Angiokinase inhibition of VEGFR-2, PDGFR and FGFR and cell growth inhibition in lung cancer: Design, synthesis, biological evaluation and molecular docking of novel azaheterocyclic coumarin derivatives

Article abstract of DOI:10.1016/j.bmcl.2021.128258

The present work represents the design and synthesis of some azaheterocyclic coumarin derivatives which are evaluated as anti-lung cancer agents. Ten out of the twenty azaheterocyclic compounds showed superior activity than the standard drug staurosporine against non-small cell lung cancer (A549). Representing the four different azaheterocyclic series, compounds 4a, 5d, 6e, and 7d, which demonstrated IC_50s of 2.38, 2.39, 1.05 and 3.98 μM, respectively, each exhibiting the best cytotoxicity in its group, were selected for further assessment of their toxicity on normal lung cells (WI-38). Compound 4a was selected for further investigations because it remarkably revealed less cytotoxicity (IC₅₀ = 53.76 μM) than 7d (IC₅₀ = 19.95 μM) on (WI-38) compared to staurosporine (IC₅₀ = 24.41 μM). 4a was assessed for its ability to inhibit the angiokinases VEGFR-2, PDGFR, FGFR and the growth factor EGFR, remarkably it showed better VEGFR-2, PDGFR, FGFR inhibition than the reference drugs used and exhibited as well noticeable EGFR inhibition. Going further, 4a was capable of arresting the cell cycle at pre-G1 phase and S phase and inducing apoptosis. Moreover, the capability of the target 4a to interact with the key amino acids of VEGFR-2 binding site was detected by molecular docking. Finally, the in silico physicochemical properties of 4a were studied.

