Insight on a new indolinone derivative as an orally bioavailable lead compound against renal cell carcinoma

Article abstract of DOI:10.1016/j.bioorg.2021.104985

A series of novel 3-indolinone-thiazolidinones and oxazolidinones 4a-k was synthesized via molecular hybridization approach and sequentially evaluated to explore its cytotoxic activity. The cytotoxicity screening pointed toward the N-cyclohexyl thiazolidinone derivative 4f that revealed promising renal cytotoxicity against CAKI-1 and UO-31 renal cancer cell lines with IC₅₀ values 4.74 and 3.99 μM, respectively, which were comparable to those of sunitinib along with good safety threshold against normal renal cells. Further emphasis on compound 4f renal cytotoxicity was achieved via different enzyme assays and CAKI-1 and UO-31 cell cycle analysis. The results were supported by in silico studies to explore its physicochemical, pharmacokinetic and drug-likeness properties. Finally, compound 4f was subjected to an in vivo pharmacokinetic study through two different routes of administration showing excellent oral bioavailability. This research represents compound 4f as a promising candidate against renal cell carcinoma.

Full text of DOI:10.1016/j.bioorg.2021.104985

Bioorganic Chemistry 112 (2021) 104985
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Insight on a new indolinone derivative as an orally bioavailable lead
compound against renal cell carcinoma
,
Marwa A. Fouada *, Mayssoune Y. Zakib, Raghda A. Lotfyb, Walaa R. Mahmouda
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, El-Kasr El-Eini Street, P.O. Box 11562 Cairo, Egypt
b Applied Organic Chemistry Department, National Organization for Drug Control and Research (NODCAR), Giza, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
A series of novel 3-indolinone-thiazolidinones and oxazolidinones 4a-k was synthesized via molecular hybridi-
zation approach and sequentially evaluated to explore its cytotoxic activity. The cytotoxicity screening pointed
toward the N-cyclohexyl thiazolidinone derivative 4f that revealed promising renal cytotoxicity against CAKI-1
and UO-31 renal cancer cell lines with IC50 values 4.74 and 3.99 µM, respectively, which were comparable to
those of sunitinib along with good safety threshold against normal renal cells. Further emphasis on compound 4f
renal cytotoxicity was achieved via different enzyme assays and CAKI-1 and UO-31 cell cycle analysis. The results
were supported by in silico studies to explore its physicochemical, pharmacokinetic and drug-likeness properties.
Finally, compound 4f was subjected to an in vivo pharmacokinetic study through two different routes of
administration showing excellent oral bioavailability. This research represents compound 4f as a promising
candidate against renal cell carcinoma.
Indolinone
Sunitinib
Renal cell carcinoma
PDGFR
CDK
Cell cycle arrest
Oral bioavailability
1. Introduction
researches reported sunitinib therapy resistance which necessitates
further emphasis on other potential candidates for treating RCC
Cancer describes a condition where cellular changes cause the un-
controlled growth and division of cells with more than 1.9 million ex-
pected new cases in 2021 [1]. Kidney cancer represents the third tumor
of the urinary tract most frequently diagnosed in adults [2]. Renal cell
carcinoma (RCC) is the most common type of kidney cancer. It is highly
aggressive in nature and is the most lethal type among the urologic
malignancies with estimated 13.780 deaths in 2021 [1]. The patho-
genesis of RCC involves alteration in genes associated with angiogenesis
and consequently this type of tumor is usually rich in vasculature [3,4].
Accordingly, current RCC treatments rely on tyrosine kinase inhibitors
that target the VEGF signaling axis associated with angiogenesis process
and consequently, inhibitors of VEFGR (vascular endothelial growth
factor receptor) and PDGFR (platelet-derived growth factor receptor)
became the glowing hope as first line treatment of RCC [5].
[12–20].
Accordingly, it deemed of interest to focus on other anti-VEGF 3-
substituted indolinone drugs as semaxanib II [21,22], nintedanib III
[23] and orantinib IV [24] with pronounced inhibitory activity. In
addition to the above mentioned facts, 1H-indole-2,3-dione (isatin) de-
rivatives are valuable precursors with variable biological activities
[25,26] and pronounced anticancer activity with different mechanisms
[27–33] against variable types of tumors as exemplified by compounds
V-IX with potent antitumor activity such as renal carcinoma [34],
metastatic advanced solid tumors [35], liver carcinoma [36] and colon
carcinoma [37,38]. These mentioned data pointed toward this class of
compounds as promising antitumor scaffolds (Fig. 1). In the same vein,
hybridization of indolinone moiety with other five or six membered
heterocycles would augment the expected antitumor activity
[28,38–40].
In 2006, the indolinone-based drug sunitinib I was the first anti-
angiogenic drug approved by the US FDA for the treatment of imatinib-
resistant advanced RCC [6,7]. Sunitinib is considered as multi inhibitor
of tyrosine kinases including VEGFR, PDGFR and c-KIT [8] and hence, it
is considered as the first-line standard treatment for RCC displacing the
traditional immunotherapy technique [9–11]. Unfortunately, several
In the light of the aforementioned findings, it seemed beneficial to
incorporate the indolinone ring together with suitable 5-membered
heterocycles. Accordingly, the aim of this work was to design and syn-
thesize different indolinone-N-substituted thiazolidinone/oxazolidinone
hybrids with promising anticancer activity and efficient
* Corresponding author.
E-mail address: marwa.fouad@pharma.cu.edu.eg (M.A. Fouad).
https://doi.org/10.1016/j.bioorg.2021.104985
Received 18 January 2021; Received in revised form 5 May 2021; Accepted 10 May 2021
Available online 12 May 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
pharmacokinetic profile that would be better or comparable to sunitinib
that is currently developing resistance against RCC.
groups in compounds 4b-d and 4i, and the characteristic pattern of the
allylic protons in compound 4e. In the same vein, multiplet signals in-
tegrated for the ten cyclohexyl protons were observed in the range of
1.25–2.40 and 1.13–3.56 ppm in compounds 4f and 4j, respectively.
Meanwhile, an increase in the integration of the aromatic protons
confirmed the additional phenyl rings in compounds 4 g and 4 k. 13C
NMR of all the compounds were in accordance with their proposed
carbon skeleton confirming the cyclization step showing the signal of the
CH2 carbon at 30.2–38.4 ppm and the added carbonyl carbon in the
range of 169.0–174.4 ppm.
2. Results and discussion
2.1. Chemistry
The indolinone-based compounds 4a-k were synthesized according
to Scheme 1 where firstly, different isocyanates/thiocyanates were
reacted with hydrazine hydrate to form semi/thiosemicarbazide de-
rivatives 1a-k that were found to be in accordance with their reported
data [41–46]. The formed intermediates 1a-k were then reacted with
the key compound isatin 2 in absolute ethanol in presence of few drops
of glacial acetic acid [47] to obtain different hydrazine carboxamide/
thioamide intermediates 3a-k [47–52]. Finally, cyclization of the open
structures 3a-k was achieved via the reaction with chloroacetic acid in
glacial acetic acid and fused sodium acetate to get the final compounds
4a-k. Interestingly, the structure of the 5-membered ring obtained var-
ied according to the reaction conditions, where conducting the reaction
in glacial acetic acid afforded the thiazolidinone 4a-g and oxazolidinone
4h-k derivatives [53,54] while performing the reaction under basic
conditions such as pyridine [55] or potassium hydroxide [56] afforded
the thioxoimidazolidinone/imidazolidinedione derivatives.
2.2. Biological evaluation:
2.2.1. Antiproliferative activity
2.2.1.1. In vitro anticancer activity as primary single high dose (10 µM)
screening. Initially, all the final synthesized compounds 4a-k were
selected to be screened at a single high dose (10 µM) against a panel of
sixty cancer cell lines by the NCI Developmental Therapeutic Program
(www.dtp.nci.nih.gov) to evaluate their cytotoxicity at concentration
10 µM against sixty cancer cell lines, for more information see Table S1
(Appendix A).
The examination of the growth inhibition results revealed that
generally the thiazolidinone derivatives (4a-g) showed a broader spec-
trum than the oxazolidinone derivatives (4 h-k) which exhibited weak
growth inhibition % (GI %). All the compounds demonstrated cytotox-
ic behavior activity against the ovarian and renal cell lines with com-
pound 4f exhibiting the broadest spectrum cytotoxic activity among all
the screened compounds, with different degrees of GI % against nearly
all the screened tumor cell types. This compound displayed a focused
–
The IR spectra of compounds 4a-k revealed additional C
O
–
stretching band along with that of indolinone in the range of 1666–1747
cmꢀ 1
.
1H NMR spectra of all the final compounds 4a-k displayed a
singlet signal corresponding to the two protons of CH2 group of the
thiazolidinone/oxazolidinone ring at 4.0–4.37 ppm confirming the
cyclization process, in addition to the appearance of aliphatic signals
matching the expected pattern of CH3, C2H5, butyl, and chloroethyl
Fig. 1. Structure of indolinone-based antitumor drugs (I-IV) and compounds V-IX.
2

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
Scheme 1. Reaction conditions: (a) Hydrazine hydrate 85%, ethanol, stir, room temperature, 30 min., (b) Absolute ethanol, glacial acetic acid, reflux, 2-44 h, (c)
Chloroacetic acid, glacial acetic acid, fused sodium acetate, reflux, 6-35 h.
activity against colon KM12, melanoma UACC-62 and ovarian IGROV1
and SK-OV-3 cancer cell lines with GI %= 84.9, 60, 65.9, 74.1, respec-
tively, for more information, see Table S1 (Appendix A). Moreover,
compound 4f demonstrated significant clustered cytotoxic activity
against 5 renal cancer cell lines namely A498, ACHN, CAKI-1, RXF393
and UO-31 with GI % = 69.5, 59.6, 77.7, 78.4 and 61.4, respectively
(For more information see Figure S1, See Appendix A). In addition,
Compounds 4d and 4 g showed moderate GI % against 11 and 9 cancer
cell lines in the range of 30 – 56 % and 31–50%, respectively.
of these compounds was well noticeable on the growth of these cell lines,
(Table 1).
Generally, the cytotoxic activity is significantly increased by isosteric
replacement of the oxygen atom in the oxazolidinone derivatives (4 h-k)
(GI %= 5.87–11.65) with a sulfur atom in the thiazolidinone derivatives
(4a-g) (GI %= 16.61–69.38). Comparing the derivatives substituted
with an aliphatic side chain, it was observed that its elongation to four
carbons as in 4d (GI %= 23.94–47.31) afforded more enhanced activity
than two carbons as in 4c (GI %= 17.58–40.98), more than one carbon
as in 4b (GI %= 1.08–33.56). On the other side, incorporation of a
double bond in the aliphatic side chain generally decreased the cytotoxic
activity as shown in the thiazolidinone derivative (4e) (GI %=
20.21–35.30). In addition, it was noticed that alicyclic substitution
2.2.1.2. SAR studies. The structure–activity relationship of the synthe-
sized final compounds was investigated for A498, ACHN, CAKI-1,
RXF393 and UO-31 renal cell lines, since the inhibition effect of most
Table 1
Growth inhibition % (GI %) of compounds 4a-k on A498, ACHN, CAKI-1, RXF393 and UO-31 renal cell lines.
Compound
X
R
A498
NTa
ACHN
CAKI-1
RXF 393
UO-31
Mean
4a
4b
4c
4d
4e
4f
S
S
S
S
S
S
H
16.940
13.209
23.099
24.823
20.207
59.687
23.980
22.989
40.980
44.043
35.297
77.748
0
40.299
33.557
39.488
47.315
32.844
61.444
20.305
16.615
28.925
35.204
27.919
69.380
CH3
C2H5
C4H9
12.239
17.580
35.894
22.476
69.556
1.083
23.477
23.943
28.772
78.464
–
–
CH2CH CH2
4g
S
27.854
31.254
50.388
31.801
44.632
37.186
4h
4i
O
O
O
H
NTa
0
8.280
3.540
0
34.790
14.000
10.296
11.652
5.867
9.613
-CH2CH2Cl
4.426
15.073
0
10.911
15.758
4j
3.988
2.948
4k
O
10.528
0
17.070
6.292
14.976
9.773
a
NT = not tested.
3

