Novel series of 6-(2-substitutedacetamido)-4-anilinoquinazolines as EGFR-ERK signal transduction inhibitors in MCF-7 breast cancer cells

Article abstract of DOI:10.1016/j.ejmech.2018.06.024

Epidermal growth factor receptor (EGFR) signaling pathway has been previously investigated for its significant role in the progression of different types of malignant tumors, where development of small molecules targeting EGFR is well known strategy for design of antitumor agents. Herein, we report the design and synthesis of two series of 6-(2-substitutedacetamido)-4-anilinoquinazolines (6a-x and 13a-d) as EGFR inhibitors. All the newly synthesized quinazoline derivatives were in vitro evaluated for their anti-proliferative activity towards MCF-7 (Breast Cancer) and HepG2 (Hepatocellular carcinoma) cell lines. In particular, compound 6n showed significant inhibitory activity against MCF-7 and HepG2 cell lines (IC₅₀ = 3 and 16 μM, respectively), compared to that of Erlotinib (IC₅₀ = 20 and 25 μM, respectively). Western blotting of 6n at MCF-7 cell line revealed the dual inhibitory activity of 6n towards diminishing the phosphorylated levels for EGFR and ERK. Also, ELISA assay confirmed the anti-EGFR activity of compound 6n (IC₅₀ = 0.037 μM). Finally, a molecular docking study showed the potential binding mode of 6n within the ATP catalytic binding site of EGFR, exhibiting similar binding mode to EGFR inhibitor Erlotinib.

Full text of DOI:10.1016/j.ejmech.2018.06.024

European Journal of Medicinal Chemistry 155 (2018) 782e796
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Novel series of 6-(2-substitutedacetamido)-4-anilinoquinazolines as
EGFR-ERK signal transduction inhibitors in MCF-7 breast cancer cells
,
,
Rania S.M. Ismail a, Sahar M. Abou-Seri b **, Wagdy M. Eldehna c *, Nasser S.M. Ismail d,
Sara M. Elgazwi e, Hazem A. Ghabbour f g, Mahmoud Salama Ahmed h,
,
Fathi T. Halaweish e, Dalal A. Abou El Ella i
,
j
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Egyptian Russian University, Badr City, P.O. Box 11829, Cairo, Egypt
b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini Street, P.O. Box 11562, Cairo, Egypt
c Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kafrelsheikh University, Kafrelsheikh, 33516, Egypt
d Pharmaceutical Chemistry Department, Faculty of Pharmaceutical Sciences and Pharmaceutical Industries, Future University in Egypt, Cairo, Egypt
e Department of Chemistry and Biochemisty, South Dakota State University, Brookings, SD, 57007, USA
f Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh, 11451, Saudi Arabia
g Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, 35516, Egypt
h Department of Pharmaceutical Chemistry, Faculty of Pharmacy, The British University in Egypt, Al-Sherouk City, Cairo, Egypt
i Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Abbassia, P.O. Box 11566, Egypt
j Faculty of Pharmacy, Nahda University, New Beni Suef (NUB), 62511, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
Epidermal growth factor receptor (EGFR) signaling pathway has been previously investigated for its
significant role in the progression of different types of malignant tumors, where development of small
molecules targeting EGFR is well known strategy for design of antitumor agents. Herein, we report the
design and synthesis of two series of 6-(2-substitutedacetamido)-4-anilinoquinazolines (6a-x and 13a-d)
as EGFR inhibitors. All the newly synthesized quinazoline derivatives were in vitro evaluated for their
anti-proliferative activity towards MCF-7 (Breast Cancer) and HepG2 (Hepatocellular carcinoma) cell
lines. In particular, compound 6n showed significant inhibitory activity against MCF-7 and HepG2 cell
Received 18 March 2018
Received in revised form
6 June 2018
Accepted 8 June 2018
Available online 19 June 2018
Keywords:
Anilinoquinazolines
ERK
lines (IC50 ¼ 3 and 16
m
M, respectively), compared to that of Erlotinib (IC50 ¼ 20 and 25
mM, respectively).
Western blotting of 6n at MCF-7 cell line revealed the dual inhibitory activity of 6n towards diminishing
the phosphorylated levels for EGFR and ERK. Also, ELISA assay confirmed the anti-EGFR activity of
EGFR
compound 6n (IC50 ¼ 0.037
mM). Finally, a molecular docking study showed the potential binding mode
of 6n within the ATP catalytic binding site of EGFR, exhibiting similar binding mode to EGFR inhibitor
Synthesis
Docking
Erlotinib.
