Design, synthesis, biological evaluation and dynamics simulation of indazole derivatives with antiangiogenic and antiproliferative anticancer activity

Article abstract of DOI:10.1016/j.bioorg.2018.10.071

VEGFR-2 has a pivotal role in promoting cancer angiogenesis. Herein, two series of novel indazole-based derivatives were designed, synthesized and evaluated for their in vitro inhibitory action against VEGFR-2 kinase enzyme. The second series 11a-e exhibited better potency than the first one 7a-d and 8a-f. Compounds 11b, 11c and 11e exhibited the most potent action, with IC₅₀ of 5.4 nM, 5.6 nM and 7 nM, respectively. As a measure of cellular VEGFR-2 inhibition, compounds 11b and 11c showed strong inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 80% and 99.6% inhibition at 10 μM concentration, respectively. Attempting to interpret SAR of the synthesized compounds, and provide a basis for further optimization; a comprehensive modeling study was implemented. Molecular docking, dynamics simulation and free energy calculation of the synthesized compounds along with known VEGFR-2 inhibitors were applied. The study illustrated the effect of several factors on VEGFR-2 inhibition, such as the interaction with solvent accessible region of the enzyme, the presence of NH linker and the degree of conformational restriction. Finally, our compounds were evaluated for their in vitro anti-proliferative effect against the full NCI panel of cancer cell lines, where compounds 11a and 11c displayed mean GI% of 93 and 130%, respectively, and showed partly a better behavior than the FDA approved drug sorafenib, with respect to activity (GI₅₀) and safety (LC₅₀) against several cell lines. Thus, compound 11c represents a promising candidate for cancer treatment through antiangiogenic dependent and antiangiogenic independent modes of action.

Full text of DOI:10.1016/j.bioorg.2018.10.071

BioorganicChemistry82(2019)340–359
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Design, synthesis, biological evaluation and dynamics simulation of indazole
derivatives with antiangiogenic and antiproliferative anticancer activity
Nevine M.Y. Elsayeda, Rabah A.T. Seryaa, Mai F. Tolbab, Marawan Ahmedc, Khaled Barakatc,d,e
,
⁎
Dalal A. Abou El Ellaa,f, Khaled A.M. Abouzida,g,
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt
b Pharmacology and Toxicology Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt
c Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta, Canada
d Li KaShing Institute of Virology, University of Alberta, Edmonton, Alberta, Canada
e Li KaShing Applied Virology Institute, University of Alberta, Edmonton, Alberta, Canada
f Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Nahda University, Beni Suef, Egypt
g Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Menoufia, Egypt
A R T I C L E I N F O
A B S T R A C T
Keywords:
Indazole
VEGFR-2 has a pivotal role in promoting cancer angiogenesis. Herein, two series of novel indazole-based de-
rivatives were designed, synthesized and evaluated for their in vitro inhibitory action against VEGFR-2 kinase
enzyme. The second series 11a-e exhibited better potency than the first one 7a-d and 8a-f. Compounds 11b, 11c
and 11e exhibited the most potent action, with IC50 of 5.4 nM, 5.6 nM and 7 nM, respectively. As a measure of
cellular VEGFR-2 inhibition, compounds 11b and 11c showed strong inhibition of human umbilical vein en-
dothelial cells (HUVEC) proliferation with 80% and 99.6% inhibition at 10 μM concentration, respectively.
Attempting to interpret SAR of the synthesized compounds, and provide a basis for further optimization; a
comprehensive modeling study was implemented. Molecular docking, dynamics simulation and free energy
calculation of the synthesized compounds along with known VEGFR-2 inhibitors were applied. The study illu-
strated the effect of several factors on VEGFR-2 inhibition, such as the interaction with solvent accessible region
of the enzyme, the presence of NH linker and the degree of conformational restriction. Finally, our compounds
were evaluated for their in vitro anti-proliferative effect against the full NCI panel of cancer cell lines, where
compounds 11a and 11c displayed mean GI% of 93 and 130%, respectively, and showed partly a better behavior
than the FDA approved drug sorafenib, with respect to activity (GI50) and safety (LC50) against several cell lines.
Thus, compound 11c represents a promising candidate for cancer treatment through antiangiogenic dependent
and antiangiogenic independent modes of action.
Dynamics
VEGFR-2
Anticancer
1. Introduction
monoclonal antibodies against VEGF, as Bevacizumab [10], and small-
molecule tyrosine kinase inhibitors [11] as Sorafenib [12], sunitinib
Antiangiogenic anticancer therapy was first introduced as a rational
approach for cancer treatment, when Folkman and colleagues illu-
strated the urgency of neovascularization for tumors to grow beyond a
limited volume (1–2 mm3) [1–5]. Vascular endothelial growth factor
(VEGF) family are the chief proangiogenic factors, which are secreted
by the cancerous cells, where VEGF-A is the major mediator of angio-
genesis [6–8]. VEGF family members target three correlated receptors
(VEGFR 1–3), activating several signaling pathways. VEGFR-2 is the
principal receptor of VEGF-A; it is responsible for vascular endothelial
cell development, proliferation, invasion and migration [7,9]. Anti-
angiogenic agents targeting VEGF/VEGFR-2 signaling pathway include
[13], pazopanib [14], cabozantinib [15], lenvatinib [16], and apatinib
[17].
Apart from the antiangiogenic effect of inhibiting VEGFR-2 sig-
naling pathway, VEGFR-2 blockade can exert direct antiproliferative
effect against cancer cell lines that express VEGFR-2 receptors on their
surface. VEGFR-2 inhibitors induce apoptosis of these cell lines in vitro.
They block the VEGFR-2 downstream signaling pathways, as PI3K/
AKT, and Ras-MEK-ERK pathways [18,19]. VEGFR-2 receptors over-
expression is characteristic of certain limited number of cell lines of
various tumors, such as, colon [20], gall bladder [21], non-small cell
lung [22,23], breast [24], prostate [25], and melanoma [26,27], human
⁎ Corresponding author at: Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo 11566, Egypt.
E-mail address: khaled.abouzid@pharma.asu.edu.eg (K.A.M. Abouzid).
https://doi.org/10.1016/j.bioorg.2018.10.071
Received 26 August 2018; Received in revised form 26 October 2018; Accepted 31 October 2018
Availableonline02November2018
0045-2068/©2018ElsevierInc.Allrightsreserved.

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
hepatocellular carcinoma (HCC) [28], renal, ovarian and cervical [29]
cancer and hematologic malignancies [18,30]. However, with respect
to the complex nature of cancer disease, it is not surprising that anti-
VEGFR-2 monotherapy–in several cases-exhibited little effect on cancer
progression, due to the emerging resistance in some individuals or the
resistant nature of some cancers [31].
restriction strategy and explore the influence of rigidification on the
biological activity of both type I and type II inhibitors, using various
modeling techniques.
Conformational rigidification has been employed as a successful
strategy in the drug design and development process [44,45]. The ef-
ficiency of conformational restriction is attributed to its ability to lower
the entropic cost of binding of the conformationally restricted deriva-
tive to the target, as compared to the more flexible derivative. Ad-
ditionally, rigid derivatives tend to be more selective and metabolically-
resistant than flexible ones. These advantages have been supported by
the comparison of rigid and flexible derivatives with respect to biolo-
gical activity, selectivity and pharmacokinetics [46].
The VEGFR-2 catalytic domain toggles between active and inactive
states, through the movement of the DFG motif (Asp-Phe-Glu) of the
activation lobe [32,33]. Movement of the DFG loop allows the phenyl
ring of the phenylalanine amino acid to rotate around the CeN bond of
aspartate, displacing itself for > 10 Å into the ATP binding pocket,
voiding a back hydrophobic pocket. As the phenyl ring of phenylalanine
partially occupies the space usually utilized by ATP for performing
phosphorylation, the DFG-out conformation inhibits ATP binding and
thus, renders the kinase in an inactive state [34]. Both conformations
can be targeted by VEGFR-2 tyrosine kinase inhibitors (TKIs) [35].
VEGFR-2 TKIs can be classified into two main types, type I and type II
inhibitors [36]. Type I inhibitors target the active DFG-in conformation
and bind the ATP binding site of the kinase. In contrast, type II in-
hibitors bind to the inactive DFG-out conformation through interacting
with the ATP binding site as well as the hydrophobic back pocket
[37,38]. It is noteworthy that some VEGFR-2 TKIs were classified as
type III inhibitors [39,40], where they exclusively occupy the back
pocket of the DFG-out conformer, without extending to the ATP site
[41].
In a previous study, we reported the development of a series of
pyrimidine based VEGFR-2 inhibitors [47]. Compound A was the most
active compound in which conformational restriction approach was
applied (IC50 = 148 nM).
Herein, two rigidification strategies were adopted, as shown in
Fig. 2. The first strategy involved incorporation of the N1 of urea
moiety in an indazole ring, to give the target compounds 7a-d and 8a-f.
This strategy has been previously implemented to yield highly potent
derivatives, where naphthalene, benzoxazine and indole rings have
been used in rigidification [48–54]. The second strategy was based
upon restricting the rotation of the central aromatic ring by in-
corporating it in an indazole ring that is attached to the hinge binder
directly at N1 position of the indazole ring, to give target compounds
and 11a-e. Such approach was adopted before in the design of type I
inhibitors, using indole [55]. Although it didn’t yield highly active
agent (IC50 > 300 nM), we believed – based on the aforementioned
observation – that potent type II inhibitors may be produced if careful
structural modulation was adopted. In order to fully assess the influence
of each strategy on the activity of type I and type II inhibitors, rigidi-
fication was also implemented in the design of parent compounds (6b
and 9), that lack the aryl urea group that interact with the back hy-
drophobic pocket of VEGFR-2 kinase enzyme, and thus, they are unable
to target the inactive conformation of VEGFR-2 kinase.
Cautious inspection of several VEGFR-2 potent inhibitors reveals
certain common key interaction features that can be summarized, as
shown in Fig. 1:
(A) Hinge binder: It consists of a flat aromatic ring system with a hy-
drogen bond acceptor for interaction with hinge region. The hy-
drogen bond acceptor makes an essential hydrogen bond with NH
of Cys919, while the aromatic ring is normally involved in π-π
interaction with phenyl ring of Phe918 [42].
(B) Extra central aromatic ring system: It is located in the proper po-
sition to provide π-π interactions with Phe1047 (as in vandetanib
or π-cation interaction with Lys 868 (as in pazopanib, axitinib and
linifanib) [42,43].
The indazole ring is as an attractive choice for the rigidifiaction
purpose. It can also yield compounds with acceptable solubility, PK
properties [56], synthetic feasibility and versatility [57]. Indazole was
previously implemented to design inhibitors that follow our first
strategy, however, it yielded compounds with mediocre activity
(IC50 = 2.3 µM) [54]. Herein, we proved that structural optimization
can give more potent indazole-based compounds.
(C) Features targeting the back hydrophobic pocket (in type II in-
hibitors only): They are responsible for bridging hydrogen-bond
network between with CO of Glu885 and the NH of Asp1046 in-
volved in the DFG loop move. In the same time, they bear an
aromatic ring with hydrophobic substitution that extends to occupy
the back hydrophobic pocket. Aryl urea and substituted benzamide
are most commonly used to satisfy these requirements (as in sor-
afinib and linifanib) [34,42].
Quinazoline was our selected hinge binder. Several quinazoline-
based VEGFR-2 inhibitors have been reported in literature [44,58–67].
Quinazoline exhibits optimum binding to the hinge region of the ki-
nases [68], as it is capable of forming two hydrogen bonds with the
hinge amino acid Cys919 and it is anchored in the hinge region by Pi-Pi
interaction with Phe918.
In this study, the main objective was to design novel potent in-
dazole-based VEGFR-2 kinase inhibitors, applying conformational
Fig. 1. Vandetanib, Sorafinib and Linifanib with their main pharmacophoric features. Hinge binders are indicated by blue dots, the central aromatic group is included
in orange circle and the aryl urea derived group is indicated by pink dots.
341

