Pyridine-derived VEGFR-2 inhibitors: Rational design, synthesis, anticancer evaluations, in silico ADMET profile, and molecular docking

Article abstract of DOI:10.1002/ardp.202100085

Novel pyridine-derived compounds (5–19) were designed and?synthesized, and their anticancer activities were evaluated against HepG2 and MCF-7 cells, targeting the VEGFR-2 enzyme. Compounds 10, 9, 8, and 15 were found to be the most potent derivatives against the two cancer cell lines,?HepG2 and MCF-7, respectively, with IC₅₀ = 4.25 and 6.08 μM, 4.68 and 11.06 μM, 4.34 and 10.29 μM, and 6.37 and 12.83 μM. Compound 10 displayed higher activity against HepG2 cells than sorafenib (IC₅₀ = 9.18 and 5.47 μM, respectively) and doxorubicin (IC₅₀ = 7.94 and 8.07 μM, respectively). It also showed higher activity than doxorubicin against MCF-7 cells, but lower activity than sorafenib. Compounds 9, 8, and 15 displayed higher activities than sorafenib and doxorubicin against HepG2 cells but exhibited lower activities against MCF-7 cells. Compound 10 potently inhibited VEGFR-2 at an IC₅₀ value of 0.12 μM, which is nearly equipotent to sorafenib (IC₅₀ = 0.10 μM). Compounds 8 and 9 exhibited very good activity with the same IC₅₀ value of 0.13 μM. The six most potent derivatives, 6, 9, 8, 10, 15, and 18, were tested for their cytotoxicity against normal Vero cells. Compounds 6, 8, 9, 10, 15, and 18 are, respectively, 1.13, 3.74, 4.18, 3.64, 2.81, and 2.00 times more toxic to HepG2 and 2.06, 1.58, 1.76, 2.54, 1.40, and 2.69 times more toxic to MCF-7 breast cancer cells than in normal Vero cells.

Full text of DOI:10.1002/ardp.202100085

Received: 3 March 2021
Revised: 2 April 2021
Accepted: 6 April 2021
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DOI: 10.1002/ardp.202100085
F U L L P A P E R
Pyridine‐derived VEGFR‐2 inhibitors: Rational design,
synthesis, anticancer evaluations, in silico ADMET profile, and
molecular docking
Nashwa M. Saleh1 | Adel A.‐H. Abdel‐Rahman2
| Asmaa M. Omar2
|
Mohamed M. Khalifa3
| Khaled El‐Adl3,4
1Department of Chemistry, Faculty of Science,
Al‐Azhar University (Girls Branch), Cairo,
Egypt
Abstract
Novel pyridine‐derived compounds (5–19) were designed and synthesized, and their
anticancer activities were evaluated against HepG2 and MCF‐7 cells, targeting the
VEGFR‐2 enzyme. Compounds 10, 9, 8, and 15 were found to be the most potent
derivatives against the two cancer cell lines, HepG2 and MCF‐7, respectively, with
IC50 = 4.25 and 6.08 µM, 4.68 and 11.06 µM, 4.34 and 10.29 µM, and 6.37 and
12.83 µM. Compound 10 displayed higher activity against HepG2 cells than sor-
afenib (IC50 = 9.18 and 5.47 µM, respectively) and doxorubicin (IC50 = 7.94 and
8.07 µM, respectively). It also showed higher activity than doxorubicin against MCF‐
7 cells, but lower activity than sorafenib. Compounds 9, 8, and 15 displayed higher
activities than sorafenib and doxorubicin against HepG2 cells but exhibited lower
activities against MCF‐7 cells. Compound 10 potently inhibited VEGFR‐2 at an IC50
value of 0.12 µM, which is nearly equipotent to sorafenib (IC50 = 0.10 µM). Com-
pounds 8 and 9 exhibited very good activity with the same IC50 value of 0.13 µM.
The six most potent derivatives, 6, 9, 8, 10, 15, and 18, were tested for their
cytotoxicity against normal Vero cells. Compounds 6, 8, 9, 10, 15, and 18 are,
respectively, 1.13, 3.74, 4.18, 3.64, 2.81, and 2.00 times more toxic to HepG2 and
2.06, 1.58, 1.76, 2.54, 1.40, and 2.69 times more toxic to MCF‐7 breast cancer cells
than in normal Vero cells.
2Department of Chemistry, Faculty of Science,
Menoufia University, Shebin El‐Koam, Egypt
3Department of Pharmaceutical Medicinal
Chemistry and Drug Design, Faculty of
Pharmacy (Boys), Al‐Azhar University, Cairo,
Egypt
4Department of Pharmaceutical Chemistry,
Faculty of Pharmacy, Heliopolis University for
Sustainable Development, Cairo, Egypt
Correspondence
Khaled El‐Adl, Department of Pharmaceutical
Medicinal Chemistry and Drug Design,
Faculty of Pharmacy (Boys), Al‐Azhar
University, Nasr City 11884, Cairo, Egypt.
Email: eladlkhaled74@yahoo.com,
eladlkhaled74@azhar.edu.eg, and
khaled.eladl@hu.edu.eg
Nashwa M. Saleh, Department of Chemistry,
Faculty of Science, Al‐Azhar University (Girls
Branch), Youssef Abbas Str., 6th District,
Nassr City, PO Box 11754, Cairo 11765,
Egypt.
Email: drnashwamostafa@azhar.edu.eg and
drnashwa78@yahoo.com
K E Y W O R D S
2‐cyanoacetohydrazone, anticancer agents, molecular docking, pyridines, VEGFR‐2 inhibitors
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1
INTRODUCTION
use in cancer treatment (https://www.cancer.gov/about-cancer/
treatment/types/targeted-therapies/targeted-therapies-fact-sheet; http
Targeted cancer remedies have been developed in an attempt to avoid
the side effects of standard chemotherapy. Inhibitors of signal trans-
duction, angiogenesis inhibitors, apoptosis inducers, hormone therapies,
modulators of gene expression, immunotherapies, and toxin delivery
molecules are various targeted therapies that have been approved for
s://emedicine.medscape.com/article/1372666-overview). Protein tyr-
osine kinases inhibitors (PTKIs) are among the first discovered and ap-
proved targeted drug therapies. Under both normal and pathological cell
conditions, protein tyrosine kinases (PTKs) are well‐established key
players in controlling most cellular processes, including metabolism,
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progression of the cell cycle, transcription, cytoskeletal rearrangement,
cell movement, differentiation, and apoptosis.[1–3] There are over 518
identified human protein kinases up‐to‐date encoded within the human
genome, thus representing approximately 1.7% of all the human
genes.[1,3] Among these kinases is the vascular endothelial growth factor
receptor‐2 (VEGFR‐2), which is well recognized to be associated with the
progression and development of many types of cancers and angiogenesis.
VEGF‐2 is a key growth factor in tumor angiogenesis. A conformational
change in VEGFR‐2 was induced through the binding of VEGF to VEGFR‐
2, followed by receptor dimerization and phosphorylation of tyrosine
residues. VEGFR‐2 transmits its angiogenic signal via cell surface re-
ceptors located on the host vascular endothelial cells, which have in-
tracellular TK activity. VEGF signaling through VEGFR‐2 has been shown
to play a key role in tumor angiogenesis regulation.[4,5] Expression of
VEGF is enhanced in several types of human tumors, and its expression
levels are associated with poor prognosis and clinical stage in solid tu-
mors patients.[5–8] Therefore, VEGF/VEGFR‐2 signaling is an attractive
therapeutic target in cancer treatment. Consequently, the biological ra-
tionale and the effectiveness of small‐molecule VEGFR‐2 inhibitors in
interfering with tumor‐induced signals have been well established.[9–12]
On the basis of the different reported VEGFR‐2 crystal structures,
VEGFR‐2 inhibitors can be classified into three main types: Type I in-
hibitors are able to block the active “DFG‐in” conformation of the re-
ceptor by occupying the adenosine triphosphate (ATP)‐binding region
forming a hydrogen bond with the hinge region amino acid Cys919. Type
II inhibitors occupy the ATP‐binding site and extend over the gate area
into the adjacent allosteric hydrophobic back pocket of the inactive
“DFG‐out” conformation. Type III inhibitors accommodate the allosteric
hydrophobic back pocket of VEGFR‐2 in the inactive “DFG‐out” con-
formation, blocking the receptor through hydrophobic interactions.[13]
Study of SAR and common pharmacophoric features shared by,
for example, sorafenib (I) and lenvatinib (II),[14] and various VEGFR‐2
inhibitors revealed that most VEGFR‐2 inhibitors shared four main
pharmacophoric features[15,16] (Figure 1), which are (1) a hinge re-
gion binding moiety “head,” which is a heterocycle that occupies the
adenine region in the ATP‐binding pocket with H‐bond donor and/or
acceptor capabilities to interact with Cys919 (colored red), (2) a
“linker,” which is a segment of three to four chemical bonds that
extends over the gatekeeper residue (colored green), (3) a hydrogen‐
bonding moiety that is required to achieve hydrogen bond interac-
tion with the Asp1046 in the conserved DFG motif and Glu885 of
the αC helix (colored purple), and (4) a “tail” segment typically con-
sisting of a hydrophobic moiety that occupies the allosteric hydro-
phobic back pocket created by the DFG‐out flip (colored blue).[16,17]
As shown in Figure 2, the pyridine skeleton had been widely
used in VEGFR inhibitors. Small‐molecule ATP‐competitive inhibitors
of VEGFR‐2 (Type I inhibitors) based on a pyridine nucleus have
demonstrated high activity as antitumor agents like apatinib (III),
motesanib (IV), and sorafenib (I), as displayed in Figure 2.[17–21] In
2020, AbdelHaleem et al.[22] designed a series of new pyridine
scaffolds that were biologically evaluated for their inhibitory activity
against VEGFR‐2. The most potent compound (V) (Figure 2) dis-
played very good anticancer activities against two prostate cancer
cell lines, namely PC3 and DU145, and two breast cancer cell lines,
namely MCF‐7 and MDA‐MB435, with IC50 values of 55, 8.5, 0.5, and
96 nM, respectively. It also inhibited VEGFR‐2 at an IC50 value of
0.19 nM.[22] Moreover, the bioisostere pyrimidine derivative com-
pound (VI) (Figure 2) displayed very good anticancer activities and
potently inhibited VEGFR‐2 at an IC50 value of 1.97 µM.[23]
The emergence of tumor resistance to the effect of currently
clinically used small‐molecule tyrosine kinases inhibitors (TKIs)
opens the door for the exploration of new chemotypes. The chemical
entities bearing a 3‐cyano‐4‐aryl‐pyridin‐2‐one moiety or its 2‐amino
bioisostere have received considerable attention owing to their
ability to elicit cytotoxic antiproliferative activities employing var-
ious mechanisms of actions specially kinase inhibition
mechanism.[22,24–26]
In view of the above‐mentioned findings and based on our
continuous efforts to develop new anticancer agents,[27–29] espe-
cially VEGFR‐2 inhibitors,[30–39] it is deemed of interest to initiate a
research work directed at the design and synthesis of a new series of
potential pyridine‐derived cytotoxic compounds. The design of these
FIGURE 1 Representation of type II vascular endothelial growth factor receptor‐2 (VEGFR‐2) inhibitors in the VEGFR‐2 active site

