, enzyme selectivity, and cellular effects.
J Med Chem 2003;46:222–36.
6. Zhao P, Li Y, Gao G, et al. Design, synthesis and biological
evaluation of N-alkyl or aryl substituted isoindigo deriva-
tives as potential dual cyclin-dependent kinase 2 (CDK2)/
glycogen synthase kinase 3b (GSK-3b) phosphorylation
inhibitors. Eur J Med Chem 2014;86:165–74.
7. Patel DS, Dessalew N, Iqbal P, Bharatam PV. Structure-
based approaches in the design of GSK-3 selective inhibi-
tors. Curr Protein Pept Sci 2007;8:352–64.
8. Arfeen M, Patel R, Khan T, Bharatam PV. Molecular dynam-
ics simulation studies of GSK-3b ATP competitive inhibitors:
understanding the factors contributing to selectivity. J
Biomol Struct Dyn 2015;33:2578–93.
9. Echalier A, Endicott JA, Noble ME. Recent developments in
cyclin-dependent kinase biochemical and structural studies.
Biochim Biophys Acta 2010;1804:511–9.
0. 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.
1. Czelen P. Inhibition mechanism of CDK-2 and GSK-3beta
by a sulfamoylphenyl derivative of indoline-a molecular
dynamics study. J Mol Model 2017;23:230.
2. Pacureanu L, Avram S, Bora A, et al. Portraying the selectiv-
ity of GSK-3 inhibitors towards CDK-2 by 3D similarity and
molecular docking. Struct Chem 2019;30:911–23.
8
8
0. Inomata M, Nishioka Y, Azuma A. Nintedanib: evidence for
its therapeutic potential in idiopathic pulmonary fibrosis.
Core Evid 2015;10:89–98.
6
6
6
1. Jagtap AD, Chang PT, Liu JR, et al. Novel acylureidoindolin-
2
-one derivatives as dual Aurora B/FLT3 inhibitors for the
treatment of acute myeloid leukemia. Eur J Med Chem
014;85:268–88.
2
8
2. Yousefian M, Ghodsi R. Structure-activity relationship stud-
ies of indolin-2-one derivatives as vascular endothelial
growth factor receptor inhibitors and anticancer agents.
Arch Pharm (Weinheim) 2020;353:e2000022.
83. Eldehna WM, El Kerdawy AM, Al-Ansary GH, 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.
6
6
8
4. Laufer R, Forrest B, Li SW, et al. The discovery of PLK4
inhibitors: (E)-3-((1H-Indazol-6-yl)methylene)indolin-2-ones
as novel antiproliferative agents. J Med Chem 2013;56:
6
7
7
7
7
6
069–87.
8
5. Wang HC, Jagtap AD, Chang PT, et al. Bioisosteric replace-
ment of an acylureido moiety attached to an indolin-2-one
scaffold with a malonamido or a 2/4-pyridinoylamido moi-
ety produces a selectively potent Aurora-B inhibitor. Eur J
Med Chem 2014;84:312–34.
8
6. Zhong Y, Xue M, Zhao X, et al. Substituted indolin-2-ones
as p90 ribosomal S6 protein kinase 2 (RSK2) inhibitors:
molecular docking simulation and structure-activity rela-
tionship analysis. Bioorg Med Chem 2013;21:1724–34.
7. Aneja B, Khan NS, Khan P, et al. Design and development
of Isatin-triazole hydrazones as potential inhibitors of
microtubule affinity-regulating kinase 4 for the therapeutic
management of cell proliferation and metastasis. Eur J Med
Chem 2019;163:840–52.
8
3. Ko HW, Kim EY, Chiu J, et al. A hierarchical phosphorylation
cascade that regulates the timing of PERIOD nuclear entry
reveals novel roles for proline-directed kinases and GSK-
3
1
beta/SGG in circadian clocks.
2664–75.