Full text of DOI:10.1016/j.bmcl.2021.128258

Bioorg. Med. Chem. Lett. 48 (2021) 128258
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Angiokinase inhibition of VEGFR-2, PDGFR and FGFR and cell growth
inhibition in lung cancer: Design, synthesis, biological evaluation and
molecular docking of novel azaheterocyclic coumarin derivatives
a
,
*
b
c
d
Eman Y. Ahmed , Weam S. Elserwy , Mohamed F. El-Mansy , Aya M. Serry ,
e
e
e
e
Abdelrahman M. Salem , Andrew M. Abdou , Basel A. Abdelrahman , Kenzi H. Elsayed ,
Moaaz R. Abd Elaziz
e
a
Chemistry of Natural Compounds Department, Pharmaceutical and Drug Industries Research Division, National Research Centre. Dokki, Cairo, Egypt
b
Chemistry of Natural and Microbial Products Department, Pharmaceutical and Drug Industries Research Division, National Research Centre. Dokki, Cairo, Egypt
c
Organometallic and Organometalloid Chemistry Department, Chemical Industries Division, National Research Centre. Dokki, Cairo, Egypt
d
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Modern University for Technology and Information, Cairo, Egypt
e
Faculty of Biotechnology, October University for Modern Sciences and Arts, Cairo, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
The present work represents the design and synthesis of some azaheterocyclic coumarin derivatives which are
evaluated as anti-lung cancer agents. Ten out of the twenty azaheterocyclic compounds showed superior activity
than the standard drug staurosporine against non-small cell lung cancer (A549). Representing the four different
azaheterocyclic series, compounds 4a, 5d, 6e, and 7d, which demonstrated IC50s of 2.38, 2.39, 1.05 and 3.98 µM,
respectively, each exhibiting the best cytotoxicity in its group, were selected for further assessment of their
toxicity on normal lung cells (WI-38). Compound 4a was selected for further investigations because it remarkably
revealed less cytotoxicity (IC50 = 53.76 µM) than 7d (IC50 = 19.95 µM) on (WI-38) compared to staurosporine
Azaheterocyclic coumarin derivatives
Lung cancer
Angiogenesis
Cell cycle inhibition
Apoptosis
Molecular modeling
Pharmacokinetics
(
IC50 = 24.41 µM). 4a was assessed for its ability to inhibit the angiokinases VEGFR-2, PDGFR, FGFR and the
growth factor EGFR, remarkably it showed better VEGFR-2, PDGFR, FGFR inhibition than the reference drugs
used and exhibited as well noticeable EGFR inhibition. Going further, 4a was capable of arresting the cell cycle at
pre-G1 phase and S phase and inducing apoptosis. Moreover, the capability of the target 4a to interact with the
key amino acids of VEGFR-2 binding site was detected by molecular docking. Finally, the in silico physico-
chemical properties of 4a were studied.
Lung cancer is a malignant tumor with distinct clinical and patho-
logical features that can be classified into small cell lung cancer (SCLC)
and non-small cell lung cancer (NSCLC) according to histopathology.
NSCLC is the most common type of lung cancer and affects about 8 of
every 10 people with lung cancer. The most common subtypes of NSCLC
are adenocarcinoma, squamous cell carcinoma and large cell carcinoma.
Most NSCLC are adenocarcinomas that are often formed in the alveoli.
factor (VEGF), platelet-derived growth factor (PDGF) and fibroblast
growth factor (FGF) which are crucial to blood vessel formation,
integrity and maintenance. Tumor cells often overexpress the three
angiokinases VEGFR, PDGFR and FGFR altogether so that they can
escape any VEGF inhibition by up regulating the other two proangio-
genic factors PDGF and FGF. 5 This finding highlights the importance
of developing multi-targeted tyrosine kinase inhibitors that have the
ability to block several key receptors. Nintedanib is an orally available,
potent and selective triple angiokinase inhibitor that blocks the activ-
ities of VEGFR, PDGFR and FGFR and was approved as a second-line
treatment of NSCLC. In addition, nintedanib has received approval for
the treatment of idiopathic lung fibrosis. The structure started as an
indolinone lead compound that was modified to an indolinone
–10
1
,2
Tumor cells require a blood supply to grow, without blood vessels
small tumors may remain dormant for months or even years. However,
tumors can develop their own blood supply by a process known as
angiogenesis. 3 Angiogenesis is mainly driven by three angiokinase
pathways, via three signaling molecules: vascular endothelial growth
,4
*
Corresponding author.
E-mail address: eyam_ha@yahoo.com (E.Y. Ahmed).
https://doi.org/10.1016/j.bmcl.2021.128258
Received 17 April 2021; Received in revised form 1 July 2021; Accepted 5 July 2021
Available online 9 July 2021
0
960-894X/© 2021 Elsevier Ltd. All rights reserved.

E.Y. Ahmed et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128258
Fig. 1. Optimizing nintedanib as a triple angiokinase inhibitor.
Fig. 2. The rationale of designing new coumarin lead compounds as multi-targeted tyrosine kinase inhibitors.
derivative BIBF1000 which was finally optimized to become nintedanib
and tumor cell growth are expected to be even more effective anticancer
therapies than agents that act on only one of these pathways. Neratinib,
a quinoline derivative, is a second generation EGFR tyrosine kinase in-
hibitor that is used in treating patients of non-small cell lung carci-
nomas, Fig. 2. 13
BIBF1120), Fig. 1. 1 Comparing nintedanib to the promising derivative
1
(
in this work, the molecular docking study revealed a superimposition
pattern and significant binding interactions that allowed for the sub-
stitution of the indolinone ring in the parent compounds with the
coumarin ring and incorporating different azaheterocyclic scaffolds that
are linked to the substituted benzene rings. Moreover, the pharmaco-
kinetic study revealed that the promising derivative showed no violation
of Lipinski’s rule regarding the molecular weight, hydrogen bonds and
lipophilicity.
As our ongoing interest in natural compounds as kinase inhibitors,
1
4–17
we herein investigate the ability of coumarin as a privileged
bioactive naturally occurring scaffold, 1
8,19
to substitute the indolinone
and quinoline scaffolds in a search for new potential multi-targeted ki-
nase inhibitors, Fig. 2. The effect of incorporating pyridine, oxopyr-
imidine, thiopyrimidine and pyrazole azaheterocyclic rings which were
reported to exhibit their anticancer activity through tyrosine kinases
On the other hand, some tumors also require activation of certain
growth factors receptors such as epidermal growth factor receptor
EGFR) in order to grow and spread throughout the body. 12 Therefore
inhibition,
that are overexpressed in NSCLC, 2
14,15,20–23
on targeting VEGFR-2, PDGFR, FGFR and EGFR
(
4–27
multi-targeted tyrosine kinase inhibitors that block both angiogenesis
was delineated as well.
2

