Discovery of new coumarin-based lead with potential anticancer, cdk4 inhibition and selective radiotheranostic effect: Synthesis, 2d & 3d qsar, molecular dynamics, in vitro cytotoxicity, radioiodination, and biodistribution studies

Article abstract of DOI:10.3390/molecules26082273

Novel 6-bromo-coumarin-ethylidene-hydrazonyl-thiazolyl and 6-bromo-coumarin-thiazolylbased derivatives were synthesized. A quantitative structure activity relationship (QSAR) model with high predictive power r² = 0.92, and RMSE = 0.44 predicted five compounds; 2b, 3b, 5a, 9a and 9i to have potential anticancer activities. Compound 2b achieved the best ?G of –15.34 kcal/mol with an affinity of 40.05 pki. In a molecular dynamic study 2b showed an equilibrium at 0.8 ? after 3.5 ns, while flavopiridol did so at 0.5 ? after the same time (3.5 ns). 2b showed an IC₅₀ of 0.0136 μM, 0.015 μM, and 0.054 μM against MCF-7, A-549, and CHO-K1 cell lines, respectively. The CDK4 enzyme assay revealed the significant CDK4 inhibitory activity of compound 2b with IC₅₀ of 0.036 μM. The selectivity of the newly discovered lead compound 2b toward localization in tumor cells was confirmed by a radioiodination biological assay that was done via electrophilic substitution reaction utilizing the oxidative effect of chloramine-t.¹³¹ I-2b showed good in vitro stability up to 4 h. In solid tumor bearing mice, the values of tumor uptake reached a height of 5.97 ± 0.82%ID/g at 60 min p.i.¹³¹ I-2b can be considered as a selective radiotheranostic agent for solid tumors with promising anticancer activity.

Full text of DOI:10.3390/molecules26082273

molecules
Article
Discovery of New Coumarin-Based Lead with Potential
Anticancer, CDK4 Inhibition and Selective Radiotheranostic
Effect: Synthesis, 2D & 3D QSAR, Molecular Dynamics, In
Vitro Cytotoxicity, Radioiodination, and Biodistribution Studies
Mona O. Sarhan 1, Somaia S. Abd El-Karim 2 , Manal M. Anwar 2, Raghda H. Gouda 3, Wafaa A. Zaghary 3,
*
and Mohammed A. Khedr 3,
*
1
Labelled Compounds Department, Hot Lab Centre, Atomic Energy Authority, Cairo 13759, Egypt;
monasarhan@windowslive.com
Department of Therapeutic Chemistry, National Research Centre, Dokki, Cairo 12622, Egypt;
somaia_elkarim@hotmail.com (S.S.A.E.-K.); manal.hasan52@live.com (M.M.A.)
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, P.O. Box 11795,
EinHelwan, Cairo 13759, Egypt; raghdahassan@yahoo.com
2
3
*
Correspondence: wafaa.zaghary@pharm.helwan.edu.eg (W.A.Z.);
mohammed_abdou@pharm.helwan.edu.eg (M.A.K.)
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: Novel 6-bromo-coumarin-ethylidene-hydrazonyl-thiazolyl and 6-bromo-coumarin-thiazolyl-
based derivatives were synthesized. A quantitative structure activity relationship (QSAR) model
Citation: Sarhan, M.O.; Abd
El-Karim, S.S.; Anwar, M.M.; Gouda,
R.H.; Zaghary, W.A.; Khedr, M.A.
Discovery of New Coumarin-Based
Lead with Potential Anticancer,
CDK4 Inhibition and Selective
with high predictive power r2 = 0.92, and RMSE = 0.44 predicted five compounds; 2b
,
3b
, 5a, 9a and
9i to have potential anticancer activities. Compound 2b achieved the best
∆G of –15.34 kcal/mol
with an affinity of 40.05 pki. In a molecular dynamic study 2b showed an equilibrium at 0.8 Å
after 3.5 ns, while flavopiridol did so at 0.5 Å after the same time (3.5 ns). 2b showed an IC50 of
Radiotheranostic Effect: Synthesis, 2D
& 3D QSAR, Molecular Dynamics, In
Vitro Cytotoxicity, Radioiodination,
and Biodistribution Studies. Molecules
2021, 26, 2273. https://doi.org/
10.3390/molecules26082273
0.0136
CDK4 enzyme assay revealed the significant CDK4 inhibitory activity of compound 2b with IC50 of
0.036 M. The selectivity of the newly discovered lead compound 2b toward localization in tumor
cells was confirmed by a radioiodination biological assay that was done via electrophilic substitution
reaction utilizing the oxidative effect of chloramine-t. 131I-2b showed good in vitro stability up to
µM, 0.015 µM, and 0.054 µM against MCF-7, A-549, and CHO-K1 cell lines, respectively. The
µ
4 h. In solid tumor bearing mice, the values of tumor uptake reached a height of 5.97
±
0.82%ID/g
at 60 min p.i. 131I-2b can be considered as a selective radiotheranostic agent for solid tumors with
Academic Editors: Qiao-Hong Chen
and Diego Muñoz-Torrero
promising anticancer activity.
Received: 15 March 2021
Accepted: 9 April 2021
Published: 14 April 2021
Keywords: coumarin; synthesis; molecular dynamics; radioiodination; CDK4
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
1. Introduction
Cancer disease is the second largest cause of death globally. Based on the WHO survey,
around 9.6 million deaths occurred in 2018 and is predicted to reach 13.1 million by 2030
specially in children [
target for treating cancer [
various tumors due to its overexpression in different tumor cells [16
is featured as a potential strategy for targeted treatment of several cancers leading to
cell cycle arrest within the G1 phase 1 [19 20]. During the last decade, three generations
of CDK inhibitors, have been developed [21] (Figure 1). The absence of appropriate
balance between the efficiency and the safety of the first-generation pan-CDK inhibitors,
such as flavopiridol [22], and seliciclib [23], because of their multi-target activity was the
main reason for the development of more selective CDK inhibitors, such as milciclib [24],
dinaciclib [25], CYC-065 [26], and AT7519 [27] (Figure 1). In comparison with non-specific
CDK suppressors, CDK4/6 inhibitors prevent the off-target toxicity and produce a specified
1–5
]. Inhibition of cyclin-dependent kinases (CDKs) is a promising
15]. CDK4 is a key factor in initiation and promoting of
18]. CDK4 inhibition
6–
–
Copyright:
© 2021 by the authors.
,
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Molecules 2021, 26, 2273. https://doi.org/10.3390/molecules26082273
https://www.mdpi.com/journal/molecules

Molecules 2021, 26, 2273
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therapeutic window since they do not inhibit the CDKs that regulate the normal cell
cycle [28]. Accordingly, the recent research has been directed towards the third-generation
CDK4/6 inhibiting drugs, palbociclib, ribociclib, and abemaciclib [29–32]. However, these
drugs lack of sensitivity to CDK4/6 monotherapy [33,34].
Figure 1. Cell cycle CDK inhibitors: FDA approved drugs and under clinical evaluation.
Chromene and its isosteric scaffold coumarin are considered to be the most versatile
chemical rings for design and discovery of potential anticancer drugs. Flavopiridol (alvo-
cidib; Figures 1 and 2) is a semisynthetic flavone showing modest potency and selectivity
for CDKs 1, 2, 4, 6, 7, and 9. Treatment of cells with flavopiridol leads to delay in S-phase,
followed by arresting the cell cycle at G2 phase [35]. Flavopiridol
in multiple clinical trials, as a monotherapy and in combinations, but with detected low
activity and high toxicity [35 36]. In vitro biological studies showed that the chromene–triazole–
I had been utilized
,
coumarin triads II and III was potential fluorescent suppressors of CDK2/CDK4 induced tumors
with potent activity against human cervical cancer cell line (HeLa) (Figure 2). Both molecules
represented high selectivity towards CDK2 and CDK4. The detected significant binding
energies of the triads were referred to their strong hydrophobic interaction between the
target proteins and the bulky coumarin-triazole-chromene moieties. This supported them
to be encapsulated into the hydrophobic pockets of CDK2 and CDK4. Furthermore, there

