Scaffold hopping and redesign approaches for quinazoline based urea derivatives as potent VEGFR-2 inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.103961

In our attempt to discover effective anticancer agents, three series of novel quinazoline-based compounds have been designed, synthesized and tested as VEGFR-2 inhibitors. Five quinazoline ?2-carboxamide derivatives (5d, 5e, 5 h, 5i, 5j) revealed potent nanomolar VEGFR-2 inhibition with IC₅₀ values of 12.1, 40.3, 15.5, 13.1 and 57.4 nM, respectively, superior to that of the reference drug sorafenib (IC₅₀ 78.9 nM). Additionally, the quinazoline 2-carboxylate derivative bearing a fluorine substituent in middle ring (7a) showed IC₅₀ values of 14.8 nM. Most of the new synthesized compounds are examined on NCI sixty cell lines of human cancer cells. Furthermore, molecular modeling study was administered to realize any clarification of the binding mode in the inactive VEGFR-2 conformation that demonstrates compatible binding modes similar to those of sorafenib and regorafenib. Finally, several ADME descriptors were calculated through a predictive kinetic study.

Full text of DOI:10.1016/j.bioorg.2020.103961

Journal Pre-proofs
Scaffold hopping and redesign approaches for quinazoline based urea deriva-
tives as potent VEGFR-2 inhibitors
Soha R. Abd El Hadi, Deena S. Lasheen, Dalia H. Soliman, Eman Z. Elrazaz,
Khaled A.M. Abouzid
PII:
S0045-2068(20)30547-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.103961
YBIOO 103961
Reference:
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Bioorganic Chemistry
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20 May 2020
Please cite this article as: S.R. Abd El Hadi, D.S. Lasheen, D.H. Soliman, E.Z. Elrazaz, K.A.M. Abouzid,
Scaffold hopping and redesign approaches for quinazoline based urea derivatives as potent VEGFR-2 inhibitors,
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Scaffold hopping and redesign approaches for quinazoline
based urea derivatives as potent VEGFR-2 inhibitors
Soha R. Abd El Hadi a*, Deena S. Lasheenb, Dalia H. Solimana,c, Eman Z.
b,d
Elrazaz b, Khaled A. M. Abouzid *
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr city, Cairo,
P.O. Box 11829, Egypt.
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Abbassia, Cairo, P.O.
Box 11566, Egypt.
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al Azhar University, Cairo, P.O. Box 11471,
Egypt
d Department of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Sadat City, Sadat City,
32897, Egypt.
Abstract
In our attempt to discover effective anticancer agents, three series of novel quinazoline-
based compounds have been designed, synthesized and tested as VEGFR-2 inhibitors. Five quinazoline -
2-carboxamide derivatives (5d, 5e, 5h, 5i, 5j) revealed potent nanomolar VEGFR-2 inhibition with IC50
values of 12.1, 40.3, 15.5, 13.1 and 57.4 nM, respectively, superior to that of the reference drug
sorafenib (IC50 78.9 nM). Additionally, the quinazoline 2-carboxylate derivative bearing a fluorine
substituent in middle ring (7a) showed IC50 values of 14.8 nM. Most of the new synthesized compounds
are examined on NCI sixty cell lines of human cancer cells. Furthermore, molecular modeling study was
administered to realize any clarification of the binding mode in the inactive VEGFR-2 conformation that
demonstrates compatible binding modes similar to those of sorafenib and regorafenib. Finally, several
ADME descriptors were calculated through a predictive kinetic study.
Keywords: Quinazoline; VEGFR-2; inhibitory activity; docking study; ADME.
*Corresponding author: Khaled A.M. Abouzid, E-mail: khaled.abouzid@pharma.asu.edu.eg
and Soha R. Abd El Hadi, E-mail: soha-ramadan@eru.edu.eg

1 Introduction:
Agreeing with World Health Organization statistics (WHO), Cancer is considered the second
lethal disease around the world and accounted for 8.8 million demise in 2015. The number of latest
cases is predicted to upward thrust by concerning 70% over the next two decagons [1,2]. The
treatment of human malignancies continues to be a significant task for medicinal chemists, given
the substantial efforts and investments in this studies area. An important part in experimental
oncology research comprises the validation of targets controlling certain signaling pathways
involved in cancer cells proliferation and propagation. Therefore, there is a continuous urge to
develop new chemotherapeutic agents that may possess different mechanisms of action in
anticancer therapy.
Protein kinases represent the largest group of cellular targets for directed anticancer
therapy. Due to its predominant function in tumor angiogenesis, VEGFR has been well established
as a tool for cancer treatment [3].
Angiogenesis is crucial for neoplasm progression as cancer cells have a comparatively high
metabolic demand for chemical element and nutrients so as to survive [4]. Moreover, the tube
network of arteries and capillaries permits tumors metastasis to alternative body organs. Neoplasm
vasculature is very chaotic, broken, and ‘leaky’ thanks to magnified transcellular gaps,
fenestrations, inter-endothelial junctions and absent basement membrane [5].
VEGFR-2, conjointly referred to as FLK-1 in mice and KDR in humans, is that the
principal negotiator of epithelium cell permeableness, proliferation, invasion, and migration [6].
Ligand interaction with extracellular VEGFR-2 domain could induce kinase-active heterodimer
formation [7]. Consequently, ATP-binding site is exposed and autophosphorylated VEGFR-2 is
formed followed by activation of many downstream pathways, such as PI3K, p38MAPK, FAK,
Src, Akt that are sometimes overexpressed in several tumors. Also, VEGFR-2 is overexpressed in
many malignancies including ovarian, renal, melanoma, and medulloblastoma [8].
This stimulated research efforts towards therapeutic approaches focused on VEGFR-2 signal
inhibition exploitation specific drugs.
Hitherto, many attempts to modify sorafenib with the aim of identifying better multiple-
targeted drugs. Small-molecule VEGFR-2 inhibitors have arised as promising antiangiogenic
agents for treatment different types of cancer, which act by competing with adenosine triphosphate
(ATP) binding site of the VEGFR-2 intracellular kinase domain, through preventing the

intracellular cellular signaling pathway. To date, many small molecules of VEGFR-2 enzyme
inhibitors are approved for clinical use in treatment of different types of cancer as (Sorafenib [9],
Regorafenib [10], Lenvatinib [8], Cabozantinib, Sunitinib, Vandetanib, Cediranib Pazopanib and
Axitinib) [11]. (Figure 1).
In recent years, 5 of the thirty kinase inhibitors approved by the FDA have been shown to
contain the quinazoline framework [12]. This core has actually been considered a privileged
structure for inhibiting ATP-dependent kinases. The rationale for this study was supported the
employment of quinazoline scaffold as a pharmacophoric moiety to develop novel VEGFR-2
inhibitors. Particularly, sorafenib, regorafenib, Vandetanib and Cediranib were considered as lead
compounds for the design of these new targets (Figure 1).
X
H
H
Cl
H
N
H
N
N
N
N
O
N
N
N
O
N
Cl
O
O
O
O
N
CF3
N
H
N
H2N
H2N
S
N
O
H
O
O
x=H: Sorafenib ( IC50=90nM)
X=F: Regorafenib (IC50=4.2nM)
Pazobanib (IC50=30nM)
Lenvatinib (IC50=4nM)
H
N
N
O
Cl
H
N
H
H
S
O
N
N
N
N
H
O
O
O
N
F
N
F
O
O
H
O
N
N
H
Sunitinib (IC50=80nM)
Axitinib (IC50=0.25nM)
Cabozantinib (IC50=0.035nM)
F
Br
NH
F
HN
N
O
O
O
N
O
O
N
N
N
N
Cediranib (IC50=0.4nM)
Vandetanib (IC50=40nM)
Figure 1: Structure of FDA and some clinically approved VEGFR-2 Inhibitors with their corresponding
IC50 values.