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Fig. 2. The design strategies for the target compounds. Site of rigidification is marked with red color, hinge binder is colored in blue, aryl urea is colored in green, and
solvent accessible region is colored in pink.
2. Results and discussion
compound 10 that was directly used in the following reaction. Com-
pound 10 was reacted with the corresponding isocyanate to yield
compounds 11a-e.
2.1. Chemistry
The synthetic routes adopted for synthesis of the target compounds
are outlined in Schemes 1–3. Diazotization of 2-methyl-4-nitroaniline 1
i yielded 5-nitroindazole 2 [69]. 5-aminoindazole 3 was obtained
through the catalytic reduction of compound 2. Nucleophilic substitu-
tion reaction between 3 and 2,4-dichloropyrimidine was employed to
obtain compound 4. Phenylisocyanate was added to compound 4 to
give compound 5.
2.2. Kinase inhibition assay
In Table 1, it is obvious that there is significant difference in activity
between series 7a-d and series 8a-f even when they share the same
substituents on the benzamide ring. This was attributed to the presence
of dimethoxy substitution at positions 6 and 7 of the quinazoline ring
that are supposed to extend to the solvent accessible region of the en-
zyme giving superior potency to series 8a-f over series 7a-d. Ex-
amination of the activity of compounds 8a-f reveals pronounced var-
iation depending on the substituent on the benzamide ring, where
compound 8a-with 3-chloro and 4-methyl substitution-was the most
active compound while other compounds showed intermediate to
strong activity. Also, the thio derivative 8f showed comparable activity
to 8a, suggesting that S in 8f maintains the H-bond with Asp1046 as O
found in other compounds. There is a remarkable difference in activity
As outlined in Scheme 2, Compounds 6a and 6b were constructed
via the reaction between 5-aminoindazole 3 and the appropriate qui-
nazoline derivative. Then, compounds 7a-d and 8a-f were synthesized
using the same procedure mentioned under the synthesis of compound
5 by mixing equimolar amounts of 6a or 6b and the corresponding
isocyanate.
As shown in Scheme 3, compound 2 was reacted with 6,7-di-
meethoxy quinazoline in presence of NaH, to give compound 9. The
nitro group in compound 9 was reduced using H2/Pd mixture to give
between compound
5 and compound 8e. This can indicate the
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N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Scheme 1. Reagents and conditions: (a) NaNO2, gl. AcOH, r.t., 72 h, 72%, (b) H2, Pd/C, 10%, methanol, 40 °C, 30 psi, 3 h, 97.2%, (c) 2,4-dichloropyrimidine, TEA,
ethanol, reflux, 5hrs, 78.59%, (d) phenylisocyanate, dry THF, r.t., 24 h, 66.4%.
inefficiency of 2-chloropyrimidine as hinge binder, when compared to
6,7-dimethoxyquinazoline. Unlike 8a-f, the series 11a-e displayed an
excellent activity profile, where compounds 11b, c and e had IC50 of
5.4, 5.6 and 7 nM, respectively, showing superior potency against
VEGFR-2 than sorafenib (IC50 = 90 nM [12]), while 11a exhibited a
relatively low activity (IC50 = 83 nM), and 11d exerted the weakest
inhibitory action among the series (mean% inhibition = 86%). Thus, it
can be concluded that increasing the bulkiness of the terminal phenyl
ring by disubstitution (11b and 11c) or trisubstitution (11e) is favor-
able for VEGFR-2 inhibitory activity. Nevertheless, using large bulky
substituents (as in compound 11d) may hinder the binding to the en-
zyme. Compounds 6b and 9 lacked the aryl urea moiety, yet they dis-
played interesting activity profiles when compared with analogous
compounds that carried the aryl urea group. While compound 6b
endothelial cells) proliferation inhibition assay at a concentration of
10 µM. (HUVECs) cell line is an ideal model system for studying the
cellular antiangiogenic effect in vitro, as they are good representatives
of the behavior of vascular endothelial cells in vivo. Besides, they are
relatively easy to harvest from large blood vessels [70,71]. As shown in
Table 2., 11b and 11c had 80% and 99.6% inhibition, respectively,
indicating that they can exert cellular antiangiogenic effect based upon
their action against VEGFR-2 kinase. Later, IC50 against HUVEC cell line
was determined for the most active compound 11c, where it showed
IC50 of 87.7 nM, with sorafenib as a reference (IC50 = 160 nM).
2.4. In vitro anticancer activity
Compounds with pronounced inhibitory potency against VEGFR-2
(8a, 8f, 11a, 11b, 11c, and 11e) were submitted to the National Cancer
Institute (NCI) Developmental Therapeutic Program (www.dtp.nci.nih.
gov). Four compounds 8a, 11a, 11b, and 11c were selected to be
screened for in vitro anticancer action. The selection was based on
several factors, including: the novelty of the structure, drug-likeness,
absence of problematic moieties (such as nitro, nitroso and flexible
acyclic group) and the likeability of the compound to display an anti-
neoplastic action, based on its structure [72]. The assays were executed
according to the protocol of the Drug Evaluation Branch, NCI, Bethesda
tested [73].
showed good activity (mean% inhibition = 100%), compound
9
showed compromised inhibitory action against the enzyme (mean%
inhibition = 46%), when compared with series 11a-e, supporting the
claim that our second design strategy is not suitable for developing
potent type I VEGFR-2 kinase inhibitors, while it is efficient in the
design of type II VEGFR-2 inhibitors with good activity.
2.3. In vitro HUVEC Anti-proliferative assay
The most active compounds against VEGFR-2 kinase (11b and c)
were preliminarily evaluated for HUVEC (human umbilical vein
The compounds showed variable anticancer activity. Compounds 8a
Scheme 2. Reagents and conditions: (a) isopropanol, reflux, 3hrs, 4-chloroquinazoline (for compound 6a), 48.92% – 4-chloro-6,7-dimethoxyquinazoline (for com-
pound 6b), 81.54%, (b) isocyanates or isothiocyanate, THF or DMF, r,t., 24 h, 10.51–88%.
343

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Scheme 3. Reagents and conditions: (a) 4-chloro 6,7-dimethoxyquinazoline, NaH, DMF, 0 °C, 6 h, 83.58%, (b) H2, Pd/C 10%, 40 °C, 50 psi, THF, overnight. (c) Aryl
isocyanate, DCM, r.t., 24 h, 34–44.5%.
and 11b had fair anticancer activity, with their mean percentages
growth inhibition (GI%) equal 13%, and 8%, respectively. However, 8a
exerted profound inhibitory action against (GI% > 50%) against
NSCLC cell line NCI-H322M (60%), and breast cancer cell line MDA-
MB-468 (90%). Similarly, 11b had elicited selective anticancer activity
against colon cancer cell line KM12 (74%), (see Supplementary file). In
the light of these results, 11b-which had potent action against VEGFR-
2-showed a promising potential to be developed into a selective potent
agent targeting colon cancer in personalized therapy. It is noteworthy
that compounds 8a and 11b exhibited promising VEGFR-2 inhibition
with IC50 values of 148 and 5.4 nM respectively, while showed poor
antiproliferative activity, with mean percentages growth inhibition (GI
%) of 13%, and 8%. This discrepancy in activity may indicate that both
compounds exhibit their antitumor effect by selective action on en-
dothelial cells rather than cancer cells via targeting targeting en-
dothelial VEGFR-2 receptors leading to hampered angiogenesis and
limiting blood supplies to the growing tumors [74]. Otherwise, this
discrepancy may be attributed to the inability of these compounds to
accumulate in the cancerous cells due to their physicochemical prop-
erties, thus they are unable to penetrate cell membrane, as observed in
some previous cases [75,76].
activity of 11a and 11c, the results were compared to those of sorafenib
(which were downloaded from https://dtp.cancer.gov/discovery_
development/nci-60/). Sorafenib is
a multi-kinase inhibitor [12],
which is approved as a treatment for renal cell carcinoma, hepatocel-
lular carcinoma, and thyroid cancer [31]. It is This comparison illu-
strated that 11a had better antiproliferative potency than sorafenib
through most of the NCI-60 panel, where 11a displayed a lower GI50
than that of sorafenib against 52 cell lines of different types. Specifi-
cally, 11a was more potent against renal cancer cell lines than sor-
afenib. With respect to safety, 11a had higher LC50 than sorafenib at 23
cell lines, showing the most superior safety profile against CNS cell
lines. In addition, it maintained better safety than sorafenib with re-
spect to two of renal cell lines (SN12C and CAKI-1). In a similar way,
11c exhibited greater potency than sorafenib against 39 cancer cell
lines of all types (including all renal cell lines), maintaining better
safety in 19 cell lines. 11c had excellent safety profiles against leukemia
and colon cancer cell lines. Based on the aforementioned facts, 11a and
11c had comparable in vitro anticancer activity to sorafenib, with a
reasonable safety profiles, thus, they can be developed into clinical
antitumor candidates.
Interpreting the in vitro anticancer behavior of 11a and 11c was not
a straightforward process. In NCI panel of cell lines, several cell lines
overexpress VEGFR-2, such as breast cell lines: T-47D, MCF7, and MDA-
MB-231 [78,79], melanoma cell line: MDA-MB-435 [80], leukemia cell
line: HL-60 [81], and colon cancer cell lines: HCT-116, HT29 [20]. The
remaining cell lines exhibit variable levels of VEGFR-2 expression [67].
Generally, some tumors display high degree of sensitivity towards anti-
VEGFR-2 therapy such as renal cell carcinoma, and ovarian tumors. On
the contrary, other cell lines are known for their high resistance to
VEGFR-2 inhibitory therapy, such as prostate cancer, and melanoma
[31]. As a result, the inhibitory potency of the compounds against
VEGFR-2 kinase can’t interpret their superior wide spectrum in vitro
anticancer activity, and further investigation was required. Compound
11c showed several advantages as: the superior anti VEGFR-2 potency,
the wide spectrum of in vitro anticancer activity (as illustrated by its
high mean GI% of 130%), its antiproliferative activity against the re-
sistant cell line NCI/ADR-RES of ovarian cancer, that exceeded that of
sorafenib, and the acceptable level of safety – as demonstrated by the
high LC50 values against several cell lines – compared to sorafenib. Of
special interest, 11c had superior safety to sorafenib with respect to
leukemia and colon cell lines, suggesting that it can avoid two of the
significant side effects of sorafenib, which are leukopenia and GIT
distress [12,82]. Thus, it represents an excellent anticancer candidate
for personalized therapy and resistant cancerous subtypes. Thus, it was
chosen for further investigation of its angiogenic – independent mode of
action. PC-3 prostate cancer cell line was chosen for the study, since it is
Table 3 shows the results of screening of the most potent derivatives
(11a and 11c) for their growth inhibition against the panel of cell lines.
On the others side, 11a and 11c showed high mean %GI (93 and 130%
respectively). 11a and 11c exerted a profound antiproliferative effect
against the full NCI panel of cell lines (%GI = or > 50%). Conse-
quently, both compounds were evaluated against the NCI panel of
tumor cell lines at five levels of concentration such that three para-
meters were calculated for each compound: GI50, TGI and LC50 –
against each cell line, as presented in Tables 4. GI50 is the concentra-
tion that showed 50% growth inhibition, TGI (tumor growth inhibition)
reflects cytostatic activity, while LC50 (50% lethal concentration) re-
presents the cytotoxicity parameter for compounds.
Compounds 11a and 11c exerted significant potency against the full
panel of cell lines. Compound 11a showed a submicromolar activity
against 23 cancer cell lines of different types where it exhibited its
highest potency against T-47D breast cancer cell line (GI50 = 183 nM).
Also, it had one digit micromolar GI50 on the rest of cell lines panel that
ranged from 1.01 to 5.22 μM. Compound 11c displayed nanomolar GI50
against two cell lines: KM12 colon cancer cell line (50 nM) and SF-539
CNS cancer cell line (612 nM). It had one digit micromolar GI50 on the
rest of cell lines panel that ranged from 1.24 to 4.44 μM. Interestingly,
11c elicited a good antiproliferative effect against the multi-drug re-
sistant ovarian cancer cell line (NCI/ADR-RES) [77] with GI50 of
2.36 µM, maintaining excellent safety profile (LC50 > 100 µM).
Attempting to achieve a better evaluation for the in vitro anticancer
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N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Table 1
In vitro VEGFR-2 inhibition data of synthesized compounds, showing mean % VEGFR-2 inhibition and IC50 values.*
Compound
R
VEGFR −2% Inhibition (10 μM)
IC50 (nM)
NT
5
6
6b
100
NT
7a
7b
−4
7
NT
NT
7c
7d
4
NT
NT
10
8a
8b
100
48
148
NT
8c
8d
86
50
NT
NT
(continued on next page)
345