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FIGURE 2 Reported pyridine‐derived vascular endothelial growth factor receptor‐2 inhibitors and our derivatives
derivatives was directed by the idea of cross‐hybridization with the
pharmacophoric elements of the cytotoxic agents: sorafenib, lenva-
tinib, apatinib, motesanib, compound (V), and compound (VI)
(Figure 2). The pyridine bioisosteres, pyran and 1,2‐diazepine scaf-
folds, were introduced to yield derivatives of general structures 22
and 23 (Figure 2). These derivatives are designed as antiproliferative
agents with potential ATP‐competitive kinase inhibition (Type I in-
hibitors) where the pyridine ring will occupy the ATP‐binding region
forming a hydrogen bond with the hinge region amino acid Cys919.
The designed hydrophobic moieties extend to occupy different hy-
drophobic pockets to increase binding affinities toward the VEGFR‐2
receptor.
for their in vitro antiproliferative activities against two human
tumor cell lines, namely hepatocellular carcinoma (HCC) type
(HepG2) and breast cancer (Michigan Cancer Foundation‐7
[MCF‐7]).
VEGFR‐2 was reported to be substantially upregulated in HepG2
cells in a dose‐dependent manner with the stimulation of the hepatocyte
growth factor (HGF), which is involved in cell proliferation, invasion, and
angiogenesis of hepatocellular carcinoma (HCH).[40–42] Blockade of
VEGFR‐2 signaling revealed a marked inhibition on both the growth and
metastasis of HCC. Also, VEGFR‐2 was found to be crucial to cell sur-
vival and regulates endothelial differentiation in the breast cancer cells
(MCF‐7).[42,43] Overexpression of VEGFR‐2 receptors in breast cancer
cells has been documented to be a contributor in resistance of such
cancer type to the chemotherapeutic effect of tamoxifen.[44]
Multiple structural manipulations were performed to study
SAR implications of these transformations on the studied
pharmacological activities of these derivatives. The adopted mo-
lecular manipulations and design strategy involved varying sub-
stituents with different hydrophobic and electronic nature. In
general, the designed compounds were synthesized and evaluated
Moreover, the most potent compounds were tested for their in
vitro cytotoxicity against the normal Vero cells. The obtained results
prompted us to make further examinations to achieve deep insight
into the mechanism of action of the synthesized compounds.

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Molecular docking studies were carried out to assess the binding
interactions of the target compounds with VEGFR‐2 active sites.
Moreover, the most active cytotoxic compounds that showed pro-
mising IC50 values against the two cancer cell lines were subjected to
further investigation for their tyrosine kinase inhibitory activities
against VEGFR‐2.
and 11.95 ppm attributed to the CH2 and NH, respectively, and
singlet signal at 8.33 ppm corresponding to (CH═N) group. In ad-
dition, the 13C NMR spectrum showed characteristic signals at δ
24.85 for (CH2), 116.45 for (C≡N), and 159.53 and 192.18 ppm
assigned to (CH═N) and (C═O), respectively. The targeted com-
pound 1,6‐diamino‐4‐(2‐bromophenyl)‐2‐oxo‐1,2‐dihydropyridine‐
3,5‐dicarbonitrile 5 was synthesized by two pathways (Scheme 1).
The first pathway was through the formation of the key inter-
mediate Nʹ‐(2‐bromobenzylidene)‐2‐cyanoacetohydrazide 4 with
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2
RESULTS AND DISCUSSION
malononitrile in the presence of piperidine as
a catalyst.
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2.1
Rationale and structure‐based design
The second pathway consists of condensation of O‐
a
bromobenzaldehyde with the active methylene of malononitrile to
afford 2‐(2‐bromobenzylidene)malononitrile 2. An alcoholic solu-
tion of 2 and 3 was heated under reflux to obtain the targeted
compound 5 (Scheme 1). The proposed mechanism of formation of
1,6‐diamino‐pyridine‐3,5‐dicarbonitrile derivative (5) is illustrated
in Figure 5.[48,49]
Our pyridine derivatives have the essential pharmacophoric features
of a VEGFR‐2 inhibitor. The presence of pyridine hetero ring alone as
in compound 15 and/or the pyridine ring fused to triazole moietie as
in compound 10 were designed to replace the pyridine ring of the
reference ligand sorafenib. These moieties were designed to occupy
the ATP‐binding pocket of the VEGFR‐2 receptors. The pyridine
moiety in compound 15 occupied the hydrophobic ATP‐binding
pocket formed by Leu1035, Cys919, Phe918, Glu917, Val848,
Leu840, and Arg833 (Figure 3). The triazolo[1,5‐a]pyridine moiety in
compound 10 occupied the hydrophobic ATP‐binding pocket also
(Figure 4).
The structure of compound 5 was proved on the basis of analytical
and spectral data. Thus, the IR spectrum of compound 5 showed char-
acteristic absorption bands at 3407, 3313, and 3210 cm−1 due to two
amino groups (2 NH2), 2207 and 1676 cm−1 for two cyano groups
(2 C≡N) and carbonyl group (C═O), respectively. Also, the 1H NMR
spectrum of compound 5 revealed the presence of two singlet signals
that were exchangeable with D2O at δ 5.70 and 8.60 ppm due to the
N–NH2 and C–NH2 protons, respectively. As a result of the difference in
nucleophilicity, hydrazide β‐nitrogen (N–NH2) reacts faster with the
electron‐deficient carbon than the second amino group (C–NH2). In ad-
dition, the 13C NMR spectrum showed characteristic signals at 75.40 and
114.59 ppm for C3 and C5 of pyridine, 116.01 and 116.41 ppm for two
cyano groups, 159.48 and 166.93 ppm assigned to (C–NH2) and (C═O),
respectively. Compound 5 was further indicated from its mass spectrum
that showed the molecular ion peak at m/z 330, which agreed with the
molecular formula C13H8BrN5O and supported the identity of the new
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2.2
Chemistry
The synthetic strategies followed to achieve the target compounds
are seen in Schemes 1–4. Synthesis of Schiff's base 4 as a key
intermediate[45–47] in excellent yield was carried out by con-
densation of equimolar amounts of O‐bromobenzaldehyde 1 with
2‐cyanoacetohydrazide 3[45–47] in absolute ethanol with few drops
of glacial acetic acid as a catalyst (Scheme 1). The infrared (IR)
spectrum of compound 4 revealed a characteristic absorption
band at 3190 cm−1 (NH), 2264 cm−1 (C≡N), and 1675 cm−1 (C═O).
Also, the 1H NMR (nuclear magnetic resonance) spectrum of
compound 4 showed the presence of two singlet signals at 4.22
compound
4‐(2‐bromophenyl)‐2‐oxo‐1,2,3,4‐tetrahydropyridine‐3,5‐
dicarbonitrile (5). Conformation of the structure of 6 is carried out by
careful data of its spectral analysis. Its 1H NMR spectrum showed
characteristic signals at δ 3.03 and 3.06 ppm for C4–H and C3–H of the
FIGURE 3 Superimposition of compound
15 and sorafenib inside the binding pocket of
vascular endothelial growth factor receptor‐2