2 Rationale and design:
Crystal structure of the complex between sorafenib and VEGFR-2, reveals that the pyridine
moiety forms hydrophobic interactions with a front hydrophobic pocket occupied by specific
amino acids residues [13,14]. Earlier studies published by our research group [15] have also shown
that hydrophobic interaction with the kinase front pocket complements the hydrogen bonding with
the hinge zone, raising residence time, that may be a key consider up the drug's efficiency and its
in vitro effectiveness [16].
Motivated by the aforementioned findings, scaffold hopping and redesign approaches were
applied to develop three series of novel quinazoline derivatives based mainly on ring fusion with
a more hydrophobic skeleton, quinazoline, to allow additional hydrophobic interaction with
residues of amino acid at the entrance of the hinge region.
Moreover, ester or amide functionalities were appended to this quinazoline core, at position
2, to explore their ability to form additional hydrogen bonding in the hinge region, particularly,
with Cys919.The design also comprised a bioisosteric modification strategy, where the hydrogen
bond donor–acceptor pair, was retained through the urea moiety targeting interaction with Glu885
and Asp1046 residues mimicking that of sorafenib. Furthermore, the connector atom between the
quinazoline core and the central phenyl ring was maintained by either O or NH, based on reported
SAR, in order to explore the preferred connector atom. Finally, different substitution patterns (R)
on terminal phenyl ring was explored, aiming to study their interactions with the terminal allosteric
hydrophobic pocket.
With the endeavor to achieve an increase in the activity mimicking that of regorafenib, a
fluorine atom was introduced at the position 2 of the central aromatic ring in quinazoline ester
derivatives with the aim to raise activity, being fluorine atom is an electron withdrawing group
(Figure 2). In this research, the targeted compounds were designed, synthesized and their
repressive activity against VEGFR-2 as well as sixty NCI cell lines was evaluated.

Gate area
Asp1046
Sorafenib
O
-OOC
N
H
Cl
F3C
O
O
N
N
NH
H
Allosteric
Hydrophobic
pocket
N
O
O
H
O
N
H
O
Glu885
Cys919
Gate area
Gate area
Asp1046
Asp1046
Hyrdophobic Pocket
Hyrdophobic Pocket
O
O
-OOC
Leu840
Ala866
Leu840
-OOC
N
N
Ala866
Val848
H
H
Val848
H
X
R
O
O
N
Leu1035
R
N
N
O
N
O
N
Allosteric
Hydrophobic
pocket
N
N
H
H
N
H
Leu1035
N
Allosteric
Hydrophobic
pocket
H
R1
H
O
N
O
O
N
H
O
O
O
O
N
H
Cys919
Glu885
Cys919
Glu885
Amide derivatives
Ester derivatives
Figure 1: Schematic representation of the targeted quinazoline derivatives.
3 Results and Discussion:
3.1 Chemistry:
Scheme 1 depicted synthetic pathways for the preparation of the target compounds. Final
compounds incorporating substituted urea moiety were obtained utilizing the corresponding
intermediates, that were synthesized in step with the rumored procedures [17,18]. Urea
intermediates were accomplished by two alternative ways. The first involved the reaction of p-
nitrophenylisocyanate with different substituted anilines in dry THF and catalytic amount of
triethylamine for 22-24h at room temperature to give the nitrodiarylurea derivatives[19] which were

reduced to their corresponding amino derivatives using 10% Pd-C as the catalyst, dissolved in
methanol to yield the 4-aminophenylsubstitutedphenyl ureas[20,21] The second method involved
stirring p-aminophenol with different substituted isocyanates in dioxane for 4-5h at room
temperature to form the corresponding 4-hydroxyphenylsubstitutedpheny urea derivatives [22].
The quinazoline scaffold was synthesized by applying classical Niementowski reaction. The
quinazoline-4-one-2-carboxylate (2) was afforded through cyclization of 2-aminobenzamide (1)
via its reflux with diethyl oxalate for 48h [23].
Chlorination of (2) was performed by refluxing it with POCl3 to produce the corresponding
4-chloro analog (3) which was used strictly in the next reaction due to its high reactivity and
sensitivity to moisture. Urea based target N-quinazoline ester and amide derivatives (4 a-j, 5a-j
,6a-e and 7a,b) were obtained by a classic nucleophilic substitution reaction between
chloroquinazoline (3) and the 4-aminophenylureas in isopropyl alcohol by dropwise addition of
concentrated hydrochloric acid to accelerate reaction rate in 62−71 yield%[24].
Preparation of the amides 5a-j was achieved through two methods either by hydrolysis of
the ester derivatives (4a-j) using LiOH [25] to their corresponding acids which were then
converted to acyl chlorides by excess thionyl chloride[26]. The freshly prepared acid chlorides
were then reacted with a solution of 40% methyl amine in methanol/THF at 5oC for 2-5h to afford
amides 5a-j. The other method was direct fusion for ester derivatives (4a-j) with 40% methylamine
in oil bath at 1800C which resulted in better yield[27].
On the other hand, the 4-substitutedphenoxy quinazoline derivatives (6a-e) were achieved by
reacting chloroquinazoline (3) with hydroxyphenyl urea derivatives in acetonitrile at 75-80oC
using Cs2CO3 to provide the desired compounds (6a-e, 7a,b) in good yields (67-70%)[28].
The titled compounds structure was actually elucidated via spectral data and elemental
1
analyses. H-NMR spectra for the synthesized esters 4a–j, 6a-e and 7a,b showed signals that
appeared downfield in the range (9.25-9.83) ppm assignable to the urea exchangeable protons,
while the ester protons appeared in the aliphatic range around 4.34-4.32 ppm for quartet CH2 of
ester and 1.31-1.29 ppm for triplet CH3 of ester. Regarding compound (4j) additional protons were
shown as singlet peak at δ 2.33 ppm of the extra methyl group. Compound 4f and 6b showed
singlet peak at δ 3.79 ppm of the methoxy group. The molecular weights of the titled compounds
were matched with their calculated ones. FT-IR spectra showed the characteristic bands of N-H

group around 3300 cm-1, C-H aromatic at around 3100 cm-1, C=O of the ester around 1730 cm-1,
C=O of the amide around 1680 cm-1 and C=N around 1630 cm-1.
Moreover, 13C NMR spectra for quinazoline derivatives 4a-j, 6a-e and 7a,b displayed
characteristic signals resonating at the range of δ 168.10-168.23 ppm attributable for the carbonyl
carbon of the ester functionality, the signals due to the ester methylene carbon in compounds 4a-
j, 6a-e and 7a,b appeared at the range δ 62.20-14.51 ppm. The extra methyl group carbon
appeared at δ 21.70 ppm as seen in 4d, 6b and 7a.
In addition, the structure of the titled compounds (5a-j) were verified by spectral and
1
elemental analysis, H NMR spectra of the amide products (5a-j) revealed the presence of the
characteristic peak of NH of urea and appearance of exchangeable NH peak of carboxamide group
13
around δ 9.30 ppm. Also C NMR spectra for amide quinazoline derivatives were detected at
expected chemical shift, whereas the methyl protons of amides 5a-j appeared at δ 26.54 ppm. FT-
IR spectra showed the characteristic bands of N-H group around 3300 cm-1, C-H aromatic at
around 3100 cm-1, C=O of the amide around 1685 cm-1 and C=N around 1640 cm-1. The molecular
weights of the titled compounds were matched with their calculated ones.