Molecules 2021, 26, 2273
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is also a possibility of hydrogen bonding and
π
–cation interactions between the inhibitor
molecules and CDK4 enhancing the binding affinities [37]. A recent study showed that
the newly synthesized 4-flourophenylacetamide-acetyl coumarin (4-FPAC) IV produced
cytotoxic effect and arrested metastasis in A549 as well as cell cycle progression at the
G0/G1 by modulating p21, CDK2, and CDK4 expression [38] (Figure 2).
Figure 2. Examples of chromene and coumarin compounds of CDK4 inhibiting activity.
Furthermore, the thiazole moiety is used in the discovery of new lead antitumor
compounds [39]. Banyu Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd
(Tsukuba, Japan) applied various structural modifications [40] in order to discover the
2-aminothiazole compounds Va, Vb, and Vc as selective and potent inhibitors of CDK4
with IC50 values of 4.2, 34, and 9.2 nmol/L, respectively exhibiting potent antiproliferative
activities against various cancer cell lines (Figure 3). The optimization studies showed that
the compounds VIa, VIb, VIc exhibited potent antiproliferative activity against HCT-116
and PC-6 cancer cell lines with potent selectivity [41]. Moreover, Tadesse and his team work
developed the thiazole product VIIa which had a potent activity on CDK4 and CDK6 (Ki = 7
and 42 nmol/L, respectively). Additionally, VIIa exhibited antiproliferative activity against
a panel of human cell lines, and arrested the cell cycle at G1 phase in M249 and M249R
melanoma cell lines [42]. Further structural optimization led to the formation of the thiazole-
pyrimidine compounds VIIb and VIIc which remained as CDK4/6 inhibitors (Ki = 4/30
and 2/55 nmol/L, respectively) with selectivity over CDKs 1, 2, 7, and 9 [43] (Figure 3).
Other modifications showed that the two N-aryl aminothiazoles VIII, IX produced in vitro
CDK4 inhibition activity with IC50 = 26 nM and IC50 = 233 nM, respectively, with in vivo
anticancer activity [44] (Figure 3).
Figure 3. Different thiazole-based compounds of CDK4 inhibitory activity.

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The molecular hybridization of two or more bioactive pharmacophores is considered
to be an optimistic strategy for the design and discovery of novel anticancer drugs [45,46].
This study has been focused on the development of two new sets of hybridized compounds
to be evaluated as potential anticancer agents against some cancer cell lines with possible
CDK4 kinase inhibitory activity. The first set (X) was bearing 6-bromocoumarin nucleus in
conjugation with different substituted thiazolidine/thiazoline/thiazole rings via ethylhy-
drazone linker which has been confirmed to enhance the antiproliferative activity of various
compounds [47,48]. While in the other set (XI) the thiazole ring was directly conjugated
with 6-bromocoumarin ring at its C-4 position and substituted with various heterocyclic
or aromatic rings via the hydrazone linker at its C2 position (Figure 4). All the prepared
conjugates were subjected to a QSAR-based prediction, then examined for their cytotoxic
effect against a panel of cancer cell lines by MTT assay. Thereafter, the compounds which
showed high prediction with the most efficient activity were investigated for their CDK4
inhibitory activity. Molecular docking and dynamic simulations were applied to evaluate
the binding modes and stability in the CDK4 active site in order to justify their inhibitory.
The selectivity toward tumor cells can be evaluated by radio-iodination assay [49]. The
selectivity of compound 2b with promising cytotoxic and CDK4 inhibition activity was
confirmed by a radio-iodination assay. The radiolabeling study confirmed that 2b can be
used as a potential imaging probe for tumors.
Figure 4. The design strategy of the new coumarin-thiazole-based compounds.
2. Results and Discussion
2.1. Chemistry
The Synthetic processes of the intermediates and target compounds were accom-
plished according to the steps illustrated in Schemes 1 and 2. Characterization and the
purity determination of the new target compounds were performed by melting points
determination, spectroscopic methods (IR, 1H NMR, 13C NMR, and mass spectra) in ad-
dition to elemental analysis. Compound 3-acetyl-6-bromo-2H-chromen-2-one (1) was
obtained by a Knoevenagel condensation reaction when 6-bromo salicylaldehyde was al-
lowed to react with ethyl acetoacetate in the presence of few drops of piperidine according
to the reported method [50
2H-chromen-3-yl)ethylidene)-N-methyl (phenyl) hydrazine-1-carbothioamide 2a,b were
prepared by the reaction of with N-methyl (phenyl)hydrazine-1-carbothioamide in ab-
solute ethyl alcohol acidified with a catalytic amount of acetic acid in accordance to the
documented method [54 56]. Thiazolidin-4-one derivatives 3a,b were obtained via the
–53]. Furthermore, the intermediates 2-(1-(6-bromo-2-oxo-
1
–

Molecules 2021, 26, 2273
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treatment of the intermediate carbothioamides 2a,b with ethyl bromoacetate in ethanol
containing anhydrous sodium acetate (Scheme 1). IR spectra of the new compounds 3a,b
showed the characteristic absorption bands of 2 C=O of coumarin and thiazolidine rings at
1744, 1700 cm−1. While 1H NMR spectra of 3a,b revealed a singlet signal at
δ
2.09–2.35 ppm
with three protons integration attributed to the hydrazone linker—CH3 (another singlet
signal at 3.20 ppm due to the N-CH3 protons of 3a), a singlet at 3.97–4.12 ppm assigned
for methylene protons of thiazolidine—CH2 as well as different singlet and doublet signals
at the range
7.42–8.17 ppm related to the aromatic protons. 13C NMR spectra of 3a,b
represented two signals at 17.30 and 32.6 ppm related to the hydrazone linker– H3
and the thiazolidine- H2 carbons (for 3a; a third singlet should be appeared 29.91 ppm
due to N- H3 of thiazolidine ring), the aromatic carbons appeared at 116.8–160.9 ppm
and the two C=O groups of the coumarin and thiazolidine rings appeared at 165.20
δ
δ
δ
δ
C
C
δ
C
δ
δ
and 172.80 ppm. Furthermore, the reaction of the carbothioamides 2a,b with phenacyl
bromide and p-bromophenacyl bromide in refluxing ethanol led to the preparation of the
corresponding thiazoline compounds 4a,b and 5a,b (Scheme 1). IR spectra of the new
derivatives 4a,b and 5a,b revealed a characteristic absorption band at the range 1744–1728
1
cm−1 assigned for the coumarin-C=O group. H NMR spectra of 4a,b and 5a,b compounds
were characterized by the appearance of two singlets at the regions
δ 2.07–2.34 ppm and δ
6.46–6.76 ppm displayed for the hydrazone linker—CH3 and the methine proton of the
thiazoline-H5, an additional singlet with three protons integration was recognized at
3.31, 3.45 ppm corresponding to (-N-CH3) of the compounds 4a
5a, respectively. Also, 13
NMR spectral data of compounds 4a, 5a showed the hydrazone— H3 and -N- H3 at
δ
C
δ
,
C
C
16.90, 19.20, 34.11, 35.01 ppm, respectively, the aromatic carbons appeared at
δ 100.80–160.0
ppm as well as the coumarin—C=O groups appeared at δ 175.61 ppm.
Scheme 1. Synthesis of new 6-bromo-3-(substituted-thylthiazolidin-2-ylidene)hydrazineylidene)ethyl)-coumarin deriva-
tives.