J
Neurosci 2010;30:
8
8. Dweedar HE, Mahrous H, Ibrahim HS, Abdel-Aziz HA.
Analogue-based design, synthesis and biological evaluation
of 3-substituted-(methylenehydrazono)indolin-2-ones as
anticancer agents. Eur J Med Chem 2014;78:275–80.
7
4. Leclerc S, Garnier M, Hoessel R, et al. Indirubins inhibit
glycogen synthase kinase-3 beta and CDK5/p25, two pro-
tein kinases involved in abnormal tau phosphorylation in

JOURNAL OF ENZYME INHIBITION AND MEDICINAL CHEMISTRY
285
8
9. Hoessel R, Leclerc S, Endicott JA, et al. Indirubin, the active 106. Acikgoz E, Guler G, Camlar M, et al. Glycogen synthase kin-
constituent of a Chinese antileukaemia medicine, inhibits
cyclin-dependent kinases. Nat Cell Biol 1999;1:60–7.
0. Damiens E, Baratte B, Marie D, et al. Anti-mitotic properties
ase-3 inhibition in glioblastoma multiforme cells induces
apoptosis, cell cycle arrest and changing biomolecular
structure. Spectrochim Acta A Mol Biomol Spectrosc 2019;
209:150–64.
9
of indirubin-3’-monoxime, a CDK/GSK-3 inhibitor: induction
of endoreplication following prophase arrest. Oncogene 107. Rashid MS, Mazur T, Ji W, et al. Analysis of the role of
001;20:3786–97.
GSK3 in the mitotic checkpoint. Sci Rep 2018;8:14259.
2
9
9
9
1. Marko D, Schatzle S, Friedel A, et al. Inhibition of cyclin- 108. Gorczyca W. Cytometric analyses to distinguish death proc-
esses. Endocr Relat Cancer 1999;6:17–9.
dependent kinase 1 (CDK1) by indirubin derivatives in
human tumour cells. Br J Cancer 2001;84:283–9.
2. Liang C, Xia J, Lei D, et al. Synthesis, in vitro and in vivo
antitumor activity of symmetrical bis-Schiff base derivatives
of isatin. Eur J Med Chem 2014;74:742–50.
1
09. Davis ST, Benson BG, Bramson HN, et al. Prevention of
chemotherapy-induced alopecia in rats by CDK inhibitors.
Science 2001;291:134–7.
1
10. Bertrand JA, Thieffine S, Vulpetti A, et al. Structural charac-
terization of the GSK-3beta active site using selective and
non-selective ATP-mimetic inhibitors. J Mol Biol 2003;333:
3. Meijer L, Skaltsounis AL, Magiatis P, et al. GSK-3-selective
inhibitors derived from Tyrian purple indirubins. Chem Biol
3
93–407.
2003;10:1255–66.
1
11. Attia MI, Eldehna WM, Afifi SA, et al. New hydrazonoindo-
lin-2-ones: Synthesis, exploration of the possible anti-prolif-
erative mechanism of action and encapsulation into PLGA
microspheres. Plos One 2017;12:e0181241.
9
9
4. Khanam H, Shamsuzzaman , Bioactive Benzofuran deriva-
tives: a review. Eur J Med Chem 2015;97:483–504.
5. Miao Y-h, Hu Y-h, Yang J, et al. Natural source, bioactivity
and synthesis of benzofuran derivatives. RSC Adv 2019;9:
1
12. Abo-Ashour MF, Eldehna WM, Nocentini A, et al. Novel
hydrazido benzenesulfonamides-isatin conjugates: synthe-
sis, carbonic anhydrase inhibitory activity and molecular
modeling studies. Eur J Med Chem 2018;157:28–36.
13. Lee J, Khanapure SP, Kim H-O, et al. Efficient synthesis of a
benzo[b]furan building block. Synth Commun 2010;40:
27510–40.
9
6. Hassan GS, Georgey HH, George RF, Mohamed ER. Aurones
and furoaurones: biological activities and synthesis. Bull
Faculty Pharm Cairo Univ 2018;56:121–7.