© 2018 Elsevier Masson SAS. All rights reserved.
1. Introduction
signaling transduction pathways [2]. EGFR is a member of tyrosine
kinases, when ligand binds to the extracellular domain, confor-
Epidermal Growth Factor Receptor (EGFR) plays pivotal role in
the regulation of the cellular functions such as cell growth, survival,
proliferation, differentiation, and apoptosis [1]. EGFR activation
occurs via binding of the endogenous ligands; epidermal growth
factor (EGF), transforming growth factor-a (TGF-a), amphiregullin,
betacellulin, heparin binding EGF, and epiregulin; resulting in the
mational change happens, leading to EGFR activation via homo-
dimerization or heterodimerization. EGFR dimerization activates its
intrinsic intracellular protein-tyrosine kinase activity [3]. As a
result, autophosphorylation of several tyrosine residues occured
leading to downstream activation for Mitogen Activated Protein
Kinase (MAPK) signaling transduction pathway via phosphoryla-
tion of a cascade of downstream proteins [3].
Different cancer types (as hepatocellular carcinoma, breast
cancer, non-small cell lung cancer, pancreatic cancer, colorectal
carcinoma, melanoma, and glioblastoma), have shown significant
mutations in several members of MAPK pathway; including EGFR,
* Corresponding author.
** Corresponding author..
E-mail addresses: sahar.shaarawy@pharma.cu.edu.eg (S.M. Abou-Seri),
wagdy2000@gmail.com (W.M. Eldehna).
https://doi.org/10.1016/j.ejmech.2018.06.024
0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
783
RAF, MEK, and ERK, [4e6]. Emerging new chemical scaffolds as
potential drug candidates targeting the phosphorylation cascade
pathway of protein kinases created plausible rational for treatment
of cancer via inhibition of cell growth and induction of apoptosis
[7].
Development of kinase inhibitors was rationalized based on the
fundamental understanding of the presence of ATP as the endog-
enous substrate binding to the kinases to aid in the phosphoryla-
tion process along the whole cascade. Hence, most of the small
molecule kinase inhibitors have been emerged to be ATP compet-
itive inhibitors binding to the ATP catalytic active site [7]. Different
chemical classes were developed to be biologically evaluated for
their potential to inhibit kinases in order of treatment of cancer [8].
4-Anilinoquinazoline scaffold has emerged as an important leading
one for developing effective EGFR inhibitors, exemplified by the
clinically approved drugs; Gefitinib I, Erlotinib II, Lapatinib III,
Afatinib IV and Vandetanib V [9e14] (Fig. 1).
Initially, the 4-anilinoquinazoline core was retained, followed by
introduction of three different modifications. The first modification
focused on introducing variety of hydrophilic solubilizing moieties
(as pyrrolidino, piperidino, morpholino, N-methylpiperazino) to
the C-6 position of the quinazoline core through acetamide spacer
(-CH2-CONH-) to elucidate their impact on the activity. The second
modification included introduction of different electron with-
drawing groups (as CF3, Cl, or Br) to 30- and/or 40-positions of the 4-
anilino moiety (6a-x). The third modification focused on extending
the anilino moiety of the first series 6a-x with a benzyloxy tail to
afford the second series 13a-d, in an attempt to increase the hy-
drophobic interaction with EGFR hydrophobic back pocket in
analogy to Lapatinib.
The newly synthesized 4-anilinoquinazoline analogs were
evaluated in enzyme-based and cell-based assays for their ability to
inhibit EGFR and the proliferation against MCF-7 and HepG2 cancer
cell lines expressing EGFR. In addition we also investigated their
effect on the activation of EGFR by western blot.