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Scheme 2. Synthesis of new 3-(2-(2-benzylidenehydrazineyl)thiazol-4-yl)-6-bromo-coumarin derivatives.
On the other hand, the target thiazolidinone compounds 6a,b were obtained via thia-
Michael addition reaction between maleic anhydride employed as a Michael acceptor and
the semicarbazides 2a,b, in refluxing toluene (Scheme 1). The IR spectra of both derivatives
represented absorption bands at the range 3441–3449 cm−1 related to OH groups and at
1
the range 1721–1675 cm−1 contributed to C=O groups. Furthermore, H NMR spectra
of compounds 6a,b revealed the two methylene protons of the acetic side chain as two
multiplets at
4.39 ppm was conferred to the thiazolidinone-H5 proton. The three protons of -CH3 of
6a,b resonated at 2.35, 2.09 ppm, respectively, -N-CH3 of 6a at 3.20 ppm, the aromatic
protons resonated at 7.40–8.17 ppm, whereas, the hydroxyl proton appeared as a D2O
exchangeable downfield signal at
12.75 ppm. 13C NMR spectra of 6a,b represented signals
at 17.3, 30.0, 37.2, 43.20 ppm due to the carbons of hydrazone-CH3, -N-CH3 (for 6a), CH2,
δ 2.87–2.95 and 3.04–3.09 ppm, whereas the triplet signal recognized at
δ
δ
δ
δ
δ
and thiazolidinone-C5, respectively. The carbonyl carbons appeared as two singlets at the
range δ 172.20–174.50 ppm.
The reaction of different aromatic/heterocyclic aldehydes with thiosemicarbazide
accomplished the corresponding substituted arylidene-hydrazine-1-carbothioamides 7a
57−63, which in turn were allowed to react with 6-bromo-3-(2-bromoacetyl)-2H-chromen-2-
one ( ) in absolute ethanol to give the corresponding coumarin—thiazole derivatives 9a
(Scheme 2).
IR spectra of the compounds 9a
range of (33418–3503 cm−1) and (1675–1728 cm−1) contributing to NH and C=O groups,
–I
8
–I
–i showed absorption stretching bands within the

Molecules 2021, 26, 2273
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respectively. Furthermore, 1H NMR chemical shifts investigated the aromatic protons, the
coumarin-H4 and the azomethine proton at the range
protons resonated downfield as a singlet signals at the range
exchangeable). In addition, a singlet signal with three protons integration assigned for CH3
of compounds 9b 9h at 2.33, 2.37 ppm, respectively, whereas the six protons related to
-N(CH3)2 group of the compound 9e appeared as a singlet signal at 3.00 ppm. On the
other hand, the six methoxy protons of compound 9c exemplified two singlet signals at
3.80, 3.82 ppm. Also, 13C NMR spectra of the compounds 9a
exhibited the aromatic
and -C=O carbons at the region of 106.30–175.50 ppm. Furthermore, -CH3, -N(CH3)2,
2OCH3 carbons of compounds 9b 9h 9e 9c were presented as singlets at 21.60, 14.21,
δ
7.00–8.50 ppm, while the -NH
δ
11.97–12.77 ppm (D2O
,
δ
δ
δ
–i
δ
,
,
,
δ
41.0, 55.81 ppm, respectively. Mass spectra represented the molecular ion peaks of the new
compounds which concurred with their molecular structures.
2.2. Quantitative Structure Activity Relationship model (QSAR-Model)
Quantitative structure activity relationship (QSAR) studies are applied to discover the
hidden features within the compounds that may be correlated with biological activity [57].
Thiazolyl-hydrazono-coumarin hybrids were reported to have potential anticancer and
potent cyclin dependent kinase inhibition activity [47]. Here, the QSAR model was done
using this series to be used for prediction of the biological activity of the newly synthesized
compounds. The QSAR model was created where the reported 16 thiazolyl-hydrazono-
coumarin compounds were used as a training set and our new synthesized compounds
(Table 1) were used as a test set. All molecular descriptors available in MOE 2016.08 58 [58
]
were computed looking for the descriptor/s that will result in the lowest root mean square
error (RMSE) and highest coefficient of determination for training set prediction r2 that can
confirm its powerful predictive power.
Table 1. Prediction of the pIC50 values for the synthesized compounds.
Scheme I
Scheme II
Predicted pIC50
Compound
Predicted pIC50
6.734
Compound
2a
2b
3a
3b
4a
4b
5a
5b
6a
6b
9a
9b
9c
9d
9e
9f
8.475
6.871
7.053
7.809
6.554
6.736
7.980
6.947
8.365
8.525
6.541
8.687
6.168
12.524
8.889
9g
9h
9i
12.493
7.840
7.381
In order to discover the descriptor/s that may be correlated with the biological activity,
we had to convert all IC50 biological values of the reported compounds into pIC50 values
to have numerical data that can be handled statistically. Then a database was created
with all chemical structures of the previously reported active compounds and their pIC50
values were added to the database as well. Molecular descriptors in MOE are classified
into physical, 2D, and 3D descriptors sets. Each set has a number of related descriptors,
as a result each set was evaluated separately. After calculation of each descriptor set for
the built database, a correlation plot was created for each descriptor to find out which one
will have a direct linear correlation with pIC50. The best linear model should achieve a
higher coefficient r2 value close to 1.00 and lowest root mean square error (RMSE) value.
In our case, the linear model (Figure 5) achieved the best r2 = 0.92 and lowest RMSE = 0.44

Molecules 2021, 26, 2273
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when BCUT_PEOE (2D descriptors) were applied. The BCUT_PEOE_0 member of these
descriptors was irreversibly proportional to the biological activity. This kind of descriptors
is responsible for the atomic partial charge distribution. It can have a direct effect on the
ability of the compounds to form H-Bonds, and an irreversible effect with the hydrophobic
feature of the compounds (highly hydrophobic compounds are less active).
Figure 5. Linear correlation between the experimental and predicted pIC50.
2.3. QSAR-Based Prediction of the Anticancer Activity of the New Compounds
Now the QSAR-model is ready to be used for prediction of the anticancer activity of
the synthesized compounds by comparing their chemical structures to those in the QSAR-
model database then the predictive pIC50 values were computed to the new compounds
and analyzed to select those close to the rage of the active reported compounds so, they
were predicted to be active (Table 1). All compounds showed promising predicted activity
however, few compounds (2b, 3b, 5a, 9a, 9i) showed predicted pIC50 values in the range
of (8–9) which was the range of the top ranked potent compounds in the previously
reported work. As a result, these five compounds were selected for further docking,
cyclin-dependent enzyme assay.
2.4. Molecular Docking Results
In a previously reported work [47] thiazol-hydrazono-coumarin hybrids showed
promising cytotoxic effect with CDK2 inhibitory activity. In this work we tried to investigate
the effect of CDK4 inhibition on the activity of coumarin-based derivatives. In order to
perform the docking study we attempted to identify the active site of CDK4 that can
be used for ligand docking. Unfortunately, the only reported CDK4 crystal structure in
protein data bank has no complexed ligand (pdb code = 2W9Z). Flavopiridol has been
reported to inhibit CDK2 and was co-crystallized with CDK2 (pdb code = 6GUB). Sequence
alignment between CDK2 and CDk4 in order to determine the identity percentage and
it was found to be 42.7%, that can confirm the high identity specially in the binding site
residues (Figure 6A) The crystal structure of CDK2 in complex with flavopiridol and CDk4
has been superimposed to identify the binding site of flavopiridol to be used for docking
(Figure 6B). Flavopiridol was kept in the binding site of CDK4 and this model was be used
for docking. The analysis of the docking (Table 2) showed that compound 2b revealed a
hydrogen bond formed between –NH of the side chain and -C=O of Asp99. The presence
of the electronegative “S” atom supported the intramolecular hydrogen bonding formation
within compound 2b and helped in the formation of a stable conformation that was able to
interact with both Asp99 and Glu144 (Figure 7A). Both C=O group and the oxygen atom
of coumarin ring interacted with Val96. In addition, the electron acceptor group -C=N
showed interaction with Ala33 (Figure 7B). Hydrophobic interactions formed between the
coumarin ring that came in front of the His95 imidazole ring (Figure 7B). Compound 3b

Molecules 2021, 26, 2273
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showed hydrophobic interactions with both Ala33 and His95. It also, had a hydrogen bond
with Asp158 (Figure 7C). Compound 5a showed two hydrogen bonds with Asp97 and
Glu144 (Figure 7D).
Figure 6. (A) Sequence alignment of CDK4 (green) and CDK2 (yellow) with 42.7% identity. (B) Structure superimposition
of CDK2 and CDK4 crystal structures to show the binding site.
Figure 7. Best docking poses of compounds: (A) 2b pose I, (B) 2b pose II, (C) compound 3b, and
(D) compound 5a.