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Table 1 (continued)
Compound
R
VEGFR −2% Inhibition (10 μM)
IC50 (nM)
NT
8e
94
8f
100
163
9
46
NT
83
11a
100
11b
11c
11d
100
100
86
5.4
5.6
NT
11e
100
7
*Staurosporine (1 µM)a
Sorafenibb
100
–
NT
90 [12]
NT: not tested.
* In a 96-well plate, each row contains a positive control (no compound) and negative control (background).
a
Staurosporine was used as a reference in the VEGFR-2 inhibition assay. Staurosporine was also evaluated at 10 nM and 100 nM concentrations, and it showed
85% and 98% inhibition, respectively.
b
Reported values.
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N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Table 2
Table 3
HUVEC% proliferation inhibition of compounds 11b and 11c.
Percent inhibition of the growth of NCI 60 cancer cell lines exerted by final
compounds (11a, and 11c) at concentration of 10 μM.
Compound/NSC no Cell name
Compound
HUVEC% inhibition at 10 µM
IC50 (µM)
11a 781,954
11c 781,956
11b
80
NT
11c
99.6
84.7
–
0.0877
–
0.003
0.01
Leukemia
CCRF-CEM
HL-60(TB)
K-562
Doxorubicina
Sorafenib
77
116
61
96
84
73
78
85
89
91
100
90
0.16
The experiment was done in absence of the compounds, and the observed
growth was considered 100% growth (0%inhibition).
MOLT-4
RPMI-8226
SR
a
Doxorubicin was used as reference, with IC50 = 0.2241 µM. The con-
centrations used were: 0.01 µM, 0.1 µM, 1µ, and 10 µM.
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
76
100
116
–
102
–
resistant to VEGFR-2 inhibitory therapy – as previously mentioned [31]
– therefore, it is suitable for evaluating the angiogenic independent
modes of action of 11c. The study followed two strategies: 1 – in-
vestigating the effect of 11c on kinases that are downstream of VEGFR-
2 and other receptor tyrosine kinases (RTK), 2 – evaluating the proa-
poptotic effect of 11c.
HOP-62
HOP-92
120
114
97
157
163
154
146
154
99
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
69
73
NCI-H522
80
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
2.5. Assessment of the effect on Akt and ERK phosphorylation
–
–
66
94
79
71
95
50
85
Activation of PI3K/Akt pathway is a common feature for cancer
progression [83–86]. Upregulation of the mitogen activated protein
kinase (MAPK)/ERK pathway is another important feature associated
with progression and poor prognosis of cancer. ERK1/2 phosphoryla-
tion is often correlated with higher tumor grade of cancer and tumor
recurrence [87,88]. Both pathways are known to be downstream for
VEGFR-2 and other RTK [89,90].
76
136
117
100
132
HT29
KM12
SW-620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
Melanoma
LOX IMVI
MALME-3 M
M14
78
73
99
55
112
–
135
167
171
90
ELISA assays indicated that treatment of PC-3 cells with compound
11c significantly reduced Akt phosphorylation by 52.5% (P < 0.001)
compared to untreated cells. On the other hand, treatment of cells with
compound 11c reduced ERK phosphorylation by 7.9% compared to
control, as shown in Fig. 3.
158
–
98
178
144
141
131
127
171
183
110
169
114
93
2.6. Effect on caspase-3 activity
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
68
104
115
60
The PI3K/Akt pathway also plays a key role in cell survival and
resistance to apoptosis [91]. Therefore the compound 11c was tested
for its pro-apoptotic effects. Caspase-3 activation is a final common step
for caspase-cascade activation through both the intrinsic and the ex-
trinsic pathways. Its activation ultimately leads to apoptosis therefore it
is known as the key executioner of apoptosis [92,93].To assess the pro-
apoptotic effects of the tested compounds the activity of caspase-3 was
evaluated in the collected cell lysates. Treatment of PC-3 cells with
compound 11c significantly (P < 0.0001) boosted caspase-3 activity
by 3 folds compared to control indicating that it possesses a proa-
poptotic activity (Fig. 4).
71
76
Ovarian Cancer
IGROV1
94
64
93
75
50
61
96
158
165
132
133
97
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
131
125
The aforementioned data indicated that the antiproliferative effect
of compound 11c is at least partly due to its inhibitory effect on Akt.
Additionally, compound 11c exhibited pro-apoptotic activity as evi-
denced by the increased level of active caspase-3. Further investigation
of the full biological profile of 11c is one of our future perspectives.
Renal Cancer
786–0
72
143
123
162
91
A498
133
83
ACHN
CAKI-1
RXF 393
SN12C
TK-10
84
184
96
187
96
141
144
114
117
2.7. Methods and computational details
UO-31
Prostate Cancer
2.7.1. Docking simulations against the DFG-out and DFG-in protein
conformations
PC-3
118
89
157
98
DU-145
Molecular docking calculations were carried out using the most
recent version of smina [94], a version of AutodockVina that offers
better control on docking and scoring parameters [95]. Two X-ray
crystal structures have been selected to carry out the docking simula-
tions, PDB codes: 4ASE [33] and 3WZD [96]. These two structures were
selected particularly because of being with high resolution and for
having a smaller number of missing residues than other crystal struc-
tures (see supporting info table, S1). The first crystal structure
Breast Cancer
MCF7
120
108
119
75
135
174
105
127
101
171
130
MDA-MB-231/ATCC
HS 578 T
BT-549
T-47D
107
167
93
MDA-MB-468
MEAN % Growth inhibition
347

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Table 4
Table 4 (continued)
Five dose assay results obtained for compounds 11a and 11c upon testing
against the 60 cancer cell lines of NCI screening program (the values are given
by µM).
Cell Name
11a
TGI
11c
GI50
LC50
GI50
TGI
LC50
Cell Name
11a
11c
BT-549
1.55
0.183
1.18
12.0
1.37
3.35
41.1
43.0
9.51
1.97
2.39
2.17
4.17
7.17
4.52
8.83
67.3
9.41
T-47D
GI50
TGI
LC50
GI50
TGI
LC50
MDA-MB-468
Leukemia
CCRF-CEM
HL-60(TB)
K-562
aUnderlined values of GI50 refer to superior potency to sorafenib. Underlined
values of LC50 refer to superior safety to sorafenib.
2.56
1.57
2.86
0.86
0.94
2.03
13.6
4.52
15.5
3.56
12.0
15.3
55.5
16.6
45.5
20.2
86.1
89.0
4.01
4.44
3.02
2.04
2.11
2.75
42.3
85.0
10.8
11.4
7.52
> 100
> 100
> 100
> 100
> 100
> 100
> 100
b
NT = not tested.
MOLT-4
RPMI-8226
SR
Non-Small Cell Lung Cancer
A549/ATCC
EKVX
1.79
8.74
7.30
10.0
4.15
4.06
4.40
11.4
16.4
2.39
35.2
30.5
35.1
21.7
33.5
32.0
37.5
94.7
NT
2.69
3.06
1.91
1.92
2.44
1.93
2.23
2.08
2.50
5.75
9.84
3.76
4.18
4.80
4.24
5.47
4.57
5.99
18.2
33.3
7.41
9.13
9.43
9.33
23.0
10.2
29.0
1.59
HOP-62
1.30
HOP-92
0.707
1.07
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
1.30
0.741
0.706
0.527
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
1.32
4.53
11.2
4.82
11.1
10.9
5.27
17.9
18.9
37.7
19.4
43.0
33.0
78.3
60.8
2.32
2.61
2.80
1.93
1.98
0.05
2.26
5.58
8.87
10.3
4.87
4.29
9.66
4.76
47.0
1.88
44.3
1.41
58.4
Fig. 3. Effect of compounds 11c on Akt (a) and ERK (b) phosphorylation and
0.579
0.615
0.207
3.07
55.9
activation using ELISA.
HT29
9.31
KM12
> 100
> 100
SW-620
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
1.55
0.73
0.23
2.29
0.191
1.27
11.2
5.87
17.3
17.7
5.69
3.23
69.1
26.7
> 100
74.1
37.7
8.18
2.44
1.38
0.612
2.66
1.24
1.50
7.32
2.94
2.47
9.58
3.41
3.06
26.5
6.29
6.58
31.5
9.32
6.21
Melanoma
LOX IMVI
MALME-3M
M14
0.723
0.502
1.10
1.40
1.34
1.38
5.22
2.32
1.23
2.75
2.55
4.22
33.9
3.52
4.33
21.3
5.13
6.27
8.76
NTb
1.97
1.72
1.65
1.84
2.31
1.57
2.09
3.23
1.70
3.76
3.89
3.58
4.29
5.01
3.24
3.88
6.79
3.44
7.17
8.79
7.77
10.0
12.6
6.70
7.21
26.3
6.94
18.6
> 100
9.24
> 100
60.6
18.0
87.4
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Fig. 4. Effect of compound 11c on caspase-3 activity in PC-3 prostate cancer
cells. Data are mean
SD, (n = 3).
Ovarian Cancer
IGROV1
0.427
2.08
1.01
1.24
5.17
2.52
0.42
4.62
13.1
4.25
13.8
28.3
17.6
4.6
59.7
40.6
28.7
77.2
> 100
73.9
25.6
2.05
1.92
1.92
1.83
2.77
2.36
2.04
4.66
3.70
3.89
4.31
7.07
6.23
4.03
13.4
7.11
7.90
10.7
48.6
> 100
7.99
represents a DFG-out conformation of VEGFR-TK bound to Tivozanib
(PDB code: 4ASE) [33], whereas the second crystal structure represents
a DFG-in conformation of VEGFR-TK bound to Lenvatinib (PDB code:
3WZD) [96]. The two crystal structures were first downloaded from the
protein data bank and prepared using the protein preparation wizard in
the Schrodinger software package [42]. Ligand atoms were deleted and
missing atoms were added and titrable groups were adjusted at a pH of
7. Protein structures were then saved as PDB files and converted to
PDBQT files and used as inputs for smina. Ligand structures were pre-
pared using the ligprep [97] module of Schrodinger and saved as mol2
files for use in smina. In addition to the 16 novel inhibitors under in-
vestigation, we have also included 5 known VEGFR-2-TK crystallized
ligands to be used as a benchmarking set to our novel inhibitors and to
assess the performance of our computational protocol. These ligands
are Lenvatinib (PDB code: 3WZD) [96], Sorafenib (PDB code: 3WZE)
[96], Tivozanib (PDB code: 4ASE) [33], a naphthamide inhibitor (PDB
code: 3B8Q) [53] and a benzoxazine inhibitor (PDB code: 2RL5) [98].
For simplicity, these ligands will be denoted as LEN, SOR, TIV, NAP and
BEN, respectively. With the exception of LEN that targets a DFG-in
conformation of VEGFR-2-TK, all other ligands are known to target a
DFG-out conformation.
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal Cancer
786-0
1.30
0.21
1.63
2.08
1.04
1.14
0.724
0.499
4.34
0.896
11.1
12.1
2.76
5.78
2.56
2.84
19.1
11.7
36.2
51.3
7.34
> 100
7.43
16.5
1.56
2.37
1.45
2.30
1.53
2.10
2.38
2.04
3.52
6.24
3.25
5.94
3.29
4.78
6.45
4.17
7.95
23.4
7.27
25.0
7.06
13.5
31.4
8.55
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate Cancer
PC-3
1.09
3.36
11.4
10.7
75.4
1.55
3.20
3.24
11.9
6.76
DU-145
0.867
> 100
Breast Cancer
MCF7
0.234
1.39
1.90
4.92
3.37
> 100
58.1
1.85
1.96
2.0
4.56
4.03
7.95
> 100
8.32
MDA-MB-231/ATCC
HS 578T
0.299
> 100
> 100
348