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
exhibited a pronounced growth inhibition in the thiazolidinone deriv-
ative (4f) (GI %= 59.69–78.46) much more than its oxazolidinone iso-
stere (4j) (% GI = 2.95–15.76). This effect is moderately decreased by
ring aromatization as shown in the thiazolidinone derivative 4 g (GI %=
27.85–50.39) when compared to the cyclohexyl substituted derivative
(4f); an effect that is not well observed with the oxazolidinone derivative
(4 k) (GI %= 0–17.07).
Table 3
Selectivity index of compound 4f toward normal renal cell line against five renal
cancer lines.
Compound
RPTEC/TERT1
Selectivity Index a
(IC50, µM)
A498
ACHN
CAKI-1
RXF393
UO-31
4f
28.89 ± 2.22*
18.78 ± 0.69
3.57
3.58
1.65
3.46
6.09
3.41
1.23
1.78
7.24
6.39
Sunitinib
* IC50 is significantly different from that of sunitinib at P < 0.05.
2.2.1.3. In vitro cytotoxic activity and selectivity against five renal cancer
cell lines. The most active compound 4f was further evaluated for its
inhibition activity against five renal cancer lines as well as normal renal
cells. The previous NCI screening at a single compound dose pointed
toward the clustered renal cytotoxicity of compound 4f. Accordingly,
IC50 values for compound 4f were calculated against the most vulnerable
renal cancer cell lines namely A498, ACHN, CAKI-1, RXF393 and UO-31
as well as the corresponding RPTEC/TERT1 normal renal cells normal
with respect to the reference drug, sunitinib, adopting an MTT colori-
metric screening assay [57] to explore both its potential toxicity and
selectivity. Sunitinib was the best choice here not only because of its
nucleus structural similarity to compound 4f but also for its prominent
renal cytotoxicity. (Tables 2 and 3).
a
Selectivity index = IC50 on normal cells/IC50 on tumor cells.
Table 4
IC50 values (nM) of compound 4f and sunitinib against angiogenesis promoting
enzymes VEGFR2, PDGFRα and PDGFRß.
Compound
VEGFR2
PDGFR
α
PDGFRß
IC50 [nM]
IC50 [nM]
IC50 [nM]
4f
452.53 ± 10.81*
38 ± 3.92
280.916 ± 17.10*
69 ± 13.92
45.013 ± 2.10*
55 ± 1.61
Sunitinib
*IC50 is significantly different from that of sunitinib at P < 0.05.
2.2.1.5. Cell cycle analysis and apoptosis study:. The promising IC50
values of compound 4f against CAKI-1 and UO-31 renal cancer cell lines
urged us to conduct its cell cycle analysis on both CAKI-1 and UO-31
renal cancer cell lines as well as an apoptotic study by Annexin V-
based flow cytometric analysis.
Clearly, the given results in Table 2 showed that the IC50 values of
compound 4f were all significantly different than those of sunitinib in
the five renal cancer cell lines. In addition, the results revealed the su-
perior cytotoxic activity of compound 4f against CAKI-1 and UO-31
renal cancer cell lines compared to the rest of the investigated cell
lines with IC50 values = 4.74 and 3.99 µM compared to sunitinib IC50
values = 5.51 and 2.94 µM, respectively corresponding to 0.86 and 1.35-
folds relative to sunitinib. In addition, the results in Table 3 indicated
that the best selectivity indices were against CAKI-1 and UO-31 renal
cancer cells with 6.09 and 7.24-fold selectivity among the five-screened
compounds with selectivity 1.54-fold lower than sunitinib in the normal
renal cell line.
2.2.1.6. Cell cycle analysis on CAKI-1 and UO-31 renal cancer cell lines.
In this part, the effect of compound 4f on different cell cycle phases of
CAKI-1 and UO-31 renal cancer cell lines was investigated by treating
the cells with concentration equals to its IC50 value (5.03 and 4.39 µM,
respectively). Fig. 2 clarified that exposure of CAKI-1 and UO-31 renal
cancer cells to compound 4f resulted in significant cell cycle arrest at
G2/M and pre-G1 phases with increase by 3.17 and 8.6 folds and by 9.51
and 13.86 folds, respectively. This was accompanied with concurrent
reduction in the percentage of cells at G0-G1 and S phases by approxi-
mately 0.49 and 0.46 folds and at the S phase by approximately 0.71 and
0.97 folds, respectively compared to the renal cancer cell without any
treatment [72,73].
2.2.1.4. Antiangiogenic activity preliminary study. Angiogenesis is the
physiological process of the growth of new blood vessels from preex-
isting ones under normal condition [58]. Likewise, the progression and
metastasis of any tumor depends on the development of new blood
vessels in and around the tumor where they could supply adequate ox-
ygen and nutrition to the tumor cells [59–62]. The strong correlation
between RCC and angiogenesis is well established [62–64] where mis-
coding of VHL, the tumor suppressor gene, usually results in enhanced
expression of certain growth factors such as vascular endothelial growth
factor (VEGF) and platelet derived growth factor (PDGF) [65] and
consequently promoting abundant vascularization and aberrant activa-
tion of signaling pathways leading to cell proliferation and inhibition of
apoptosis [66]. It’s worth mentioning that despite the fact that activa-
tion of both PDGFR isomers evoke mitogenic signals but it was proved
2.2.1.7. Annexin-V FTIC apoptotic study. The apoptotic effect of com-
pound 4f was studied using Annexin-V FTIC/PI dual staining assay at its
IC50 concentration on CAKI-1 and UO-31 renal cancer cell lines. The
results revealed the apoptotic impact of compound 4f displaying a
pronounced increase in the percentage of positive apoptotic cells in
(Upper Right + Lower Right) quadrants from 1.52% to 18.2% and from
1.52% to 23.97%, respectively in CAKI-1 and UO-31 renal cell lines
which comprises 11.97- and 15.76- fold increase with respect to the
control, respectively. In the same context, compound 4f caused 2.94-
and 6-fold increase in its necrotic ability compared to the control,
(Fig. 3).
that stimulation of PDGFRα inhibits chemotaxis of fibroblasts and
smooth muscle cells while PDGFRβ activation potently stimulates
fibroblast chemotaxis [67,68]. Therefore, the pronounced renal cyto-
toxicity of compound 4f can be rationalized by proving its significant
antiangiogenic activity against PDGFRß isoform with IC50 value com-
parable to the well-known antiangiogenic reference drug sunitinib
[68–71]. (Table 4)
2.2.1.8. In vitro CDK inhibitory activity:. Cyclin-dependent kinases
(CDKs) play crucial roles as regulators of cell progression through
different phases of the cell cycle and are regulated by phosphorylation
and activated by their association with cyclins [74]. Cyclin-dependent
kinase 1 enzyme (CDK1) is considered as central cell cycle regulator
that drives cells through G2 phase and mitosis [75,76] while the role of
CDK2 enzyme in G1 to S checkpoint activation is well documented [77].
Simultaneously, the cell cycle analysis results of compound 4f together
with its renal cytotoxicity pathed the road to explore its CDK inhibitory
activity in its different isoforms; CDK1/cyclin A, CDK1/cyclin B and
CDK2/ cyclin A enzymes using the well-known CDK inhibitor, roscovi-
tine as the reference drug. The IC50 values were calculated using
nonlinear regression analysis of their inhibition curves as shown in
Table 2
IC50 values (µM) of compound 4f and sunitinib against five renal cancer cell
lines.
Compound
A498
ACHN
CAKI-1
RXF393
UO-31
4f
8.1 ±
0.31*
5.24 ±
0.18
17.47 ±
0.46*
4.74 ±
0.19*
5.51 ±
0.26
23.57 ±
0.71*
3.99 ±
0.11*
2.94 ±
0.10
Sunitinib
5.42 ±
0.22
10.55 ±
0.32
*
IC50 is significantly different from that of sunitinib at P < 0.05.
4