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Table 2. Docking results obtained by MOE 2016.08.
Docking by MOE 2016.08
Computed Affinity
Affinity pKi
30.27
Compound
∆G (kcal/mol)
2a
−10.97
−15.34
−11.04
−15.28
−10.75
−10.64
−15.07
−10.08
−11.52
−11.34
−12.10
−11.63
−11.95
−11.68
−11.86
−12.03
−11.70
−11.75
−12.24
−19.91
2b
40.05
3a
29.57
3b
39.78
4a
29.68
4b
30.77
5a
39.94
5b
30.98
6a
32.72
6b
32.82
9a
34.71
9b
34.36
9c
34.48
9d
33.90
9e
34.39
9f
34.52
9g
34.12
9h
9i
34.33
35.11
Flavopiridol
45.50
Compound 9a and 9i shared a common pose of interactions where they formed two
hydrogen bonds with Lys35, a hydrogen bond with Asp158, Ala33, and Val96 (Figure
8A,B). Docking of flavopiridol showed more computed affinity (45.50) and better free
energy of binding ( 19.91 kcal/mol) than that of compound 2b. Flavopiridol showed
−
hydrophobic interactions with Ala33, Phe93, Val20, and Ile12 in addition to a hydrogen
bond with Asp158 (Figure 8C). In another pose, flavopiridol illustrated a hydrogen bond
with -NH of Asp158 and another hydrogen bond that was formed between the oxygen
atom of 3-hydroxy group of piperidinyl moiety and the -NH group of His95 (Figure 8D).
2.5. Molecular Dynamic Simulations
Molecular dynamic simulation study is usually applied to evaluate the strength and
stability of binding of the promising compounds. The top-ranked compound in both;
QSAR-based prediction, and docking score was 2b. It was subjected to drastic conditions
in which high temperature and pressure in addition to movement and rotation of all bonds
and angles were applied over 10 ns period of time. The results of these MD study were
compared to the reference flavopiridol (Figure 9). The oscillations of compound 2b was
parallel to that flavopiridol. However, flavopiridol was more stable as its equilibrium
was reached at RMSD of 0.5 Å after 3.5 ns. While compound 2b reached the equilibrium
at RMSD of 0.8 Å after the same time 3.5 ns and this was interesting and confirmed the
binding stability of our compound. Additionally, it confirmed the previous prediction of
the high biological activity of this compound.

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Figure 8. Best docking poses of compounds (
A
)
9a, (B)
9i, (
C
) flavopiridol pose I, and (
D)
flavopiridol
pose II.
Figure 9. MD simulations of both the best active compound 2b (blue) and flavopiridol (red).
2.6. Biological Evaluation of the Synthesized Compounds
2.6.1. In Vitro Anticancer Effect and Structure Activity Relationship
The cytotoxic activity of the compounds was tested against three cell lines MCF-7 Cell
line (Breast cancer), A-549 (human alveolar basal epithelial cells), and CHO-K1(Ovarian
cell line). All the compounds exhibited moderate cytotoxic activity, however, compound
2b showed the best cytotoxic effect on the tested cell lines (Table 3). Despite the appar-

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ent promising activity of the compounds they showed lower activity when compared
to flavopiridol.
Table 3. Cytotoxic effect of the scheme I (6-bromo-3-(substituted-thylthiazolidin-2-ylidene)
hydrazineylidene)ethyl)-coumarin derivatives.) against MCF-7, A-549 and CHO-K1 cell lines.
MCF-7
(IC50, µM)
A-549
(IC50, µM)
CHO-K1
(IC50, µM)
Compound
2a
0.161
0.0314
0.138
0.116
0.360
0.861
0.102
0.305
0.055
0.055
0.00445
0.451
0.015
0.560
0.056
0.537
0.784
0.054
0.305
0.095
0.077
0.00114
0.604
0.054
0.735
0.127
0.757
0.968
0.075
0.414
0.131
0.101
0.00241
2b
3a
3b
4a
4b
5a
5b
6a
6b
Flavopiridol
Studying the effect of structure variation on the anticancer activity (Figure 10), it
appeared that the presence of a thiourea moiety had a significant effect on the activity
whereas its inclusion in a ring system such as thiazolidinone (3a), thiazoline (4b) markedly
decreased the cytotoxic activity. Substitution on the thiourea group also appeared to
possess a major impact on the anti-cancer activity. N-phenyl thiourea moiety appeared
to have the optimum activity. While the 3-phenylthiazolidin-4-one (3b), diphenylthia-
zoline (4b), 3-methyl-4-phenylthiazol (4a), 4-bromophenyl-3-phenylthiazol (5b), 4-oxo-3-
phenylthiazolidin-5-yl-acetic acid (6b), 1-methylthiourea (2a), methylthiozolidinone (3a),
methyl-4-bromophenyl-thiazoline (5a), and methyl-thiazolidinone-acetic acid (6a) had a
negative effect on the anticancer activity.
Figure 10. Structure activity relationship of compound 2b that is correlated with cytotoxicity activity.
On the other hand, the second compound series containing 2-benzylidenehydrazinyl-
thiazole scaffold showed generally lower activity when compared to the first series. Addi-

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tion of any substitution on the phenyl ring e.g., 4-methyl (9b), 3,4-dimethoxy (9c), 4-nitro
(9d) 4-flouro (9f), decreased the anticancer activity. Only the unsubstituted phenyl deriva-
tive (9a) showed the lowest IC50 value against all three cell lines (Table 4). On the other
hand, replacement of the phenyl substitution with furan decreased the cytotoxic activity
when compared to compound 9d. However, a nitro-furan ring offered relatively good
cytotoxic activity when compared to the rest of the compounds in the series (Figure 11).
Table 4. Cytotoxic effect of the scheme II (3-(2-(2-benzylidenehydrazineyl)thiazol-4-yl)-6-bromo-
Coumarin derivatives.) against MCF-7, A-549 and CHO-K1 cell lines.
MCF-7
(IC50, µM)
A549
(IC50, µM)
CHO-K1
(IC50, µM)
Compound
9a
0.060
0.295
0.479
0.780
0.432
0.276
0.488
0.534
0.124
0.00445
0.034
0.386
0.244
0.513
0.242
0.200
0.441
0.34
0.091
0.522
0.649
0.901
0.507
0.495
0.744
0.574
0.189
0.00241
9b
9c
9d
9e
9f
9g
9h
9i
0.075
0.00114
Flavopiridol
Figure 11. Structure activity relationship of compound 9a.
2.6.2. In Vitro Activity against CDK4 Enzyme
Compounds with the highest in-vitro cytotoxicity were tested for their CDK4 en-
zyme inhibition. The results are presented in Table 5. Compound 2b showed the highest
inhibitory activity against CDK4 enzyme.
Table 5. CDK4 enzyme inhibition for the selected compounds.
Compound
IC50 (µM)
0.036
2b
3b
0.127
5a
0.142
9a
9i
0.131
0.193
Flavopiridol
0.001