Structure-Activity Relationship (SAR) studies for the 4-
anilinoquinazoline-based EGFR inhibitors suggested that: The
quinazoline heterocyclic moiety binds in the ATP-binding pocket
and forms a hydrogen bond with the NH group of Met769 (Met793)
in the hinge region. Erlotinib binds to the active conformation while
2. Results and discussion
2.1. Chemistry
Lapatinib binds to the displaced inactive aC-out conformation of
EGFR. The anilino group of Gefitinib, Erlotinib, or Lapatinib extends
into a hydrophobic region of the kinase domain of EGFR that is
adjacent to the adenine-binding site (the back pocket), as shown in
The synthetic pathways adopted for the preparation of the in-
termediates and target 4-anilinoquinazolines are depicted in
Schemes 1 and 2. Synthesis was initiated in Scheme 1 by reacting 5-
nitroanthranilonitrile 1 with DMF-DMA to afford the formamidine
intermediate 2, which cyclized upon reaction with different ani-
lines in refluxing glacial acetic acid into 6-nitro-4-
anilinoquinazolines 3a-f through Dimroth rearrangement. Reduc-
tion of the latter using sodium dithionite/ammonia mixture yielded
the 6-amino-4-anilinoquinazolines 4a-f, which was subsequently
acylated with 2-chloroacetyl chloride in dioxane to afford the cor-
responding 2-chloroacetamido derivatives 5a-f. The target com-
pounds 6a-x were prepared through a nucleophilic substitution on
5a-f with the appropriate heterocyclic secondary amines in dry
acetonitrile in the presence of anhydrous potassium carbonate and
a catalytic amount of potassium iodide (Scheme 1).
In Scheme 2, the N-(4-arayloxyphenyl)acetamides 8a and b
were prepared by o-alkylation of paracetamol 7 with appropriate
benzyl halide. This was followed by hydrolysis of the protected
amino group of compounds 8a, b using NaOH to give the benzy-
loxyanilines 9a, b as white solid and an oily residue, respectively,
which used directly in cyclization of the formamidine 2 in refluxing
glacial acetic acid to afford 6-nitro-4-(benzyloxyanilino)quinazo-
lines 10a, b. Then, the target compounds 13a-d were obtained
adopting the general synthetic pathway for synthesis of 6a-x. The
Fig. 2. This hydrophobic site extends only to the
b5-sheet con-
taining the Thr790 gate keeper residue and to Thr855 on top of the
activation segment. The anilino moiety of Lapatinib contains a 3-
fluorobenzyloxy extension that displaces the
position. As a result of the larger size of Lapatinib, the hydrophobic
pocket next to the adenine-binding site extends to the 4-sheet
(Leu778), the C-helix (Met766), the top of the 7-activation
aC-helix to its out
b
a
b
segment loop (Leu856), and the activation segment (Phe856)
[15e19].
Utilizing the reported key binding features between the EGFR
and 4-anilinoquinazoline template and as a part of our ongoing
efforts to assemble novel small molecules targeting kinases
[20e24], herein we present two new series of 6-substituted-4-
anilinoquinazolines (6a-x and 13a-d) as promising candidates for
EGFR inhibition. Our strategy is directed towards designing a va-
riety of ligands with diverse chemical properties hypothesizing that
the potency and selectivity of these molecules might be enhanced
by attaching new moieties at C6 of substituted 4-
anilinoquinazolines and/or by varying the aniline substitution
pattern, Fig. 3.
Gefitinib, Erlotinib and Lapatinib were used as lead compounds.
Fig. 1. Chemical structure for the clinically approved 4-anilinoquinazoline EGFR inhibitors I-V.

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R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
Fig. 2. Pharmacophore model of (A) Erlotinib II and (B) Lapatinib III along with catalytic domain of EGFR kinase.
Fig. 3. Rational design of the target 4-anilinoquinazolines (6a-x and 13a-d).
nitro group in compounds 10a, b reduced into the corresponding 6-
aminoquinazoline derivatives 11a, b using sodium dithionite/
ammonia, which subsequently acylated with 2-chloroacetyl chlo-
ride into the 2-chlrolacetamides 12a, b then reacted with the
appropriate alicyclic amine to yield 13a-d.