H
N
H
N
R1
O
HN
N
O
N
O
iii
NH2
NH2
R1
O
4,5
4a-j
1
a
b
c
d
e
f
g
h
i
H
3-F
4-F
3-CH3
3-OCH3
4-OCH3
4-Cl
3-CF3
3-Cl-4-Cl
3-CF3-4-Cl
iva
ivb
ivc
H
H
i
N
N
R1
O
N
O
HN
N
NH
N
O
H
N
2
j
O
5a-j
O
ii
H
N
H
N
R1
R1
Cl
N
6
O
O
v
H
3-CH3
3-OCH3
3-Cl-4-CH3
3-CF3-4-Cl
a
b
c
d
e
N
N
O
O
3
N
O
6a-e
O
O
F
H
N
H
N
R1
O
N
vi
N
R1
7
a
b
O
3-CH3
3-OCH3
O
7a,b
Scheme 1. Reagents and conditions: (i) diethyl oxalate, 150oC, 48 hrs, 86% (ii) POCl3, reflux, 5 -
7 hr, 40-50%, (iii) 4- aminophenyl urea derivatives, isopropyl alcohol, catalytic amount of Conc
HCl, reflux, 5-7 hrs, 54-68%;(iva) Li(OH), ethyl acetate, THF (ivb) SOCl2,drops of DMF,
reflux, 7-10 hrs (ivc) 40%Methylamine solution, Methanol/THF at 0oC,4-5 hrs , 35-56%.(v) 4-
hydroxyphenyl urea derivatives, Cs2CO3, acetonitrile,55-60oC,6-8hrs,54-64%.(vi) 2-Fluoro4-
hydroxyphenyl urea derivatives, Cs2CO3, acetonitrile, 55-70oC,8-10hrs,46-49%

3.2 Biological evaluation
3.2.1 In vitro VEGFR-2 tyrosine kinase activity
3.2.1.1 Initial screening at a single dose of 10 μM concentration.
The VEGFR-2 tyrosine kinase assays were performed at Thermo Fischer Scientific, USA
(www.thermofischer.com/selectscreen). The assay was performed to test the designed compounds
' VEGFR-2 inhibitory activity, using the Z'-LYTE biochemical assay applies a fluorescence-based,
coupled-enzyme method, focusing on the differential sensitivity of phosphorylated and non-
phosphorylated peptides to proteolytic cleavage. The strength of FRET-peptide phosphorylation
can be calculated from the emission ratio. The emission percentage will persist small when the
complex of FRET-peptide is phosphorylated (i.e., no inhibition of kinase), and large when the
complex of FRET-peptide is non-phosphorylated (i.e., kinase inhibition) [29]. All targeted
compounds were tested against VEGFR-2 kinase in a single point concentration assay at 10 μM to
measure their inhibitory activity against sorafenib as a reference compound. The compounds
showed either moderate or excellent inhibitory activity against VEGFR-2 kinases as shown in
Table 1.

Table 1: Percent inhibition at 10uM against VEGFR-2 achieved by the urea-based
derivatives (4a-j, 5a-j, 6a-e,7a-b):
Y
H
H
N
N
R1
O
X
N
N
R2
O
Compd ID
4a
R1
R2
X
Y
VEGFR-2(%)
49%
H
OEt
NH
H
4b
4c
4d
4e
4f
3-F
4-F
3-CH3
3-OCH3
4-OCH3
4-Cl
OEt
OEt
OEt
OEt
OEt
OEt
NH
NH
NH
NH
NH
NH
H
H
H
H
H
H
18%
30%
105%
71%
26%
24%
4g
4h
4i
4j
5a
5b
3-CF3
3-Cl-4-Cl
3-CF3-4-Cl
H
OEt
OEt
OEt
NHCH3
NHCH3
NH
NH
NH
NH
NH
H
H
H
H
H
83%
79%
10%
19%
57%
3-F
5c
5d
5e
5f
4-F
NHCH3
NHCH3
NHCH3
NHCH3
NHCH3
NH
NH
NH
NH
NH
H
H
H
H
H
37%
108%
99%
33%
47%
3-CH3
3-OCH3
4-OCH3
4-Cl
5g
5h
5i
5j
3-CF3
3-Cl-4-Cl
3-CF3-4-Cl
NHCH3
NHCH3
NHCH3
NH
NH
NH
H
H
H
110%
101%
101%
6a
6b
6c
H
OEt
OEt
OEt
O
O
O
H
H
H
20%
19%
109%
3-CH3
3-OCH3
6d
6e
7a
3-Cl-4-CH3
3-CF3-4-Cl
3-CH3
OEt
OEt
OEt
O
O
O
H
H
F
79%
71%
110%
7b
3-OCH3
OEt
O
F
114%
97%
Sorafenib

3.2.1.2 Measurement of potential enzyme inhibitory activity (IC50)
Promising candidates demonstrating VEGFR-2 inhibition percentage above 90 percent at
10μM concentration were prompted us to further examine their dose-related enzymatic inhibition
of VEGFR-2 at 5 different concentrations either (0.5 nM – 5 nM – 50 nM – 0.5 μM – 5 μM) or (1
nM – 10 nM – 100 nM – 1 μM – 10 μM) to evaluate their IC50 values.
Nine of the tested compounds displayed potent VEGFR-2 inhibitory activity with IC50
values in nanomolar range. The quinazoline 2- carboxamide based derivatives (5d), have a m-tolyl
substituted urea tail showed the most potent nanomolar VEGFR-2 inhibition (IC50 of 12.1 nM)
suggesting that the m-tolyl substitution is optimum for activity (Table 2).
Table 2: IC50 values against VEGFR-2 achieved by the most active derivatives:
Compd ID
VEGFR-2(%)
105%
108%
99%
IC50(nM)
254
4d
5d
12.1
40.3
15.5
13.1
57.4
150
5e
5h
110%
101%
101%
109%
110%
114%
97%
5i
5j
6c
7a
14.8
44.4
7b
Sorafenib
78.9

5d
IC50=78
IC50=12.1
5i
7a
IC50=13.1
IC50=14.8
5h
5e
IC50=40.3
IC50=15.5
Figure 3: Inhibitory concentration effect (IC50) of the targeted compounds on the VEGFR-
2 kinase activity.