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2.6.3. Radioiodination of Compound 2b
Compound 2b showed the most promising anticancer activity in the previous in-vitro
assays. Its tagging with a radiotracer (131I) was planned to study its in vivo distribu-
tion and selective localization at tumor site. Radioiodination of 2b was achieved via
electrophilic substitution reaction utilizing the oxidative effect of chloramine-t (CAT). To
achieve optimized reaction conditions and obtain the highest percentage of 131I-2b (highest
radiochemical yield, RCY), various factors were studied. These factors are, the amount of
oxidizing agent, the amount of 2b, pH of the reaction and the reaction time. The amount of
CAT used showed to influence the % RCY as depicted in Figure 12A. Starting with only
25
µg of CAT the RCY was 25.74 + 3.23% this yield was increased proportionally with
amount of CAT to reach 91.84 + 5.82% when 50
µg of CAT was used. A further increase
in the CAT amount caused a significant drop in the RCY, which can be attributed to the
formation of chlorinated by-products [59]. Similarly, the amount of 2b had the same effect
on the RCY (Figure 12B). The RCY was only 8.01 + 1.33% when 100
µg of 2b was used
and roused to 91.84 + 5.82% at 500 g of 2b. The pH of the reaction was found to be 6
µ
without the use of a buffer solution. In this study dil. HCl and 0.01 M NaOH was used to
alter the pH of the reaction. The best RCY (91.84 + 5.82%) was achieved at pH 2. The RCY
decrease by increasing the pH towards more basic values to reach only 14.40 + 3.11% at
pH 7 (Figure 12C).
Figure 12. Dependence of RCY on various factors: (
reaction; (D) reaction time.
A) CAT amount; (B) 2b amount; (C) pH of the
Finally, the reaction time was found to be crucial in obtaining the RCY (Figure 12D).
The RCY was 60.09 + 5.11 after 15 min and reached its optimum at 45 min. At longer
reaction time the RCY slightly declined to reach 87.2 + 5.05%.
2.6.4. Chromatographic Determination of the RCY
The RCY was evaluated using thin layer chromatography (TLC). Two mobile phases
were used for achieving good separation between 131I-2b and free 131I and to ensure
accurate determination of the RCY. Chloroform:methanol was used as the first mobile

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phase; free 131I had a retention factor (Rf) of 0.2 and 131I-2b had Rf of 0.5. The second
mobile phase was chloroform: ethanol (9:1). The Rf for 131I and 131I-2b were 0.3 and 0.5,
respectively. A blank reaction was used to establish the Rf of free 131I.
2.6.5. Purification of 131I-2b by Column Chromatography
The separation of 131I-2b to its precursor 2b and from other radioactive species was
achieved via column chromatography. Silica column was used for the purification process
using a mixture of chloroform: methanol (4.8: 0.2) as a mobile phase. The eluate was
monitored by TLC. 131I-2b was eluted first (Rt = 1 min) followed by 131I at 3.5 min (Figure 13).
Cold unreacted 2b appeared at Rt 1.75 min as detected by TLC.
Figure 13. Purification of 131I-2b by silica column.
2.6.6. In Vitro Stability of 131I-2b
To ensure the stability of the radioiodinated compound, column purified 131I-2b was
kept in a dark vial to avoid the effect of light. A sample was withdrawn at various time
points (2, 4, 6 and 8 h). 131I-2b showed good stability up to 4 h where the change in RCY
was only
(Figure 14).
−
0.87%. After 12 h and 24 h the change in RCY was
−
11.82% and
−
26.21%
Figure 14. In vitro stability of 131I-2b.

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2.6.7. Biodistribution Study
Pure 131I-2b was injected in both normal and solid tumor bearing mice to establish the
normal route of 131I-2b distribution and to monitor its localization at tumor site. 131I-2b
showed a comparable distribution pattern in both normal and tumor bearing mice. As can
be observed the 131I-2b showed rapid clearance from the blood where the initial uptake
was 2.71
±
0.93% injected dose/g (% ID/g) 15 min post injection (p.i) which gradually
declined by time to reach a value of 0.4 ± 0.018% ID/g, 240 min p.i.
The liver and kidney uptake clearly established the metabolic and excretory pathway
of 131I-2b. The liver uptake started at 5.20
±
0.83 at 15 min p.i and started to increase
gradually to reach its maximum at 120 min p.i (14.29 2.85%ID/g) followed by a decline
in the uptake afterwards as the drug was cleared from the body. Likewise, the kidneys
started with low uptake (0.86 0.21%ID/g at 15 min p.i) and started to increase gradually
with time to reach a highest of 7.46 ± 1.96%ID/g at 120 min p.i (Figure 15).
±
±
Figure 15. Radioactivity uptake of liver, kidney and blood at different time points p.i.
The biodistribution of 131I-2b is demonstrated in Figure 16. Thyroid uptake is consid-
ered as an indicator for the in vivo stability of radioiodinated compounds. Low thyroid
uptake indicates that the compound is resistant to in vivo de-iodination59. A low uptake of
radioactivity was observed at 15 min p.i. (0.67
±
0.06% ID/g) which increased to reach a
peak of 8.27
±
1.09% ID/g at 240 min p.i. which indicated gradual in vivo de-iodination
of 131I-2b.
In solid tumor bearing mice, the values of tumor uptake were promising starting with
uptake of 4.5 0.54% ID/g at 15 min p.i. which continued to increase to reach a height of
5.97 0.82% ID/g at 60 min p.i. At longer time points, the uptake decreased gradually to
reach 0.47 0.15% ID/g. On the other hand, normal muscle showed negligible uptake at
all time points (Figure 17).
±
±
±

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Figure 16. Biodistribution of 131I-2b in normal mice at different time points p.i.
Figure 17. Radioactivity uptake in blood, tumor muscle and normal muscle at different time points p.i.
Tumor: normal tissue ratio is used as a parameter to evaluate preferential uptake and
tumor targeting of a radiolabeled agent [60]. The calculated values of ratio of (tumor:blood)
(T/B) and ratio of (Tumor: Normal muscle) (T/NM) were; 1.66 and 2.75 at 15 and 30 min
p.i, respectively Table 6. Compared with blood, the radioactivity uptake was1.66 and 2.75
at 15 and 30 min p.i, respectively. This ratio reached its peak (3.75) at 60 min p.i. and