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FIGURE 4 Superimposition of compound
10 and sorafenib inside the binding pocket of
vascular endothelial growth factor receptor‐2
SCHEME 1 Synthetic route for the formation of 1,6‐diaminopyridones 5 and 6
pyridine ring. O‐Amino heterocyclic carboxamides are a good nucleophilic
center to produce fused nitrogen heterocyclic rings.[50,51]
acetic acid and one molecule of water (Scheme 2). The IR and 1H
NMR spectra of compound 8 confirmed the absence of the two NH2
groups. The IR spectrum showed characteristic absorption bands at
1690 cm−1 for (C═O pyridone) and 2126 cm−1 for (2 C≡N). The 1H
NMR spectrum revealed the presence of methyl group at δ 1.29 (CH3
triazole) and at 23.05 ppm in 13C NMR spectra.
Cyclization of 5 with ethyl chloroformate was carried out to
yield
7‐(2‐bromophenyl)‐5‐oxo‐3,5‐dihydro‐[1,2,4]triazolo[1,5‐a]
pyridine‐6,8‐dicarbonitrile 7. New triazolopyridone is formed by
the reaction of the starting compound 5 with some monoelec-
trophilic reagents such as acetic anhydride. Heterocyclization of
Reaction of product
5 with benzaldehyde afforded 7‐(2‐
compound
5
with acetic anhydride under reflux yielded 7‐(2‐
bromophenyl)‐5‐oxo‐2‐phenyl‐1,2,3,5‐tetrahydro‐[1,2,4]triazolo[1,5‐
a]pyridine‐6,8‐dicarbonitrile (9) (Scheme 2). 1H NMR spectral
data for compound 9 showed singlet signal at δ 4.21 ppm, assigned to
bromophenyl)‐2‐methyl‐5‐oxo‐3,5‐dihydro‐[1,2,4]triazolo[1,5‐a]
pyridine‐6,8‐dicarbonitrile 8 (Scheme 2). Formation of compound 8
occurs via a nucleophilic attack of the exocyclic amino group of 5 on
the keto group of 1,3‐dicarbonyls, followed by triazole ring closure
through intramolecular cyclization by the loss of one molecule of
CH‐triazole, in addition to two singlet signals at
δ 9.26 and
10.71 ppm, indicating the presence of two NH groups. The [1,2,4]
triazolo[1,5‐a]pyridine derivative 10 was obtained by stirring of 9

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SCHEME 2 Synthetic route for the formation of compounds 5–11
with trifluoroacetic acid, as shown in (Scheme 2). N‐Deamination of
the diaminopyridinone derivative 5 was carried out under mild
conditions. 1H NMR spectra of 10 revealed the absence of the me-
thylene proton signals and presence of one deuterium oxide ex-
changeable signal at δ 8.46 ppm for NH. 13C NMR spectrum of these
compounds exhibited two signals for cyano group at 116.26 and
116.64 ppm, as well as Ar‐C at a range of 128.78–148.52 ppm; fi-
nally, C═O appeared at 166.36 ppm.
2216 cm−1 for cyano groups (C≡N), and presence of new absorption
bands at 1651 cm−1 due to a carbonyl group (C═O). Also, the 1H NMR
spectrum of compound 13 revealed triplet and quarter signals at 1.19
and 4.30 ppm attributed to the ester proton and the presence of two
singlet signals that were exchangeable with D2O at 5.70 and 9.05 ppm
due to the N–NH2 and C–NH2 protons, respectively. 13C NMR spectrum
was characterized by signals at 159.20 and 165.82 ppm corresponding to
two carbonyl carbon atoms, signals at 116.39 ppm assigned to (C–CN),
and 120.75 ppm assigned to the cyano group. Treating 4 with p‐
methoxybenzylidene malononitrile in the presence of triethylamine gave
6‐amino‐1‐[(2‐bromobenzylidene)amino]‐4‐(4‐methoxyphenyl)‐2‐
oxo‐1,2,3,4‐tetrahydropyridine‐3,5‐dicarbonitrile (15) (Scheme 3).
The reaction may proceed as nucleophilic addition to the active
double bond, followed by cycloaddition of the amino group of
(NH–NH2) with concomitant dehydration to produce the target
compound 15, which appears more likely than compound 16 on
the basis of elemental analysis and spectral data. IR spectrum of
15 revealed characteristic absorption bands at 3347 and
3202 cm−1 due to bands of amino groups (NH2), 2215 cm−1 for
cyano groups (C≡N), and presence of new absorption bands at
1699 cm−1 assignable to a carbonyl group (C═O). The 1H NMR
spectrum of compound 15 showed singlet signals at
Thus, upon treating 5 with sodium nitrile in aqueous acetic acid,
the expected product 11 was obtained, rather than the unexpected
tetrazolopyridine derivatives 12 (Scheme 2). Elemental analysis and
spectral data confirmed the proposed structure of 11. Its IR spec-
trum showed stretching vibration bands for NH/NH2 around 3300
and 3406 cm−1, cyano group at 2210 cm−1, and C═O at 1691 cm−1
,
respectively.
However, reaction of 4 with ethyl cyanoacetate in the presence of
triethylamine afforded ethyl‐1,2‐diamino‐4‐(2‐bromophenyl)‐5‐cyano‐6‐
oxo‐1,6‐dihydropyridine‐3‐carboxylate 13 rather than 1‐amino‐4‐argio‐6‐
hydroxy‐2‐oxo‐1,2‐dihydropyridine‐3,5‐dicarbonitrile 14 (Scheme 3). The
structure of compound 13 was proved on the basis of analytical and
spectral data. The IR spectrum of 13 revealed a characteristic absorption
band at 3399 cm−1 and 3290 cm−1 due to bands of amino groups (2 NH2),

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SCHEME 3 Synthetic route for the formation of the expected products 13 and 15
SCHEME 4 Cyclization of 4 with different nucleophilic reagents to obtain compounds 17–19