3.2.1.3 Structure activity Relationship:
At 10 µM concentration, nine quinazoline-2-carboxylate and quinazoline-2-
carboxamide derivatives incorporating a substituted biaryl urea motif linked via an ether or
NH linker to the parent scaffold (4d, 5d, 5e, 5h, 5i, 5j, 6c,7a and 7b) demonstrated highly
potent VEGFR-2 inhibition of 99-100%. Moreover, significant inhibition ranging between
71-83 % was exhibited by compounds (4e, 4h, 4i, 6d and 6e). Finally compounds (4a, 5b
and 5g) showed moderate inhibition between 47-57%.
The SAR outcomes of the NH-linked urea based derivatives (series 4 and 5) highlighted
that incorporating a carboxamide moiety at position 2 of quinazoline scaffold, replacing the
ester functionality, resulted in marked increase in enzymatic potency (5a-j vs. 4a-j), which is
probably attributed to the extra H-bond with Cys919 that is suggested to be crucial for potent
VEGFR-2 inhibition. Thus, five quinazoline-2-carboxamide derivatives (5d, 5e, 5h, 5i and
5j) exhibited potent nanomolar VEGFR-2 inhibition with IC50 values of 12.1, 40.3, 15.5, 13.1
and 57.4 nM, respectively, superior to that of sorafenib (IC50 78.9 nM).
Incorporating different substituents at position 3 or/and 4 of the terminal phenyl ring
was well tolerated. The highest VEGFR-2 inhibitory activity was usually noticed with the
meta substituted derivatives and the 3,4 disubstituted ones. The 3-CH3 derivatives (5d, 7a)
(IC50 12.1 and 14.8 nM, respectively), the 3-CF3 derivative (5h) (IC50 15.5 nM) and 3,4-
dichloro derivatives (5i) IC50 13.1 nM ) showed highest potencies. Furthermore, 3-OCH3
analogues (5e, 6c, 7b) (IC50 40.3, 150 and 44.4 nM, respectively) as well as the 3-CF3-4-Cl
derivative (5j, IC50 57.4 nM) also exhibited nanomolar VEGFR-2 inhibition.
Replacement of NH–linker with an ether isostere resulted in fluctuating effect on
enzymatic inhibition. Thus, increased potency was observed only in the 3-OCH3 and 3-CF3-
4-Cl analogues (6c, 6e vs. 4e, 4j) (109%, 71% compared to 71%, 10%, respectively). While,
surprisingly the 3-CH3 ether linked derivative (6b) did not show any significant inhibition
(19%) compared to its NH-linked analogue (4d, 105%, IC50 254 nM).
It is noteworthy, that bioisosteric replacement in the middle phenyl ring of the biaryl
urea tail with a fluorine substituent to mimic regorafenib resulted in compounds (7a , 7b)
showing potent nanomolar VEGFR-2 inhibition of 14.8 and 44.4 nM, respectively preferable
to their unsubstituted analogues (4d, 4e) and superior to tested reference drug sorafenib.

3-F substitution increase
activity of ester derivatives
than unsubstituted one
Y=H or F
Y
Terminal substituted phenyl ring is
essential for activity and
substitution at 3 or 4 position or
both is well tolerated and the
3-methyl substitution is the optimum
for activity
H
H
Both O and NH linkers
are well tolerated
N
N
R1
O
X=NH or O
X
N
N
R2
Substitution at position
2 with amide or ester moiety
increase activity as it forms extra
hydrogen bond with Cys 919
O
R2=OEt or NHCH3
Figure 4: Structure activity relationship for the targeted quinazoline derivatives.
3.2.2 In-vitro antiproliferative activity assay against NCI 60-cell line panel
Sixteen of the targeted compounds have been selected by the National Cancer Institute
(NCI) Developmental Therapeutics Program (DTP), division of cancer treatment and diagnosis,
NIH, Bethesda, Maryland, USA (www.dtp.nci.nih.gov), namely (4a, 4b, 4c, 4d, 4e, 4f, 4h, 4g, 5c,
5e, 5g, 5h, 5i, 5j, 6a, 6b). Selection of the screening representing the various chemotypes of this
work was based on the ability of the submitted compounds to feature diversity to the NCI small
molecule compound collection[30]. This screening procedure uses 60 separate cell lines of human
tumors, divided into nine subpanels; leukemia, melanoma, lung, colon, liver, ovary, kidney,
prostate, and breast cancer, respectively. All selected compounds were tested on the full NCI 60
cell panel at initial single dose 10 μM inhibition level assay. Analysis of the NCI-60 results showed
that some compounds have sensible activity against some of the investigated cell lines. The 3-
OCH3 biarylurea derivative having 2-NHCH3 at quinazoline scaffold (IC50=40nM) (5e) showed
considerable cell growth inhibition with mean growth inhibition percent of 10.29%. It exhibited
good anti-proliferative activity against some NCI cell panel, namely; the leukemic CCRF-CEM,
K-562, MOLT-4 and RPMI-8226 cancer cell lines with growth inhibition 44.73, 30.65 and
40.99%, respectively , the CNS cancer SF-539 , SNB-75 and U251 cell lines with 47.80 % ,
41.37% and 25.55% inhibition, respectively, the ovarian cancer OVCAR-8 cell line with 20.49%
inhibition, renal cancer cell line UO-31 with 32.44% inhibition. Finally, it showed good activity
against the breast cancer HS 578T and T-47D cell lines with 23.85% and 37.29%, respectively.

Moreover, it 3-CF3 analogue (5h) also showed non-significant cell growth inhibition with
mean growth inhibition percent of 8.91%. It exhibited good antiproliferative activity against the
leukemic CCRF-CEM, K-562, MOLT-4 and RPMI-8226 cancer cell lines with growth inhibition
36.27, 34.12 and 36.40%, respectively, non-small lung cancer HOP-62 and NCI-H522 cell lines
with 20.03% and 23.94%, respectively, the CNS cancer SF-539 cell line with 32.99 % inhibition,
respectively, the ovarian cancer SK-OV-3 cell line with 20.44% inhibition, renal cancer cell line
CAKI-1 and UO-31 with 26.11% and 40.86% inhibition. Inhibition percent of 21.03% on prostate
cancer cell line PC-3. Finally, it showed notable activity against the breast cancer MDA-MB-
231/ATCC and T-47D cell lines with 20.32% and 29.06%, respectively.
Ongoing with our kinase results, 3-Cl,4-Cl analogue (5i) showed good antiproliferative
activity against the leukemic CCRF-CEM, K-562, MOLT-4 , RPMI-8226 and SR 22.34 cancer
cell lines with growth inhibition 28.35, 27.02 , 24.81 and 22.34%, respectively, renal cancer cell
line TK-10 with 26.91% inhibition. Finally, it showed notable activity against the breast cancer
T-47D cell lines with 33.03% (Table 3).
On the contrary, poor antiproliferative activity displayed by other synthesized compounds
despite their potent VEGFR activity may be attributed to their selectivity towards VEGFR kinase,
since VEGFR is not highly expressed in the tested cell lines. The NCI 60-cell line testing is not a
true reflection to VEGFR-2 inhibition, as VEGFR is mainly relevant to angiogenesis not
cytotoxicity and this may explain the poor cytotoxicity relative to the excellent enzymatic
inhibition. Experiments for angiogenesis are usually performed on HUVEC not cancer cells.