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declined to 0.76 at 120 min p.i. As for T/NM ratio, 131I-2b showed high preferential uptake
when compared to normal muscle. The lowest T/NM value (7.12) achieved 120 min p.i.
and highest value of 94.04 at 60 min p.i. Comparing T/NM for 131I-2b with other tumor
targeting agents, such as 177Lu-DTPA-DG (5.07 at 60 min p.i) [61], 125I-antiTLR5 mAb
(6.481 at 48 h) [62], and 99mTc-dioxime (5.14 at 30 min) [63], indicating its high targeting
ability as well as the fact that 131I-2b can be considered as a good radiotheranostic agent
for solid tumors.
Table 6. Prediction of some of ADMET properties of hit compounds using admetSAR server.
Models
2b
3b
5a
9a
9i
Blood-Brain Barrier
BBB+
BBB+
BBB+
BBB+
BBB-
Absorption
Metabolism
Human Intestinal Absorption
Renal Organic Cation Transporter
CYP450 3A4 Substrate
CYP450 1A2 Inhibitor
CYP450 2D6 inhibitor
HIA+
HIA+
HIA+
HIA+
HIA+
Non-inhibitor
Non-substrate
Inhibitor
Non-inhibitor
Substrate
Inhibitor
Non-inhibitor
Substrate
Inhibitor
Non-inhibitor
Non-substrate
Inhibitor
Non-inhibitor
Substrate
Inhibitor
Non-inhibitor
Inhibitor
Non-inhibitor
Inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Non-inhibitor
Inhibitor
CYP450 3A4 Inhibitor
Human Ether-a-go-go-Related Gene
Inhibition I
Weak inhibitor
Non-inhibitor
Weak inhibitor
Non-inhibitor
Weak inhibitor
Non-inhibitor
Weak inhibitor
Non-inhibitor
Weak inhibitor
Non-inhibitor
AMES toxic
Human Ether –a-go-go Related Gene
Inhibition II
Non AMES
toxic
Non AMES
toxic
Non AMES
toxic
Non AMES
toxic
AMES Toxicity
Carcinogens
Non-
carcinogens
Non-
carcinogens
Non-
carcinogens
Non-
carcinogens
Non-
carcinogens
Toxicity
Honey Bee Toxicity
Acute Oral Toxicity
Low HBT
III
Low HBT
III
Low HBT
III
Low HBT
III
Low HBT
III
Carcinogenicity (three class)
Rat Acute Toxicity (LD50, mol/kg)
Aqueous solubility (Log s)
Non-required
2.6533
Non-required
2.4214
Non-required
2.3609
Non-required
2.5460
Danger
2.4924
−3.8523
−4.2840
−4.0964
−4.2184
−4.2955
3. In Silico Evaluation of ADMET
Different predictive qualitative ADMET models were applied to study the effect of
different physicochemical properties of the synthesized compounds on their expected ab-
sorption, metabolism, and toxicity using admetSAR server (Table 6). Except for compound
9i, all the tested compounds showed the ability to cross the blood brain barrier (BBB).
In addition, all the compounds had good predicted human intestinal absorption (HIA).
Compounds 3a
, 5a, and 9i were predicted to be substrate for CYP450 3A4. On the other
hand, all the compounds were predicted to be inhibitors to CYP450 1A2. Compounds 2b
,
9a, and 9i showed predicted inhibitory activity for CYP450 3A4. The four compounds
showed non-toxic profile as predicted by the studied parameters.
4. Materials and Methods
4.1. Chemical Synthesis
All melting points are uncorrected and were taken in open capillary tubes using a
Stuart melting point apparatus. Elemental microanalyses were carried out at the Regional
Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. Mass spectra
(MS) were performed at 70 e.v by GCMS-QP1000 EX spectrometer using the electron
ionization technique (EI) at the Regional Center for Mycology and Biotechnology, Al-Azhar
University, Cairo, Egypt. Infrared spectra (IR) were recorded (KBr discs) on a Shimadzu
FT-IR 8201 PC spectrophotometer, faculty of Science, Cairo University (Cairo, Egypt). NMR
spectra were recorded on a mercury spectrometer at 300 MHz or Bruker NMR spectrometer

Molecules 2021, 26, 2273
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1
at 400 MHz. H NMR spectra were run at 300 or 400 MHz, while 13C spectra were run
at 75 MHz at the Faculty of Pharmacy, Ain shams University, Cairo, Egypt. Chemical
shifts were expressed in
δ (ppm) downfield from TMS as an internal standard. Follow-
up of the reactions and checking the purity of the compounds were made by TLC on
silica gel-precoated aluminum sheets (Type 60, F 254, Merck, Darmstadt, Germany) using
hexane/ethylacetate (3:1, v/v) and the spots were detected by exposure to a UV lamp at
254 nanometer for few seconds and by iodine vapor. The chemical names given for the
prepared compounds are according to the IUPAC system.
Synthesis of 2-bromo-1-(1H-benzo[d]imidazol-2-yl)-1-ethanone (3) was achieved ac-
cording to the reported method [47,50–53].
General procedure for synthesis of 2-(1-(6-bromo-2-oxo-2H-chromen-3-yl)ethylidene)-N-
methyl/phenylhydrazine-1-carbothioamide 2a,b
To a hot solution of the 3-acetyl-6-bromo-2H-chromen-2-one (1.8 mmol) in absolute
ethanol (10 mL), 4-methyl/phenyl-thiosemicarbazide (1.8 mmol) and five drops of concen-
trated HCl were added. The mixture was refluxed for 14–17 h. The reaction mixture was
allowed to cool, the formed precipitate was filtered, dried and recrystallized from absolute
ethanol to afford the title compounds 2a,b.
General procedure for synthesis of 2-((1-(6-bromo-2-oxo-2H-chromen-3-yl)ethylidene)
hydrazino)-3-methyl/phenyl-thiazolidin-4-one 3a,b
To a hot solution of the 2a,b (1.4 mmol) in absolute ethanol (20 mL), ethyl bromoacetate
(0.2 g, 1.5 mmol) and anhydrous sodium acetate (0.2 g, 2.8 mmol) were added. The reaction
mixture was refluxed for 7–12 h. The formed precipitate was filtered, washed with water,
dried, and recrystallized from the appropriate solvent to afford the corresponding title
compound 3a,b.
General procedure for synthesis of 6-Bromo-3-(1-((3-methyl/phenyl-4-phenylthiazol-2(3H)
ylidene)hydrazineylidene) ethyl)-2H-chromen-2-one 4a,b
To a hot solution of the 6-bromocoumarin compound 2a,b (1.4 mmol) in absolute
ethanol (20 mL), phenacylbromide (0.3 g, 1.4 mmol) and anhydrous sodium acetate (0.2 g,
2.8 mmol) were added. The reaction mixture was refluxed for 9 h. The formed precipitate
was filtered then washed with water, filtered, dried, and recrystallized from the proper
solvent to afford the corresponding the title compounds 4a,b.
General procedure for synthesis of bromo-3-(1-((4-(4-bromophenyl)-3-methyl/phenylthiazol-
2(3H)-ylidene) hydrazono)ethyl)-2H-chromen-2-one 5a,b
To a hot solution of compound 2a,b (1.2 mmol) in absolute ethanol (20 mL), p-
bromophenacylbromide (0.3 g, 1.2 mmol) and anhydrous sodium acetate (0.2 g, 2.4 mmol)
were added. The reaction mixture was refluxed for 19 h. After reaction completion, the
formed precipitate was filtered while hot then washed with water, filtered, dried, and
crystalized from the proper solvent to accomplish the corresponding derivatives 5a,b.
General procedure for synthesis of 2-(2-((1-(6-bromo-2-oxo-2H-chromen-3-yl)ethylidene)
hydrazono)-3-methyl/phenyl-4-oxothiazolidin-5-yl)acetic acid 6a,b
To a hot solution of compound 2a,b (1.2 mmol) in toluene (20 mL), maleic anhydride
(1.2 mmol) was added. The reaction mixture was refluxed for 16 h till reaction completion,
then left to cool. The formed precipitate was filtered, dried, and recrystallized from the
proper solvent to afford the corresponding title compounds 6a,b.
General procedure for synthesis of 1-(substituted) thiosemicarbazides (Schiff bases) 7a–i
The synthesis of thiosemicarbazones was carried out according to the reported proce-
dures 57–63 and the brief procedure is provided in (Supplementary Materials).