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
The search space was limited to a 20 * 20 * 20 Å3 box around the
original ligand-binding site with and an exhaustiveness search para-
meter of 20 (default is 8). The best 3 scoring poses for each ligand were
selected and visually inspected for a proper filling of the designated
binding site. Poses that do not satisfy proper interaction with the hinge
residue, Cys919, were discarded. If none of the best three poses exhibits
the Cys919, we continue our search until a proper pose is found.
Successful poses were subjected to molecular dynamics (MD) simula-
tions and binding free energy calculations using the AMBER-MMGBSA
module [99]. Unless otherwise stated, only the top AMBER-MMGBSA
pose is reported.
2.7.2. Molecular dynamics simulations and binding free energy estimation
Simulated protein-ligand complexes were prepared with tleap using
the AMBER-FF99SB [100] forcefield for the protein and the GAFF for-
cefield for the ligands [101]. Missing ligand charges were calculated
using the AM1-BCC method of antechamber [102]. Each complex was
then immersed in a cubic 12 Å3 box of TIP3 water and neutralized with
counter ions and put to a 150 mM salt concentration by adding the
proper number of Na+ and Cl− ions. Each system was then subjected
to several rounds of minimization and equilibration simulations. In-
itially, 20,000 steps of energy minimizations were conducted in four
consecutive rounds in which ligands and protein atoms are constrained
to their original 3D coordinates and constrains are gradually relieved
from 100, 50, 5, 1 kcal/mol. This was followed by a non-constrained
energy minimization for 5000 steps. Each system was then brought to
300 K by a gradual heating NVT simulation over a 50,000 steps con-
strained simulation. This was followed by 4 constrained NPT equili-
bration simulations of 25,000 steps in which protein constrains were
gradually reduced from 5, 1, 0.1 and 0.01 kcal/mol constrains followed
by a final 25,000 steps un-constrained equilibration simulation. Pro-
duction simulations were carried out for 5 * 1 ns simulations and tra-
jectories were merged at the end to give a single 5 ns MD simulation
trajectory. Periodic boundary conditions and an integration time step of
1 fs were used for all simulations. Long-range electrostatic interactions
were computed using the Particle Mesh Ewald summation method
[103] and the short-range electrostatic interactions were truncated
after 9 Å. The binding free energies were calculated using the AMBER-
MMGBSA module and averaged over the 5 ns simulation trajectories.
MD simulations were carried out using the GPU implementation of
pmemd (pmemd.cuda) of the most recent version of AMBER bio-mo-
lecular simulation software.
Fig. 5. The (a) 3D and the (b) 2D ligand interaction of LEN bound to the DFG-in
conformation of the VEGFR-2 catalytic domain (PDB code: 3WZD). LEN forms a
very strong H-bond interaction with Cys919 in addition to Glu885 and
Asp1046.
2.7.3. The binding modes of the novel inhibitors compared to known
VEGFR-TK ligands
In the current study and as we were interested to determine the
targeted conformations by our novel compounds, two X-ray crystal
structures have been selected to predict the binding modes. Each
structure typifies a distinct conformation of the VEGFR-2 catalytic do-
main. The first structure, PDB code: 3WZD represents an active DFG-in
conformation, whereas the second structure, PDB code: 4ASE represents
an inactive DFG-out conformation of the VEGFR-2 catalytic domain.
Fig. 5 displays the structure of the DFG-in conformation, PDB code:
3WZD, whereas Fig. 6 displays a structure for the DFG-out conforma-
tion, PDB code: 4ASE, with the co-crystallized ligands Lenvatinib (LEN)
and Tivozanib (TIV), respectively. As we can see in the figures, LEN
binds to the DFG-in conformation and forms a strong H-bond with the
hinge residue Cys919 backbone amide group and extends only partially
to the side pocket. On the other hand, TIV forms H-bond with the hinge
residue Cys919, extends the bulky phenyl-methylisoxazol group deeply
into the side hydrophobic pocket and interacts with side pocket lipo-
philic residues, such as Ile888, Ile892, Val898, Leu1019 and Ile1044.
Both ligands also form additional H-bonds with Asp1046 and Glu885 in
the side pocket through their urea motif.
To gain more insight into the atomistic details of the interaction of
the novel inhibitors in comparison with known VEGFR-2 TK binders,
we have given the three-dimensional (3D) and the two-dimensional
(2D) ligand interaction diagrams for the most three active inhibitors
tested in vitro, compound 11b (IC50 5.4 nm), compound 11c (IC50
5.6 nm) and compound 11e (IC50 7 nm) in Fig. 7. As expected and being
bulky, these inhibitors seems to exclusively target the DFG-out con-
formations of VEGFR-2-TK. All inhibitors are able to interact very
strongly with the hinge residue, Cys919. The urea moieties in the side
group are capable of forming very productive H-bonding interactions
with the main chain amide nitrogen of Asp1046 and the side chain of
Glu885. The substituted chloro-phenyl ring extends deeply in the hy-
drophobic pocket forming additional lipophilic interactions with
nearby hydrophobic residues, such as Ile889, Ile892 and Leu1019.
To assess the stability of the generated complexes during the MD
simulation, the RMSD for the protein Cα and ligands heavy atoms of the
349

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
2.7.4. Structure activity relationship
Based on the exceptionally high potency of the three most potent
derivatives, compound 11b (IC50 5.4 nm), compound 11c (IC50 5.6 nm)
and compound 11e (IC50 7 nm), three main conclusions can be drawn.
First, it seems that an optimum size of substituents on the terminal phenyl
ring provides the best overall potency for the inhibitors to reach the single
digit nanomolar range. For example, chlorine-substituted aryl derivatives
exhibit much higher potencies than bromine-substituted derivatives in the
non-NeH linker containing derivatives. Typical examples are compounds
11b (IC50 5.4 nm) and 11c (IC50 5.6 nm) that show 100% inhibition of
enzymatic activities at 10 μM compared to 11d, which shows 86% in-
hibition at the same concentration. Also, the unsubstiuted phenyl ring in
11a, results in a significantly lower IC50 value of 83 nm compared to
compounds 11c and 11b. The second conclusion is that the presence of
the NeH linker significantly reduces the activity, presumably due to en-
tropic reasons. In other words, the conformational constraint introduced
by the direct attachment of the indazolyl group enhances the potency of
the compounds. This is typically the case when a derivative from the NeH
linker containing derivatives, such as compound 8b (48% inhibition),
compound 8a (100% inhibition, IC50 148 nm) and compound 8d (50%
inhibition) are compared with their corresponding non-NeH linker con-
taining analogues, which are compound 11b (100% inhibition, IC50
5.4 nm), compound 11c (100% inhibition, IC50 5.6 nm) and compound
11e (100% inhibition, IC50 7 nm), respectively. Herein, an important
conclusion can be drawn. In most reported VEGFR-2 type II inhibitors
NH-linker-based derivatives exhibited mediocre activity profiles, as
compared with the O-linker-based ones, where using O-linker increased
activity by several folds [53,58,59,61,104–106]. Our second rigidification
strategy represents another option for promoting activity, in addition to
using O-linker.
Another very interesting observation was the significance of the
presence of a 6,7-di-substituted quinzaoline ring. As discussed in earlier
studies for VEGFRs TKIs as well as other TKIs, the presence of these 6,7-
di-substituted quinazoline tails stabilizes the formed protein-ligand
complexes, presumably through a pushing effect on the ligand to tightly
pack the side hydrophobic pocket [104,107–109]. We have examined
this hypothesis for our new derivatives by monitoring the MD simula-
tion to see if the absence of these tails will disturb the formed com-
plexes. Indeed for the complexes formed by compounds 7a-d that lack
the 6,7-quinazoline tails, we observed that during the MD simulations,
the ligands partially migrate from the binding pocket, loosening the
very important anchoring H-bond with the hinge residue, Cys919 (see
supplementary file for examples). To quantify this behavior, we have
calculated the % occupancy of the H-bond formed by the quinazoline
nitrogen with Cys919 during the 5 ns MD for all inhibitors under study.
Percentage occupancy values are given in Table 5 together with the
binding energies as well as the in vitro assay data. As we can see in the
table, all inhibitors that lack the 6,7-quinazoline tails have significantly
lower occupancies for this Cys919 H-bond compared to their tail con-
taining analogues. For example, the % occupancy of this H-bond for
compound 7a (−4% inhibition) is given by 0.44%, for compound 7d
(10% inhibition) is given by 0.24%, for compound 7b (7% inhibition) is
given by 1.60% and for compound 7c (4% inhibition) is given by
0.44%. It is noteworthy to mention here that we also examined other
poses with higher binding energies for these compounds to make sure
that this phenomenon is not an artifact of the simulation and we ob-
served the same behavior. In contrast to this very low occupancy values
of the weak inhibitors, 7a-d for H-bonding with Cys919, we have ex-
amined the occupancy values for the strong inhibitors and we indeed
found that potent inhibitors enjoys a very high occupancy values. For
example, the most active inhibitors 11b, 11c and 11e exhibit occu-
pancies of 88.4%, 87.88% and 95.52%, respectively. The table also
gives the occupancy values for the H-bond formed with Asp1046.
Nevertheless, based on the data listed on the table, it can be concluded
that the occupancy values of this H-bond plays a much less critical role
in determining the activity of the compounds.
Fig. 6. The (a) 3D and the (b) 2D ligand interaction of TIV bound to the DFG-
out conformation of the VEGFR-2 catalytic domain (PDB code: 4ASE). TIV
forms a very strong H-bond interaction with Cys919 in addition to Glu885 and
Asp1046.
protein ligand complexes formed with compounds 11b, 11c, 11e, and
7c (one of the weakest inhibitors) have been calculated over the 5 ns
MD trajectory. Protein atoms reach a stable MD trajectory in the first
0.5 ns of the simulation with minimal fluctuations after that with an
average RMSD value of approximately 2.0 Å. Maximum RMSD fluc-
tuations is reached by the Cα atoms of complex formed with compound
7c which reaches a maximum of 2.7 Å. In contrast to the relatively
stable RMSD trajectories for the protein atoms, RMSD values for ligand
atoms show obvious variations between the most active compounds
that are compounds 11b, 11c and 11e and the weak inhibitor, com-
pound 7c. Also, the ligands atoms of compounds 11b, 11c and 11e
exhibit very stable RMSD values of below 1.0 Å in contrast to the atomic
RMSD value of compound 7c, which displays an average RMSD value of
approximately 1.7 Å and a maximum of 2.2 Å (see supplementary).
350