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
Fig. 2. Flow cytometric analysis of CAKI-1 and UO-31 renal cancer cell lines untreated and treated with compound 4f.
Table 5. Closer look on the results located in the nM range indicates that
compound 4f exhibited 6.1, 0.19 and 1.64-fold inhibitory potency to-
ward CDK1/cyclin A, CDK1/cyclin B and CDK2/cyclin A enzymes,
respectively compared to the corresponding value of roscovitine with
significant statistical difference at P < 0.05. These results support the
previous results of the apoptotic study with the marked increase in the
percentage of positive apoptotic cells and complements its previous
antiangiogenic activity against PDGFRß isoform in comparison to
sunitinib.
metabolism of arachidonic acid, [84] which together with the inhibition
of CYP1A2 can generate problems in the metabolism of hormones [85]
and cholesterol synthesis [86,87].
Finally, compound 4f complies with the drug-likeness properties
with no violation of Lipinski (Pfizer), [88] Ghose, [89] Veber (GSK),
[90] Egan (Pharmacia) [91] and Muegge (Bayer) [92] filters specified by
the major pharmaceutical companies. In addition, this compound ex-
hibits high drug-likeness and bioavailability scores. One alert for Brenk
problematic fragments [93] and for Pan Assay Interfering substances
(PAINS) [94] were reported due to the presence of the imine fragment.
Accordingly, compound 4f is not only with promising biological efficacy
but also with encouraging pharmacokinetic and physicochemical
properties (Table 6).
2.3. In silico studies
2.3.1. Drug likeness profile
Physicochemical properties are important aspects to consider in drug
design and drug development [78]. They affect both pharmacokinetics
and pharmacological properties leading ultimately to modification in
the biological activity. In this context, the physicochemical properties as
well as the drug-like nature profile of compound 4f were computed
using SwissADME online web tool provided by the Swiss Institute of
Bioinformatics (SIB) [79]. A summary of these predictions is shown in
Table 6. The compound exhibits a predicted logPo/w in a range of
1.65–3.31, moderate water solubility without blood brain barrier
permeability (BBB) and thus no anticipated CNS side effects. A probable
high gastrointestinal absorption is predicted, as confirmed later in the in
vivo pharmacokinetic results. In addition, it is not expected to be a
substrate of the p-glycoprotein, so it is not disposed drug-resistance due
to its efflux mechanism used by many tumor cells [80]. Another
important fact that could be concluded from table 6 is that out of the five
predicted CYPs, compound 4f inhibited three isoforms namely CYP2C9,
CYPIA2 and CYP2C19 while it wasn’t able to inhibit two isoforms
namely CYP2D6 and CYP3A4. It is worth mentioning that CYP2C9 has
great impact on the metabolism of important drug families as NSAIDs
[81], antihypertensives [82] and some CNS neurotransmitters as sero-
tonin [83] while CYP2C19 inhibition could lead to alterations in the
2.3.1.1. Molecular docking study. By searching the protein data bank
(PDB), a co-crystallized PDGFRβ protein structure with an inhibitor was
not available. The only available proteins were apoprotein structures for
PDGFRβ transmembrane segment and in complex with PDGF without
inhibitor (PDB ID 2L6W [95] and 3MJG [96], respectively). On the other
hand, a co-crystallized of PDGFR
α with an inhibitor was available in the
protein data bank [97]. It was reported in literature that the tyrosine
kinase domain of PDGFRα and the tyrosine kinase domain of PDGFRβ
share strong sequence homology [98] and this was also confirmed using
UniProt (https://www.uniprot.org/) [99,100] by aligning the two pro-
teins with the code P16234 for human PDGFRα and P09619 for human
PDGFRβ resulting in 43.226% identity, 485 identical positions and 323
similar positions, For more information, (Appendix A). Accordingly, it
was suggested to use the available co-crystallized PDGFRα as surrogate
for PDGFRβ in order to highlight the possible interactions that could
possibly be the reason behind the activity of compound 4f and to vali-
date the ability of the synthesized compound to fit in the kinase domain.
Molecular docking simulations were performed to study the binding
pattern of compound 4f in the active site of PDGFRα and this pattern was
compared to that of the marketed indolinone derivative, sunitinib I. X-
5

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
Fig. 3. Apoptotic effect of compound 4f on CAKI-1 and UO-31 renal cancer cell lines via Annexin V-FTIC positive staining technique. The four quadrants are known
as (LL: viable, LR: early apoptosis, UR: late apoptosis, UL: necrosis).
binding interactions of the co-crystallized ligand, as confirmed by the
Table 5
obtained RMSD (1.11 Å) between the native ligand and the docked one.
IC50 values (nM) of compound 4f and roscovitine against the three CDK
As shown in Figs. 4 and 5, the amine and the carbonyl groups of the
isoforms.
sunitinib indole form two hydrogen bonds with the carbonyl group and
Compound
CDK1/cyclin A
IC50 [nM]
CDK1/cyclin B
IC50 [nM]
CDK2/cyclin A
IC50 [nM]
the NH group of the third hinge residue, Cys677, respectively. It also
interacts hydrophobically with the residues near the ceiling of the
adenine pocket including Leu599 and Val607 [101,102]. Similarly,
compound 4f forms one hydrogen bond with the NH group of Cys677
through the carbonyl group of the indolinone moiety, in addition to a
hydrophobic interaction with two amino acids: Leu599 and Gly680.
These similar interactions are reflected in the comparable docking
scores of the new indolinone derivative, 4f (-14.13445 kcal/mol), which
4f
61.014 ± 2.20*
372 ± 5.79
3424.048 ± 54.09*
650 ± 16.4
316.402 ± 7.35*
520 ± 4.99
Roscovitine
*IC50 is significantly different from that of roscovitine at P < 0.05.
ray crystal structure of PDGFRα (PDB ID: 5GRN, resolution 1.87 Å) [97]
that is in complex with an inhibitor, WQ-C-159, was downloaded from
the Protein Data Bank. First, the molecular docking setup was validated
by carrying out re-docking of the ligand, WQ-C-159, in the vicinity of the
displayed potent PDGFRα inhibitory activity comparable to sunitinib
(-15.09492 kcal/mol). According to the sequence homology between the
two isoforms of PDGFR, it can be concluded that the higher inhibition
PDGFRα active site. The applied docking protocol demonstrated to be
activity of compound 4f towards PDGFRβ more than PDGFR
α
might be
suitable for the presented docking study by reproducing the same
Table 6
Molecular properties of compound 4f predicted using SwissADME website.
Parameter
Result
Parameter
Result
Parameter
Result
Consensus Log P
ESOL, Ali and Silicos-IT Class
GI absorption
2.51
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Lipinski #violations
Ghose #violations
Veber #violations
Egan #violations
Yes
No
No
0
Muegge #violations
Bioavailability Score
PAINS #alerts
0
Moderately soluble
0.55
1
High
No
BBB permeant
Brenk #alerts
1
P-gp substrate
No
0
Leadlikeness #violations
Synthetic Accessibility
0
CYP1A2 inhibitor
CYP2C19 inhibitor
Yes
Yes
0
3.41
0
6

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
Fig. 4. 2D Diagram of the interaction of the docked (A) sunitinib and (B) 4f in the active site of PDGFR
α
(PDB ID: 5GRN) using Molecular Operating Environment
(MOE, 10.2008) software.
7

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
Table 7
Sprague-Dawley rat pharmacokinetic profile for compound 4f after oral and
intravenous administration.
Parameter
p.o.
i.v.
Rat no.
6
6
Dose level (mg/kg)
t1/2 (h)
15
5
7.862
18.569
–
2281.052
7100.526
10491.398
12767.714
476.581
T
max (h)
2
C
max (ng/mL)
6095.899
32821.686
36305.602
4686.533
413.159
115.353%
AUC0-t (ng.h/mL)
AUC0-∞ (ng.h/mL)
Vd (mL/kg)
CL (mL/h.kg)
F (%)
7000
6000
5000
4000
3000
2000
1000
i.v.
p.o.
Above IC90
0
0
5
10
15
20
25
30
Time (hr)
Fig. 6. Time-dependent plasma concentrations of 4f after oral and intravenous
administration to male Sprague-Dawley rats.
respectively), was higher than the IC90 value required for the cytotoxic
activity against CAKI-1 and UO-31 renal cancer cell line even after 24 h,
as shown in Fig. 6. These important findings showed that after oral
dosing in rats, compound 4f has not only an outstanding cytotoxic ac-
tivity but also good pharmacokinetic properties with excellent oral
bioavailability.
Fig. 5. 3D Representation of the interaction of the docked (A) sunitinib and (B)
4f in the active site of PDGFRα (PDB ID: 5GRN) using Molecular Operating
Environment (MOE, 10.2008) software.
3. Conclusion
justified by certain extra interactions than those shown in this molecular
docking analysis with certain amino acid residues present in PDGFRβ
only (Figs. 4 and 5).
In conclusion, compound 4f is a potent scaffold against renal carci-
noma with excellent physiochemical, pharmacokinetic profile and
interesting PDGFR/CDK inhibition activity. Accordingly, compound 4f
could be considered as a promising candidate for further preclinical and
clinical studies as an anticancer agent for treatment of renal cell
carcinoma.
2.4. In vivo PK profile of compound 4f
Pharmacokinetic parameters of the most potent compound 4f was
determined in male Sprague-Dawley rats, Table 7 and Fig. 6. The com-
pound was administered intravenously (i.v.) at 5 mg/kg or orally (p.o.)
at 15 mg/kg. Comparing the two routes of administration, it was found
that compound 4f demonstrated a longer half-life (t1/2) of 18.6 h after i.
v. administration than that after oral dosing (7.9 h) and this can be
correlated to the difference in the two values of volume of distribution
(Vd) following i.v. and oral administration (12767.7 and 4686.533 mL/
kg, respectively). Comparable plasma clearance (CL) values were ob-
tained following the two routes of administration (476.6 mL/h.kg after i.
v. administration and 413.2 mL/h.kg after oral administration).
Importantly, compound 4f possessed an excellent oral bioavailability (F)
of ~ 100% leading to a good oral exposure showed by AUC of 36305.6
ng.h/mL and a high maximum plasma concentration (Cmax) of 6095.9
ng/mL after oral dosing. These results confirmed the high gastrointes-
tinal absorption as predicted using the SwissADME web tool. The rat
plasma 4f concentration h (126.57 and 307.14 ng/mL for i.v. and p.o.,
4. Experimental
4.1. Chemistry
4.1.1. General
All solvents and reagents were commercially available and used
without further purification. Compounds 1a-k [41–46], 3a-h [47–51],
3k [52], 4a [39] were prepared as reported in the literature. For more
information, (Appendix A).
4.1.2. General procedure for preparation of compounds 3i and 3j
To a hot solution of isatin 2 (0.7 g, 5 mmol) in absolute ethanol (10
mL) containing few drops of glacial acetic acid either the semicarbazide
derivative 1i or 1j (5 mmol) dissolved in absolute ethanol (10 mL) was
8