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General procedure for the synthesis of 3-(2-(2-(substituted)hydrazinyl)thiazol-4-yl)-6-
bromo-2H-chromen-2-one 9a–i
6-Bromo-3-(2-bromoacetyl)-2H-chromen-2-one (0.69 g, 0.002 mol) and the appropriate
thiosemicarbazone (0.002 mol) were dissolved in ethanol (10 mL). The reaction mixture was
refluxed for 5–7 h, monitored by TLC. The formed precipitate was filtered while hot, dried
and recrystallized from the proper solvent to afford the corresponding title compounds
9a–i, respectively.
4.2. QSAR Study
All structures were built and saved in a database (mdb format). The pIC50 were
computed. The energy of built compounds was minimized. MOE 2016.08 molecular
descriptors were calculated for all compounds. The previously reported 16 synthesized
compounds [47] were used as a training set and our newly synthesized compounds were
considered as a test set. A QSAR model was created. All redundant descriptors with
low variance among the different compounds were removed. Multiple linear regression
was used as the machine learning algorithm for linear model generation which removes
co-linear descriptors by default.
4.3. Molecular Docking
The crystal structure of CDK4 in complex with flavopiridol that has been resulted
from superimposition of (CDK2 + Flavopiridol) (pdb code = 2W9Z) and CDK4 (pdb
code = 6GUB). The resulted (CDK4 + Flavopiridol) model was used for molecular docking
and the protein was prepared prior to the docking process by MOE 2016.08 in which
minimization were done. Hydrogen atoms were added, and the docking site was identified
and selected. All compounds were built and saved as (moe). Rigid receptor was used as a
docking protocol. Both receptor and solvent were kept as a “receptor”. Triangle matcher
was used as a placement method. Two rescoring were computed, rescoring 1 was selected
as London dG. Rescoring 2 was selected as affinity. Force field was used as a refinement. All
coordinates were derived from pdb and all interactions were observed with the conserved
residues. The docking protocol used the triangle method as a placement method with
timeout of 300 s, and number of return poses as 1000. London dG was used as a rescoring
method. Force field was used as a refinement method by applying MMFF94x.
4.4. Molecular Dynamic Simulations
All molecular dynamic simulations were conducted by MOE 2016.08. The best con-
formations from each docking process were kept inside the active site. The quality of
the temperature-related factors, protein geometries, and electron density were tested. All
hydrogens were added, and energy minimization was computed. The solvent molecules
that were in the system were deleted before solvation and salt atoms were added to ensure
complete neutralization of the biomolecular system. The solvent atoms were added to
surround the biomolecular system (protein-ligand complex) in a spherical shape. The
protein-ligand complex was surrounded by a sphere shape of solvent (water) and NaCl
was used as a salt to neutralize charged system. Amber 10:EHT was selected as a force
field in the potential setup step. All Van der Waals forces, electrostatics, and restraints were
enabled. The heat was adjusted in order to increase the temperature of the system from 0
to 300 K which was followed by equilibration and production for 300 ps. The simulation
was conducted over a 10 ns period of time.
4.5. Cytotoxic Evaluation
Mammalian cell lines: MCF-7 cells (human breast cancer) cell line, A-549 (human
Lung Carcinoma) and CHO-K1 cells (hamster ovary cancer) cell line were obtained from
the American Type Culture Collection (ATCC, Rockville, MD). Fetal bovine serum, RPMI-
1640, HEPES buffer solution, L-glutamine, gentamycin and 0.25% Trypsin-EDTA were
purchased from Lonza (Belgium). All reagents and chemicals were of pharmaceutical pure

Molecules 2021, 26, 2273
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grade. Non-carrier added Na131I solution (pH = 7) was purchased from the Research and
Production Facility, The Egyptian Atomic Energy Authority. Chemicals were purchased
from Sigma Aldrich and Lonza. Monitoring of chemical reactions was done by using
analytical TLC with Merck 60 F-254 silica-gel plates (Merck, Germany), and visualization
was done with ultraviolet light.
Cytotoxicity study was performed at the Regional Centre for Mycology and Biotech-
nology (RCMB), Al-Azhar University. Mammalian cell lines: human breast cancer cell
line (MCF-7 cells), human colon cancer cell line (HCT-116 cells), and human lung cancer
cell line (A-549 cells) were obtained from VACSERA Tissue Culture Unit, Cairo, Egypt.
The cells were propagated in Dulbecco’s modified Eagle’s medium supplemented with
10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer, and 50 µg/mL
gentamycin. All cells were maintained at 37 ◦C in a humidified atmosphere with 5% CO2
and were subcultured two times a week. For the cytotoxicity assay, the cells were seeded in
96- well plate at a cell concentration of 1
×
104 cells per well in 100 µL of growth medium.
Fresh medium containing different concentrations of the test sample was added after 24 h
of seeding. Serial two-fold dilutions of the tested chemical compound were added to
confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon,
◦
NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37 C
in a humidified incubator with 5% CO2 for a period of 48 h. Three wells were used for
each concentration of the test sample. Control cells were incubated without test sample
and with or without DMSO. The little percentage of DMSO present in the wells (maximal
0.1%) was found not to affect the experiment. After incubation of the cells for at 37 ◦C,
various concentrations of sample were added, and the incubation was continued for 24 h,
and viable cells yield was determined by a colorimetric method. In brief, after the end
of the incubation period, media were aspirated, and the crystal violet solution (1%) was
added to each well for at least 30 min. The stain was removed, and the plates were rinsed
using tap water until all excess stain is removed. Glacial acetic acid (30%) was then added
to all wells and mixed thoroughly, and then the absorbance of the plates was measured
after gently shaken on a microplate reader, using a test wavelength of 490 nm. All results
were corrected for background absorbance detected in wells without added stain. Treated
samples were compared with the cell control in the absence of the tested compound. All
experiments were carried out in triplicate. The cell cytotoxic effect of each tested com-
pound was calculated. The optical density was measured with the microplate reader to
determine the number of viable cells, and the percentage of viability was calculated as
(1
−
(ODt/ODc))
×
100% where ODt is the mean optical density of wells treated with the
tested sample and ODc is the mean optical density of untreated cells. The relation between
surviving cells and drug concentration is plotted to get the survival curve of each tumor
cell line after treatment with the specified compound. The 50% inhibitory concentration
(IC50), the concentration required to cause toxic effects in 50% of intact cells, was estimated
from graphic plots of the dose response curve for each conc [64,65].
4.6. CDK4 Enzyme Inhibition Assay
Reaction Biology Corp. Kinase HotSpotSM service (http://www.reactionbiology.
com, accessed on 1 April 2019) was used for screening of tested compounds. CDK4
Kinase inhibitory activities were assessed by the HotSpot assay platform, which contained
specific kinase/substrate pairs along with required cofactors. Base reaction buffer: 20 mM
Hepes (pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mM
Na3VO4, 2 mM DTT, 1% DMSO. Testing compounds were dissolved in 100% DMSO to
specific concentration. The serial dilution was conducted in DMSO. The reaction mixture
containing the examined compound and 33P-ATP was incubated at room temperature for
2 h and radioactivity was detected by the filter-binding method. Kinase activity data were
expressed as the percent remaining kinase activity in test samples compared to vehicle
(dimethyl sulfoxide) reactions.