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Fig. 7. The 3D and 2D ligand interaction diagrams of compounds 11b (upper panel), compound 11c (middle panel) and compound 11e (lower panel) bound to the
DFG-out conformation of VEGFR2 TK domain. All inhibitors are capable of forming strong interaction.
351

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Table 5
The complete list of compounds studied with their calculated AMBER-MMGBSA binding energies, important H-bond %occupancies as well as the in vitro measured %
inhibition at 10 μM and and the IC50 values. The table also gives the Pearson correlation of the calculated binding energies and the IC50 data, which is given by 0.64.
With the exception of LEN and 5, which are predicted to bind to the DFG-in conformation 3WZD crystal structure, all other inhibitors are predicted to bind to the
4ASE that typifies a DFG-out conformation [7,110–113].
Compound name Binding
Cys919 H-bond
Average Cys919 H-
Asp1046 H-bond
Average Asp1046 H-
IC50 (nM) % Inhibition at
energy
occupancy (%)
bond (Ang.)
occupancy (%)
bond (Ang.)
10 μM
NAP1
BEN2
11e
8d
−58.23
−56.97
−52.72
−48.26
−49.83
−52.48
−48.35
−47.71
−46.95
−46.17
−43.10
−45.86
−42.26
−48.79
−42.49
−39.44
−39.66
−37.49
−35.54
−38.46
–22.46
97.44
97.44
95.52
95.72
94.44
95.04
63.6
3.059
3.058
3.103
3.133
3.14
99.24
99.52
99.96
100
2.936
2.934
2.817
2.852
2.822
2.968
2.856
2.98
0.5
0.5
7
–
–
100
50
100
–
–
8a
100
148
0.16
–
TIV3
8b
3.067
3.118
3.107
3.133
3.213
3.109
3.126
3.141
3.089
3.41
96.4
99.88
84.96
99.96
99.2
48
100
100
–
11c
11a
SOR4
8c
87.88
95.76
87.6
5.6
83
90
–
2.872
2.904
2.897
3.011
3.109
2.894
2.893
2.951
2.872
3.195
2.9
96.44
88.4
97.48
62.0
86
100
86
–
11b
11d
LEN5
7a
5.4
–
67.92
95.04
0.44
60.6
98.44
99.84
99.56
98.68
94.8
4
–
−4
7
7b
1.6
3.385
3.136
3.11
–
7d
2.96
–
10
100
4
8f
98.0
163
–
7c
0.44
3.149
3.084
3.305
97.8
8e
2.08
97.04
2.98
–
94
6
5
26.76
–
rPearson with IC50 0.64
2.7.5. The AMBER-MMGBSA binding energies of the novel inhibitors and
the known ligands
restriction strategies. The second design strategy yielded more active
compounds that act as type II kinase inhibitors, supporting the rationale
of the design. Compounds 11b, 11c and 11e were the most active
compounds with IC50 of 5.4, 5.6 and 7 nM respectively. Molecular dy-
namics study were made to illustrate the difference in behaviour be-
tween the most active compounds 11b, 11c and 11e and one of the
weakest inhibitors 7c. Compound 7c showed high RMSD values for the
ligand atoms in comparison to the low RMSD values for compounds
11b, 11c and 11e. Also, the dynamics study showed the effect of 6,7-
disubstitution on quinazoline ring. It demonstrated that this dis-
ubstitution that extend to the solvent accessible area stabilized the
compound in the binding site and increased the % occupancy of the
essential hydrogen bond with Cys919. Additionally, this work supports
a relatively novel design strategy for VEGFR-2 inhibitors that avoids
using the unfavourable NH linker. Instead the linker is included in a
ring, yielding a less flexible structure. In Fig. 8, we introduce a gra-
phical representation of the SAR of the most potent series in this work
11a-e.
Table 5 also lists the calculated AMBER-MMGBSA binding energy
data for the new inhibitors under study as well as the known VEGFR-2
TKIs ligands. Including these known ligands in our calculations was
very important to act as positive controls for the new inhibitors and to
benchmark the results. It is noteworthy to mention here that the high
flexibility of VEGFR-2 TKIs implies that in addition to considering the
binding affinity of the ligands to the target in order to quantify the
potency, it is also important to take binding kinetics measures into
account. Examples of these measures to determine the binding kinetics
is the residence time (mins), the association (kon, S−1 M−1) and dis-
sociation (koff, S−1) rate constants. This is different from the other more
commonly used equilibrium constants, such as the equilibrium dis-
sociation constant Kd (nmol/L) or IC50 values that are typically used as
measures of drug potency. In the current study, we will consider the
IC50 values as the main measure for drug potency to be correlated with
the calculated binding energies.
As can be seen in Table 5, the AMBER-MMGBSA data achieves a good
correlation (rpearson = 0.64) with the in vitro measured IC50 values for the
compounds. All known VEGFR-2 TKIs exhibit low binding energies with
NAP and BEN have the lowest binding energies among the whole list of
inhibitors under investigation, with values given by −58.23 kcal/mol and
−56.97 kcal/mol, respectively. Compound 11e, the third most potent
compound in our novel series of compounds (100% inhibition,
IC50 = 7 nM), exhibits the lowest binding energy in our series,
−52.72 kcal/mol. Also, very weakly active compounds, exhibit very poor
binding energies as well. For example, compound 7c (4% inhibition ex-
hibits the highest binding energy among the whole list of studied inhibitors,
that is −35.54 kcal/mol. However, binding energy failed to correlate with
the activity of some compounds such as 7a, 8b, and 8d. On the other side,
the occupancy of Cys919 H- bond was better correlated with the activity,
highlighting its pronounced effect on VEGFR-2 inhibitory action.
In order to assess the in vitro cellular antiangiogenic activity, com-
pounds 11b and 11c were tested against HUVEC cell line, where they
showed 80% and 99.6% inhibitory activity respectively.
In vitro anticancer activity was evaluated for 4 compounds selected
by NCI. Compounds 11a and 11c showed remarkable mean % GI
against NCI-60 panel of cell lines of 93% and 130%, respectively, and
they were selected for further five-dose evaluation. As compared to
sorafenib, both compounds showed superior potency and safety profiles
against numerous cell lines. Further investigation of the underlying
mechanism of in vitro antiproliferative action demonstrated that the
mechanism is a combination of VEGFR-2 kinase inhibition, interfering
with the downstream AKT pathway, and induction of apoptosis through
the action on caspase-3.
In brief, in this work, conformational restriction was employed to
develop a potent VEGFR-2 inhibitor: 11c (IC50 = 5.6 nM), that can
exert anticancer activity through angiogenesis-dependent and in-
dependent modes of action, with reasonable safety. Computational
methods were implemented to develop SAR of the synthesized com-
pounds, which can facilitate future optimization. Thus, 11c is a suitable
candidate for further development and preclinical studies.
3. Conclusion
Two series of indazole-based compounds were designed, synthe-
sized and biologically evaluated, using two different conformational
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Fig. 8. SAR representation of our novel series of compounds. The figure shows 11c in the binding site of VEGFR-2 kinase.
4. Experimental
refluxed for 3 h, where a heavy yellow ppt of HCl salt of the product
was formed. The mixture was, then, cooled to r.t., filtered and the ppt.
was washed thoroughly with isopropanol and ether and heated with
mixture of 65 mL of water and ethanol with ratio (4:1) that was basified
with aqueous ammonia. Mixture was cooled and filtered to yield the
product that was washed with water and dried.
4.1. Chemistry
All chemicals used were purchased from Aldrich (USA), Alfa-Aesar
Organics (USA) and SD-fine (India) and used without further purifica-
tion. Melting points determined in one end open capillary tubes using
Stuart Scientific apparatus and were uncorrected. 1HNMR spectra were
recorded in δ scale given in ppm on a Joel 300 MHz or 400 MHz
spectrophotometer and referred to TMS at Microanalytical Center at
Cairo University or the faculty of Pharmacy, Cairo University respec-
tively. 13C spectra were run at 126 MHz in dimethylsulfoxide
(DMSO‑d6). Chemical shifts are quoted in δ and were related to that of
the solvents at Microanalytical Center at Cairo University. Analytical
thin layer chromatography (TLC) was performed on silica gel 60 F254
packed on Aluminium sheets, purchased from Merck (Merck,
Darmstadt, Germany), the developing solvents were DCM/MeOH (9:1),
with visualization under U.V. light (254 nm). FT-IR spectra were re-
corded on a Perkin-Elmer spectrophotometer (Ain Shams University).
Elemental analyses were performed at Al-azhar university.
4.1.2.1. N-(1H-indazol-5-yl)quinazolin-4-amine (6a). The product was
separated as off- white solid, (0.96 g, 48.92%) m.p.: 232–234 °C. 1H
NMR (300 MHz, DMSO‑d6) δ 13.19 (s, 1H, NH), 11.18 (s, 1H, NH), 8.82
(d, J = 8.3 Hz, 1H, ArH), 8.76 (s, 1H, ArH), 8.13 (s, 1H, ArH),
8.09–7.98 (m, 2H, ArH), 7.96 (d J = 8.6, 1H, ArH), 7.77–7.63 (m,
2H, ArH), 7.62 (s, 1H, ArH).
4.1.2.2. N-(1H-indazol-5-yl)-6,7-dimethoxyquinazolin-4-amine
(6b). The product was separated as off- white solid,(1.6 g, 81.54%)
m.p.: 266–268 °C. 1H NMR (300 MHz, DMSO‑d6) δ12.99 (s, 1H, NH),
9.50 (s, 1H, NH), 8.40 (s, 1H, ArH), 8.13 (s, 1H, ArH), 8.07 (s, 1H, ArH),
7.87 (s, 1H, ArH), 7.64 (d, J = 8.2, 1H, ArH), 7.55 (d, J = 8.2, 1H,