Spectral data supported the structures of the target 4-
anilinoquinazolines (6a-x and 13a-d), where IR spectra revealed
the presence of bands of the carbonyl group at 1662-1705 cmꢀ1,
while the anilino NH and the amidic NH bands were detected be-
tween 3100 and 3500 cmꢀ1. Their 1H NMR spectra showed protons
of acetamide spacer (-COCH2-) as singlet signal, shifted up-field
(around
secondary amine. In addition, 1H NMR spectra of 13a-d revealed a
singlet signal at 5.04e5.18 ppm due to benzylic CH2 protons. For
d 3.1 ppm) because of the shielding effect of heterocyclic
d
compounds (6a, 6e, 6i, 6m, 6q, 6u, 13a and 13c), the pyrrolidine
methylene protons appeared as two broad singlet signals around

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
785
Scheme 1. Synthesis of target 4-anilinoquinazolines (6a-x). Reagents and conditions: (i) DMF-DMA, reflux, 90 min; (ii) Substituted aniline, glacial acetic acid, reflux, 2 h; (iii)
Sodium dithionite, ammonia, reflux, 15 min; (iv) ClCH2COCl, dioxane, 0 ꢁC, 30 min; (v) Heterocyclic secondary amine, K2CO3, KI, acetonitrile, reflux, 6e8 h.
Scheme 2. Synthesis of target 4-anilinoquinazolines (13a-d). Reagents and conditions: (i) Sodium ethoxide, stir, rt; then benzyl bromide or 3-fluorobenzyl bromide, reflux, 1hr;
(ii) NaOH, H2O, EtOH, 7 h; (iii) Intermediate 2, glacial acetic acid, reflux, 2 h; (iv) Sodium dithionite, ammonia, reflux, 15 min; (v) ClCH2COCl, dioxane, 0 ꢁC, 30 min; (vi) Alicyclic 2ꢁ
amine, K2CO3, KI, acetonitrile, reflux, 6 h.
1.7 and 2.6 ppm. On the other hand the 1H NMR spectra of com-
pounds (6b, 6f, 6j, 6n, 6r and 6v) showed three signals of the
piperidine protons around 1.4, 1.6 and 2.5 ppm. In case of com-
pounds (6c, 6g, 6k, 6o, 6p, 6w, 13b and 13d), two sets of triplet
Compounds (6d, 6h, 6l, 6o, 6q and 6x) showed the N-methyl
piperazine protons at around 2.1 ppm for the methyl group as
singlet and two signals around 2.4 and 2.5 ppm as triplets.
The 13C NMR spectra also confirmed the presence of (-COCH2-)
spacer by two types of signals, one for CH2 at range
d
d
signals of the morpholine appeared at
d
around 2.25 and 3.6 ppm.
a d

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R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
Table 2
59.8e62.4 ppm and the other for the carbonyl group at a
168.5e169.5 ppm. Elucidation of pyrrolidine moiety in compounds
6m, 6q and 13c showed two signals at 23.9 and 54.2 ppm, while
the piperidine ring in compound 6b showed three signals at 23.9,
25.9 and 54.6 ppm. The morpholine carbons in compounds 6c, 6w
and 13d were detected as signals at 53.7 and 66.5 ppm, while the
N-methyl piperazine in compound 6l appeared as three signals at
45.7, 52.7 and 54.4 ppm.
d range
IC50 values for the inhibitory activity of compounds
(6i, 6n-p and 13c) against EGFR.
d
Compound
IC50 (nM)a
EGFR
d
d
6i
6n
6o
6p
13c
Erlotinib
32
37
98
32
71
73
2
2
5
3
6
5
d
2.2. Biological evaluation
a
IC50 values are the mean SD of three separate
2.2.1. In vitro anti-proliferative activity against EGFR-expressing
cancer cell lines
experiments.
The in vitro anti-proliferative activity of the newly prepared 4-
anilinoquinazolines (6a-x and 13a-d) was examined against two
EGFR-expressing cancer cell lines; MCF-7 (Breast Cancer) and
HepG2 (Hepatocellular carcinoma) cell lines using the 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
colorimetric assay [25]. Erlotinib was used as a reference drug. The
results were expressed as median growth inhibitory concentration
indicated that compound 6f (IC50 ¼ 12.00 0.16
be the most potent derivative, being two times more active than
Erlotinib (IC50 ¼ 25 1.50 M). Also, compounds 6i, 6j, 6n and 6o
possessed higher anti-proliferative activities than the reference
drug 16.00 3.95, 16.00 0.15 and
(IC50 ¼ 18.00 2.47,
19.00 0.95 M, respectively). Beside, compounds 6a, 6p, 6x and
13c were almost equipotent to Erlotinib (IC50 z 25 M). On the
other hand, evaluation of the anti-proliferative activity in MCF-
7 cell line revealed that compound 6n (IC50 ¼ 3.00 0.10 M) was
the most potent counterpart against MCF-7 as it was six times more
mM) was found to
m
m
m
(IC50) values, which represent the compound concentration
required to produce a 50% inhibition of cell growth after 48 h of
incubation, compared to untreated controls (Table 1).
m
Investigations of the anti-proliferative activity against HepG2
Table 1
Anti-proliferative activity of target compounds 6a-x and 13a-d towards HepG2 and MCF-7 cancer cell lines.