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
added. After then, the reaction mixture was heated to reflux for 2 h and
the crystalline solid formed was collected by filtration, washed with hot
ethanol then by ether to give to give both compounds in a pure form.
6.89 (d, 1H, H7-indoline, J = 7.8), 7.04 (t, 1H, H5-indoline, J = 7.5), 7.37
(t, 1H, H6-indoline, J = 7.6), 8.13 (d, 1H, H4-indoline, J = 7.5), 10.75 (s,
1H, NH, D2O exchangeable), 13C NMR (DMSO‑d6) δ ppm: 12.3 (CH3),
21.3 (CH2), 32.9 (COCH2), 111.1, 117.1, 123.1, 128.5, 133.6, 144.0,
4.1.2.1. N-(2-Chloroethyl)-2-(2-indolinone-3-ylidene)
hydrazine-1-
148.9, 165.6, 172.9, 173.1 (2C = O), Anal. Calcd. for C13H12N4O2S
(288.33): C, 54.16; H, 4.20; N, 19.43%; Found: C, 54.37; H, 4.38; N,
19.62%.
carboxamide (3i):. Buff powder, (yield 78%), m.p. 246–247 ◦C; reac-
tion time 31 h; IR (KBr,
ν
cmꢀ 1): 3325, br. 3213 (NHs), 3097 (CH aro-
matic), 2939 (CH aliphatic), 1670, 1654 (2C = O); 1H NMR (DMSO‑d6,
400 MHz) δ ppm: 3.80 (t, 2H, CH2, J = 6.2), 4.90 (t, 2H, CH2, J = 6.1),
6.65 (s, 1H, NH, D2O exchangeable), 7.21–7.81 (m, 4H, indoline-H),
10.55 (s, 1H, NH, D2O exchangeable), 11.35 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6) δ ppm: 41.7, 44.1, 111.3, 112.9,
4.1.3.4. 3-Butyl-2-[(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-4-one
(4d). Brown powder, (yield 65%), m.p. 246–248 ◦C; reaction time 31 h;
IR (KBr,
ν
cmꢀ 1): 3182 (NH), 3089 (CH aromatic), 2958 (CH aliphatic),
1732 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm: 0.93 (t, 3H,
CH3, J = 7.3), 1.35–1.41 (m, 2H, CH2), 1.68–1.71 (m, 2H, CH2), 3.86 (t,
2H, CH2, J = 7.3), 4.08 (s, 2H, CH2), 6.89 (d, 1H, H7-indoline, J = 7.8),
7.01 (t, 1H, H5-indoline, J = 7.5), 7.37 (t, 1H, H6-indoline, J = 7.2), 8.12
–
–
126.9, 127.3, 128.7, 134.9, 139.8, 152.8, 158.9 2 (C O); Anal. Calcd.
for C11H11ClN4O2 (266.69): C, 49.54; H, 4.16; N, 21.01%; Found: C,
49.81; H, 4.40; N, 20.87%.
(d, 1H, H4-indoline, J = 7.5), 10.75 (s, 1H, NH, D2O exchangeable); 13
C
4.1.2.2. N-Cyclohexyl-2-(2-oxoindolin-3-ylidene)hydrazine-1-carbox-
NMR (DMSO‑d6) δ ppm: 14.0 (CH3), 20.0 (CH2), 28.9 (CH2), 32.9
(COCH2), 43.5 (CH2), 111.0, 117.4, 122.4, 128.4, 133.4, 144.7, 149.1,
165.2, 172.8, 172.9 (2C = O), Anal. Calcd. for C15H16N4O2S (316.38): C,
56.95; H, 5.10; N, 17.71%; Found C, 57.11; H, 5.34; N, 17.58%.
amide (3j). Bright yellow powder, (yield 69%), m.p. 229–231 ◦C; re-
action time 31 hr; IR (KBr,
ν
cmꢀ 1) : 3325 (2 NH), 3032 (CH aromatic),
2927 (CH aliphatic), 1620 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ
ppm: 1.27–1.34 (m, 5H, cyclohexyl-H), 1.55–1.72 (m, 5H, cyclohexyl-
H), 1.83–1.85 (m, 1H, cyclohexyl-H), 6.90 (d, 1H, H7-indoline, J =
7.8), 7.03 (t,1H, H5-indoline, J = 7.6), 7.32–7.39 (m, 2H, H6-indoline +
NH, D2O exchangeable), 8.19 (d, 1H, H4-indoline, J = 7.6), 10.30 (s, 1H,
NH, D2O exchangeable), 10.74 (s, 1H, NH, D2O exchangeable). Anal.
Calcd. for C15H18N4O2 (286.33): C, 63.16; H, 6.52; N, 19.34%; Found: C,
63.01; H, 6.28; N, 19.65%.
4.1.3.5. 3-Allyl-2-[(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-4-one
(4e). Dark green powder, (yield 75%), m.p. 254–256 ◦C; reaction time
35 h; IR (KBr,
ν
cmꢀ 1): 3429 (NH), 3086 (CH aromatic), 2981 (CH
aliphatic), 1732 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm: 4.13
(s, 2H, CH2), 4.48 (d, 2H, CH2, J = 4.6), 5.22 (d, 1H, =CH2, J = 10.5),
5.26 (d, 1H, =CH2, J = 17.4), 5.89–5.98 (m, 1H, CH), 6.88 (d, 1H,
H7-indoline, J = 7.7), 7.02 (t,1H, H5-indoline, J = 7.6), 7.36 (t, 1H,
H6-indoline, J = 7.6), 8.11 (d, 1H, H4-indoline, J = 7.6), 10.74 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO‑d6) δ ppm: 32.9 (N-CH2), 45.6
(CH2), 110.9, 117.3, 117.7, 122.6, 128.8, 131.4, 133.4, 144.7, 149.3,
165.2, 172.1, 172.6 (2C = O), Anal. Calcd. for C14H12N4O2S (300.34): C,
55.99; H, 4.03; N, 18.66%; Found C, 56.13; H, 4.29; N, 18.53%.
4.1.3. General procedure for preparation of compounds (4a-k)
An equimolar mixture of 3a-k (0.01 mol) and monochloroacetic acid
(0.49 g, 0.01 mol) with anhydrous sodium acetate (0.82 g, 0.01 mol) in
glacial acetic acid (20 mL) was refluxed for 6–35 h. Then, the reaction
mixture was allowed to cool to room temperature and poured into ice
water. The solid was filtered, washed with water and finally recrystal-
lized from ethanol to give compounds 4a-k.
4.1.3.6. 3-Cyclohexyl-2-[(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-
4-one (4f). Orange powder, (yield 89%), m.p. 288–290 ◦C; reaction time
4.1.3.1. 2-[(2-Oxoindolin-3-ylidene)hydrazono]thiazolidin-4-one
(4a)
35 h; IR (KBr,
ν
cmꢀ 1): 3414 (NH), 3086 (CH aromatic), 2931 (CH
[39]. Dark orange powder, (yield 60%), m.p. 208–210 ; reaction time 6
aliphatic), 1728 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm:
1.25–1.40 (m, 4H, cyclohexyl-H), 1.71–1.74 (m, 4H, cyclohexyl-H),
1.87–1.91 (m, 2H, cyclohexyl-H), 2.34–2.40 (m, 1H, cyclohexyl-H),
4.03 (s, 2H, CH2), 6.91 (d, 2H, H7-indoline, J = 7.8), 7.04 (t, 1H,
H5-indoline, J = 7.3), 7.39 (t, 1H, H6-indoline, J = 7.9), 8.19 (d, 1H,
H4-indoline, J = 7.5), 10.77 (s, 1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6) δ ppm: 25.3, 26.0, 28.05, 32.6 (CH2), 56.3, 111.3, 117.2,
122.4, 127.8, 133.5, 144.7, 148.6, 165.3, 173.1, 173.8 (2C = O), MS (m/
z %): 342 (M+, 82.35%), Anal. Calcd. for C17H18N4O2S (342.42): C,
59.63; H, 5.30; N, 16.36%; Found: C, 59.85; H, 5.41; N, 16.59%.
h; IR (KBr,
ν
cmꢀ 1): 3417, 3174 (2 NH), 3028 (CH aromatic), 2889 (CH
aliphatic), 1716 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm: 4.01
(s, 2H, CH2), 6.88 (d, 1H, H7-indoline, J = 7.6), 7.04 (t, 1H, H5-indoline,
J = 7.4), 7.35 (t, 1H, H6-indoline, J = 7.6), 8.22 (d, 1H, H4-indoline, J =
7.8), 10.71 (s, 1H, NH, D2O exchangeable), 11.18 (s, 1H, NH, D2O
exchangeable); 13C NMR (DMSO‑d6) δ ppm: 34.0 (CH2), 110.8, 117.5,
122.4, 128.4, 132.4, 133.6, 144.9, 165.1, 173.2, 174.4 (2C = O); Anal.
Calcd. for C11H8N4O2S (260.27): C, 50.76; H, 3.10; N, 21.53%; Found: C,
51.02; H, 3.36; N, 21.31%.
4.1.3.2. 3-Methyl-2-[(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-4-
4.1.3.7. 2-[(2-Oxoindolin-3-ylidene)hydrazono]-3-phenylthiazolidin-4-
one (4b). Dark orange powder, (yield 64%), m.p. > 300; reaction time 9
one (4g). Brownish orange powder, (yield 87%), m.p. >300 ◦C ; reaction
h; IR (KBr,
ν
cmꢀ 1): 3159 (NH), 3062 (CH aromatic), 2970 (CH
time 24 h; IR (KBr,
ν
cmꢀ 1): 3186 (NH), 3086 (CH aromatic), 2981
aliphatic), 1732,1716 (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm:
3.32 (s, 3H, CH3), 4.06 (s, 2H, CH2), 6.88 (d, 1H, H7-indoline, J = 7.7),
7.04 (t, 1H, H5-indoline, J = 7.5), 7.37 (t, 1H, H6-indoline, J = 7.6), 8.17
(aliphatic CH), 1728 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm:
4.22 (s, 2H, CH2), 6.65 (t, 1H, Ar-H, J = 7.8), 6.80 (d, 2H, Ar-H, J = 7.7),
7.22–7.27 (m, 3H, 2Ar-H + H7-indoline), 7.49 (d, 1H, H5-indoline, J =
7.2), 7.54–7.66 (m, 2H, H4+ H6-indoline), 10.69 (s, 1H, NH, D2O
(d, 1H, H4-indoline, J = 7.5), 10.75 (s, 1H, NH, D2O exchangeable); 13
NMR (DMSO‑d6) δ ppm: 30.2 (CH3), 33.0 (CH2), 110.9, 117.4, 122.6,
C
exchangeable); 13C NMR (DMSO‑d6) δ ppm: 33.4 (COCH2), 110.9,
129.0, 133.3, 144.7, 149.2, 165.2, 172.9, 173.0 (2C = O); Anal. Calcd.
for C12H10N4O2S (274.30): C, 52.55; H, 3.67; N, 20.43%; Found: C,
52.81; H, 3.90; N; 20.19%.
117.1, 122.1, 128.5, 129.5, 129.6, 133.4, 135.4, 144.5, 149.8, 165.2,
172.4, 172.9 (2C = O), Anal. Calcd. for C17H12N4O2S (336.37): C, 60.70;
H, 3.60; N, 16.66%; Found: C, 60.89; H, 3.76; N, 16.90%.
4.1.3.3. 3-Ethyl-2-[(2-oxoindolin-3-ylidene)hydrazono]thiazolidin-4-one
4.1.3.8. 2-[(2-Oxoindolin-3-ylidene)hydrazono]oxazolidin-4-one (4h).
(4c). Light orange powder, (yield 66%), m.p. > 300 ◦C ; reaction time 9
Reddish brown powder, (yield 67%), m.p. >300; reaction time 33 h; IR
h; IR (KBr,
ν
cmꢀ 1): 3429 (NH), 3082 (CH aromatic), 2970 (CH
(KBr,
ν
cmꢀ 1): 3178, 3143 (2 NH), 3082 (CH aromatic), 2962 (CH
aliphatic), 1732, 1705 (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm:
1.27 (t, 3H, CH3, J = 7.0), 3.90 (q, 2H, CH2, J = 6.9), 4.07 (s, 2H, CH2),
aliphatic), 1693, 1666 (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ ppm:
9