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FIGURE 5 The proposed mechanism of
formation of 1,6‐diamino‐pyridine‐3,
5‐dicarbonitrile derivative 5
3.22 ppm attributed to methoxy protons and the presence of
singlet signal that was exchangeable with D2O at δ 5.62 ppm due
to the presence of amino group, providing good evidence for the
suggested open chain structure 15 instead of the cyclic [1,2,4]
triazolo structure 16. Furthermore, its 13C NMR spectrum ex-
hibited three signals at 116.16, 117.04, and 157.09 ppm due to
cyano and carbonyl groups, respectively, in addition to signals
between 114.17 and 133.87 ppm corresponding to aromatic
carbons.
3192 and 3404 cm−1 and the C≡N group at 2213 cm−1. The 1H
NMR spectrum of compound 19 showed the mixture of keto‐enol
tautomers.
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2.3
Docking studies
Molsoft software was used in all modeling experiments. Each ex-
periment used VEGFR‐2 downloaded from the Brookhaven Protein
Data Bank (PDB ID 3B8Q).[22]
In turn, quinoline derivative 17 was obtained upon treating 4
with anthranilic acid in refluxing dioxane and triethylamine
(Scheme 4). The chemical structure of 17 was established from its
spectral data that showed bands characteristic for C═N, NH, and
C═O groups (Scheme 4). Cyclocondensation of acetohydrazone
(4) with salicylaldehyde in dioxane and triethylamine as a base
achieved the chromene derivative 18 (Scheme 4). The IR spectra
provided the presence of sharp stretching absorption bands
characteristic for NH, CO, and C═N group frequencies and the
absence of C≡N absorption and carbonyl band for δ‐lactone,
which confirm the imino coumarin structure. IR spectrum of
compound 18 exhibited the characteristic absorption bands as
follows: 3199 and 1614 cm−1 for NH, and C═N, respectively. The
1H NMR spectrum of compound 18 shed further light on the
assigned structure as it displayed the characteristic signals for
NH, CH═N, and aromatic protons. The 1H NMR spectrum pro-
vided two exchangeable signals due to NH protons. The higher δ
values of NHCO signal at 11.95 ppm and singlet signals for imino
═NH proton at 8.94 ppm were observed. The formation of
chromenone derivative 18 was attributed to the initial Knoeve-
nagel condensation of the active methylene of compound 4 with
aldehydic carbonyl group of salicylaldehyde, followed by an in-
tramolecular cyclization via nucleophilic addition of the phenolic
OH group to the cyano function to furnish the target compound
The binding site of the VEGFR‐2 receptor reveals a large space
bounded by a membrane‐binding domain, which serves as an entry
channel for the substrate to the active site (Figure 6). All studied
ligands have a similar position and orientation inside the recognized
active site. Most of our derivatives had a good binding affinity to-
ward the VEGFR‐2 receptor, which is explained through the obtained
results of free energy of binding (ΔG) (Table 1).
The proposed binding mode of sorafenib revealed an
affinity value of −84.12 kcal/mol and four H‐bonds. The N‐
methylpicolinamide moiety was stabilized by the formation of two
H‐bonds with the essential amino acid Cys919 where the pyridine
N atom formed one H‐bond with the NH of Cys919 (2.94 Å),
whereas its NH group formed one H‐bond with the carbonyl of
Cys919 (2.07 Å). The urea linker formed one H‐bond with the key
amino acid Glu885 (2.40 Å) through its NH group and one H‐bond
with Asp1046 (2.08 Å) through its carbonyl group. The N‐
methylpicolinamide moiety occupied the hydrophobic ATP‐binding
pocket formed by Leu1035, Lys920, Cys919, Phe918, Glu917,
Val848, and Leu840. Moreover, the central phenyl ring occupied
the hydrophobic pocket formed by Cys1045, Leu1035, Thr916,
Lys868, and Val848. Furthermore, the hydrophobic 3‐
trifluromethyl‐4‐chlorophenyl moiety attached to the urea linker
occupied the hydrophobic pocket formed by Asp1046, Cys1045,
His1026, Ile892, Ile888, and Glu885 (Figure 7).
(Scheme 4). When
4
was allowed to react with 2‐
cyanoacetohydrazide, it gave 1,2‐diazepine derivatives 19
(Scheme 4). The assignment of the structure of compound 19 was
inferred from their spectroscopic data. Thus, the IR spectrum
displayed the stretching absorption bands for the NH2 group at
The proposed binding mode of compound 10 is virtually the
same as that of sorafenib. It revealed an affinity value of −84.08 kcal/
mol and three H‐bonds. The triazolo[1,5‐a]pyridine moiety was sta-
bilized by the formation of two H‐bonds with Cys919. The NH at

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FIGURE 6 Superimposition of some
docked compounds inside the binding pocket
of vascular endothelial growth factor
receptor‐2
TABLE 1 The calculated free energy of binding (ΔG in kcal/
mole) for the ligands
pyridine moiety occupied the hydrophobic ATP‐binding pocket
formed by Cys1045, Leu1035, Lys920, Cys919, Phe918, Glu917,
Val848, and Leu840. Furthermore, the 2‐bromophenyl ring attached
at position‐7 occupied the hydrophobic groove formed by Asp1046,
Cys1045, His1026, Thr916, Lys868, and Val848 (Figure 8). These
interactions of compound 10 may explain its high anticancer activity.
The proposed binding mode of compound 8 is virtually the
same as that of 10. It revealed an affinity value of −83.72 kcal/
mol and three H‐bonds. The triazolo[1,5‐a]pyridine moiety was
stabilized by the formation of two H‐bonds with Cys919. The NH
at position‐3 formed one H‐bond with the carbonyl of Cys919
with a distance of 2.31 Å, whereas the carbonyl group at position‐
5 formed another H‐bond with the NH group of Cys919 (1.94 Å).
Moreover, the CN group at position‐6 formed the third H‐bond
with Thr916 (1.40 Å). The methyl group at position‐2 occupied
the hydrophobic groove formed by Lys920, Phe918, and Leu840.
The triazolo[1,5‐a]pyridine moiety occupied the hydrophobic
ATP‐binding pocket formed by Cys1045, Leu1035, Lys920,
Cys919, Phe918, Glu917, Val848, and Leu840. Furthermore, the
2‐bromophenyl ring attached at position‐7 occupied the
Compound
ΔG (kcal/mol)
−78.95
Compound
ΔG (kcal/mol)
−69.05
5
13
6
−77.31
15
−81.76
7
−69.56
17
−79.13
8
−83.72
18
−80.08
9
−81.89
19
−69.75
10
11
−84.08
Sorafenib
−86.12
−70.16
position‐3 formed one H‐bond with the carbonyl of Cys919 with a
distance of 2.37 Å, whereas the carbonyl group at position‐5 formed
another H‐bond with the NH group of Cys919 (2.08 Å). Moreover,
the CN group at position‐6 formed the third H‐bond with Thr916
(1.36 Å). The phenyl moiety at position‐2 occupied the hydrophobic
groove formed by Lys920, Phe918, and Leu840. The triazolo[1,5‐a]
FIGURE 7 Predicted binding mode for
sorafenib with vascular endothelial growth
factor receptor‐2. H‐bonded atoms are
indicated by dotted lines

10 of 18
SALEH ET AL.
|
FIGURE 8 Predicted binding mode for 10
with vascular endothelial growth factor
receptor‐2
|
In vitro cytotoxic activity
hydrophobic groove formed by Asp1046, Cys1045, His1026,
Thr916, Lys868, and Val848 (Figure 9). These interactions of
compound 8 may explain its high anticancer activity.
2.4
Antiproliferative activity of the newly synthesized derivatives
5–19 was examined against two human tumor cell lines, namely,
hepatocellular carcinoma (HepG2) and breast cancer (MCF‐7),
using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bro-
mide colorimetric assay, as described by El‐Helby et al.[52] Sor-
afenib and doxorubicin were incorporated in the experiments as
reference cytotoxic drugs. The results were expressed as growth
inhibitory concentration (IC50) values, and they are summarized in
Table 2. From the obtained results, it was explicated that most of
the prepared compounds displayed excellent‐to‐good growth in-
hibitory activity against the tested cancer cell lines. In particular,
compounds 10, 9, 8, and 15 were found to be the most potent
derivatives over all the tested compounds against the two HepG2
The proposed binding mode of compound 15 is virtually the
same as that of 10. It revealed an affinity value of −83.13 kcal/mol
and three H‐bonds. The pyridine ring was stabilized by the formation
of three H‐bonds. The NH2 at position‐6 formed two H‐bonds with
Thr916 (2.48 Å) and Glu917 (2.31 Å). Moreover, the CN group at
position‐5 formed the third H‐bond with Cys919 (1.50 Å). The 4‐
methoxyphenyl moiety at position‐3 occupied the hydrophobic
groove formed by Lys920, Cys919, Phe918, Leu840, and Lys838. The
pyridine moiety occupied the hydrophobic ATP‐binding pocket
formed by Leu1035, Cys919, Phe918, Glu917, Val848, Leu840, and
Arg833. Furthermore, the 2‐bromophenyl ring attached at position‐7
occupied the hydrophobic groove formed by Asp1046, Cys1045,
His1026, Thre916, Glu885, Lys868, and Val848 (Figure 10). These
interactions of compound 15 may explain its high anticancer activity.
and
MCF‐7
cancer
cell
lines
with
IC50 = 4.25
0.03, 6.08 0.06 µM, 4.68 0.06, 11.06 0.09 µM, 4.34 0.04,
FIGURE 9 Predicted binding mode for 8
with vascular endothelial growth factor
receptor‐2