7. Eldehna WM, Nocentini A, Elsayed ZM, et al. Benzofuran-
based carboxylic acids as carbonic anhydrase inhibitors
and antiproliferative agents against breast cancer. ACS
Med Chem Lett 2020;11:1022–7.
8. Xiang Y, Hirth B, Asmussen G, et al. The discovery of novel
benzofuran-2-carboxylic acids as potent Pim-1 inhibitors.
Bioorg Med Chem Lett 2011;21:3050–6.
1
9
3
390–6.
1
14. 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 Enzyme Inhibit Med Chem 2018;33:
9
9
6
86–700.
9. Salom ꢀe C, Narbonne V, Ribeiro N, et al. Benzofuran deriva-
1
1
15. Sabt A, Abdelhafez OM, El-Haggar RS, et al. Novel couma-
rin-6-sulfonamides as apoptotic anti-proliferative agents:
synthesis, in vitro biological evaluation, and QSAR studies.
J Enzyme Inhibit Med Chem 2018;33:1095–107.
16. Mahdy HA, Ibrahim MK, Metwaly AM, et al. Design, synthe-
sis, molecular modeling, in vivo studies and anticancer
evaluation of quinazolin-4(3H)-one derivatives as potential
VEGFR-2 inhibitors and apoptosis inducers. Bioorg Chem
tives as a novel class of inhibitors of mTOR signaling. Eur J
Med Chem 2014;74:41–9.
1
00. Salom ꢀe C, Ribeiro N, Chavagnan T, et al. Benzofuran deriva-
tives as anticancer inhibitors of mTOR signaling. Eur J Med
Chem 2014;81:181–91.
01. Abd El-Karim SS, Anwar MM, Mohamed NA, et al. Design,
synthesis, biological evaluation and molecular docking
studies of novel benzofuran-pyrazole derivatives as anti-
cancer agents. Bioorg Chem 2015;63:1–12.
02. Mosmann T. Rapid colorimetric assay for cellular growth
and survival: Application to proliferation and cytotoxicity
assays. J Immunol Methods 1983;65:55–63.
03. Cherukupalli S, Chandrasekaran B, Krystof V, et al.
Synthesis, anticancer evaluation, and molecular docking
studies of some novel 4,6-disubstituted pyrazolo[3,4-d]pyri-
midines as cyclin-dependent kinase 2 (CDK2) inhibitors.
Bioorg Chem 2018;79:46–59.
1
2
020;94:103422.
1
1
17. Eldehna WM, Hassan GS, Al-Rashood ST, et al. Synthesis
and in vitro anticancer activity of certain novel 1-(2-methyl-
1
1
6
-arylpyridin-3-yl)-3-phenylureas as apoptosis-inducing
agents. J Enzyme Inhibit Med Chem 2019;34:322–32.
18. Elmetwally SA, Saied KF, Eissa IH, Elkaeed EB. Design, syn-
thesis and anticancer evaluation of thieno[2,3-d]pyrimidine
derivatives as dual EGFR/HER2 inhibitors and apoptosis
inducers. Bioorgan Chem 2019;88:102944.
119. Sabt A, Eldehna WM, Al-Warhi T, et al. Discovery of 3,6-
disubstituted pyridazines as a novel class of anticancer
agents targeting cyclin-dependent kinase 2: synthesis, bio-
logical evaluation and in silico insights. J Enzyme Inhibit
Med Chem 2020;35:1616–30.
1
1
04. Oakes V, Wang W, Harrington B, et al. Cyclin A/Cdk2 regu-
lates Cdh1 and claspin during late S/G2 phase of the cell
cycle. Cell Cycle 2014;13:3302–11.
05. Martin A, Odajima J, Hunt SL, et al. Cdk2 is dispensable for
cell cycle inhibition and tumor suppression mediated by 120. Protein Data Bank. https://www.rcsb.org/ [last accessed10
p27(Kip1) and p21(Cip1). Cancer Cell 2005;7:591–8. Oct 2020].