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
4.05 (s, 2H, CH2), 6.84 (d, 1H, H7-indoline, J = 7.6), 6.97 (t,1H,
H5-indoline, J = 7.6), 7.34 (t, 1H, H6-indoline, J = 7.4), 9.06 (d, 1H,
H4-indoline, J = 7.9), 10.88 (s, 2H, 2NH, D2O exchangeable); 13C NMR
(DMSO‑d6) δ ppm: 35.5 (CH2), 110.0, 121.6, 122.1, 129.7, 133.0, 133.8,
144.5, 169.4 (2C = O), Anal. Calcd. for C11H8N4O3 (244.21): C, 54.10;
H, 3.30; N, 22.94%; Found: C, 53.89; H, 3.47; N, 22.82%.
μ
M) for 24 h. The reported methodology of MTT colorimetric assay was
then applied [57].
4.2.2. Antiangiogenic activity preliminary study
The enzyme inhibition assay of compound 4f against VEGFR2,
PDGFRα and PDGFRß was performed at the Egyptian company for the
production of vaccines, sera, and drugs VACSERA, Giza, EGYPT. This
was carried out using BPS Bioscience® VEGFR2 (KDR) Kinase and
4.1.3.9. 3-(2-Chloroethyl)-2-[(2-oxoindolin-3-ylidene)hydrazono]oxazoli-
din-4-one (4i). White powder, (yield 60%), m.p. 225 ◦C; reaction time
PDGFR
α (D842I) Assay Kits with PDGFRα (D842I), GST-Tag (BPS
cmꢀ 1): 3305 (NH), 3097 (CH aromatic), 2943 (CH
Bioscience) or Recombinant Human PDGFRß, GST-tagged (Creative
BioMart) according to the manufacturer’s manual. The assay was carried
12 h; IR (KBr,
ν
aliphatic), 1747, 1666 (2C = O); 1H NMR (DMSO‑d6 400 MHz) δ ppm:
3.57 (t, 2H, CH2, J = 6.5), 3.98 (t, 2H, CH2, J = 5.8), 4.37 (s, 2H, CH2),
6.49–6.63 (m, 2H, H5-indoline and H7-indoline), 7.70–7.81 (m, 2H,
H4-indoline and H6-indoline), 8.8 (1H, NH, D2O exchangeable); 13C NMR
(DMSO‑d6) δ ppm: 38.4 (COCH2), 44.1 (CH2), 64.1 (CH2), 118.5, 127.0,
137.3, 145.2, 158.4, 158.7, 159.0, 159.1, 167.7, 170.8 (2C = O), Anal.
Calcd. for C13H11ClN4O3 (306.71): C, 50.91; H, 3.62; N, 18.27%, Found:
C, 51.23; H, 3.86; N, 18.49%.
at concentrations 10–10000 nM for VEGFR2 and PDGFR
α
, and at
1–1000 nM for PDGFRß using 20 μL of the diluted enzyme (1 ng/μL).
(For more information, see Appendix A)
4.2.3. Cell cycle analysis and apoptosis study
Cell cycle analysis of compound 4f was achieved using propidium
iodide (PI) flow cytomertric analysis according to the reported proced-
ure [106,107]. An apoptotic study using the Annexin V-FITC Apoptosis
Detection Kit (K101-25, BioVision®, Mountain View, Canada), was
performed on compound 4f at the Egyptian company for the production
of vaccines, sera, and drugs VACSERA, Giza, EGYPT. The assay was
measured at its IC50 concentration value on both CAKI-1 and UO-31
renal cells after 24 h in three successive steps; according to the manu-
facturer’s instructions.
4.1.3.10. 3-Cyclohexyl-2-[(2-oxoindolin-3-ylidene)hydrazono]oxazolidin-
4-one (4j). Bright yellow powder, (yield 94%), m.p. 226–227 ◦C; reac-
tion time 9 h; IR (KBr,
ν
cmꢀ 1): 3170 (NH), 3093 (CH aromatic), 2931
(aliphatic CH), 1716, 1693 (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ
ppm: 1.13–1.35 (m, 6H, cyclohexyl H), 1.58–1.81 (m, 4H, cyclohexyl
H), 3.55–3.56 (m, 1H, cyclohexyl H), 4.07 (s, 2H, CH2), 6.93 (d, 1H,
H7-indoline, J = 7.7), 7.08 (t, 1H, H5-indoline, J = 7.5), 7.32 (t, 1H,
H6-indoline, J = 7.6), 7.63 (d, 1H, H4-indoline, J = 7.3), 11.88 (s, 1H,
NH, D2O exchangeable); 13C NMR (DMSO‑d6) δ ppm: 25.2, 25.5, 33.0
(COCH2), 49.1, 111.2, 120.7, 120.8, 122.7, 130.8, 131.5, 141.7, 153.5,
163.1 (2C = O), Anal. Calcd. for C17H18N4O3 (326.36): C, 62.57; H, 5.56;
N, 17.17%; Found: C, 62.34; H, 5.70; N, 17.43%.
4.2.4. In vitro CDK inhibitory activity
IC50 values of compound 4f were estimated using enzyme-linked
immunosorbent assay kits for CDK1a (Cloud clone prob.®), CDK1b
(Cell signaling Technology®) and CDK2a (Bioscience.com) at the
Egyptian company for the production of vaccines, sera, and drugs
VACSERA, Giza, EGYPT following the kits suppliers protocols. The assay
was carried at concentrations 10–10000 nM for CDK1b and CDK2a using
4.1.3.11. 3-(4-Fluorophenyl)-2-[(2-oxoindolin-3-ylidene)hydrazono]oxa-
enzyme concentration (~5 ng/
μ
L) and at 1–1000 nM for CDK1a using
zolidin-4-one (4k). Light yellow powder, (yield 77%), m.p. 263–265 ◦C;
ν
cmꢀ 1): 3294 (NH), 3093 (CH aromatic),
enzyme concentration (~3.5 ng/
μ
L). (For more information, see Ap-
reaction time 6 h; IR (KBr,
2912 (aliphatic CH), 1670 br. (2C = O); 1H NMR (DMSO‑d6, 400 MHz) δ
ppm: 4.0 (s, 2H, CH2), 7.07–7.11 (m, 3H, 2 Ar-H + H7-indoline),
7.44–7.54 (m, 3H, 2 Ar-H + H5-indoline), 7.95–8.05 (m, 1H, H6-indo-
line), 8.68 (s, 1H, NH, D2O exchangeable), 8.82–8.86 (m, 1H, H4-indo-
line). 13C NMR (DMSO‑d6) δ ppm: 34.3 (COCH2), 115.4, 115.6, 115.8,
120.5, 120.6, 120.7, 120.8, 136.4, 136.43, 153.2, 156.62, 156.66,
159.0, 169.0 (2C = O), Anal. Calcd. for C17H11FN4O3 (338.30): C, 60.36;
H, 3.28; N, 16.56%; Found: C, 60.48; H, 3.39; N, 16.80%.
pendix A)
4.3. In silico studies
4.3.1. Drug likeness profile
The pharmacokinetic and physicochemical data of compound 4f was
calculated using the free online web tool swissADME (http://swissadme.
ch/index.php#undefined) [108].
4.3.2. Docking study
4.2. Biological evaluation
Molecular docking studies were performed using the Molecular
Operating Environment (MOE, 10.2008) software. Minimization was
done with MOE until an RMSD gradient of 0.05 kcal∙molꢀ 1Åꢀ 1 with
MMFF94x forcefield. Partial charges were automatically calculated. The
4.2.1. Antiproliferative activity
4.2.1.1. In vitro anticancer activity as primary single high dose (10 µM)
screening. The preliminary cytotoxicity study for the synthesized com-
pounds 4a-k was determined as described in the protocol of the National
Cancer Institute (NCI), Bethesda, USA against a panel of 60 cell lines
[103,104]. Cell lines was exposed to the compounds for 48 h using
sulforhodamine B (SRB) protein assay [105] and then cell viability and
growth was determined, as previously described [103,104].
X-ray crystallographic structure of PDGFRα, co-crystallized with WQ-C-
159 was retrieved from the Protein Data Bank, https://www.rcsb.org/,
(PDB code: 5GRN) [109]. The enzyme was prepared by removing the
water molecules. Protonation of the enzyme was done using protonate
3D protocol in MOE with the default parameters. Docking protocol was
carried out using Triangle Matcher placement method while London dG
scoring function were used for the docking protocol and the produced
poses were refined using forcefield. Redocking of the native ligand into
the active site was carried out with the purpose of docking setup vali-
dation. After then, the validated setup was used to predict the possible
binding pose of compound 4f to be compared to that of sunitinib as
reference compound and so its affinity to the target enzyme.
4.2.1.2. MTT assay for cytotoxicity. The assay was performed at the
Egyptian company for the production of vaccines, sera, and drugs
VACSERA, Giza, EGYPT. Standard MTT colorimetric method was used to
test the IC50 of compound 4f against five renal cancer cell lines, A498,
ACHN, CAKI-1, RXF393 and UO-31 and one normal renal cell line,
RPTEC/TERT1, which were obtained from the American Type Culture
Collection (ATCC) (Rockville, MD). Cell lines were incubated in RPMI-
1640 medium with 10% fetal bovine serum at 37 ◦C. The cells were
incubated with different concentration of the compound (0.39 – 100
4.4. Pharmacokinetic study
Pharmacokinetic properties of compound 4f were determined
following i.v. and p.o. administration in male Sprague-Dawley rats
10