ArH), 7.17 (s, 1H, ArH), 3.96 (s, 3H, OCH3), 3.93 (s, 3H, OCH3).
Compounds 2, 3 and 4 were prepared according to the procedures
reported in the literature [69,114,115].
4.1.3. General procedure for the preparation of target compounds (7a–d)
Compound 6a (0.2 g, 0.765 mmol) was dissolved in dry THF
(20 mL), to which isocyanate (0.765 mmol) was added. The mixture
was stirred at r.t. for 24 hrs and then, it was filtered and the ppt. was
washed with excess THF and ether and dried to give the titled com-
pounds 7a-d.
4.1.1. 5-((2-Chloropyrimidin-4-yl)amino)-N-phenyl-1H-indazole-1-
carboxamide (5)
To a solution of 4 (0.2 g, 0.814 mmol) in dry THF (15 mL) phenyl
isocyanate (0.097 g, 0.814 mmol) was added. The resulted solution was
stirred at room temperature for 24 h, then, solvent was removed under
vacuo and resulting solid was washed thoroughly with methanol and
diethyl ether to give the titled compound as pale pink powder (148 mg,
49.84%). m.p.: > 250 °C, 1H NMR (300 MHz, DMSO‑d6) δ 10.34 (s, 1H,
NH), 10.29 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.32 (s, 1H, ArH), 8.26 (d,
J = 9, 1H, ArH), 8.19 (d, J = 5.9, 1H, ArH), 7.80 (d, J = 8.5, 2H, ArH),
7.65 (d, J = 9, 1H, ArH), 7.39 (t, J = 8.5, 2H, ArH), 7.15 (t, J = 8.5,
1H, ArH) 6.82 (d, J = 5.9, 1H, ArH), Anal. Calcd for C18H13ClN6O: C,
59.27; H, 3.59; N, 23.04, Found: C, 59.42; H, 3.63; N, 23.17.
4.1.3.1. N-(3-Chloro-4-methylphenyl)-5-(quinazolin-4-ylamino)-1H-
indazole-1-carboxamide (7a). The product was separated as pale yellow
powder. (72 mg, 21.93%) m.p.: 216–218 °C.1H NMR (300 MHz,
DMSO‑d6) δ 11.67 (s, 1H, NH), 10.50 (s, 1H, NH), 8.91 (s, 1H, ArH),
8.76 (d, J = 8.3 Hz, 1H. ArH), 8.17 (s, 1H, ArH), 8.16–8.08 (m, 1H,
ArH), 8.05 (s, 1H, ArH), 7.95–7.83 (m, 2H, ArH), 7.67 (d, J = 8.6 Hz,
1H, ArH), 7.59 (d, J = 8.9 Hz, 1H, ArH), 7.44 (d, J = 8.1 Hz, 1H, ArH),
7.38 (s, 1H, ArH), 7.21 (d, J = 7.9 Hz, 1H, ArH), 2.32 (s, 3H, CH3).
4.1.2. General procedure for preparation of target compounds (6a) and
(6b)
4.1.3.2. N-(3,4-Dichlorophenyl)-5-(quinazolin-4-ylamino)-1H-indazole-1-
carboxamide (7b). The product was separated as white powder. (40 mg,
11.63%) m.p.: 226–229 °C, 1H NMR (300 MHz, DMSO‑d6) δ 11.75 (s,
1H, NH), 10.73 (s, 1H, NH), 8.97 (s, 1H, ArH), 8.78 (d, J = 8.5 Hz, 1H,
Mixture of 5-aminoindazole 3 (1 g, 7.51 mmol) and the appropriate
quinazoline derivative (7.51 mmol) in isopropanol (38 mL) was
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ArH), 8.61 (s, 1H, ArH), 8.44 (d, J = 9.0 Hz, 1H, ArH), 8.25 (d,
J = 1.5 Hz, 1H, ArH), 8.18 (d, J = 2.3 Hz, 1H, ArH), 8.13 (t,
J = 8.3 Hz, 1H, ArH), 7.95–7.88 (m, 3H, ArH), 7.85 (dd, J = 9.0,
2.3 Hz, 1H, ArH), 7.61 (dd, J = 8.9, 1.5 Hz, 1H, ArH).
1H, ArH), 6.90 (s, 1H, ArH), 4.15–3.81 (m, 12H, OCH3). Anal. Calcd for
C
26H23ClN6O5: C, 58.38; H, 4.33; N, 15.71, Found: C, 58.54H, 4.37; N,
15.87.
4.1.4.5. 5-((6,7-Dimethoxyquinazolin-4-yl)amino)-N-phenyl-1H-indazole-
1-carboxamide (8e). The product was separated as off-white powder.
(52 mg, 18.97%) m.p.: 216–218 °C, 1H NMR (300 MHz, DMSO‑d6) δ
11.10 (s, 2H, NH), 8.59 (s, 1H, ArH), 8.09 (s, 1H, ArH), 8.02 (s, 1H,
ArH), 7.92 (s, 1H, ArH), 7.59 (d, J = 8.7 Hz, 1H, ArH), 7.49 (d,
J = 8.9 Hz, 1H, ArH), 7.42–7.20 (m, 5H, ArH), 7.15 (s, 1H, ArH),
3.91 (s, 6H, OCH3). FT-IR (ύ max, cm−1): 3362 (NH), 3090 (CH
aromatic) 2991 (CH aliphatic), 1705 (C]O), 1625 (C]C), Anal. Calcd
for C24H20N6O3: C, 65.45; H, 4.58; N, 19.08; O, 10.90, Found: C, 65.57;
H, 4.56; N, 19.17; O, 10.88. MS: (Mwt.:440:m/z, 441 [M+, (38.16%)],
440 (43.42%), 98 (100%).
4.1.3.3. N-(3-Bromophenyl)-5-(quinazolin-4-ylamino)-1H-indazole-1-
carboxamide (7c). The product was separated as pale yellow powder.
(109 mg, 31%) m.p.: 214–218 °C, 1H NMR (300 MHz, DMSO‑d6) δ 11.67
(s, 1H, NH), 10.58 (s, 1H, NH), 8.97 (s, 1H, ArH), 8.76 (d, J = 8.5 Hz,
1H, ArH), 8.44 (d, J = 8.7, 1H, ArH), 8.24 (s, 1H, ArH), 8.15–8.04 (m,
2H, ArH), 7.88–7.82 (m, 2H, ArH), 7.60 (d, J = 8.8 Hz, 1H, ArH), 7.63
(d, J = 8.8 Hz, 1H, ArH), 7.40 (d, J = 8.7 Hz, 1H, ArH), 7.25–7.13 (m,
2H, ArH), Anal. Calcd for C22H15BrN6O: C, 57.53; H, 3.29; N, 18.30,
Found: C, 57.71; H, 3.25; N, 18.3.9.
4.1.3.4. N-(5-Chloro-2,4-dimethoxyphenyl)-5-(quinazolin-4-ylamino)-
1H-indazole-1-carboxamide (7d). The product was separated as yellow
powder. (320 mg, 88%) m.p.: 220–222 °C, 1H NMR (300 MHz,
DMSO‑d6) δ 11.66 (s, 1H, NH), 10.50 (s, 1H, NH), 8.88 (s, 1H, ArH),
8.76 (d, J = 8.5 Hz, 1H, ArH), 8.23–7.99 (m, 3H, ArH), 7.97–7.77 (m,
2H, ArH), 7.72–7.50 (m, 2H, ArH), 7.38 (s, 1H, ArH), 6.94 (s, 1H, ArH),
3.98 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), Anal. Calcd for C24H19ClN6O3:
C, 60.70; H, 4.03; N, 17.70, Found: C, 60.84; H, 4.07; N, 17.83.
4.1.4.6. 5-((6,7-Dimethoxyquinazolin-4-yl)amino)-N-phenyl-1H-indazole-
1-carbothioamide (8f). The product was separated as white powder.
(38 mg, 13.37%) m.p.: 208–211 °C, 1H NMR (300 MHz, DMSO‑d6) δ
11.22 (s, 1H, NH), 9.2 (s, 1H, NH), 8.78 (s, 1H, ArH), 8.13 (s, 1H, ArH),
8.10 (s, 1H, ArH), 7.98 (s, 1H, ArH), 7.66 (d, J = 8.9 Hz, 1H, ArH), 7.53
(d, J = 8.9 Hz, 1H, ArH), 7.47–7.33 (m, 5H, ArH), 7.27 (s, 1H, ArH),
3.99 (s, 6H, OCH3).
4.1.4. General procedure for preparation of target compounds (8a-f)
Compound 6b (0.2 g, 0.622 mmol) was dissolved in dry DMF
(7.5 mL), to which appropriate isocyanate or isothiocyanate
(0.622 mmol) was added. The mixture was stirred at r.t. for 24 h,
poured on ice, stirred for 30 min. and filtered. The resulted ppt was
washed with acetic acid, then excess water and left to dry, then washed
with ether and dried to give the titled compounds 8a-f.
4.1.5. 6,7-Dimethoxy-4-(5-nitro-1H-indazol-1-yl)quinazoline (9)
To a solution of 5-nitroindazole 2 (3 g, 18.39 mmol) in dry DMF
(55 mL) cooled to 0 °C, NaH (0.735 g, 18.39 mmol, 60% in mineral oil)
was added. The mixture was stirred at r.t. for 2 h, then cooled to 0 °C once
more before addition of 4-chloro-6,7-dimethoxyquinazoline (4.13 g,
18.39 mmol). The mixture was stirred for 4 h, then quenched with water
and filtered. The resulted residue was crystallized from methanol and
washed with ether to yield the product as dark orange powder. (5.4 g,
83.58%), m.p.: 236–239 °C, 1H NMR (300 MHz, DMSO‑d6) δ 9.06 (s, 1H,
ArH), 8.96 (s, 1H, ArH), 8.87 (d, J = 2.2 Hz, 1H, ArH), 8.64 (d,
J = 9.1 Hz, 1H, ArH), 8.40 (d, J = 9.1 Hz, 1H, ArH), 8.32 (s, 1H, ArH),
7.47 (s, 1H, ArH), 4.02 (s, 3H, OCH3), 3.92 (s, 3H, OCH3). FT-IR (ύ max,
cm−1): 3111 (CH aromatic), 2835 (CH aliphatic), 1616 (C]C), 1567,
1344 (NO2), Anal. Calcd for C17H13N5O4: C, 58.12; H, 3.73; N, 19.93; O,
18.22, Found: C, 58.29; H, 3.71; N, 20.18; O, 18.31
4.1.4.1. N-(3-Chloro-4-methylphenyl)-5-((6,7-dimethoxyquinazolin-4-yl)
amino)-1H-indazole-1-carboxamide (8a). The product was separated as
yellow powder.(50 mg, 16.43%) m.p.: 196–198 °C. 1H NMR (400 MHz,
DMSO‑d6) δ 10.58 (s, 1H, NH), 9.81 (s, 1H, NH), 8.58 (s, 1H, ArH), 8.53
(s, 1H, ArH), 8.45 (d, J = 2.1 Hz, 1H, ArH), 8.35 (d, J = 8.9 Hz, 1H,
ArH), 8.15 (s, 1H, ArH), 7.92 (d, J = 9 Hz, 1H, ArH), 7.87–7.82 (m, 1H,
ArH), 7.34 (s, 1H, ArH), 7.22 (s, 1H, ArH), 7.17 (d, J = 8.0 Hz, 1H,
ArH), 3.99 (s, 3H,OCH3), 3.96 (s, 3H,OCH3), 1.92 (s, 3H, CH3). Anal.
Calcd for C25H21ClN6O3: C, 61.41; H, 4.33; N, 17.19, Found: C, 61.59;
H, 4.31; N, 17.32.
4.1.6. 1-(6,7-Dimethoxyquinazolin-4-yl)-1H-indazol-5-amine (10)
A mixture of compound 9 (5 g, 14.23 mmol) and 10% Pd/C (0.5 g.)
in THF (200 mL) was heated to 40 °C under H2 atmosphere (30 psi) for
12 h, after which the mixture was filtered and the filtrate was evapo-
rated under vacuo to yield the titled compound as yellow solid (3.3 g,
72.16%) to be used in situ in the following step. The purity of the
compound was monitored using TLC (DCM/Methanol 9:1).
4.1.4.2. N-(3,4-Dichlorophenyl)-5-((6,7-dimethoxyquinazolin-4-yl)
amino)-1H-indazole-1-carboxamide (8b). The product was separated as
yellow powder.(72 mg, 22.71%) m.p.: 202–205 °C . 1H NMR (300 MHz,
DMSO‑d6) δ 11.20 (s, 1H, NH), 9.32 (s, 1H, NH), 8.74 (s, 1H, ArH), 8.10
(m, 2H, ArH), 7.96 (s, 1H, ArH), 7.85 (s, 1H, ArH), 7.49–7.18 (m, 4H,
ArH), 6.84 (s, 1H, ArH), 3.97 (s, 6H, OCH3).
4.1.7. General procedure for preparation of target compounds (11a-e)
Compound 10 (0.3 g, 0.93 mmol) is dissolved into DCM (40 mL),
and then the appropriate isocyanate (0.93 mmol) is added to the solu-
tion. The mixture is stirred at room temperature for 24 hrs and then the
precipitate formed was filtered out and crystallized from mixture of
THF and DCM to give the titled products (11a-e).
4.1.4.3. N-(3-Bromophenyl)-5-((6,7-dimethoxyquinazolin-4-yl)amino)-
1H-indazole-1-carboxamide (8c). The product was separated as pale
yellow powder. (48 mg, 14.85%) m.p.: 198–201 °C, 1H NMR (300 MHz,
DMSO‑d6) δ 11.22 (s, 1H, NH), 9.05 (s, 1H, NH), 8.78 (s, 1H, ArH), 8.14
(s, 1H, ArH), 8.09 (s, 1H, ArH), 7.98 (s, 1H, ArH), 7.72–7.60 (m, 2H,
ArH), 7.53 (d, J = 8.8 Hz, 1H, ArH), 7.49–7.33 (m, 1H, ArH), 7.29 (s,
1H, ArH), 7.25–7.13 (m, 2H, ArH), 3.99 (s, 6H, OCH3). Anal. Calcd for
4.1.7.1. 1-(1-(6,7-Dimethoxyquinazolin-4-yl)-1H-indazol-5-yl)-3-
phenylurea (11a). The product was separated as pale yellow powder,
(140 mg, 34%) m.p.: 251–254 °C, 1H NMR (300 MHz, DMSO‑d6) δ 9.09