Compound
R
X
n
IC50 (m
M)a
HepG2
MCF-7
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
13a
13b
13c
13d
Erlotinib
3-CF3
3-CF3
3-CF3
3-CF3
3-Cl
3-Cl
3-Cl
3-Cl
4-Cl
CH2
CH2
O
N-CH3
CH2
CH2
O
N-CH3
CH2
CH2
O
N-CH3
CH2
CH2
O
N-CH3
CH2
CH2
O
N-CH3
CH2
CH2
O
N-CH3
CH2
O
CH2
O
0
1
1
1
0
1
1
1
0
1
1
1
0
1
1
1
0
1
1
1
0
1
1
1
0
1
0
1
e
25.20 1.05
>100
>100
>100
>100
12.00 0.16
>100
>100
18.00 2.47
16.00 3.95
>100
>100
>100
16.00 0.15
19.00 0.95
25.30 1.08
>100
>100
>100
>100
>100
>100
>100
15.00 1.10
>100
>100
>100
>100
>100
16.00 0.20
>100
4-Cl
4-Cl
4-Cl
>100
15.00 1.10
12.00 0.70
3.00 0.10
15.00 1.08
10.00 0.70
>100
>100
>100
>100
>100
>100
>100
>100
>100
25.00 2.40
25.00 3.60
>100
3-Br
3-Br
3-Br
3-Br
4-Br
4-Br
4-Br
4-Br
4-Cl-3-CF3
4-Cl-3-CF3
4-Cl-3-CF3
4-Cl-3-CF3
H
>100
>100
25.30 0.06
>100
>100
25.05 4.20
>100
25 1.50
H
F
F
e
e
20 0.93
a
IC50 values are the mean SD of three separate experiments.

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
787
potent than Erlotinib (IC50 ¼ 20 0.93
m
M). Furthermore, com-
In a similar vein to the anti-proliferative activity of the target 4-
anilinoquinazolines, replacing the 4-Cl substituent in compound
6i (IC50 ¼ 32 2 nM) with the 3-fluorobenzyloxy extension in
compound 13c (IC50 ¼ 71 6 nM) resulted in 2-fold decrease in
inhibitory activity against EGFR. Moreover, the 3-bromo-anilino-
quinazoline derivatives featuring a piperidinyl 6n (IC50 ¼ 37 2)
or N-methylpiprazinyl 6p moiety (32 3 nM) were more potent
EGFR inhibitors than the morpholinyl counterpart 6o
(IC50 ¼ 98 5 nM).
pounds 6c, 6i, 6l, 6m, 6o and 6p possessed superior activity to
Erlotinib with IC50 values ranging from 10.00 0.70 to
16.00 0.20 mM. Only five compounds (6i, 6n-6p, and 13c) showed
activity against both HepG2 and MCF-7 cancer cell lines.
It is worth noting that grafting 3-Br substituent to the 4-anilino
moiety enhanced the anti-proliferative activity against HepG2 and
MCF-7 cell lines, especially when accompanied with piperidinyl
moiety on the acetamide spacer at 6-position, otherwise 3-CF3 and
3-Cl substituents mostly resulted in inactive members. Also,
incorporation of electron withdrawing groups at 4-position is not
favorable for the anti-proliferative activity, except for the 4-Cl
substituent which is well tolerated. Furthermore, extending the
aniline moiety with a benzyloxy group, as in compounds 13,
resulted in a decreased or abolished activity.