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
G. McMahon, J.M. Cherrington, In vivo antitumor activity of SU11248, a novel
tyrosine kinase inhibitor targeting vascular endothelial growth factor and
platelet-derived growth factor receptors: determination of a pharmacokinetic/
pharmacodynamic relationship, Clin. Cancer Res. 9 (2003) 327–337.
[9] R.J. Motzer, T.E. Hutson, P. Tomczak, M.D. Michaelson, R.M. Bukowski, O. Rixe,
S. Oudard, S. Negrier, C. Szczylik, S.T. Kim, I. Chen, P.W. Bycott, C.M. Baum, R.
A. Figlin, Sunitinib versus interferon alfa in metastatic renal-cell carcinoma,
N. Engl. J. Med. 356 (2007) 115–124.
(250–300 g) obtained from the Laboratory Animal Center, Faculty of
Pharmacy, Cairo University. Approval of all experimental procedures
was obtained from the Research Ethics Committee for experimental and
clinical studies at Faculty of Pharmacy, Cairo University. The animals
were placed in cages with free access to food and water. Compound 4f
was dissolved with the aid of 5% tween 80 in normal saline. Animals
were randomly divided into two groups (n = 4). The first group was
orally dosed with 15 mg/kg of compound 4f by gastric gavage. The
second group was given a dose of 5 mg/kg by gastric gavage injection
[10] M. Rizzo, C. Porta, Sunitinib in the treatment of renal cell carcinoma: an update
on recent evidence, Therap. adv. urol. 9 (2017) 195–207.
[11] B.I. Rini, T.E. Hutson, R.A. Figlin, M.J. Lechuga, O. Valota, L. Serfass,
B. Rosbrook, R.J. Motzer, Sunitinib in Patients With Metastatic Renal Cell
Carcinoma: Clinical Outcome According to International Metastatic Renal Cell
Carcinoma Database Consortium Risk Group, Clin. Genitourin. Cancer 16 (2018)
298–304.
into the tail vein. After then, blood samples (200 μL) were withdrawn
from the orbital venous plexus at the following time intervals: 30 min
and 1, 2, 3, 4, 5, 6, 8 and 24 h (p.o.); 5, 20, 40 min and 1, 2, 4, 6, 8 and
24 h (i.v.). Whole blood samples were collected in heparinized tubes and
the plasma was immediately centrifuged (4000 rpm, 10 min, 4 ◦C) and
then stored at ꢀ 20 ◦C until analysis. For preparation of the calibration
curve, compound 4f was dissolved in acetonitrile at a concentration of
0.5 mg/mL (Working solution), and then seven calibration standards
[12] S. Giuliano, Y. Cormerais, M. Dufies, R. Grepin, P. Colosetti, A. Belaid, J. Parola,
A. Martin, S. Lacas-Gervais, N.M. Mazure, R. Benhida, P. Auberger, B. Mograbi,
G. Pages, Resistance to sunitinib in renal clear cell carcinoma results from
sequestration in lysosomes and inhibition of the autophagic flux, Autophagy 11
(2015) 1891–1904.
[13] L. Zhou, X.D. Liu, M. Sun, X. Zhang, P. German, S. Bai, Z. Ding, N. Tannir, C.
G. Wood, S.F. Matin, J.A. Karam, P. Tamboli, K. Sircar, P. Rao, E.B. Rankin, D.
A. Laird, A.G. Hoang, C.L. Walker, A.J. Giaccia, E. Jonasch, Targeting MET and
AXL overcomes resistance to sunitinib therapy in renal cell carcinoma, Oncogene
35 (2016) 2687–2897.
ranging from 50 to 9000 ng/mL of 4f were prepared by adding 10
μ
L of
serial dilutions from the working solution to 90
μL of drug free rat
[14] P.J. Siska, K.E. Beckermann, W.K. Rathmell, S.M. Haake, Strategies to overcome
therapeutic resistance in renal cell carcinoma, Urol. Oncol. 35 (2017) 102–110.
[15] H. Butz, Q. Ding, R. Nofech-Mozes, Z. Lichner, H. Ni, G.M. Yousef, Elucidating
mechanisms of sunitinib resistance in renal cancer: an integrated pathological-
molecular analysis, Oncotarget 9 (2017) 4661–4674.
plasma. The calibration standards and the plasma samples were
extracted by protein precipitation using acetonitrile. The concentrations
of compound 4f in the extracted standards and plasma samples were
quantified by LC-UV with a reversed-phase column (Waters Spherisorb
ODS column (150 × 4.6 mm, 5
μ
m) (column temperature = 45 ± 2 ◦C)
[16] N. Yamaguchy, M. Osaki, K. Onuma, T. Yumioka, H. Iwamoto, T. Sejima,
H. Kugoh, A. Takenaka, F. Okada, Identification of MicroRNAs Involved in
Resistance to Sunitinib in Renal Cell Carcinoma Cells, Anticancer Res. 37 (2017)
2985–2992.
and acetonitrile: 0.1% triethylamine (50:50, v/v) as the mobile phase.
The flow rate was maintained at 1 mL/min and UV detection at 341 nm.
The pharmacokinetics parameters were calculated using WinNonlin
Software 7.0 [110,111].
[17] S.P. Robinson, J.K.R. Boult, N.S. Vasudev, A.R. Reynolds, Monitoring the Vascular
Response and Resistance to Sunitinib in Renal Cell Carcinoma In Vivo with
Susceptibility Contrast MRI, Cancer Res. 77 (2017) 4127–4134.
[18] P. Makhov, S. Joshi, P. Ghatalia, A. Kutikov, R.G. Uzzo, V.M. Kolenko, Resistance
to Systemic Therapies in Clear Cell Renal Cell Carcinoma: Mechanisms and
Management Strategies, Mol. Cancer Ther. 17 (2018) 1355–1364.
4.5. IC50 calculations and statistical analysis
´
´
[19] A. Sanchez-Gastaldo, E. Kempf, A. Gonzalez del Alba, I. Duran, Systemic
treatment of renal cell cancer: A comprehensive review, Cancer Treat. Rev. 60
(2017) 77–89.
All IC50 calculations were carried out using Quest GraphTM IC50
Calculator, which uses a four-parameter logistic regression model [112].
The results presented herein are expressed as mean ± SD. Statistical
significance of the IC50 values was checked using GraphPad Prism 7.00
software using the Student’s t-test at P < 0.05.
[20] X. Guo, R. Li, Q. Bai, S. Jiang, H. Wang, TFE3-PD-L1 axis is pivotal for sunitinib
resistance in clear cell renal cell carcinoma, J. cell. mol. med. 24 (2020)
14441–14452.
[21] M.M. Mita, E.K. Rowinsky, L. Forero, S.G. Eckhart, E. Izbicka, G.R. Weiss,
M. Beeram, A.C. Mita, J.S. de Bono, A.W. Tolcher, L.A. Hammond, P. Simmons,
K. Berg, C. Takimoto, A. Patnaik, A phase II, pharmacokinetic, and biologic study
of semaxanib and thalidomide in patients with metastatic melanoma, Cancer
Chemother. Pharmacol. 59 (2007) 165–174.
Funding:
This research did not receive any specific grant from funding
agencies in the public, commercial, or not-for-profit sectors.
[22] J.J. Haddad, The immunopharmacologic potential of Semaxanib and new
generation directed therapeutic drugs: Receptor tyrosine kinase regulation with
anti-tumorigenensis/angiogenesis properties, Saudi pharm. j. 20 (2012) 103–123.
[23] K. Fukunaga, S. Yokoe, S. Kawashima, Y. Uchida, H. Nakagawa, Y. Nakano,
Nintedanib prevented fibrosis progression and lung cancer growth in idiopathic
pulmonary fibrosis, Respir. Case Rep. 6 (2018) 1–3.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
[24] A.D. Laird, P. Vajkoczy, L.K. Shawver, A. Thurnher, C. Liang, M. Mohammadi,
J. Schlessinger, A. Ullrich, S.R. Hubbard, R.A. Blake, T.A. Fong, L.M. Strawn,
L. Sun, C. Tang, R. Hawtin, F. Tang, N. Shenoy, K.P. Hirth, G. McMahon,
Cherrington, SU6668 is a potent antiangiogenic and antitumor agent that induces
regression of established tumors, Cancer Res. 60 (2000) 4152–4160.
[25] G. Cerchiaro, A.M.d.C. Ferreira, Oxindoles and copper complexes with oxindole-
derivatives as potential pharmacological agents, J. Braz. Chem. Soc. 17 (2006)
1473–1485.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bioorg.2021.104985.
[26] M. Kidwai, A. Jahan, N.K. Mishra, Isatins: A Diversity Orientated Biological
Profile, Med. Chem. 4 (2014) 451–468.
[27] A. Cane, M.-C. Tournaire, D. Barritault, M. Crumeyrolle-Arias, The Endogenous
Oxindoles 5-Hydroxyoxindole and Isatin Are Antiproliferative and Proapoptotic,
Biochem. Biophys. Res. Comm. 276 (2000) 379–384.
References:
[1] American Cancer Society “Cancer Facts and Figures”, (2021) 1–72.
[2] T. Alonso-Gordoa, M.L. García-Bermejo, E. Grande, P. Garrido, A. Carrato,
J. Molina-Cerrillo, Targeting Tyrosine kinases in Renal Cell Carcinoma: “New
Bullets against Old Guys, Int. J. Mol. Sci. 20 (2019) 1–24.
[28] D. Havrylyuk, N. Kovach, B. Zimenkovsky, O. Vasylenko, R. Lesyk, Synthesis and
anticancer activity of isatin-based pyrazolines and thiazolidines conjugates, Arch.
Pharm. 344 (2011) 514–522.
[29] K.L. Vine, L. Matesic, J.M. Locke, M. Ranson, D. Skropeta, Cytotoxic and
anticancer activities of isatin and its derivatives: a comprehensive review from
2000–2008, Anticancer Agents Med. Chem. 9 (2009) 397–414.
[30] G. Krishnegowda, A.S. Prakasha Gowda, H.R. Tagaram, K.F. Carroll, R.B. Irby, A.
K. Sharma, S. Amin, Synthesis and biological evaluation of a novel class of isatin
analogs as dual inhibitors of tubulin polymerization and Akt pathway, Bioorg.
Med. Chem. 19 (2011) 6006–6014.
[3] V.E. Reuter, S.K. Tickoo, Differential diagnosis of renal tumours with clear cell
histology, Pathology 42 (2010) 374–783.
[4] S. Nabi, E.R. Kessler, B. Bernard, T.W. Flaig, E.T. Lam, Renal cell carcinoma: a
review of biology and pathophysiology, F1000, Research 7 (2018) 307.
[5] L. Mologni, Development of RET kinase inhibitors for targeted cancer therapy,
Curr. Med. Chem. 18 (2011) 162–175.
[6] M. Song, Recent developments in small molecule therapies for renal cell
carcinoma, Eur. J. Med. Chem. 142 (2017) 383–392.
[31] K.L. Vine, L. Belfiore, L. Jones, J.M. Locke, S. Wade, E. Minaei, M. Ranson, N-
alkylated isatins evade P-gp mediated efflux and retain potency in MDR cancer
cell lines, Heliyon, 2 (2016) 1-23.
[7] V.R. Adams, M. Leggas, Sunitinib malate for the treatment of metastatic renal cell
carcinoma and gastrointestinal stromal tumors, Clin. Ther. 29 (2007) 1338–1353.
[8] D.B. Mendel, A.D. Laird, X. Xin, S.G. Louie, J.G. Christensen, G. Li, R.E. Schreck,
T.J. Abrams, T.J. Ngai, L.B. Lee, L.J. Murray, J. Carver, E. Chan, K.G. Moss, J.
O. Haznedar, J. Sukbuntherng, R.A. Blake, L. Sun, C. Tang, T. Miller, S. Shirazian,
[32] Sonam Varun, R. Kakkar, Isatin and its derivatives: a survey of recent syntheses,
reactions, and applications, MedChemComm. 10 (2019) 351–368.
11