E.Y. Ahmed et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128258
Scheme 1. Reagents and reaction conditions : (a) phosphorous oxycholoride ,acetic acid, reflux 30 m; (b) different substituted benzaldehyde, absolute ethanol,
stirring, rt, 24 h; (c) Amonium acetate, malononitrile, absolute ethanol, reflux 2-4 h; (d) urea,absolute ethanol, 20% potassium hydroxide , reflux 6 h; (e) thiourea,
absolute ethanol, 20% potassium hydroxide , reflux 6h; (f) EtOH, hydrazine hydrate, reflux 6 h.
3
-Acetyl-4-hydroxycoumarin 2, prepared from 4-hydroxycoumarin 1
potassium hydroxide. Finally, the pyrazoline compounds 7a-e were
prepared by the reaction of the chalcones 3a-e with hydrazine hydrate
under reflux.
as reported, 28 was used as a starting material for the synthesis of the
target compounds in Scheme 1. Claisen-Schmidt reaction of 3-Acetyl-4-
hydroxycoumarin 2 in aqueous potassium hydroxide and ethanol at
room temperature with different substituted benzaldehydes, namely, 3-
anisaldehyde, veratraldehyde, vanillin, 4-fluor-benzaldehyde and 2-
choloro-6-fluoro benzaldehyde, led to the formation of the corre-
sponding chalcones 3a-e, which were utilized to prepare the azaheter-
The antiproliferative effect of the synthesized azaheterocyclic com-
pounds against A549 non-small cell lung cancer was detected by the
MTT assay.2
9,30
50% of the twenty target azaheterocyclic coumarin
compounds showed higher cytotoxicity against A549 cell line with IC50
values ranging from 1.05 to 7.58 M compared to the reference drug
μ
ocyclic compounds. On refluxing the
α
, β unsaturated keto derivatives
staurosporine (IC50 = 9.50 µM), the most active derivatives in each se-
ries were compounds 4a (IC50 = 2.38 µM), 5d (IC50 = 2.39 µM), 6e (IC50
= 3.98 µM) and 7d (IC50 = 1.05 µM), Table 1.
3
a-e with malononitrile in the presence of anhydrous ammonium ace-
tate and using ethanol as a solvent, the target azaheterocyclic com-
pounds 4a-e were obtained. The reaction of 3a-e with urea and/or
thiourea yielded the pyrimidine derivatives 5a-e and 6a-e, respectively,
the reaction was carried out in ethanol and in the presence of aqueous
The four most active derivatives in each azaheterocyclic series were
tested on normal lung cell line WI-38. 4a, 5d and 6e expressed high IC50
values and lower cytotoxicity towards WI-38 compared to A549 lung
3