SALEH ET AL.
11 of 18
|
FIGURE 10 Predicted binding mode for
15 with vascular endothelial growth factor
receptor‐2
10.29 0.09 µM, 6.37 0.06, and 12.83 0.13 µM, respectively.
Compound 10 exhibited higher activity against HepG2 than
sorafenib (IC50 = 9.18 0.6 and 5.47 0.3 µM, respectively) and
doxorubicin (IC50 = 7.94 0.6 and 8.07 0.8 µM, respectively).
It also exhibited higher activity than doxorubicin against
MCF‐7 cancer cell lines but lower than sorafenib. Compounds 9, 8,
and 15 exhibited higher activities than sorafenib and doxorubicin
against HepG2 but exhibited lower activities against MCF‐7
cancer cell lines.
With respect to the HepG2 hepatocellular carcinoma cell line,
compounds 18, 17, 5, 6, and 11 displayed very good anticancer ac-
tivities with IC50 = 11.79 0.13, 12.90 0.61, 14.29 0.13,
16.57 0.15, and 19.21 0.16 µM, respectively, whereas compounds
19, 7, and 13, with IC50 = 23.55 0.24, 28.20 0.25, and
30.33 0.29 µM, respectively, displayed good cytotoxicity.
Cytotoxicity evaluation against MCF‐7 cell line revealed that
compounds 18, 6, 5, 17, and 13 displayed very good anticancer
activities with IC50 = 8.77 0.07, 8.85 0.06, 11.37 0.11,
TABLE 2 In vitro cytotoxic activities of the newly synthesized compounds against HepG2, MCF‐7, and Vero cell lines, and VEGFR‐2 kinase
assay
IC50 (µM)
Compound
HepG2
MCF‐7
Vero cells
NT
VEGFR‐2
5
14.29 0.13
16.57 0.15
28.20 0.25
4.68 0.06
4.34 0.04
4.25 0.03
19.21 0.16
30.33 0.29
6.37 0.06
12.90 0.61
11.79 0.13
23.55 0.24
9.18 0.6
11.37 0.11
8.85 0.06
24.52 0.24
11.06 0.09
10.29 0.09
6.08 0.06
23.84 0.22
20.58 0.21
12.83 0.13
17.65 0.16
8.77 0.07
22.39 0.21
5.47 0.3
0.19 0.02
0.19 0.02
0.29 0.02
0.13 0.02
0.13 0.02
0.12 0.01
0.25 0.03
0.33 0.01
0.14 0.05
0.19 0.02
0.16 0.02
0.25 0.02
0.10 0.02
NT
6
18.22 0.16
NT
7
8
17.50 0.16
18.14 0.17
15.45 0.16
NT
9
10
11
13
NT
15
17.93 0.22
NT
17
18
23.59 0.12
NT
19
Sorafenib
Doxorubicin
NT
7.94 0.6
8.07 0.8
NT
Note: IC50 values are the mean SD of three separate experiments.
Abbreviations: HepG2, hepatocellular carcinoma (HCC) type; MCF‐7, Michigan Cancer Foundation‐7; NT, compounds not tested; VEGFR, vascular
endothelial growth factor receptor.

12 of 18
SALEH ET AL.
|
17.65 0.16, and 20.58 0.21 µM, respectively, whereas compounds
19, 11 and 7, with IC50 = 22.39 0.21, 23.84 0.22, and
24.52 0.24 µM, respectively, displayed good cytotoxicity.
Finally, the most potent six derivatives 6, 9, 8, 10, 15, and 18
were tested for their cytotoxicity against normal Vero cell lines. The
results revealed that the tested compounds have low toxicity against
Vero normal cells with IC50 values ranging from 15.45 to 23.59 μM.
The cytotoxicity of these compounds against the cancer cell lines
ranged from 4.25 to 16.57 μM. Compounds 6, 8, 9, 10, 15, and 18 are,
respectively, 1.13, 3.74, 4.18, 3.64, 2.81, and 2.00 times more toxic to
HePG2 cancer cell lines than to Vero normal cells. Also, compounds
6, 8, 9, 10, 15, and 18 are, respectively, 2.06, 1.58, 1.76, 2.54, 1.40,
and 2.69 times more toxic to breast cancer cell lines (MCF‐7) than
to Vero normal cells.
From the structure of the synthesized derivatives and the data
shown in Table 2, we can divide these tested compounds into three
groups. The first group is the pyridine‐derived compounds para‐
substituted with phenyl group as in 5, 6, 11, 13, and 15. The presence
of NH2 group attached to N‐1 of pyridine ring condensed with
2‐bromophenyl as in compound 15 exhibited higher activity than the
free NH2 as in 5 and 6 against HepG2, whereas in MCF‐7, it showed
lower activity than 5 and 6, respectively. The presence of amino
groups at position‐1 and 3,5‐dicyano groups as in compounds 5 and 6
displayed higher activity than only 3,5‐dicyano groups like 11 and
NH2, 3‐CN, and 5‐ethyl ester as in 13 against HepG2, respectively.
The 1,2‐dihydropyridine 5 exhibited higher activity than 1,2,3,4‐
tetrahydropyridine 6 against HepG2 cell lines, whereas compound 6
showed higher activity than 5, 15, 13, and 11 against MCF‐7,
respectively.
In the second group 7, 8, 9, and 10, the pyridine nucleus fused
with triazole one. The triazolopyridine substituted with a phenyl
group at position‐2 as in compounds 10 and 9 exhibited higher ac-
tivities than that substituted with methyl 8 and the unsubstituted
one, 7, against both HepG2 and MCF‐7 cell lines, respectively. The
1,2,3,5‐tetrahydro‐triazolopyridine 10 showed higher activities than
the 3,5‐dihydro‐triazolopyridine 9 against both HepG2 and MCF‐7
cell lines, respectively.
|
2.5
In vitro VEGFR‐2 kinase assay
Furthermore, all the synthesized compounds, 5–19, were evaluated
for their inhibitory activities against VEGFR‐2 by using an anti‐
phosphotyrosine antibody with the Alpha Screen system (Perki-
nElmer).[31,33] The results were reported as a 50% inhibition con-
centration value (IC50) calculated from the concentration–inhibition
response curve and summarized in Table 2. Sorafenib was used as a
positive control in this assay. The tested compounds displayed high‐
to‐medium inhibitory activity with IC50 values ranging from
0.12 0.01 to 0.29 0.02 µM. Among them, compound 10 was found
to be the most potent derivative that inhibited VEGFR‐2 at an IC50
value of 0.12 0.01 µM, which is nearly equipotent to sorafenib IC50
value (0.10 0.02 µM). Compounds 8 and 9 exhibited very good ac-
tivity with the same IC50 value of 0.13 0.02 µM. Also, compounds
15 and 18 exhibited very good activity with IC50 values of
0.14 0.05 and 0.16 0.02 µM. Also, compounds 6, 5, and 17 pos-
sessed very good VEGFR‐2 inhibition with the same IC50 value of
0.19 0.02 µM, which is more than half the activity of sorafenib.
Compounds 19 and 7 displayed good VEGFR‐2 inhibition with IC50
values of 0.25 0.02 and 0.29 0.02 µM, respectively. However,
compound 13 displayed moderate VEGFR‐2 inhibition with an IC50
value of 0.33 0.01 µM.
In the third group 17, 18, and 19, the pyridine nucleus was fused
to the benzene ring to form quinoline derivative 17. In compound 18,
the bioisostere pyran ring was fused to the benzene ring to form
chromene derivative. Moreover, ring expansion of the pyridine ring
occurred to form the bioisostere 1,2‐diazepine derivative 19. The
chromene derivative 18 exhibited higher activities than the quinoline
derivative 17 and 1,2‐diazepine derivative 19 against both HepG2
and MCF‐7 cell lines, respectively.
|
2.7
In silico absorption, distribution, metabolism,
excretion, and toxicity (ADMET) profile
In the present study, a computational study of the four most active
compounds (8, 9, 10, and 15) was conducted to determine the sur-
face area and other physicochemical properties according to the
directions of Lipinski's rule.[53] Lipinski suggested that the absorption
of a compound is more likely to be better if the molecule obeys at
least three out of four of the following rules: (i) HB donor groups ≤ 5;
(ii) HB acceptor groups ≤ 10; (iii) M. Wt < 500; (iv) logP < 5. In this
study, whereas the reference anticancer agent doxorubicin violates
three of Lipinski's rules, our derivatives 8, 9, 10, and 15 obey all
Lipinski's rules. All the highest active derivatives have a number of
hydrogen‐bonding acceptor groups 5 and/or 6 and only 1 and/or 2
hydrogen‐bonding donors. Also, molecular weights are less than 500
and logP less than 5 and all these values agree with Lipinski's rules.
Also, ADMET profiles of the newly synthesized derivatives were
preliminarily assessed to analyze their potentials to build up as good
medication candidates. Prediction of ADMET profiles was conducted
with the aid of pkCSM descriptors algorithm protocol.[54]
|
2.6
Structure–activity relationship (SAR)
The preliminary SAR study has focused on the effect of inhibition of
ATP‐binding hydrophobic pocket by pyridine nucleus. The pyridine‐
derived compounds occupied the ATP hydrophobic pocket and
formed H‐bonds with the essential amino acid residue Cys919.
However, different moieties with different lipophilicity and electro-
nic nature were introduced to study their effects on anticancer ac-
tivity. The presence of lipophilic 2‐bromophenyl moieties attached to
the pyridine ring increases the hydrophobic interactions with the
active site. The data obtained revealed that the tested compounds
displayed different levels of anticancer activities.