(s, 1H, NH), 8.98 (s, 1H, ArH), 8.78 (s, 1H, NH), 8.82–8.47 (m, 3H,
ArH), 8.24 (s, 1H, ArH), 7.62–7.10 (m, 6H, ArH), 6.99 (s, 1H, ArH),
4.02 (s, 3H, OCH3), 3.92 (s, 3H, OCH3).13C NMR (101 MHz, DMSO‑d6) δ
155.77, 154.40, 153.22, 152.30, 151.04, 150.23, 140.16, 139.55,
136.13, 129.26, 126.24, 124.17, 122.33, 121.69, 118.72, 116.51,
112.05, 111.76, 109.05, 107.37, 106.06, 105.53, 56.51, 56.12. Anal.
Calcd for C24H20N6O3: C, 65.45; H, 4.58; N, 19.08; O, 10.90, Found: C,
65.68; H, 4.64; N, 19.32; O, 10.88.
C
24H19BrN6O3: C, 55.50; H, 3.69; N, 16.18, Found: C, 55.61; H, 3.68; N,
16.41.
4.1.4.4. N-(5-Chloro-2,4-dimethoxyphenyl)-5-((6,7-
dimethoxyquinazolin-4-yl)amino)-1H-indazole-1-carboxamide (8d). The
product was separated as white powder. (35 mg, 10.51%) m.p.:
226–228 °C, 1H NMR (300 MHz, DMSO‑d6) δ 11.21 (s, 1H, NH), 9.46
(s, 1H, NH), 8.85 (s, 1H, ArH), 8.54 (s, 1H, ArH), 8.32 (s, 1H, ArH), 8.17
(s, 1H, ArH), 8.13–8.01 (m, 2H, ArH), 7.80 (s, 1H, ArH), 7.33–7.21 (m,
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4.1.7.2. 1-(3-Chloro-4-methylphenyl)-3-(1-(6,7-dimethoxyquinazolin-4-
yl)-1H-indazol-5-yl)urea (11b). The product was separated as buff
powder, (156 mg, 34.18%) m.p.: 253–258 °C. 1H NMR (300 MHz,
DMSO‑d6) δ 8.93 (s, 1H, ArH), 8.91 (s, 1H, NH), 8.78–8.70 (m, 2H,
NH, ArH), 8.59–8.44 (m, 2H, ArH), 8.09 (s, 1H, ArH), 7.64 (s, 1H, ArH),
7.49 (d, J = 9.2 Hz, 1H, ArH), 7.35 (d, J = 8.1 Hz, 1H, ArH), 7.22–7.14
(m, 2H, ArH), 3.98 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 2.21 (s, 3H, CH3).
FT-IR (ύ max, cm−1): 3292 (NH), 3137 (CH aromatic), 2927 (CH
aliphatic), 1638 (C]O), 1576 (C]C). Anal. Calcd for C25H21ClN6O3: C,
61.41; H, 4.33; N, 17.19, Found: C, 61.62; H, 4.30; N, 17.37.
concentration. The luminescence data were analyzed using the com-
puter software, Graphpad Prism. The difference between luminescence
intensities in the absence of Kinase (Lut) and in the presence of Kinase
(Luc) was defined as 100% activity (Lut – Luc). Using luminescence
signal (Lu) in the presence of the compound, % activity was calculated
as:
% activity = [(Lut − Lu)/(Lut − Luc)] × 100%, where Lu = the lu-
minescence intensity in the presence of the compound (all percent ac-
tivities below zero were shown zero in the table).
The values of % activity versus a series of compound concentrations
were then plotted using non-linear regression analysis of Sigmoidal
dose-response curve generated with the equation Y = B + (T − B)/
1 + 10((LogEC50-X)×Hill Slope), where Y = percent activity, B = minimum
percent activity, T = maximum percent activity, X = logarithm of
compound and Hill Slope = slope factor or Hill coefficient. The IC50
value was determined by the concentration causing a half-maximal
percent activity.
4.1.7.3. 1-(3,4-Dichlorophenyl)-3-(1-(6,7-dimethoxyquinazolin-4-yl)-1H-
indazol-5-yl)urea (11c). The product was separated as yellow powder,
(200 mg, 42%) m.p.: 233–238 °C. 1H NMR (300 MHz, DMSO‑d6) δ 9.21
(s, 1H, ArH), 9.13–8.79 (m, 3H, 2NH, 1ArH), 8.58 (d, J = 9.2 Hz, 1H,
ArH), 8.26–7.81 (m, 2H, ArH), 7.73 (d, J = 10.6 Hz, 1H, ArH),
7.86–7.45 (m, 2H, ArH), 7.35 (s, 2H, ArH), 3.95 (s, 6H, OCH3). FT-IR
(ύ max, cm−1): 3277 (NH), 3021 (CH aromatic), 2928 (CH aliphatic),
1636 (C]O), 1570 (C]C). Anal. Calcd for C24H18Cl2N6O3: C, 56.59; H,
3.56; N, 16.50, Found: C, 56.71; H, 3.60; N, 16.71.
The compounds were evaluated at the concentrations of: 1 nM,
50 nM, 100 nM, 1 µM, and 10 µM.
Concentration gap between 1 nM and 50 nM might be too big to
precisely measure the IC50 lower than 10 nM. Therefore, IC50 values of
compounds 11b, c and e were reassessed using concentrations of:
0.01 nM, 0.1 nM, 1 nM, 10 nM, and 100 nM. The results shown for these
compounds are the output of the second performed assay.
4.1.7.4. 1-(3-Bromophenyl)-3-(1-(6,7-dimethoxyquinazolin-4-yl)-1H-
indazol-5-yl)urea (11d). The product was separated as buff powder,
(120 mg, 24.75%) m.p.: 222–224 °C, 1H NMR (300 MHz, DMSO‑d6) δ
9.18 (s, 1H, ArH), 8.98 (s, 1H, ArH), 8.89 (s, 2H, NH), 8.62–8.51 (m,
2H, ArH), 8.15 (d, J = 2.1 Hz, 1H, ArH), 8.06–7.97 (m, 1H, ArH), 7.75
(d, J = 9.4 Hz, 1H, ArH), 7.53 (dd, J = 9.1, 2.2 Hz, 1H), 7.33–7.25 (m,
2H, ArH), 7.12 (s, 1H, ArH), 3.96 (s, 3H, OCH3), 3.93 (s, 3H, OCH3). FT-
IR (ύ max, cm−1): 3285 (NH), 3129 (CH aromatic), 2931 (CH
aliphatic), 1638 (C]O), 1600 (C]C). Anal. Calcd forC24H19BrN6O3:
C, 55.50; H, 3.69; N, 16.18, Found: C, 55.67; H, 3.73; N, 16.34.
4.3. In vitro HUVEC Anti-proliferative assay
This assay was also carried out in BPS Bioscience Corporation, San
Diego, CA, USA (www.bpsbioscience.com). HUVEC cells were cultured
in Medium 200 with 2% large vessel endothelial supplement (LVES)
and 1% Pen-strep. To perform the proliferation assay, HUVEC cells
were seeded at 5000 cells/50 μl/well in a 96-well black clear-bottom
tissue culture plate. Cells were incubated at 37 °C and 5% CO2 over-
night to allow them to recover and reattach. All the experiments were
done with 0.1% DMSO in the growth medium.
4.1.7.5. 1-(5-Chloro-2,4-dimethoxyphenyl)-3-(1-(6,7-
dimethoxyquinazolin-4-yl)-1H-indazol-5-yl)urea (11e). The product was
separated as bright yellow powder, (216 mg, 44.5%) m.p.: 268–270 °C,
1H NMR (300 MHz, DMSO‑d6) δ 9.56 (s, 1H, NH), 9.01 (s, 1H, ArH),
8.65 (s, 1H, ArH), 8.61 (s, 1H, ArH), 8.57 (d, J = 9.2 Hz, 1H, ArH), 8.23
(d, 2H, ArH, NH), 8.19 (d, J = 1.4 Hz, 1H, ArH), 7.49 (d, J = 9.4 Hz,
1H, ArH), 7.42 (s, 1H, ArH), 6.87 (s, 1H, ArH), 4.02 (s, 3H, OCH3),
3.96–3.92 (m, 6H, OCH3), 3.87 (s, 3H, OCH3).13C NMR (101 MHz,
DMSO‑d6) δ 154.36, 153.08, 152.27, 151.79, 151.01, 149.98, 148.16,
139.52, 136.21, 126.24, 124.82, 122.87, 121.30, 119.87, 116.57,
111.91, 108.61, 107.33, 105.57, 98.48, 56.90, 56.47, 56.08. Anal.
Calcd for C26H23ClN6O5: C, 58.38; H, 4.33; N, 15.71, Found: C, 58.54;
H, 4.38; N, 15.79.
Next day cells were treated with test compounds for 72 h. After
treatment, cell proliferation was measured by Fluorescent quantitation
of alamarBlue reagent. The alamarBlue assay incorporates a fluoro-
metric/colorimetric growth indicator based on detection of metabolic
activity. Specifically, resazurin, the active ingredient in the alamarBlue
reagent, is blue in color and virtually non-fluorescent. Upon entering
cells, resazurin is reduced to resorufin, a compound that is red in color
and highly fluorescent. Continued cell growth maintains a reduced
environment, therefore increasing the overall fluorescence and color of
the media surrounding cells. Our experiment has shown that the
fluorescence intensity of alamarBlue reagent was directly proportional
to cell number. To perform the alamarBlue assay, 10 μl of alamarBlue
reagent was added to each well and the plate was incubated at 37 °C for
an additional 2 h.
4.2. In vitro VEGFR-2 tyrosine kinase activity.
Evaluation of the in vitro VEGFR-2 kinase activity was carried out in
BPS Bioscience Corporation, San Diego, CA, USA (www.bpsbioscience.
com). The assay was performed using Kinase-Glo Plus luminescence
kinase assay kit (Promega). It measures kinase activity by quantitating
the amount of ATP remaining in solution following a kinase reaction.
The luminescent signal from the assay is correlated with the amount of
ATP present and is inversely correlated with the amount of kinase ac-
tivity. The compounds were diluted in 10% DMSO and 5 µl of the di-
lution was added to a 50 µl reaction so that the final concentration of
DMSO is 1% in all of reactions. All of the enzymatic reactions were
conducted at 30 °C for 40 min. The 50 µl reaction mixture contains
40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1 mg/ml BSA, 1 mM DTT, 10 µM
ATP, Kinase substrate and the enzyme.
Fluorescence intensity was measured at an excitation of 530 nm and
an emission of 590 nm using a BioTek SynergyTM 2 microplate reader.
The reading of the background was reported.
Fluorescent intensity data were analyzed using the computer soft-
ware, Graphpad Prism. In the absence of the compound, the fluorescent
intensity (Ft) in each data set was defined as 100%. In the absence of
cells, the fluorescent intensity (Fb) in each data set was defined as 0%.
The percent cell in the presence of each compound was calculated ac-
cording to the following equation: %cell = (F − Fb)/(Ft − Fb), where
F = the fluorescent intensity in the presence of the compound, Fb = the
fluorescent intensity in the absence of cells, and Ft = the fluorescent
intensity in the absence of the compound.
After the enzymatic reaction, 50 µl of Kinase-Glo Plus Luminescence
kinase assay solution (Promega) was added to each reaction and in-
cubate the plate for 5 min at room temperature. Luminescence signal
was measured using a BioTek Synergy 2 microplate reader.
The values of % cell versus a series of compound concentrations
were then plotted using non-linear regression analysis of Sigmoidal
dose-response curve generated with the equation Y = B+(T − B)/
((LogEC-X)×Hill
1 + 10
Slope), where Y = percent cell, B = minimum per-
50
Kinase activity assays were performed in duplicate at each
cent cell, T = maximum percent cell, X = logarithm of compound and
355