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For CDK4 enzyme inhibition, CDK4 ELISA kit, Cloud-Clone Corp, US was used. This
kit is used for the in vitro quantitative measurement of CDK4 concentrations in tissue
homogenates, cell lysates and other biological fluids with high sensitivity and specificity.
The cancer cells were treated with different amounts of each compound according to the
aforementioned protocol. In addition, the cells were lysed and then treated as instructed by
the manufacturer’s protocol. The absorbance of each well was detected at 450 nm using an
ELISA plate reader. The results are reported as half maximal inhibitory concentration (IC50).
4.7. Radioiodination of 2b
Stock solutions of 2b, Chloramine-T (CAT), and sodium thiosulfate were prepared
for use in the radioiodination reaction as follows. Amount of 5 mg of 2b was dissolved in
5 mL of DMF. For CAT stock solution, exactly 1 mg of CAT was dissolved in 1 mL absolute
ethanol. For sodium thiosulfate stock solution; amount of 15 mg sodium thiosulfate was
dissolved in 1 mL of double distilled water. Radioiodination reaction was prepared as
follows; 500
by the addition of 10
CAT solution was added (0.22
µ
L (1.20
µ
M) of 2b stock solution was placed in a dark screw cap vial followed
L of Na131I solution (equivalent to 150 MBq) then exactly 50
L of
M) the reaction pH was adjusted to 2 using 80 L of dil HCl.
µ
µ
µ
µ
The reaction mixture was vortexed and allowed to stand for 45 min at room temperature.
Afterwards the reaction was stopped using 5 µL of sodium thiosulfate solution.
4.8. Determination of the Radiochemical Yield
The radiochemical yield was determined using paper chromatography using TLC
(1 cm
×
13 cm strip). Exactly 20 µL (4.6 MBq) of the reaction mixture was placed 1 cm
away from the TLC plate base and was developed using chloroform: methanol (4.8: 0.2)
and chloroform:ethanol (9: 1). After complete development, the TLC plate was air dried
and cut into 1 cm strips. Each strip was counted in a well type γ-counter. A blank reaction
was analyzed similarly to establish the retention factor (Rf) of 131I.
4.9. Purification of 131I-2b
For the purpose of purification, a silica column was used (1 cm
of 100 µL (23.25 MBq) was injected to the column and eluted using chloroform: methanol
×
10 cm). Amount
(4.8: 0.2). The flow rate was 0.5 mL/min and eluted fractions were collected manually and
counted individually using well type
individually by TLC.
γ-counter. All the eluted fractions were also analyzed
4.10. In-Vitro Stability Study
Column purified 131I-2b was kept in a dark screw vial in dark chamber for 24 h. A
sample of the solution was withdrawn and the radiochemical yield was re-evaluated by
TLC at different time points (2, 4, 6, 12, 24 h).
4.11. Biodistribution Study
Ehrlich ascites carcinoma (EAC) was used in this study. The parent line was supplied
from the ascitic fluid of Swiss Albino mice purchased from the Egyptian National Cancer
Institute, Cairo University. Adult male Swiss albino mice weighing 22–25 g purchased
from the breeding unit of the Egyptian Organization for Biological Products and Vaccines,
Cairo, Egypt, were used in this study. Animals were on pellet diet and tap water. Animal
treatment was in accordance with the National Institute of Health Guide for Animal, as
approved by the Institutional Animal Care and Use Committee. The EAC was withdrawn
from the ascitic fluid of a mice bearing EAC (10 days old) under aseptic conditions and
diluted using physiological sterile saline solution. The diluted cell line was mixed for 5 min
by vortex shaker. To generate Ehrlich solid tumor, 200 µL of the diluted ascitic fluid was
injected intramuscularly in the right lower limb of male mice (22–25 g). Palpable solid
mass was observed 10 days after inoculation.

Molecules 2021, 26, 2273
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For commencing the biodistribution study the animals were divided into two groups
the first included normal mice (no tumor inoculation) and the second group included
tumor inoculated mice. Five individuals were used for each time point. Exactly 100 µL
(23.25 MBq) of purified 131I-2b was injected in the tail vein of each mice. Mice were
anesthetized with a mixture of xylazine (10 mg/kg) and ketamine (80 mg/kg) then weighed
and dissected. Blood was collected via cardiac puncture, and all organs were separated
and washed with physiological saline. For the tumor muscle, sample of the tumor was
isolated completely from the leg and washed with normal saline then weighed. Another
sample of the contralateral normal muscle was used as control to measure the tumor
uptake of compound 2. All the collected biological samples were placed in previously
weighed plastic containers, and the activity was counted in automatic scintillation gamma
counter. A standard dose containing the same injected amount was counted simultaneously
and accounted as 100% activity [66] The uptake was expressed as percentage injected
dose/gram tissue (%ID/g). Blood, bone, and muscles were assumed to be 7%, 10%, and
40% of the total body weight, respectively [67]. Data are reported as the mean
were analyzed using compare means function in SPSS software (Version 20.0).
±
SE. Data
4.12. In Silico Prediction of ADMET
Different absorption, distribution, metabolism, excretion and toxicity parameters
were evaluated using the qualitative models offered by the free webserver admetSAR
(http://lmmd.ecust.edu.cn/admetsar2) {Yang, 2018 #245}. The compounds were converted
to smiles format and uploaded to obtain the desired prediction.
5. Conclusions
This study showed the discovery of novel coumarin-based lead compound with
promising anticancer and selective radiotheranostic activity. Compound 2b had potential
cytotoxic activity with IC50 = 0.0136, 0.015, 0.054
cell lines, respectively. Its binding stability within CDK4 was confirmed by both docking
G = 15.34 kcal/mol) and dynamic simulations in which it achieved equilibrium at
RMSD of 0.8 Å after 3.5 ns. This was confirmed by the CDK4 enzyme assay where 2b
showed inhibitory activity of CDK4 = 0.036 M. In addition, the radio-iodinated form of
compound 131I-2b showed good in vitro stability up to 4 h, biodistribution pattern, tumor
uptake reached a height of 5.97 0.82%ID/g at 60 min p.i and promising results in terms
µM against MCF-7, A-549, and CHO-K1
(
∆
−
µ
±
of selective targeting and localization in solid tumors. A finding that will encourage to use
compound 2b as a chemotherapeutic agent, a radiotherapeutic agent, and a radio-imaging
agent for solid tumors.
Supplementary Materials: The following are available online, Figure S1 Mass spectrum of com-
pound 2b, Figure S2 Mass spectrum of compound 2a, Figure S3 Mass spectrum of compound 3a,
Figure S4 Mass spectrum of compound 3b, Figure S5 Mass spectrum of compound 4a, Figure S6
Mass spectrum of compound 4b, Figure S7 Mass spectrum of compound 5a, Figure S8 Mass spec-
trum of compound 5b, Figure S9 Mass spectrum of compound 6a, Figure S10 Mass spectrum of
compound 6b, Figure S11 Mass spectrum of compound 9a, Figure S12 Mass spectrum of compound
9b, Figure S13 Mass spectrum of compound 9c, Figure S14 Mass spectrum of compound 9d, Figure
S15 Mass spectrum of compound 9e, Figure S16 Mass spectrum of compound 9f, Figure S17 Mass
spectrum of compound 9g, Figure S18 Mass spectrum of compound 9h, Figure S19 Mass spectrum of
compound 9i.
Author Contributions: Conceptualization, W.A.Z., S.S.A.E.-K., M.A.K. and M.M.A.; methodology,
R.H.G., M.O.S., and M.A.K.; software, M.A.K.; validation, W.A.Z., S.S.A.E.-K., M.O.S., M.A.K. and
M.M.A.; formal analysis, M.O.S., W.A.Z., S.S.A.E.-K., and M.M.A.; investigation, W.A.Z., S.S.A.E.-
K., M.O.S., M.A.K. and M.M.A.; resources, M.O.S., R.H.G.; data curation, W.A.Z., and M.A.K.;
writing—original draft preparation. All authors contributed in writing the original draft. Project
administration, W.A.Z.; funding acquisition, W.A.Z., S.S.A.E.-K., and M.M.A. All authors have read
and agreed to the published version of the manuscript.

Molecules 2021, 26, 2273
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Funding: This research did not receive any specific grant from funding agencies in the public,
commercial, or not-for-profit sectors.
Institutional Review Board Statement: All the biodistribution studies were performed accord-
ing to the animal ethics guidelines of the Egyptian Atomic Energy Authority. The animal ethics
committee of Egyptian Atomic Energy Authority approved the biodistribution study performed
(EAEA/202003/185).
Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.
Conflicts of Interest: All the authors agreed on the contents of the article and post no financial,
personal, or organizational conflicting interest that may affect this research article.
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