2.2.3. Western blotting of EGFR and ERK in MCF-7 cells
The promising anti-proliferative activity of 6n prompted us to
study its effect at the molecular level investigating its potential
inhibitory effect to the targets of interest. Initially, we investigated
the potential of 6n to inhibit the phosphorylation of EGFR at 3 mM
Finally, we can deduce that the substitution pattern on the 4-
anilino moiety is a crucial element for the anti-proliferative activ-
ity of the target 4-anilinoquinazolines (6a-x and 13a-d). While
grafting a 4-benzyloxy tail decreases the activity, incorporation of
the electron withdrawing (Br) substituent at the meta position
along with piperidinyl moiety at C-6 position of the quinazoline
core through acetamide spacer (-CH2-CONH-) greatly enhances the
anti-proliferative activity particularly against breast cancer MCF-
7 cell line.
within the MCF-7 cell lysate. The phosphorylated EGFR serves to
recruit specific effector molecules to activate several signal trans-
duction pathways that have been implicated pathogenesis of many
cancers including the RAS/RAF/MEK/ERk (MAPK) and STATs path-
ways [26]. Thus, our molecular mechanistic investigation was
extended to test the effect of 6n to inhibit the phosphorylation of
the downstream kinases of these signaling pathways, ERK and
STAT3.
The western blot analysis results (Fig. 4) revealed the potential
of 6n to have inhibitory mechanism towards diminishing the
phosphorylated levels for both of EGFR and ERK, as shown in Fig. 4a
and b. However, 6n did not manage to inhibit STAT3 phosphory-
lation (Fig. 4c). These results were further supported by quantita-
tive determination of phosphorylated EGFR in MCF-7 cell lysate
using ELISA assay. The obtained data revealed that, treatment of
MCF-7 cell with 6n for 24 and 48 h diminished EGFR phosphory-
lation by about 2 and 3.3 folds compared to untreated cell (Control)
Fig. 4d.
2.2.2. In vitro EGFR kinase ELISA assay
2.2.2.1. Determination of EGFR inhibitory activity (IC50).
Compounds (6i, 6n-6p, and 13c), which showed good anti-
proliferative activity against both HepG2 and MCF-7 cancer cell
lines, were selected to evaluate their inhibitory activity against
EGFR using the Alpha Screen system (PerkinElmer, USA), in com-
parison to the clinically used Erlotinib as standard EGFR inhibitor.
The results are reported as a 50% inhibition concentration value
(IC50) and are summarized in Table 2. The tested compounds
exhibited excellent to good inhibitory activity against EGFR with
IC50 values ranging from 32 to 98 nM.
The ability of 6n to inhibit the intrinsic kinase activity of EGFR
and prevent the phosphorylation and activation of ERK, suggests
that it may play a role in suppressing the proliferation of tumors in
Fig. 4. Western blot analysis of MCF-7 cell lysate after treatment with 3
and c) Stat3 and its phosphorylated form. The loading control was -actin. d) ELISA assay for phosphorylated EGFR relative to total EGFR in MCF-7 cell lysate after treatment with
M of compound 6n for 24 and 48 h.
mM of compound 6n for 48 h: a) EGFR and its phosphorylated form, b) ERK1/2 and its phosphorylated form,
b
3
m

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Table 3
FRED Chemgauss score (Kcal/mol) based on docking along with PDB ID: 1XKK and Rocs_tanimoto combo score for (6i, 6n-p, and 13c)
based on Lapatinib as query.
Compounds
FRED Chemgauss score (kcal/mol)
Rocs_ Tanimoto Combo score
6I
6N
6O
6P
13C
Lapatinib
ꢀ12.6724
ꢀ11.6706
ꢀ11.1193
ꢀ12.3660
ꢀ6.0030
ꢀ9.4283
0.977
0.956
0.942
0.939
0.854
NA
to ꢀ9.4283 kcal/mol, compared to Lapatinib ꢀ9.4283 kcal/mol.
They shared the same binding affinity to the receptor of interest
that in terms of sharing the hydrogen bonding towards MET 793:A,
the quinzoline moiety showed hydrophobic-hydrophobic in-
teractions along with LEU 718:A, LEU 792:A, GLY 796:A, LEU 844:A
within the catalytic binding domain of EGFR, and the 2-(piperidin-
1-yl)acetamide side chain at position 6 of quinazoline ring is ori-
ented toward solvent front with piperidine ring projecting in the
solvent bulk, as shown in Fig. 5. Meanwhile, 13c showed lower
binding affinity in terms of energy profile (ꢀ6.0030 kcal/mol) and
different binding mode compared to Lapatinib.