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
Hill Slope = slope factor or Hill coefficient. The reported values re-
present the average of the three experiments. The IC50 value was de-
termined by the concentration causing a half-maximal percent activity.
calculated. Growth inhibition by 50% (GI50) was calculated by
[(Ti − Tz)/(C − Tz)j × 100 = 50, it is the drug concentration that
makes a 50% reduction in the net protein increase (as measured by SRB
staining) in control cells during the drug incubation. Concentration of
the drug that leads to total growth inhibition (TGI) was calculated by
Ti = Tz. The LC50 is the drug concentration that causes a 50% reduction
in the measured protein at the end of the drug treatment as compared to
that at the beginning. It indicates the net loss of cells after the treat-
ment. It was calculated using the formula: [(Ti − Tz)/
Tzj × 100 = −50. Values were calculated for each of these three
parameters, if the level of activity is reached. Nevertheless, when the
effect was not reached or was exceeded, the value for that parameter
was expressed as greater or less than the maximum or minimum con-
centration tested.
4.4. In vitro HUVEC IC50 determination:
The determination of IC50 of 11c against HUVEC cell line was car-
ried out at Vacsera-Egypt, where the HUVEC umbilical vein endothelial
cells, human (Life Technologies#C-003-5C) served as the cells’ source,
in Medium 200 (Life Technologies#M-200-500), with large vessel en-
dothelial supplement (LVES) (Life Technologies#A14608-01) and Pen-
step (Hyclone#SV30010). Alamar Blue (Life Technologies#DAL1025)
was used as the fluorescent reagent (see supplementary file).
4.5. In vitro Anti-proliferative activity against NCI-60 panel
4.6. Effect on Akt and ERK phsophorylation/activation
The in vitro anticancer activity of the newly synthesized compounds
was evaluated by the National Cancer Institute anticancer screening
program (National Cancer Institute Bethesda, Maryland, USA). The
human tumour cell lines of the cancer-screening panel are grown in
RPMI-1640 medium. This medium contains 5% fetal bovine serum and
2 mM L-glutamine. To achieve typical screening experiment, cells were
inoculated into 96 well microtiter plates in 100 pL. The plating densities
ranged from 5000 to 40,000 cells/well. This depended on the doubling
time of individual cell lines. After the cell inoculation, incubation of the
microtiter plates at 37 °C, 5% CO2, 95% air and 100% relative humidity
for 24 h was done, before adding the experimental drugs.
The effect of PT on AKT activation in PC-3 cells was assessed using
phospho-AKT 1/2/3 (Ser473) and phospho-ERK1/2 InstantOne™ ELISA
kit (eBiosciences, San Diego, CA). These kits were developed to detect
phosphorylation at AKT1/2/3(Ser473) and ERK1/2(Thr202/Tyr204,
Thr185/Tyr187) respectively. PC-3 cells were seeded into 96- well
plates at a density of 3 × 104 cells/well and incubated overnight to
allow for attachment. The tested compounds were added at con-
centrations equal to the IC50. After 48 h of exposure, the medium was
discarded and the wells were washed twice with Hank’s buffered salt
solution. Akt and ERK activity was determined in the cell lysates as
previously described by Tolba and Abdel-Rahman [116].
After twenty four hours, two plates of each cell line are fixed in situ
with TCA, to get a measurement of the cell population at the time of
tested compound addition for each cell line (Tz). Experimental drugs
are dissolved in DMSO at 400-fold the required final maximum test
concentration and stored frozen, before being used. At the time of drug
addition, an aliquot of the frozen concentrate is melted and diluted to
twice the desired final maximum test concentration, where the com-
plete medium contained 50 mg/ml gentamicin.
4.7. Effect on caspase-3 activity
Caspase-3 activity was evaluated using colorimetric protease assay
kit (R&D Systems, Minneapolis, MN). At the end of exposures, about
2 × 106 cells were collected and the pellet was resuspended in lysis
buffer. Protein levels were determined using BCA protein assay
(BioVision, Inc., Milpitas, CA, USA). Aliquots of 50 µl of cell lysate (total
protein, 100 µg) were incubated with 5 µl of caspase-3 colorimetric
substrate (DEVD-pNA) at 37 °C for 2 h. The activity was expressed as
optical density of the released pNA measured at 405 nm using micro-
plate reader (ChroMate-4300, Palm City, FL).
In addition, 10-fold orlog serial dilutions were prepared to provide a
total of five drug concentrations plus control. Aliquots of 100 mL of
these drug dilutions were added to the appropriate microtiter wells that
contained 100 mL of medium, to give the required final drug con-
centrations. After adding the drug, the plates are incubated for an extra
48 h at 37 °C, 5% CO2, 95% air, and 100% relative humidity. As for the
adherent cells, the assay was terminated by the adding the cold TCA.
Cells were fixed in situ by the gentle addition of 50 mL of cold 50% (w/
v) TCA (final concentration, 10% TCA) and incubated at 4 °C for
60 min. The supernatant was discarded, and the plates were washed
five times, using tap water, then, it was air dried. Sulforhodamine B
(SRB) solution (100 mL) at 0.4% (w/v) in 1% acetic acid was added to
each well, and the plates were incubated at room temperature for
10 min.
4.8. Statistical analysis
Data of caspase-3 activity, Akt and ERK phsophorylation/activation,
are presented as means
SD. Individual groups were compared using
the two-tailed independent Student's t-test. Multiple group comparisons
were carried out using one-way analysis of variance (ANOVA) followed
by the Tukey-Kramer test for post-hoc analysis. Statistical significance
was accepted at a level of P < 0.05. All statistical analyses were per-
formed using GraphPadInStat software, version 3.05 (GraphPad
Software, Inc. La Jolla, CA, USA). Graphs were sketched using
GraphPad Prism software, version 5.00 (GraphPad Software, Inc. La
Jolla, CA, USA).
After staining, the unbound dye was removed by washing five times
with 1% acetic acid and then, the plates were air dried. Bound stain was
subsequently dissolved with 10 mM trizma base, and the absorbance
was read on an automated plate reader, using a wavelength of 515 nm.
For the suspension cells, the methodology applied was the same, but the
assay was terminated through fixing the settled cells at the bottom of
the wells by slowly adding 50 mL of 80% TCA, where the final con-
centration was 16% TCA). Seven absorbance measurements were re-
corded: [time zero, (Tz), control growth, (C), and test growth in the
presence of drug at the five concentration levels (Ti)], the percentage
growth is calculated at each of the drug concentrations levels.
Percentage growth inhibition was calculated as:
Acknowlegments
The authors are grateful to the Center of Excellence for Drug
Discovery and Development Research (Funded by the STDF, project no.
5251) at the Faculty of Pharmacy, Ain Shams University, for per-
forming the NMR spectroscopy of the compounds. Also, we are grateful
to NCI, Maryland USA for carrying out cytotoxicity study.
[(Ti − Tz)/(C − Tz)] × 100 for concentrations for which Ti ≥ Tz
[(Ti − Tz)/Tz] × 100 for concentrations for which Ti < Tz.
Appendix A. Supplementary material
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2018.10.071.
For each experimental agent, three dose response parameters were
356

N.M.Y. Elsayed et al.
BioorganicChemistry82(2019)340–359
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