Table 3.Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 µM
concentration for some of targeted compounds.
Cell Growth inhibition Percent for the tested compoundsa
Panel/
Cell line
4a
4c
4d
4e
4f
5c
5e
5g
5h
5i
6a
6b
Leukemia
-
-
-
-
-
-
-
-
-
-
-
-
-
-
15.72
-
-
17.47
13.78
-
-
-
-
-
-
-
-
44.73 17.58 36.27 28.35
-
-
-
-
-
-
-
-
-
-
-
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
-
-
-
-
-
-
13.32 10.55
24.14
14.44
19.39
-
10.61
-
-
15.33
30.65
40.99
-
34.12 27.02
36.40 24.81
14.45 22.34
12.14
22.26 15.81 19.05
18.53
Non-Small Cell Lung Cancer
A549/
ATCC
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
EKVX
-
-
-
-
-
20.03
-
-
-
-
-
-
-
-
-
-
-
-
10.68
14.01
HOP-62
HOP-92
NCI-H226
10.67
12.89
19.35 20.34 18.83
-
-
-
-
11.48
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
NCI-H23
-
-
NCI-H322M
NCI-H460
NCI-H522
10.47
23.94 14.03 10.36
Colon Cancer
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
COLO205
HCC-2998
HCT-116
HCT-15
HT29
19.27
10.51
11.62
-
-
14.09
-
KM12
SW-620
-
CNS Cancer
11.20
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11.19
-
-
-
25.12
-
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
-
12.82
-
10.41
47.80
14.37
-
32.99
-
11.77
-
-
23.92
15.14
30.21
12.82
41.97 30.38
58.90 21.32 47.79
12.57
41.37 14.49 16.68 10.97 35.04
25.55
-
24.27
14.89
-
-
15.07
-
-
Melanoma
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.23 11.73 12.71 11.50
-
LOX IMVI
MALME-3M
M14
-
-
-
-
-
-
-
10.13
-
11.84
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
11.37
-
-
10.09
-
-
-
-
-
-
-
-
-
MDA-MB-435
-
-
-
-
-
-
-
-
-
10.93
-
-
-
11.48
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
-
-
-
-
-
-
-
-
16.43
19.44
-
-
-
-
-
-
11.73
10.96
11.82

Ovarian Cancer
-
-
-
-
-
-
-
-
-
-
-
-
11.27
-
-
-
-
-
-
-
-
-
11.14
-
-
-
-
-
-
-
-
-
-
-
-
-
IGROV1
-
-
-
-
14.31
-
OVCAR-3
OVCAR-4
OVCAR-5
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
OVCAR-8
NCI/ADR-
RES
20.49
-
-
-
12.23
-
13.14
-
-
-
-
-
-
-
-
SK-OV-3
11.73
-
-
20.44
Renal Cancer
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
19.09
-
-
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
33.95 37.96 33.84 26.38
-
24.78
-
-
-
50.77
-
17.14
-
-
-
42.41
-
23.25
-
-
-
-
-
-
-
-
-
-
-
16.92 17.96
16.23 16.07 26.11
12.67
-
-
11.1
-
-
-
-
-
-
-
-
-
11.49 19.28
-
-
-
26.91
31.93 19.00 32.10 25.79
12.66
19.62 32.44 20.25 40.86 19.17 19.12
23.16
Prostate Cancer
-
-
-
-
11.07 12.57
-
-
-
-
19.63
-
-
-
14.26
-
-
-
-
-
21.03
-
PC-3
DU-145
-
-
Breast Cancer
MCF7
13.35
11.65
-
-
-
-
10.48 14.11
11.82 20.40
-
-
16.80
-
-
-
15.50 16.90
20.32 12.98
-
-
-
-
MDA-MB-
231/ATCC
HS 578T
BT-549
T-47D
14.07
-
-
-
15.52 20.93
-
-
-
23.85
-
-
-
14.44
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
19.27
-
11.26
12.56
-
-
12.54 37.29 28.08 29.06 33.03
MDA-MB-
468
19.7
3
-
14.14
-
-
-
31.07
MEAN %
Growth
2.92 0.93 2.79
3.47
0.82
-0.11 10.26
1.1
8.91
8.28
3.16
1.20
inhibition
a Only GI % higher than 10% are shown.
3.3 Molecular modeling study:
3.3.1 Docking Study:
To rationalize the highest VEGFR-2 inhibitory activity of the newly synthesized
compounds, molecular docking study using C-Docker protocol in Discovery Studio 2.5 Software
was performed for the target compounds in the VEGFR-2 binding site.
The X-ray crystallographic enzyme (VEGFR-2) substrate with sorafenib complex of PDB
code (4ASD), revealed that the substituted terminal phenyl ring lied in a deep extended
hydrophobic pocket formed by the movement of Phe1047 residue of the ‘DFG’ motif to induce
the ‘DFG-out’ conformation.

Validation of the C-Docker protocol used was carried out by redocking the lead compound
(sorafenib) in the VEGFR-2 kinase active site and aligning the lead compound's bioactive X-ray
conformer with the best fitted pose obtained from the docking run. The alignment showed good
coexistence between them with RMSD=0.95A°, indicating the ability of the used docking protocol
to fetch valid docking poses. Fig.5
Figure 5: Alignment of the original ligand sorafenib (blue) and re-docked ligand (pink).
The docking study of the newly compounds into VEGFR-2 active site exposed comparable
binding modes to lead compound. Most of designed compounds demonstrated higher or similar
docking scores relative to it of the docked lead compound. Docking of the target compounds
showed that the core scaffold adopted volumes and orientation as the lead compound. Targeted
compounds satisfied the hot spots, where the hydrogen bonds with Cys919, Glu885, Asp1046
residues were established. These findings further explained the potent VEGFR-2 inhibitory
activity observed for the targeted compounds as shown in figure 6.

A
B
C
D
Figure 6: Targeted compounds binds with VEGFR binding site (PDB ID: 4ASD); A) Compound
5d in ATP binding site of VEGR-2 showing two hydrogen interactions with Cys919 residue,
hydrogen bond with Glu885; O of urea moiety makes hydrogen bond with Asp1046.B) compound
5i exhibited essential hydrogen bond and its distances with VEGFR-2 binding site; N1,NH amide
of quinazoline makes hydrogen bond interaction with Cys919 residue; NH of urea linker makes
hydrogen bond with Glu885; O of urea moiety makes hydrogen bond with Asp1046. C) Docking
pose of compound 4d demonstrate the same key interactions with VEGFR-2 binding site; N1 of
quinazoline makes hydrogen bond interaction with NH of Cys919 residue; NH of urea motif makes
hydrogen bond with Glu885; O of urea moiety makes hydrogen bond with Asp1046. D) Docking
of 7a showing the presence of HB interactions of N1 of quinazoline and NH of Cys919; NH of
urea moiety makes hydrogen bond with Glu885; O of urea moiety makes hydrogen bond with
Asp1046.

3.3.2 Field alignment study:
Molecules attracted to each other via electrostatic and Van der Waals forces; proteins verify
ligands as molecular fields instead of pure 3D arrangement of individual atoms. Thus, molecules
with similar potential fields are expected to perform similar biological activities by establishing
similar interactions inside the same binding site, even if they have different structures, since the
field patterns don't seem to be directly impressed by the 2D connectivity of the molecule, however
rather by its 3D properties [31]. Based on this theory, the field alignment technique provided by
Cresset's FieldAlign ® Module, version 2.0.1, was used to align our designed compounds with the
bioactive conformation of sorafenib (PDB: 4ASD). In FieldAlign, every molecule is represented
as Field Point Pattern (FPP), using the local extrema of four molecular fields: negative
electrostatic, positive electrostatic, surface (van der Waals interactions), and hydrophobic. The
resulting FPPs are matched and graded to provide a measure of similitude in different
conformations between a query molecule and a database molecule. Alignment of the target
compound (5d, 5h, 7a and 7b) against sorafenib co-crystallized under PDB code: 4ASD, showed
great similarity of their molecular fields that are manifested by the high alignment score ranging
from 0.790 to 0.766. The quinazoline nucleus of the target compounds is aligned with pyridine
nucleus of the (Sorafenib). In addition, the hydrophobic substituted phenyl ring, The O or NH
linkers and the biphenyl urea in the designed compounds were aligned with the terminal phenyl
ring, the O linker and the biphenyl urea moieties in both sorafenib and regorafenib with targeted
compounds (5d, 5h, 7a and 7b). It should be mentioned that compounds (5d, 5h, 7a and 7b) with
the highest inhibitory potency (IC50 =12.1, 15.5, 14.8 and 44.4 nM, respectively) represented by
purple color showed the best alignment with highest score; 0.790 0.781, 0.766, 0.775 and 0.767,
respectively [Figure 7].
B
A