SALEH ET AL.
13 of 18
|
TABLE 3 ADMET profile of the four most active compounds and doxorubicin
Parameter
8
9
10
15
Doxorubicin
Molecular properties
Molecular weight
LogP
345.167
418.254
416.238
450.296
543.525
2.50388
3.68896
3.86246
3.25126
0.0013
Rotatable bonds
Acceptors
1
2
2
4
5
5
6
5
5
12
Donors
1
2
1
1
6
Surface area
132.923
162.948
161.616
175.970
222.081
Absorption
Water solubility
CaCO2 permeability
Intestinal abs. (human)
Skin permeability
P‐glycoprotein substrate
P‐glycoprotein I inhibitor
P‐glycoprotein II inhibitor
Distribution
−3.261
0.572
83.944
−2.77
Yes
−4.051
0.45
−3.276
0.477
91.333
−2.735
Yes
−5.284
0.717
88.569
−2.831
No
−2.915
0.457
62.372
−2.735
Yes
93.321
−2.746
Yes
No
Yes
No
Yes
No
No
Yes
Yes
Yes
No
VDss (human)
−0.346
0.143
−0.318
0.018
0.089
−1.67
−0.617
0.05
−0.046
0.0
1.647
Fraction unbound (human)
BBB permeability
CNS permeability
Metabolism
0.215
−1.117
−2.118
−1.047
−1.788
−0.793
−2.144
−1.379
−4.307
CYP2D6 substrate
CYP3A4 substrate
CYP1A2 inhibitor
CYP2C19 inhibitor
CYP2C9 inhibitor
CYP2D6 inhibitor
CYP3A4 inhibitor
Excretion
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
Yes
Yes
Yes
Yes
Yes
No
No
No
No
No
No
No
No
No
Yes
No
Yes
Yes
No
Yes
No
No
Yes
Yes
Yes
Total clearance
Renal OCT2 substrate
Toxicity
−0.049
−0.243
0.035
No
0.261
Yes
0.987
No
No
No
AMES toxicity
No
Yes
Yes
No
No
Max. tolerated dose (human)
hERG I inhibitor
hERG II inhibitor
0.048
No
0.05
No
0.506
No
−0.243
No
0.081
No
Yes
Yes
Yes
No
Yes
Oral rat acute toxicity (LD50
Oral rat chronic toxicity (LOAEL)
Hepatotoxicity
)
2.569
1.114
No
2.669
1.166
No
2.641
1.061
No
2.583
1.217
No
2.408
3.339
Yes
(Continues)

14 of 18
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|
TABLE 3 (Continued)
Parameter
8
9
10
15
Doxorubicin
No
Skin sensitization
Tetrahymena pyriformis toxicity
Minnow toxicity
No
No
No
No
0.403
2.257
0.446
1.335
0.286
0.998
1.409
−0.633
0.285
4.412
Abbreviations: ADMET, absorption, distribution, metabolism, excretion, and toxicity; BBB, blood–brain barrier; CNS, central nervous system; VDss,
volume of distribution at steady state.
After assessing ADMET profiles of compounds 8, 9, 10, and 15
(Table 3), we can suggest that these derivatives have the advantage
of better intestinal absorption in humans than doxorubicin
(83.944–93.321), compared with 62.3 in the case of doxorubicin.
This preference may be attributed to the superior lipophilicity of our
designed ligands, which would make it easier to go along different
biological membranes.[55] Accordingly, they may have significant to
good bioavailability after oral administration. Studying the central
nervous system (CNS) permeability, our derivatives 8, 9, 10, and 15
demonstrated the best ability to penetrate the CNS (CNS perme-
ability values −1.67 to −2.144), whereas doxorubicin is unable to
penetrate (CNS permeability < −4.0). It is also clear that the cyto-
chrome P3A4, the main enzyme involved in drug metabolism, could
be inhibited under the effect of compounds 9, 10, and 15, whereas 8
and doxorubicin could not. This is also perhaps due to the higher
lipophilicity of our new ligands 9, 10, and 15. The total clearance is a
significant parameter in deciding dose intervals as a tool for the
assessment of excretion. Doxorubicin exhibited the highest total
clearance value as compared with other ligands. However, new li-
gands showed lower total clearance values. Thus, doxorubicin could
be excreted quicker and accordingly require shorter dosing intervals.
Dissimilar to doxorubicin, new compounds exhibited slower clear-
ance rates, which means the preference of possible extended dosing
intervals of the novel derivatives. The last parameter examined in the
ADMET profiles of our newly synthesized VEGFR‐2 inhibitors is
toxicity. As displayed in Table 3, all the new ligands showed no he-
patotoxicity drawback, unlike doxorubicin that exhibited unwanted
hepatotoxic effects. Finally, oral acute toxic doses of the new com-
pounds (LD50) are almost slightly more than the reference drug
(2.569–2.669 for our new derivatives as compared with 2.408 of
doxorubicin).
the VEGFR‐2 receptor. Inhibition of the ATP‐binding pocket of
the VEGFR‐2 receptor has an important effect on anticancer
activities. Most of the prepared compounds displayed excellent‐
to‐good growth inhibitory activity against the tested cancer cell
lines. In particular, compounds 10, 9, 8, and 15 were found to be
the most potent derivatives over all the tested compounds
against the HepG2 and MCF‐7 cancer cell lines with IC50 = 4.25
0.03, 6.08 0.06 µM, 4.68 0.06, 11.06 0.09 µM, 4.34 0.04,
10.29 0.09 µM and 6.37 0.06, 12.83 0.13 µM, respectively.
Compound 10 exhibited higher activity against HepG2 than
sorafenib (IC50 = 9.18 0.6 and 5.47 0.3 µM, respectively) and
doxorubicin (IC50 = 7.94 0.6 and 8.07 0.8 µM, respectively). It
also exhibited higher activity than doxorubicin against MCF‐7
cancer cell lines but lower than sorafenib. Compounds 9, 8, and
15 exhibited higher activities than sorafenib and doxorubicin
against HepG2 but exhibited lower activities against MCF‐7
cancer cell lines. The most potent six derivatives 6, 9, 8, 10, 15,
and 18 were tested for their cytotoxicity against normal Vero cell
lines. The results revealed that the tested compounds have low
toxicity against Vero normal cells with IC50 values ranging from
15.45 to 23.59 μM. The cytotoxicity of these compounds against
the cancer cell lines ranged from 4.25 to 16.57 μM. Compounds 6,
8, 9, 10, 15, and 18 are, respectively, 1.13, 3.74, 4.18, 3.64, 2.81,
and 2.00 times more toxic to HepG2 cancer cell lines than to
Vero normal cells. Also, compounds 6, 8, 9, 10, 15, and 18 are,
respectively, 2.06, 1.58, 1.76, 2.54, 1.40, and 2.69 times more
toxic to breast cancer cell lines (MCF‐7) than to Vero normal
cells. The tested compounds displayed high‐to‐medium inhibitory
activity with IC50 values ranging from 0.12 0.01 to
0.29 0.02 µM. Among them, compound 10 was found to be the
most potent derivative that inhibited VEGFR‐2 at IC50 value of
0.12 0.01 µM, which is nearly equipotent to sorafenib IC50 va-
lue of 0.10 0.02 µM. Compounds 8 and 9 exhibited very good
activity with the same IC50 value of 0.13 0.02 µM. Also, com-
pounds 15 and 18 exhibited very good activity with IC50 values of
0.14 0.05 and 0.16 0.02 µM. Also, compounds 6, 5, and 17
possessed very good VEGFR‐2 inhibition with the same IC50
value of 0.19 0.02 µM, which is more than half of the activity of
sorafenib. Compounds 19 and 7 displayed good VEGFR‐2 in-
hibition with IC50 values = 0.25 0.02 and 0.29 0.02 µM, re-
spectively. However, compounds 13 displayed moderate VEGFR‐
2 inhibition with an IC50 value = 0.33 0.01 µM.
|
3
CONCLUSION
In summary, 12 new pyridine‐derived compounds 5–19 have
been designed, synthesized, and evaluated for their anticancer
activities against two human tumor cell lines, hepatocellular
carcinoma (HepG2) and breast cancer (MCF‐7), targeting the
VEGFR‐2 enzyme. All the tested compounds showed variable
anticancer activities. The molecular design was performed to
investigate the binding mode of the proposed compounds with