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
[33] Y. Chang, Y. Yuan, Q. Zhang, Y. Rong, Y. Yang, M. Chi, Z. Liu, Y. Zhang, P. Yu,
Y. Teng, Effects of an isatin derivative on tumor cell migration and angiogenesis,
RSC Adv. 10 (2020) 1191–1197.
[59] Y. Cao, J. Arbiser, R.J. D’Amato, P.A. D’Amore, D.E. Ingber, R. Kerbel,
M. Klagsbrun, S. Lim, M.A. Moses, B. Zetter, H. Dvorak, R. Langer, Forty-Year
Journey of Angiogenesis Translational Research, Sci. Transl. Med. 3 (2011) 1–8.
[60] R.H. Adams, K. Alitalo, Molecular regulation of angiogenesis and
lymphangiogenesis, Nat. Rev. Mol. Cell Biol. 8 (2007) 464–478.
[34] N.R. Penthala, T.R. Yerramreddy, N.R. Madadi, P.A. Crooks, Synthesis and in
vitro evaluation of N-alkyl-3-hydroxy-3-(2-imino-3-methyl-5-oxoimidazolidin-4-
yl)indolin-2-one analogs as potential anticancer agents, Bioorg. Med. Chem. Lett.
20 (2010) 4468–4471.
[61] D. Hanahan, G. Christofori, P. Naik, J. Arbeit, Transgenic mouse models of
tumour angiogenesis: the angiogenic switch, its molecular controls, and prospects
for preclinical therapeutic models, Eur. J. Cancer 32 (1996) 2386–2393.
[62] M. Bianconi, L. Faloppi, A. Lopez-Beltran, M. Scarpelli, M. Scartozzi, L. Cheng,
R. Montironi, Clinical impact of tumoral angiogenesis on renal cell carcinoma
management: where do we stand? Exp. Rev. Pre. Med. Drug Dev. 1 (2016)
229–231.
¨
[35] O. Güzel-Akdemir, A. Akdemir, N. Karalı, C.T. Supuran, Discovery of novel isatin-
based sulfonamides with potent and selective inhibition of the tumor-associated
carbonic anhydrase isoforms IX and XII, Org. Biomolec. Chem. 13 (2015)
6493–6499.
[36] W.M. Eldehna, A. Altoukhy, H. Mahrous, H.A. Abdel-Aziz, Design, synthesis and
QSAR study of certain isatin-pyridine hybrids as potential anti-proliferative
agents, Eur. J. Med. Chem. 90 (2015) 684–694.
[63] R. Iacovelli, A. Palazzo, P. Trenta, S. Mezi, D. Pellegrino, G. Naso, E. Cortesi,
Management of metastatic renal cell carcinoma progressed after sunitinib or
another antiangiogenic treatment, Am. J. Clin. Oncol. 37 (2014) 611–615.
[64] D.Y. Heng, W. Xie, M.M. Regan, M.A. Warren, A.R. Golshayan, C. Sahi, B.J. Eigl,
J.D. Ruether, T. Cheng, S. North, P. Venner, J.J. Knox, K.N. Chi,
[37] D. Havrylyuk, B. Zimenkovsky, O. Vasylenko, A. Gzella, R. Lesyk, Synthesis of
New 4-Thiazolidinone-, Pyrazoline-, and Isatin-Based Conjugates with Promising
Antitumor Activity, Med. Chem. 55 (2012) 8630–8641.
[38] S. Wang, Y. Zhao, G. Zhang, Y. Lv, N. Zhang, P. Gong, Design, synthesis and
biological evaluation of novel 4-thiazolidinones containing indolin-2-one moiety
as potential antitumor agent, Eur. J. Med. Chem. 46 (2011) 3509–3518.
[39] P.K. Ramshid, S. Jagadeeshan, A. Krishnan, M. Mathew, S.A. Nair, M.R. Pillai,
Synthesis and in vitro evaluation of some isatin-thiazolidinone hybrid analogues
as anti-proliferative agents, Med. Chem. 6 (2010) 306–312.
C. Kollmannsberger, D.F. McDermott, W.K. Oh, M.B. Atkins, R.M. Bukowski, B.
I. Rini, T.K. Choueiri, Prognostic factors for overall survival in patients with
metastatic renal cell carcinoma treated with vascular endothelial growth factor-
targeted agents: results from a large, multicenter study, J. Clin. Oncol. 27 (2009)
5794–5799.
[65] X. Na, G. Wu, C.K. Ryan, S.R. Schoen, P.A. Di’Santagnese, E.M. Messing,
Overproduction of vascular endothelial growth factor related to von Hippel-
Lindau tumor suppressor gene mutations and hypoxia-inducible factor-1 alpha
expression in renal cell carcinomas, J. Urol. 170 (2003) 588–592.
[66] Y. Lai, Z. Zhao, T. Zeng, X. Liang, D. Chen, X. Duan, G. Zeng, W. Wu, Crosstalk
between VEGFR and other receptor tyrosine kinases for TKI therapy of metastatic
renal cell carcinoma, Cancer Cell Int. 18 (2018) 1–9.
[40] D. Kaminskyy, D. Khyluk, O. Vasylenko, L. Zaprutko, R. Lesyk, A facile synthesis
and anticancer activity evaluation of spiro[thiazolidinone-isatin] conjugates, Sci.
pharm. 79 (2011) 763–777.
[41] A. Benmohammed, O. Khoumeri, A. Djafri, T. Terme, P. Vanelle, Synthesis of
novel highly functionalized 4-thiazolidinone derivatives from 4-phenyl-3-
thiosemicarbazones, Molecules (Basel, Switzerland) 19 (2014) 3068–3083.
[42] S. Sardari, S. Feizi, A.H. Rezayan, P. Azerang, S.M. Shahcheragh, G. Ghavami,
A. Habibi, Synthesis and Biological Evaluation of Thiosemicarbazide Derivatives
Endowed with High Activity toward Mycobacterium Bovis, Iran J. Pharm. Res. 16
(2017) 1128–1140.
[67] J. Donovan, X. Shiwen, J. Norman, D. Abraham, Platelet-derived growth factor
alpha and beta receptors have overlapping functional activities towards
fibroblasts, Fibrogenesis Tissue Repair 6 (2013) 1–9.
[68] A.A. Cumpanas, A.M. Cimpean, O. Ferician, R.A. Ceausu, S. Sarb, V. Barbos,
A. Dema, M. Raica, The Involvement of PDGF-B/PDGFRbeta Axis in the
Resistance to Antiangiogenic and Antivascular Therapy in Renal Cancer,
Anticancer Res. 36 (2016) 2291–2295.
[43] M.W. Beukers, M.J. Wanner, J.K. Von Frijtag Drabbe, E.C. Kunzel, I.J.A.
P. Klaasse, G.J. Koomen, N6-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)
adenosine (TCPA), a very selective agonist with high affinity for the human
adenosine A1 receptor, J. Med. Chem. 46 (2003) 1492–1503.
[69] R. Roskoski Jr., Sunitinib: a VEGF and PDGF receptor protein kinase and
angiogenesis inhibitor, Biochem. Biophys. Res. Commun. 356 (2007) 323–328.
[44] B.X. Hu, Z.L. Shen, J. Lu, X.Q. Hu, W.M. Mo, N. Sun, D. Xu, Synthesis and
Characterization of New Thiazolidin-4-one Derivatives, Phosphorus Sulfur Silicon
Relat. Elem. 184 (2009) 523–535.
¨
[70] I. Sulzbacher, P. Birner, M. Traxler, M. Marberger, A. Haitel, Expression of
Platelet-Derived Growth Factor-α Receptor Is Associated With Tumor Progression
[45] A.A. Khan, N. Siddiqui, M.J. Akhtar, Z. Ali, M.S. Yar, Design, Synthesis, and
Biological Evaluation of 6-(2-Amino-substituted phenyl)-4-(substituted phenyl)-
1,2,4-triazine-3,5(2H,4H)-dione Derivatives as Anticonvulsant Agents, Arch.
Pharm. 349 (2016) 277–292.
in Clear Cell Renal Cell Carcinoma, Am. J. Clin. Pathol. 120 (2003) 107–112.
[71] L.F. Qi, D. Sun, J.H. Zheng, J. Du, X. Yao, Detection and clinical significance of
platelet derived growth factor-BB and microvessel density in clear cell renal cell
carcinoma, Chin. J. Oncol. 35 (2013) 672–677.
[46] L.F. Audrieth, The preparation of semicarbazide, J. Am. Chem. Soc. 52 (1930)
1250–1251.
[72] C.Y. Yu, C.L. Jerry Teng, P.S. Hung, C.C. Cheng, S.L. Hsu, G.Y. Hwang, Y.
M. Tzeng, Ovatodiolide isolated from Anisomeles indica induces cell cycle G2/M
arrest and apoptosis via a ROS-dependent ATM/ATR signaling pathways, Eur. J.
Pharmacol. 819 (2018) 16–29.
[47] H. Pervez, Z.H. Chohan, M. Ramzan, F.-U.-H. Nasim, K.M. Khan, Synthesis and
biological evaluation of some new N4-substituted isatin-3-thiosemicarbazones,
J. Enz. Inh. Med. Chem. 24 (2009) 437–446.
[73] W.M. Eldehna, M.F. Abo-Ashour, H.S. Ibrahim, G.H. Al-Ansary, H.A. Ghabbour,
M.M. Elaasser, H.Y.A. Ahmed, N.A. Safwat, Novel [(3-indolylmethylene)
hydrazono]indolin-2-ones as apoptotic anti-proliferative agents: design, synthesis
and in vitro biological evaluation, J. Enz. Inhib. Med. Chem. 33 (2018) 686–700.
[74] J.W. Harper, P.D. Adams, Cyclin-dependent kinases, Chem. Rev. 101 (2001)
2511–2526.
[48] M.D. Hall, N.K. Salam, J.L. Hellawell, H.M. Fales, C.B. Kensler, J.A. Ludwig,
G. Szakacs, D.E. Hibbs, M.M. Gottesman, Synthesis, activity, and pharmacophore
development for isatin-beta-thiosemicarbazones with selective activity toward
multidrug-resistant cells, J. Med. Chem. 52 (2009) 3191–3204.
[49] A. Qasem Ali, N.E. Eltayeb, S.G. Teoh, A. Salhin, H.K. Fun, (Z)-2-(2-Oxoindolin-3-
yl-idene)-N-phenylhydrazinecarbothio-amide, Acta Crystallogr, Sect. E.-Struct
Rep. Online 68 (2012) 962–963.
[75] M.K. Diril, C.K. Ratnacaram, V.C. Padmakumar, T. Du, M. Wasser, V. Coppola,
L. Tessarollo, P. Kaldis, Cyclin-dependent kinase 1 (Cdk1) is essential for cell
division and suppression of DNA re-replication but not for liver regeneration,
Proc. Natl. Acad. Sci. U.S.A. 109 (2012) 3826–3831.
[50] A.Q. Ali, S.G. Teoh, A. Salhin, N.E. Eltayeb, M.B. Khadeer Ahamed, A.M. Abdul
Majid, Synthesis of isatin thiosemicarbazones derivatives: in vitro anti-cancer,
DNA binding and cleavage activities, Spectrochim. Acta A Mol. Biomol. Spectrosc.
125 (2014) 440–448.
[76] N.R. Brown, S. Korolchuk, M.P. Martin, W.A. Stanley, R. Moukhametzianov, M.E.
M. Noble, J.A. Endicott, CDK1 structures reveal conserved and unique features of
the essential cell cycle CDK, Nat. Commun. 6 (2015) 1–12.
[51] G. Pelosi, C. Pelizzi, M. Belicchi Ferrari, M.C. Rodriguez-Arguelles, C. Vieito,
J. Sanmartin, Isatin 3-semicarbazone and 1-methylisatin 3-semicarbazone, Acta
Crystallogr. C 61 (2005) 589–592.
[77] I. Neganova, F. Vilella, S.P. Atkinson, M. Lloret, J.F. Passos, T. von Zglinicki, J.
E. O’Connor, D. Burks, R. Jones, L. Armstrong, M. Lako, An important role for
CDK2 in G1 to S checkpoint activation and DNA damage response in human
embryonic stem cells, Stem Cells 29 (2011) 651–659.
[52] M. Cigan, K. Jakusov, J. Donovalov, V. Szocs, A. Gaplovsk, Isatin N-
phenylsemicarbazone: effect of substituents and concentration on anion sensing
selectivity and sensitivity, RSC Adv. 5 (2014) 54072–54079.
¨
[78] H. Waterbeemd, Modern Methods of Drug Discovery, Third ed., Birkhauser Basel
[53] F.A. Ragab, N.A. El-Sayed, A.A. Eissa, A.M. El Kerdawy, Synthesis and
anticonvulsant activity of certain substituted furochromone, benzofuran and
flavone derivatives, Chem. Pharm. Bull. 58 (2010) 1148–1156.
[54] A.S. Salman, N.A. Mahmoud, M.A. Mohamed, A. Abdel-aziem, D.M. Elsisi,
Synthesis, Characterization and in vitro Cytotoxic Evaluation of Some Novel
Heterocyclic Compounds Bearing Indole Ring, Am. J. Org. Chem. 6 (2016) 39–53.
[55] N. Hussain, A. Joshi, C. Sharma, G. Talesara, A Convenient Synthesis of Some
New Indole Containing Thiazolidinone, Thiohydantoin, Triazine and its
Derivatives with Ethoxyphthalimide Moiety, Asi. J. Chem. 24 (2012) 5917–5921.
[56] E.A. Mohamed, M.M. Ismail, Y. Gabr, M. Abass, Synthesis of Some
Multiazaheterocycles as Substituents to Quinolone Moiety of Specific Biological
Activity, Chem. Papers 48 (1994) 285–292.
(2003).
[79] A. Daina, O. Michielin, V. Zoete, SwissADME: a free web tool to evaluate
pharmacokinetics, drug-likeness and medicinal chemistry friendliness of small
molecules, Sci Rep 7 (2017) 42717.
[80] http://www.swissadme.ch/index.php.
[81] J.H. Martin, E.J. Begg, M.A. Kennedy, R. Roberts, M.L. Barclay, Is cytochrome
P450 2C9 genotype associated with NSAID gastric ulceration? Br. J. Clin. Pharm.
51 (2001) 627–630.
[82] P. Hallberg, J. Karlsson, L. Kurland, L. Lind, T. Kahan, K. Malmqvist, K.P. Ohman,
¨
F. Nystrom, H. Melhus, The CYP2C9 genotype predicts the blood pressure
response to irbesartan: results from the Swedish Irbesartan Left Ventricular
Hypertrophy Investigation vs Atenolol (SILVHIA) trial, J. Hypertens 20 (2002)
2089–2093.
[57] J.M. Edmondson, L.S. Armstrong, A.O. Martinez, A rapid and simple MTT-based
spectrophotometric assay for determining drug sensitivity in monolayer cultures,
J. Tissue Cult. Methods 11 (1988) 15–17.
[83] J. Schmider, D.J. Greenblatt, L.L. von Moltke, D. Karsov, R.I. Shader, Inhibition of
CYP2C9 by selective serotonin reuptake inhibitors in vitro: studies of phenytoin
p-hydroxylation, Br. J. Clin. Pharmacol. 44 (1997) 495–498.
[58] M. Greenblatt, P. Shubi, Tumor angiogenesis: transfilter diffusion studies in the
hamster by the transparent chamber technique, J. Natl. Cancer Inst. 41 (1968)
111–124.
[84] G.J. Gross, J.R. Falck, E.R. Gross, M. Isbell, J. Moore, K. Nithipatikom,
Cytochrome P450 and arachidonic acid metabolites: Role in myocardial
ischemia/reperfusion injury revisited, Cardiovasc. Res. 68 (2005) 18–25.
12