D
C
Figure 7: [A] Field alignment of compound 5d (purple) and Sorafenib (green) showing similar molecular
fields suggesting a similar binding mode to sorafenib. [B] Compound 5h (purple) aligned to Sorafenib
(purple). [C] Field alignment of compound 7a (purple) and Regorfenib (Cyan) showing similar molecular
fields suggesting a similar binding mode to regorafenib. [D] Field alignment of compound 7b (purple) and
Regorfenib (Cyan) showing similar molecular fields suggesting a similar binding mode to regorafenib. *
Spherical field points: designed compounds, icosahedral field points: Reference compound. Field points
are condensed field pattern that make fields computationally viable. Larger field points represent stronger
points of potential interaction. Cyan: Negative field points, Red: Positive field points, Yellow: van der
Waals surface field points, Gold: Hydrophobic field points.
A significant aspect about the SAR of the designed compounds justified enough by the
molecular docking. Field alignment analysis was proceeding in trial to grasp the high activity at
position 2 of quinazoline scaffold upon inclusion of these ester or amide groups. These compounds
demonstrated comparable high inhibitory activity similar to sorafenib and regorafenib. When
comparing their molecular fields with the approved active compounds, the target compounds '
quinazoline nucleus is aligned with the pyridine nucleus of sorafenib, also the phenyl-substituted
hydrophobic terminal ring with the terminal phenyl ring in sorafenib and regorafenib. In addition,
the alignment of the hydrophobic substituted central phenyl ring with the 2-fluoro substituted
phenyl ring in regorafenib as shown in Figure 8.

A
B
D
C
Figure 8: A comparison of the hydrophobic fields of different compounds: [A] Hydrophobic field of
Sorafenib, [B] Hydrophobic field of Regorafenib, [C] Hydrophobic field of compound (5d) as a
representative example of the active amide derivatives, [D] Hydrophobic field of compound (7a) as an
example of the active phenoxyurea derivatives with 2-fluoro substitution in the central aromatic ring.

3.3.3 In silico Predictive ADME study for targeted compounds:
In order to further examine pharmacokinetics properties of synthesized compounds,
computer-aided ADMET study was performed by using Accelrys Discovery Studio 2.5 software.
These studies are based on the chemical structure of the molecule and involve the calculation of
certain parameters including atom-based Log P98 (A LogP 98), aqueous solubility value (AQ
SOl), aqueous solubility level (AQ SOl LEV), 2D polar surface area (ADMET 2D PSA), blood–
brain barrier value (BBB), blood–brain barrier level (BBB LEV), cytochrome P450 2D6
(CYP2D6), probability of cytochrome P450 2D6 (CYP PROB), logarithmic value of plasma
protein binding (PPB LOG), and plasma protein binding level (PPB LEV). The results of the
ADMET study are presented as ADMET plot, which is a 2D plot drawn using calculated PSA_2D
and A log P98 properties. BBB and human intestinal absorption (HIA) plots were drawn using all
the synthesized compounds (Fig. 9).
Figure 9: Human intestinal absorption (HIA) and Blood Brain Barrier (BBB) plot for the newly
synthesized compounds.

The results of the ADMET study are presented as ADMET-Plot, which is a 2D plot drawn
using calculated PSA_2D and A logp98 properties. Blood Brain Barrier (BBB) and Human
Intestinal Absorption (HIA) plots were drawn for all targeted compounds.
In BBB plot, all synthesized compounds were located outside the 99% and 95% ellipses.
In addition, these targeted compounds may not be ready to perforate the blood brain barrier; so the
CNS adverse effects probability is anticipated to be low.
In HIA plot, six compounds fell outside the 99% confidence ellipse (undefined), whereas
the other remaining compounds fell inside the 95-99% ellipse, thus they are predicted to have
better intestinal absorption. Interestingly, it was shown that compounds having amide moiety (5a-
j) demonstrated expected better human intestinal absorption (absorption level 0 or 1; table
2(supplementary)) compared to their ester analogues (4a-j). The aqueous solubility level was
anticipated to be one or two which suggests low aqueous solubility. The CYP2D6 value predicts
the inhibitory and non-inhibitory aspect of the titled chemical structure on Cytochrome P450 2D6
enzyme. Most of the compounds are foreseen as inhibitors of CYP2D6, so drug-drug interactions
are expected upon administration of these compounds. Two compounds are predicted as non-
inhibitor for CYP2D6, so the side effects as liver dysfunction are not expected upon administration
of these two compounds. The plasma protein-binding parameter predicts whether a compound is
highly bound to carrier proteins. So these newly compounds may be expected to reach the targeted
enzyme as its showed more than 95-90% plasma protein binding. Bioavailability radar provided
from ADME tool showed that most of the tested compounds exhibited promising predicted
physicochemical properties for oral bioavailability. PSA is a drug bioavailability aspect.
Therefore, it is assumed that actively absorbed molecules with PSA > 140 are poor in
bioavailability. All the synthesized compounds have PSA ranging from 44.26- 103.09, thus, these
values predicted to present good passive oral absorption. The ADMET study parameters are
calculated in table 4.

Table 4: Computer aided ADMET screening of the synthesized compounds:
CPD
ID
BBB_
Leva
Absorp
_Levb
AQ SOl Hepatox
CYP
2D6f
CYP
2D6
PPB_
Levh
A log
P98i
ADEM_
d
LEVc
PSA_2D
j
Probg
4a
4b
4c
4d
4e
4f
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
4
1
1
1
1
0
1
2
2
1
2
0
0
0
1
1
1
1
1
1
2
1
1
1
2
2
1
1
2
1
1
2
1
2
1
1
1
1
2
2
2
2
2
2
2
1
1
1
2
1
2
1
1
1
2
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
1
1
1
0
1
1
1
1
1
1
1
1
1
0.712
0.722
0.702
0.712
0.445
0.702
0.603
0.613
0.693
0.504
0.702
0.693
0.683
0.603
0.673
0.673
0.574
0.445
0.584
0.524
0.742
0.722
0.722
0.623
0..564
0..524
0.623
1
1
1
1
1
1
2
2
1
2
1
1
1
1
1
1
1
1
1
2
1
1
1
2
2
2
1
4.476
4.681
4.681
4.459
4.175
4.459
5.140
5.804
4.962
5.418
3.480
3.685
3.685
3.966
3.463
3.463
4.144
4.422
4.808
5.086
4.486
4.972
4.469
5.636
6.092
5.177
4.675
104.484
104.484
104.484
113.414
93.223
113.414
104.484
104.484
104.484
104.484
108.364
108.364
108.364
108.364
117.294
117.294
108.364
108.364
108.364
108.364
100.604
100.604
109.534
100.604
100.604
100.604
109.534
4g
4h
4i
4j
5a
5b
5c
5d
5e
5f
5g
5h
5i
5j
6a
6b
6c
6d
6e
7a
7b
a Blood brain barrier level; 4= undefined, 2= medium penetration, 1= high penetration.
b Absorption Level; 3= very low absorption, 2= low absorption, 1= moderate, 0= good absorption.
a Aqueous solubility level; 4= optimal, 3= good, 2= low solubility, 1= very low but soluble, 0= extremely low.
d Hepatotoxicity level; 1= toxic, 0= nontoxic.
e Hepatotoxicity probability.
f CYP2D6 inhibition; 1= likely to inhibit, 0= non inhibitor.
g Cyp2D6 inhibition probability.