SALEH ET AL.
15 of 18
|
|
4
EXPERIMENTAL
Chemistry
precipitate obtained during heating was filtered and recrystallized
from suitable solvents to give compound 5.
|
4.1
Method B: A mixture of 2‐(2‐bromobenzylidene)malononitrile 2
(2.31 g, 0.01 mol) and 2‐cyanoacetohydrazide 3 (0.99 g, 0.01 mol) in
absolute ethanol (20 ml) containing two drops of piperidine was
heated under reflux for 5 h. The pale brown precipitate obtained
during heating was filtered and recrystallized from ethanol to give
the targeted compound 5. When the reaction mother liquor was
heat‐concentrated and left to cool, another solid product crop was
obtained and known as product 6.
|
4.1.1
General
Melting points of the reaction products were determined in open
capillary tubes on an electrothermal melting point apparatus and
were uncorrected. The Fourier Transform infrared measure-
ments were recorded on a Perkin‐Elmer Model 297 IR spectro-
meter using the KBr wafer technique at the Central Laboratory
of Faculty of Science, Cairo University. The 1H NMR (400 MHz)
and 13C NMR (100 MHz) spectra were recorded on a Varian
Gemini spectrometer with chemical shift (δ) expressed in ppm
downfield using tetramethylsilane as an internal standard at the
main Defense Chemical Laboratory. Mass spectra were con-
ducted using Shimadzu GC‐MSQP 1000 EX instrument operating
at 70 eV, and the elemental analyses were performed on a
Perkin‐Elmer 2400 CHN elemental analyzer at the Micro-
analytical Center of Al‐Azhar University. All reactions were
monitored by thin‐layer chromatography (TLC) and PTLC (1‐mm
layer thickness), which were conducted using pre‐coated plates
of silica gel 60 F254 (Merck), and spots were detected using a UV
lamp (254 nm). The spots on TLC were visualized by warming
with 5% cerium ammonium molybdate in 2 N H2SO4‐sprayed
plates on a hot plate.
Dehydrogenation of 6 leads to formation of 5; the suspension of
6 (3.3 g, 0.01 mol) in trifluoroacetic acid was heated under reflux for
2 h. The solid product obtained was filtered, washed with diethyl
ether, and crystallized from ethanol to give 5 as brown crystals; yield
(2.31 g, 70%); m.p. 170°C. IR (KBr, cm−1): 3407, 3313, 3210 (2 NH2),
2957 (CH aliph.), 2207 (2 C≡N), and 1676 (C═O); 1H NMR
(400 MHz, DMSO‐d6) δ (ppm): 5.70 (s, 2H, N–NH2 exchangeable with
D2O), 7.27–7.83 (m, 4H, Ar‐H), and 8.60 (s, 2H, C–NH2 exchangeable
with D2O); 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 75.40, 114.59,
116.01, 116.41, 120.81, 124.68, 128.73, 130.54, 132.14, 134.27,
136.31, 159.48, and 166.93; Anal. calcd. for C13H8BrN5O (330.15);
C, 47.30; H, 2.44; N, 21.21%. Found: C, 47.44; H, 2.19; N, 21.38%.
1,6‐Diamino‐4‐(2‐bromophenyl)‐2‐oxo‐1,2,3,4‐tetrahydropyridine‐
3,5‐dicarbonitrile (6)
Compound 6 as brown crystals; yield (1.66 g, 50%); m.p. 200°C. 1H
NMR (400 MHz, DMSO‐d6) δ (ppm): 3.02 (d, 1H, C4–H), 3.06 (d, 1H,
C3–H), 4.56 (s, 2H, N‐NH2 exchangeable with D2O), 7.01–7.62 (m,
4H, Ar‐H), and 12.48 (s, 2H, C–NH2 exchangeable with D2O). Anal.
calcd. for C13H10BrN5O (332.16); C, 47.01; H, 3.03; N, 21.08%.
Found: C, 47.15; H, 2.79; N, 21.25%.
The InChI codes of the investigated compounds, together with
some biological activity data, are provided as Supporting
Information.
Nʹ‐(2‐Bromobenzylidene)‐2‐cyanoacetohydrazide (4)
Equimolar amounts of 2‐cyanoacetohydrazide 1 (0.99 g, 0.01 mol) and 2‐
bromobenzaldehyde 2 (1.85 ml, 0.01 mol) were synthesized according to
Bondock et al.[46] in 50 ml absolute ethanol, followed by addition of two
to three drops of glacial acetic acid. The resulting mixture was magne-
tically stirred, while cold, for an hour. The reaction was examined by TLC.
The precipitated product that formed was collected by filtration and
crystallized from ethanol to give compound 4 with high purity. Com-
pound 3 as white precipitate; yield (2.3 g, 95%); m.p. 150°C. IR (KBr,
cm−1): 3190 (NH), 3094 (CH arom.), 2960 (CH aliph.), 2264 (C≡N), 1675
(C═O), and 1587 (C═N); 1H NMR (400 MHz, deuterated dimethyl sulf-
oxide [DMSO‐d6]) δ (ppm): 4.22 (s, 2H, CH2), 7.32–7.97 (m, 4H, Ar‐H),
7‐(2‐Bromophenyl)‐5‐oxo‐3,5‐dihydro‐[1,2,4]triazolo[1,5‐a]pyridine‐
6,8‐dicarbonitrile (7)
Stirring a mixture of ethyl chloroformate/N,N‐dimethylformamide (30 ml,
1:5), compound 5 (3.3 g, 0.01 mol) was added gradually for half an hour,
and then the reaction mixture was heated under reflux for 3 h. The
reaction mixture was evaporated under reduced pressure and a solid
product was triturated with methanol. The solidified product was filtered
off and recrystallized from suitable solvents to give compound 7 as
brown crystals; yield (2.24 g, 66%), m.p. 220°C. IR (KBr, cm−1): 3322 (NH),
2951 (CH arom.), 2859 (CH aliph.), 2213 (2 C≡N), and 1675 (C═O); 1H
NMR (400 MHz, DMSO‐d6) δ (ppm): 7.09–7.92 (m, 4H, Ar‐H), 8.26 (s, 1H,
NH, exchangeable with D2O), and 8.57 (s, 1H, CH═N); 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 115.07, 116.48, 117.96, 121.81, 128.72,
130.65, 131.52, 133.33, 136.30, 137.62, 153.30, 154.39, 159.45, and
162.77. Anal. calcd. for C14H6BrN5O (340.14); C, 49.44; H, 1.78; N,
20.59%. Found: C, 49.36; H, 1.73; N, 20.47%.
8.49 (s, 1H, CH═N), and 11.95 (s, 1H, NH exchangeable with D2O); 13
C
NMR (100 MHz, DMSO‐d6) δ (ppm): 24.85, 116.45, 124.09, 127.79,
128.65, 130.57, 132.51, 133.62, 159.53, and 165.94. Anal. calcd. for
C10H8BrN3O (266.10): C, 45.14; H, 3.03; N, 15.79%. Found: C, 45.10; H,
2.98; N, 15.75%.
1,6‐Diamino‐4‐(2‐bromophenyl)‐2‐oxo‐1,2‐dihydropyridine‐3,5‐
dicarbonitrile (5)
7‐(2‐Bromophenyl)‐2‐methyl‐5‐oxo‐3,5‐dihydro‐[1,2,4]triazolo[1,5‐
a]pyridine‐6,8‐dicarbonitrile (8)
Method A: A mixture of compound 4 (2.66 g, 0.01 mol) and mal-
ononitrile (0.66 ml, 0.01 mol) in absolute ethanol (20 ml) containing
two drops of piperidine was refluxed for 5 h. The pale brown
A mixture of 5 (3.3 g, 0.01 mol) and acetic anhydride (15 ml) was
heated under reflux for 5 h. A solid product was filtered off and