M.A. Fouad et al.
Bioorganic Chemistry 112 (2021) 104985
[85] J. Mwinyi, I. Cavaco, R.S. Pedersen, A. Persson, S. Burkhardt, S. Mkrtchian,
M. Ingelman-Sundberg, Regulation of CYP2C19 expression by estrogen receptor
[98] A. Trojani, C.B. Ripamonti, S. Penco, A. Beghini, G. Nadali, E. Di Bona, A. Viola,
C. Castagnola, P. Colapietro, G. Grillo, L. Pezzetti, E. Ravelli, M.C. Patrosso,
A. Marocchi, A. Cuneo, F. Ferrara, M. Lazzarino, G. Pizzolo, R. Cairoli, E. Morra,
Molecular analysis of PDGFRA and PDGFRB genes by rapid single-strand
conformation polymorphism (SSCP) in patients with core-binding factor
leukaemias with KIT or FLT3 mutation, Anticancer Res. 28 (2008) 2745–2751.
[99] S. Pundir, M.J. Martin, C. O’Donovan, UniProt Tools, Curr. Protoc.
Bioinformatics, 53 (2016) 1.29.21-21.29.15.
α
: implications for estrogen-dependent inhibition of drug metabolism, Mol.
Pharmacol. 78 (2010) 886–894.
[86] S.-F. Zhou, L.-P. Yang, Z.-W. Zhou, Y.-H. Liu, E. Chan, Insights into the substrate
specificity, inhibitors, regulation, and polymorphisms and the clinical impact of
human cytochrome P450 1A2, The AAPS j. 11 (2009) 481–494.
[87] Q. Zhang, Z. Zhong, B. Li, Z. Liao, P. Zhao, Z. Ye, X. He, H. Wang, W. Chen,
J. Huang, Effects of different CYP2C19 genotypes on prognosis of patients
complicated with atrial fibrillation taking clopidogrel after PCI, Exp. Ther. Med.
16 (2018) 3492–3496.
[100] T.U. Consortium, UniProt: the universal protein knowledgebase in 2021, Nucleic
Acids Res. 49 (2020) D480–D489.
[101] R. Roskoski Jr., Classification of small molecule protein kinase inhibitors based
upon the structures of their drug-enzyme complexes, Pharmacol. Res. 103 (2016)
26–48.
[88] C.A. Lipinski, F. Lombardo, B.W. Dominy, P.J. Feeney, Experimental and
computational approaches to estimate solubility and permeability in drug
discovery and development settings, Adv. Drug Deliv. Rev. 46 (2001) 3–26.
[89] A.K. Ghose, V.N. Viswanadhan, J.J. Wendoloski, A Knowledge-Based Approach in
Designing Combinatorial or Medicinal Chemistry Libraries for Drug Discovery. 1.
A Qualitative and Quantitative Characterization of Known Drug Databases,
J. Comb. Chem. 1 (1999) 55–68.
[102] R. Roskoski Jr., The role of small molecule Kit protein-tyrosine kinase inhibitors
in the treatment of neoplastic disorders, Pharmacol. Res. 133 (2018) 35–52.
[103] A. Monks, D. Scudiero, P. Skehan, R. Shoemaker, K. Paull, D. Vistica, C. Hose,
J. Langley, P. Cronise, A. Vaigro-Wolff, et al., Feasibility of a high-flux anticancer
drug screen using a diverse panel of cultured human tumor cell lines, J. Natl.
Cancer Inst. 83 (1991) 757–766.
[90] D.F. Veber, S.R. Johnson, H.Y. Cheng, B.R. Smith, K.W. Ward, K.D. Kopple,
Molecular properties that influence the oral bioavailability of drug candidates,
J. Med. Chem. 45 (2002) 2615–5623.
[104] M.R. Boyd, K.D. Paull, Some practical considerations and applications of the
national cancer institute in vitro anticancer drug discovery screen, Drug Dev. Res.
34 (1995) 91–109.
[91] W.J. Egan, K.M. Merz Jr., J.J. Baldwin, Prediction of drug absorption using
multivariate statistics, J. Med. Chem. 43 (2000) 3867–3877.
[105] E.A. Orellana, A.L. Kasinski, Sulforhodamine B (SRB) Assay in Cell Culture to
Investigate Cell Proliferation, Bio. Protoc. 6 (2016) 1–9.
[92] I. Muegge, S.L. Heald, D. Brittelli, Simple Selection Criteria for Drug-like
Chemical Matter, J. Med. Chem. 44 (2001) 1841–1846.
[106] P. Pozarowski, Z. Darzynkiewicz, Analysis of cell cycle by flow cytometry,
Methods Mol. Biol. 281 (2004) 301–311.
[93] R. Brenk, A. Schipani, D. James, A. Krasowski, I.H. Gilbert, J. Frearson, P.
G. Wyatt, Lessons learnt from assembling screening libraries for drug discovery
for neglected diseases, ChemMedChem. 3 (2008) 435–544.
[107] L.C. Crowley, A.P. Scott, B.J. Marfell, J.A. Boughaba, G. Chojnowski, N.
J. Waterhouse, Measuring Cell Death by Propidium Iodide Uptake and Flow
Cytometry, Cold Spring Harb. Protoc. 2016 (2016) 647–652.
[94] J.B. Baell, G.A. Holloway, New Substructure Filters for Removal of Pan Assay
Interference Compounds (PAINS) from Screening Libraries and for Their
Exclusion in Bioassays, J. Med. Chem. 53 (2010) 2719–2740.
[108] A. Daina, V. Zoete, A BOILED-Egg To Predict Gastrointestinal Absorption and
Brain Penetration of Small Molecules, ChemMedChem. 11 (2016) 1117–1121.
[109] https://www.rcsb.org/structure/5GRN.
[95] C. Muhle-Goll, S. Hoffmann, S. Afonin, S.L. Grage, A.A. Polyansky, D. Windisch,
M. Zeitler, J. Bürck, A.S. Ulrich, Hydrophobic matching controls the tilt and
stability of the dimeric platelet-derived growth factor receptor (PDGFR) β
transmembrane segment, J. Biol. Chem. 287 (2012) 26178–26186.
[96] A.H.-R. Shim, H. Liu, P.J. Focia, X. Chen, P.C. Lin, X. He, Structures of a platelet-
derived growth factor/propeptide complex and a platelet-derived growth factor/
receptor complex, PNAS USA 107 (2010) 11307–11312.
[110] B.A. Moussa, A.A. El-Zaher, M.K. El-Ashrey, M.A. Fouad, Synthesis and molecular
docking of new roflumilast analogues as preferential-selective potent PDE-4B
inhibitors with improved pharmacokinetic profile, Eur. J. Med. Chem. 148 (2018)
477–486.
[111] B.A. Moussa, A.A. El-Zaher, M.K. El-Ashrey, M.A. Fouad, Roflumilast analogs with
improved metabolic stability, plasma protein binding, and pharmacokinetic
profile, Drug Test. Anal. 11 (2019) 886–897.
[97] M.P. Martin, R. Alam, S. Betzi, D.J. Ingles, J.Y. Zhu, E. Schonbrunn, A novel
approach to the discovery of small-molecule ligands of CDK2, Chembiochem 13
(2012) 2128–2136.
[112] “Quest GraphTM IC50 Calculator.” AAT Bioquest, Inc, 16 Jan. 2021, https
://www.aatbio.com/tools/ic50-calculator.
13