h Plasma protein binding; 2= more than 95%, 1= more than 90%, 0= less than 90%.
i Lipophilicity descriptor.
j Polar surface area; compounds must have log p value not greater than 5.0 to attain a reasonable probability of being well
absorbed.
4 Conclusion:
Novel series of quinazoline-2-carboxylate and quinazoline-2-carboxamide derivatives were
designed, synthesized and evaluated for their anti-proliferative activity against NCI sixty cell line
panels as well as in vitro VEGFR-2 inhibitory activity. Nine of the biaryl urea-based derivatives
exhibited potent dose-related VEGFR-2 inhibitory activity with IC50 values in the nanomolar
range.
Replacement of ester functionality at position 2 of quinazoline scaffold with a carboxamide
moiety resulted in remarkable increase in enzymatic potency which may be attributed to the extra
H-bond with Cys919. Thus, five quinazoline-2-carboxamide bearing a biaryl urea tail via an NH
linker (5d, 5e, 5h, 5i, 5j) having 3-CH3, 3-OCH3, 3-CF3, 3,4-dichloro and 3-CF3-4-Cl substituents
at terminal phenyl ring exhibited potent nanomolar VEGFR-2 inhibition with IC50s 12.1, 40.3,
15.5, 13.1, 57.4 nM, respectively, superior to that of reference drug sorafenib (IC50 78.9nM).
Additionally, incorporating a 3-fluoro substituent to the middle phenyl ring of the biaryl urea
tail to mimic regorafenib resulted in compounds (7a, 7b) with potent nanomolar inhibition of 14.8
and 44.4 nM also superior to sorafenib.
Compound (5e) exhibited significant cell growth inhibition on some specific NCI cell lines,
especially those of breast, renal, ovarian, CNS, and non-small cell lung cancers.
These results were further explained using molecular docking studies, which detected the
ability of the target compounds to compose essential key interactions, known to be pivotal for type
II VEGFR-2 inhibitors. Field alignment study also revealed that these compounds displayed
molecular fields similar to those of sorafenib. Also hydrophobic field around the target compounds
was similar to that of sorafenib.
5 Experimental:
5.1 Chemistry and analysis.
Starting materials and reagents were purchased from Alfa- Aesar and Loba chemie Organics
and were used without further purification. Solvents were acquired from Fisher scientific or

Sigma-Aldrich and used without further purification. Via Molecular Sieves, anhydrous methylene
chloride was dried. Chemical reactions were monitored by analytical thin layer chromatography
(TLC), on silica gel 60 F254 packed on Aluminum sheets, purchased from Merck, and were
visualized under U.V. light (254 nm). Column chromatography was conducted on silica 60 (35-70
microns). Melting points were determined by Stuart Scientific apparatus. Using DMSO as a
solvent and TMS as an internal standard signal ,1H-NMR spectra were recorded on a Bruker 400
MHz spectrometer in δ scale (ppm), at the Faculty of Pharmacy, Ain-shams and Mansoura
13
Universities. C-NMR spectra were recorded on Bruker 400MHz (at 101 MHz). EI-MS spectra
were recorded by Triple Quadrupole LC/Ms/Ms mass spectrometer API 200 (AB Sciex
Instrument) at the regional center of mycology and biotechnology at Al-Azhar University.
Elemental analyses were carried out at Al-Azhar University's regional mycology and
biotechnology center. Compounds 2, 3 and 4- amino/hydroxyphenyl urea derivatives were
prepared according to the reported procedures [18, 19, 22, 23].
5.1.1 General procedure for synthesis of Ethyl 4-((4-(3-substituted phenylureido)
phenyl)amino)quinazoline-2-carboxylate derivatives (4a-j):
To a solution of 4-chloroquinazoline derivative (3) (0.256 g, 1 mmol: 1equiv.) in 2-propanol (5
mL) , the respective 4-aminophenyl urea derivative (1 mmol;1 equiv.) and catalytic amount of
HCl (5 drops) were added. The mixture was heated under reflux for 5-7 hrs. The resultant solids
(4a-j) were collected either by filtration, washed with 2-propanol then purified by column
chromatography CH2Cl2/MeOH (9/1).
5.1.1.1 Ethyl 4-((4-(3-phenylureido)phenyl)amino)quinazoline-2-carboxylate (4a):
The titled compound was separated as red crystals (0.24 g, 58%); m.p. 250-251°C; 1HNMR (400
MHz, DMSO-d6) δ 9.84 (s, 1H, NH D2O exchangeable), 8.81 (s, 2H, NH D2O exchangeable),
8.16 (d, J = 7.6 Hz, 2H, quinazoline H), 7.93 (t, J = 8.0 Hz, 2H, quinazoline H), 7.84-7.88 (m, 4H,
ArH), 7.65 (t, J = 7.2 Hz, 2H, ArH), 7.55 (d, J = 8.8 Hz, 1H, ArH), 7.48 (d, J = 8.0 Hz, 1H, ArH),
7.27 (t, J = 8.0 Hz, 1H, ArH), 4.38 (q, J = 6.8 Hz, 2H, -CH2CH3), 1.35 (t, J = 5.6 Hz, 3H, -CH2CH3)
; FT-IR (ύ max, cm-1): 3406-3186-3136 (3NH), 3074 (CH aromatic), 2989 (CH aliphatic), 1738
(C=O ester) , 1680 (C=O amide), 1647 (C=N); MS: (Mwt.: 427.46) : m/z (% rel. Int.) , 427.29
(M+ , 30 %), 136.43 (100%); Anal. Calcd for C24H21N5O3: C, 67.44; H, 4.95; N, 16.38. Found: C,
67.73; H, 5.12; N, 16.49.