E.Y. Ahmed et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128258
Table 1
cancer cell lines. The pyridine carbonitrile 4a displayed the highest IC50.
The cytotoxic IC50 values of the compounds according to the MTT assay on A549
and WI-38.
Since compound 4a exhibited an excellent cytotoxic activity against
lung cancer cell line A549, and at the same time it showed high safety
profile towards normal lung cell line, it was selected for further
screening on different kinases.
Compound
A549IC50 (µM) ± SD
WI-38IC50 (µM) ± SD
4
4
4
4
4
5
5
5
5
5
6
6
6
6
6
7
7
7
7
7
a
b
c
2.38 ± 0.10
7.38 ± 0.38
38.50 ± 2.00
22.30 ± 1.16
46.30 ± 2.40
5.55 ± 0.29
21.70 ± 1.13
138.00 ± 7.15
2.39 ± 0.12
5.10 ± 0.26
10.20 ± 0.53
62.60 ± 3.25
36.10 ± 1.87
21.10 ± 1.10
3.98 ± 0.21
5.91±0.40
53.76 ± 3.25
The results of VEGFR-2 inhibition assay revealed the superior
inhibitory activity of compound 4a (IC50 = 0.042 µM) compared to the
reference vandetanib (IC50 = 0.069 µM). Similarly, the PDGFR inhibi-
tion activity of derivative 4a (IC50 = 0.02 µM) was more pronounced
relative to saturosporine as the reference compound which showed an
IC50 of 0.055 µM. Moreover, the selected derivative exhibited strong
FGFR inhibitory activity; showing IC50 value of 0.021 µM versus 0.027
µM for the reference compound Staurosporine. On the other hand, the
EGFR inhibitory activity of 4a was noticeable, the derivative showed
IC50 value of sub-micromolar level (0.033 µM) versus 0.015 µM for the
reference compound lapatinib.
ND
ND
d
e
a
b
c
ND
ND
ND
ND
ND
d
e
a
b
c
37.82 ± 2.29
ND
ND
ND
ND
d
e
a
b
c
ND
30.47 ± 1.84
ND
The IC50 values of the tested derivative on VEGFR-2, PDGFR-β and
FGFR, as markers of angiogensis, were consistent with the promising
cytotoxic result of 4a.
7.58±0.10
ND
8.40±0.50
ND
d
e
1.05±0.10
19.95±1.21
ND
A549 cells were subjected to 4a for 24 h at its IC50 value to inves-
tigate its impact on the cell cycle profile and apoptosis induction.
Exposure of A549 cells to 4a influenced the normal cell cycle sequence
by increasing the pre-G1 phase cells percentage by 24 folds and the
percentage of the cells at S phase by 1.5 folds, compared to the control.
Cells accumulation in pre-G1 phase, which was affirmed by the existence
of sub-G1 peaks in the cell cycle profile analysis, is likely caused by the
23.50±1.40
9.50±0.49
Staurosporine
24.41±1.84
ND*: Not Determined.
Fig. 3. Cellular mechanism of action of compound 4a. (A, B) show Cell cycle analysis of A549 after incubation with compound 4a for 24 h. DMSO diluent was used as
a control. (C, D) Induction of apoptosis after cells were exposed to 4a for 24 h.
4

E.Y. Ahmed et al.
Bioorganic & Medicinal Chemistry Letters 48 (2021) 128258
activity against A549 cancer cell line, the least cytoxicity against normal
lung cell line (WI-38), better VEGFR-2, PDGFR, FGFR inhibition than the
reference drugs and exhibited as well noticeable EGFR inhibition.
Remarkably, 4a arrested the cell cycle at pre-G1 phase and S phase and
induced apoptosis. Moreover, compound 4a was able to interact with
VEGFR-2 key amino acids exhibiting a docking score of ꢀ 7.0594 kcal/
mol kcal/mol. Finally, the in silico physicochemical properties elect 4a
as a potential drug-like lead compound.
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.
Acknowledgment
The authors extend their deep thanks and appreciation to Dr.
Mohamed El-Ashrey, Faculty of Pharmacy - Cairo University, for per-
forming the molecular docking study.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.bmcl.2021.128258.
Fig. 4. 2D diagram of compound 4a interactions within VEGFR2 binding site.
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