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recrystallized from suitable solvents to give compound 8 as pale
brown crystals; yield (3.4 g, 97%), m.p. 240°C. IR (KBr, cm−1): 3387
(NH), 3062 (CH arom.), 2970 (CH aliph.), 2126 (2 C≡N), and 1690
(C═O); 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 1.29 (s, 3H, CH3),
7.05–7.89 (m, 4H, Ar‐H), and 7.94 (s, 1H, NH, exchangeable with
D2O); 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 23.05, 114.52, 117.99,
120.38, 121.81, 127.89, 128.85, 130.49, 131.61, 133.67, 137.62,
157.86, 158.37, 158.77, and 168.32; Anal. calcd. for C15H8BrN5O
(354.17); C, 50.87; H, 2.28; N, 19.77%. Found: C, 50.78; H, 2.15;
N, 19.65%.
and 8.56 (s, 1H, NH, exchangeable with D2O); 13C NMR (100 MHz,
DMSO‐d6) δ (ppm): 115.11 (2), 120.92, 123.90, 129.04, 129.26,
131.89, 137.39, 149.62, 149.74, 149.91, 157.47, and 168.49. Anal.
calcd. for C13H7BrN4O (315.13): C, 49.55; H, 2.24; N, 17.78%. Found:
C, 49.48; H, 2.15; N, 17.72%.
Ethyl 1,2‐diamino‐4‐(2‐bromophenyl)‐5‐cyano‐6‐oxo‐1,6‐
dihydropyridine‐3‐carboxylate (13)
A mixture of 3 (2.66 g, 0.01 mol) and ethyl cyanoacetate (1.13 ml,
0.01 mol) in absolute ethanol (20 ml) containing two drops of
triethylamine was heated under reflux for 5 h, and the reaction
mixture was left to cool. The solid product obtained was filtered
off and crystallized for purification from ethanol to give 13 as
white powder; (1.9 g, 50.39%), m.p. 170°C. IR (KBr, cm−1): 3399,
3290 (2NH2), 3078 (CH arom.), 2980 (CH aliph.), 2216 (CN), and
1651 (C═O); 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 1.19 (q, 2H,
CH2), 4.30 (t, 3H, CH3), 5.70 (s, 2H, NH2 exchangeable with D2O),
7.20–7.65 (m, 4H, Ar‐H), and 9.05 (s, 2H, NH2, exchangeable with
D2O); 13C NMR (100 MHz, DMSO‐d6) δ (ppm): 14.24, 60.47,
90.96, 116.39, 120.75, 127.92, 128.92, 129.91, 130.26, 132.40,
133.24, 140.42, 158.48, 159.20, and 165.82. Anal. calcd. for
7‐(2‐Bromophenyl)‐5‐oxo‐2‐phenyl‐1,2,3,5‐tetrahydro‐[1,2,4]‐
triazolo[1,5‐a]pyridine‐6,8‐dicarbonitrile (9)
A mixture of 5 (3.3 g, 0.01 mol) and benzaldehyde (1.06 ml, 0.01 mol)
in dioxane (50 ml) containing piperidine was heated under reflux for
3 h. A solid product was filtered off and recrystallized from suitable
solvents to give compound 9 as pale brown crystals; yield (4 g,
95.7%), m.p. 130°C. IR (KBr, cm−1): 3318, 3210 (2NH), 2957 (CH
aliph.), 2314, 2207 (2 C≡N), and 1676 (C═O); 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 4.21 (s, 1H, C5–H triazol), 7.56–7.85 (m, 9H, Ar‐
H), 9.26, (s, 1H, C‐NH, exchangeable with D2O), 9.26, and 10.71 (s,
2H, N–NH+OH tautomeric exchangeable with D2O); 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 10.95, 36.07, 38.82, 101.64, 123.71
(2), 128.07 (2), 128.44 (2), 130.08 (2), 132.91 (2), 137.61 (2), 143.50,
160.25, and 173.17 (2). Anal. calcd. for C20H12BrN5O (418.25); C,
57.43; H, 2.89; N, 16.74%. Found: C, 57.39%; H, 2.77%; N, 16.65%.
C
15H13BrN4O3 (377.20): C, 47.76%; H, 3.47%; N, 14.85%. Found:
C, 47.85%; H, 3.63%; N, 14.66%.
6‐Amino‐1‐[(2‐bromobenzylidene)amino]‐4‐(4‐methoxyphenyl)‐2‐
oxo‐1,2,3,4‐tetrahydro‐pyridine‐3,5‐dicarbonitrile (15)
A
mixture of 3 (2.66 g, 0.01 mol), 2‐(4‐methoxybenzylidene)
7‐(2‐Bromophenyl)‐5‐oxo‐2‐phenyl‐3,5‐dihydro‐[1,2,4]triazolo[1,5‐
a]pyridine‐6,8‐dicarbo‐nitrile (10)
malononitrile (1.84 g, 0.01 mol), and 0.5 ml of triethylamine was re-
fluxed in 30 ml of 1,4‐dioxane for 5 h and then left to cool. The solid
product formed was collected by filtration and recrystallized from
ethanol to give compounds 15 as yellow powder; (2.25 g, 50%)
m.p. 140°C. IR (KBr, cm−1): 3347, 3202 (NH2), 3078 (CH arom.), 2973
(CH aliph.), 2215 (2 CN), and 1699 (C═O); 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 3.22 (s, 3H, OCH3), 3.81 (d, 1H, CH– Ar‐H), 3.85
(d, 1H, CH‐CN), 5.62 (s, 2H, NH2, exchangeable with D2O), 6.97–7.54
(m, 8H, Ar‐H), and 8.91 (s, 1H, CH═N); 13C NMR (100 MHz, DMSO‐
d6) δ (ppm): 25.61, 55.78, 114.17, 114.38, 114.38, 116.16, 117.04,
126.92, 127.83, 128.02, 128.49, 128.73, 129.04, 130.27, 130.61,
132.26, 132.53, 133.87, 157.09, 159.77, and 169.14. Anal. calcd. for
A suspension of 9 (0.2 g) in trifluoroacetic acid (5 ml) was stirred
at room temperature for 5 min and then poured into cold water.
The solid product was filtered off and recrystallized from suitable
solvents to give compound 10 as brown crystals; yield (4.16 g,
35.7%), m.p. 300°C. IR (KBr, cm−1): 3182 (NH), 3090 (CH arom.),
2924 (CH aliph.), 2258, 2208 (2 C≡N), 1672 (C═O), and 1611
(C═N); 1H NMR (400 MHz, DMSO‐d6) δ (ppm): 7.15–8.40 (m, 4H,
Ar‐H), and 8.46 (s, 1H, NH, exchangeable with D2O); 13C NMR
(100 MHz, DMSO‐d6) δ (ppm): 116.26, 116.64, 118.63 (2), 123.26
(2), 123.87, 128.78 (2), 128.89, 129.02 (2), 131.71, 131.77,
131.87, 137.37, 137.41, 148.52, 162.38, and 166.36. Anal. calcd.
for C20H10BrN5O (416.24); C, 57.71; H, 2.42; N, 16.83%. Found:
C, 57.68; H, 2.38; N, 16.75%.
C21H16BrN5O2 (450.30): C, 56.01%; H, 3.58%; N, 15.55%. Found: C,
56.15%; H, 3.55%; N, 15.65%.
2‐[2‐(2‐Bromobenzylidene)hydrazinyl]‐4‐oxo‐3,4‐dihydroquinoline‐
3‐carbonitrile (17)
6‐Amino‐4‐(2‐bromophenyl)‐2‐oxo‐1,2‐dihydropyridine‐3,5‐
dicarbonitrile (11)
A mixture of 3 (2.66 g, 0.01 mol) and anthranilic acid (1.38 g,
0.01 mol) in dioxane (30 ml) was refluxed for 2–6 h with a few drops
of triethylamine. A solid product was obtained after cooling the re-
action mixture to room temperature, which was filtered, dried, and
recrystallized from suitable solvents to give compound 17 ;(1. 65 g,
45%) m.p. 170°C. IR (KBr, cm−1): 3228 (NH), 3009 (CH arom.), 2996
(CH aliph.), 1701 (C═O), and 1587 (C═N); 1H NMR (400 MHz,
DMSO‐d6) δ (ppm): 1.38 (s, 1H, NH), 3.34 (s, 1H, CH), 7.44–7.99
(m, 8H, CH– aromatic ring), and 8.01 (s, 1H, CH═N); 13C NMR
To a suspension of 5 (3.3 g, 0.01 mol) in aqueous acetic acid (100 ml,
60%), sodium nitrite (0.015 mol in 5 ml water) was added. The re-
action mixture was stirred at room temperature for 2 h and then left
overnight. A solid product was filtered off and recrystallized from
suitable solvents to give compound 11 as pale brown crystals; yield
(1 g, 31.7%), m.p. 230°C. IR (KBr, cm−1): 3406, 3300 (NH2/NH), 2210
(2 C≡N), and 1691 (C═O); 1H NMR (400 MHz, DMSO‐d6) δ (ppm):
5.67 (s, 2H, NH2, exchangeable with D2O), 7.12–7.80 (m, 4H, Ar‐H),

SALEH ET AL.
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(100 MHz, DMSO‐d6) δ (ppm): 21.20, 38.43, 54.50, 116.08 (2),
129.47 (2), 129.81 (2), 131.27 (2), 131.83 (2), 133.89 (2), 155.60, and
162.27. Anal. calcd. for C17H11BrN4O (367.21): C, 55.61; H, 3.02; N,
15.26%. Found: C, 55.58; H, 2.99; N, 15.14%.
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How to cite this article: N. M. Saleh, A. A.‐H. Abdel‐Rahman, A.
M. Omar, M. M. Khalifa, K. El‐Adl. Pyridine‐derived VEGFR‐2
inhibitors: Rational design, synthesis, anticancer evaluations, in
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