5.1.1.2 Ethyl 4-((4-(3-(3-fluorophenyl)ureido)phenyl)amino)quinazoline-2-carboxylate (4b)
The titled compound was separated as red crystals (0.26 g, 60%); m.p 254-258°C; 1HNMR (400
MHz, DMSO-d6) δ 9.44 (s, 1H, NH D2O exchangeable), 8.82 (s, 1H, NH D2O exchangeable),
8.18 (d, J = 8.0 Hz, 2H, quinazoline H), 8.03 (s, 1H, NH D2O exchangeable), 7.93 (d, J = 8.8 Hz,
1H, ArH), 7.91 (s, 1H, ArH), 7.88 (d, J = 7.6 Hz, 2H, ArH), 7.83 (d, J = 8.0 Hz, 2H, ArH), 7.64
(t, J = 7.2 Hz, 3H, quinazoline 2H, ArH), 7.57 (d, J = 8.8 Hz, 1H, ArH), 4.39 (q, J = 6.8 Hz, 2H,-
CH2CH3), 1.36 (t, J = 7.2 Hz, 3H,CH2CH3) ; FT-IR (ύ max, cm-1): 3441-3340-3182 (3NH), 3070
(CH aromatic), 2981 (CH aliphatic), 1739 (C=O ester) , 1678 (C=O amide), 1631 (C=N); MS:
(Mwt.: 445.45) : m/z (% rel. Int.), 445.12 (M+, 25%), 129.47 (100%); Anal. Calcd for
C24H20FN5O3: C, 64.71; H, 4.53; N, 15.72. Found: C, 64.58; H, 4.67; N, 15.64.
5.1.1.3 Ethyl 4-((4-(3-(4-fluorophenyl)ureido)phenyl)amino)quinazoline-2-carboxylate (4c)
The titled compound was separated as red crystals (0.21 g, 54%); m.p. 252-254oC; 1HNMR (400
MHz, DMSO-d6) δ 11.22 (s, 1H, NH D2O exchangeable), 8.68 (s, 2H, NH D2O exchangeable),
8.17 (m, 2H, quinazoline H), 7.90 (m, 3H, ArH, quinazoline H), 7.83 (d, J = 8.0 Hz, 2H, ArH),
7.79 (d, J = 8.0 Hz, 2H, ArH), 7.64 (m, 3H, ArH), 4.39 (q, J = 7.2 Hz, 2H, -CH2CH3), 1.35 (t, J =
5.6 Hz, 3H, -CH2CH3) ; FT-IR (ύ max, cm-1): 3410-3259-3178 (3NH), 3070 (CH aromatic), 2916
(CH aliphatic), 1730 (C=O ester ) , 1690 (C=O amide), 1647 (C=N); MS: (Mwt.: 445.45) : m/z
(% rel. Int.), 445.95 (M+, 8%), 407.19 (100%); Anal. Calcd for C24H20FN5O3: C, 64.71; H, 4.53;
N, 15.72. Found: C, 64.58; H, 4.69; N, 15.88.
5.1.1.4 Ethyl 4-((4-(3-(m-tolyl)ureido)phenyl)amino)quinazoline-2-carboxylate (4d)
The titled compound was separated as red crystals (0.24 g, 61%); m.p. 260-261°C; 1HNMR (400
MHz, DMSO-d6) δ 9.20 (s, 1H, NH D2O exchangeable), 9.11 (s, 1H, NH D2O exchangeable),
8.17 (d, J = 8.0 Hz, 1H, quinazoline H), 8.15 (s, 1H, NH D2O exchangeable), 8.04 (t, J = 7.0 Hz,
1H, quinazoline H), 7.89 (d, J = 8.0 Hz, 2H, ArH), 7.83 (d, J = 7.8 Hz, 2H, ArH), 7.64 (t, J = 8.0
Hz, 1H, quinazoline H), 7.54 (d, J =8.8 Hz, 1H, quinazoline H), 7.30 (s, 1H, ArH), 7.26 (d, J = 8.0
Hz, 1H, ArH), 7.15 (t, J = 8.0 Hz, 1H, ArH), 6.78 (d, J = 8.0 Hz, 1H, ArH), 4.36 (q, J = 4.0 Hz,
13
2H, -CH2CH3), 2.27 (s, 3H, CH3) 1.35 (t, J = 4.0 Hz, 3H, -CH2CH3) ; C NMR (101 MHz,
DMSO) δ 160.9, 146.7, 138.4, 135.2, 129.1, 128.9, 127.7, 126.5, 126.3, 124.1, 123.9, 122.7,
119.0, 118.4, 115.7 , 62.5 ,21.9 14.5; FT-IR (ύ max, cm-1): 3406-3224-3194 (3NH), 3074 (CH

aromatic), 2989 (CH aliphatic), 1730 (C=O ester) 1681 (C=O amide), 1630 (C=N); MS: (Mwt.:
441.49) : m/z (% rel. Int.), 441.57 (M+, 26%), 298.47 (100%); Anal. Calcd for C25H23N5O3: C,
68.01; H, 5.25; N, 15.86. Found: C, 68.24; H, 5.41; N, 16.03.
5.1.1.5 Ethyl 4-((4-(3-(3-methoxyphenyl)ureido)phenyl)amino)quinazoline-2-carboxylate
(4e)
The titled compound was separated as red crystals (0.19 g, 60%); m.p. above 300°C; 1HNMR
(400 MHz, DMSO-d6) δ 9.48 (s, 1H, NH D2O exchangeable), 9.37 (s, 1H, NH D2O exchangeable),
8.86 (s, 1H, NH D2O exchangeable), 8.36 (d, J = 8.4 Hz, 1H, quinazoline H), 8.23 (t, J = 6.8 Hz,
1H, quinazoline H), 8.03 (d, J = 8.0 Hz, 1H, quinazoline H), 7.82 (d, J = 8.0 Hz, 2H, ArH), 7.62
(t, J = 8.0 Hz, 1H, quinazoline H), 7.56 (d, J = 8.0 Hz, 2H, ArH), 7.22 (s, 1H, ArH), 7.15 (t, J =8.0
Hz, 1H, ArH), 6.95 (d, J = 8.0 Hz, 1H, ArH), 6.54 (d, J = 6.4 Hz, 1H, ArH), 4.43 (q, J = 7.2 Hz,
2H, -CH2CH3), 3.73 (s,3H,OCH3) ,1.38 (t, J = 6.8 Hz, 3H, -CH2CH3) ; FT-IR (ύ max, cm-1): 3444-
3221-3194 (3NH), 3097 (CH aromatic), 2989 (CH aliphatic), 1728 (C=O ester) , 1693 (C=O
amide), 1635 (C=N); MS: (Mwt.: 457.49) : m/z (% rel. Int.), 457.79 (M+, 28 %), 142.80 (100%);
Anal. Calcd for C25H23N5O4: C, 65.64; H, 5.07; N, 15.31. Found: C, 65.49; H, 5.07; N, 15.43.
5.1.1.6 Ethyl 4-((4-(3-(4-methoxyphenyl)ureido)phenyl)amino)quinazoline-2-carboxylate (4f)
1
The titled compound was separated as red crystals (0.26 g, 63%); m.p above 300°C; HNMR
(400 MHz, DMSO-d6) δ 10.95 (s, 2H, NH D2O exchangeable), 9.46 (s, 1H, NH D2O
exchangeable), 8.83 (d, J=8.4, 1H, quinazoline H), 8.83 (d, J = 8.4 Hz, 2H, ArH), 8.05 (m, 2H,
ArH), 7.93 (d, J = 8.8 Hz, 2H, ArH), 7.83 (t, J = 6.0 Hz, 2H, ArH), 7.57 (d, J = 8.8 Hz, 3H, ArH),
4.42 (q, J = 6.8 Hz, 2H, -CH2CH3), 3.77 (s,3H,OCH3) 1.38 (t, J = 7.2 Hz, 3H, -CH2CH3) ; FT-IR
(ύ max, cm-1): 3390-3259-3101(3NH), 3016(CH aromatic), 2935 (CH aliphatic), 1743 (C=O
ester), 1701 (C=O amide), 1631 (C=N); MS: (Mwt.: 457.49) : m/z (% rel. Int.), 457.76 (M+, 28
%), 140.70 (100%); Anal. Calcd for C25H23N5O4: C, 65.64; H, 5.07; N, 15.31. Found: C, 65.78;
H, 5.30; N, 15.49.
5.1.1.7 Ethyl...4-((4-(3-(4-chlorophenyl)ureido)phenyl)amino)quinazoline-2-carboxylate (4g)
The titled compound was separated as red crystals (0.26 g, 60%); m.p. above 280°C; 1HNMR
(400 MHz, DMSO-d6) δ 9.43 (s, 1H, NH D2O exchangeable), 8.49 (s, 1H, NH D2O exchangeable),