However, the designed analogs (6i, 6n, 6o, and 6p) didn't exhibit
the expected projection towards the back pocket of the EGFR cat-
alytic domain in a different binding fashion as exhibited by Lapa-
tinib, this prompted us to evaluate the binding mode of the
designed analogs with respect to Erlotinib as a query structure in
terms of Tanimoto Combo co-efficients; whereas 6i, 6n, 6o, and 6p
showed better degree of superimposition towards Erlotinib ranging
from 1.071 to 1.019, as shown in Table 4.
The in silico molecular docking study for the designed analogs
was conducted in comparison along with Erlotinib (PDB ID: 1M17)
to exhibit comparable binding affinity of 6i, 6n, 6o, and 6p with
respect to FRED shapegauss score ranging from ꢀ10.2993
to ꢀ8.2338 kcal/mol, related to Erlotinibꢀ9.2285 kcal/mol; sharing
the same neighbouring amino acid residues for hydrophobic-
hydrophobic interactions, such as LEU 694:A, LYS 721:A, LEU
768:A, GLY 772:A, LEU 820:A, and ASP 831:A and hydrogen bonding
interactions between N- atom of quinazoline ring along with MET
769:A, as per shown for 6n in Fig. 6. Meanwhile, 13c showed lower
binding affinity in terms of energy profile (ꢀ3.8095 kcal/mol) and
different binding mode compared to Erlotinib.
Fig. 5. Visual representation of 6i (orange), 6n (grey), 6o (purple), and 6p (red), and
Lapitinib (green) towards catalytic binding domain of EGFR, where the green dotted
line showing hydrogen bonding interactions along with MET 793:A. (For interpretation
of the references to color in this figure legend, the reader is referred to the Web version
of this article.)
which ERK is deregulated by mutational activation of the upstream
components of MAPK pathway. Therefore, further investigations
are needed to explore the full antitumor spectrum of 6n.
3. In silico molecular modeling
Initially, ligand based drug design approach was employed to
evaluate the degree of superimposition of 6i, 6n, 6o, 6p, and 13c
along with Lapitinib as a query structure in terms of Tanimoto
combo co-efficients (Shape_Tanimoto þ Color_Tanimoto) ranging
from 0 to 2. Compounds 6i, 6n, 6o, 6p, and 13c showing moderate
degree of superimposition towards Lapatinib ranging from 0.977 to
0.854, as shown in Table 3. This was further suggested by con-
ducting the docking study for 6i, 6n, 6p, and 6o to exhibit better
binding affinity compared to Lapatinib (The co-crystallized ligand
for PDB ID: 1XKK) with respect to the FRED shape gauss score and
the binding mode, as shown in Table 3.
Studying two different crystal structures for EGFR catalytic
binding domain (PDB IDs: 1XKK and 1M17) was executed to validate
the binding mode for the designed analogs to suggest the potential
binding mode for (6i, 6n, 6o, and 6p) is related to that of the Erlo-
tinib, where they bind to the hinge region with no projection to-
wards the ATP back pocket to disrupt the salt bridge between the
helix aC and the conserved lysine in a similar fashion to Lapatinib .
4. Conclusions
4-Anilinoquinazoline-based analogs targeting kinases have
been previously studied, however applying the fundamental drug
design strategies to offer new chemical scaffold targeting EGFR is a
The designed analogs (6i, 6n, 6o, and 6p) exhibited better
binding affinity with lower energy profile ranging from ꢀ12.6724
Table 4
FRED Chemgauss score (Kcal/mol) based on docking along with PDB ID: 1M17 and Rocs_tanimoto combo score for (6i, 6n-p, and 13c)
based on Erlotinib as query.
Compounds
FRED Chemgauss score (kcal/mol)
Rocs_ Tanimoto Combo score
6I
6N
6O
6P
13C
Erlotinib
ꢀ10.2993
ꢀ10.1926
ꢀ8.3920
ꢀ8.2338
ꢀ3.8095
ꢀ9.2285
1.046
1.071
1.047
1.019
0.758
NA

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
789
Fig. 6. Visual representation of (A) Overlay of Erlotinib and 6n molecular structures of Tanimoto combo score ¼ 1.071. (B) Catalytic binding domain of EGFR along with 6n (Top) and
Erlotinib (Bottom), showing hydrophobic e hydrophobic interactions and the purple arrow refers to green dotted line showing hydrogen bonding interactions along with MET
769:A. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
newly adopted approach to introduce novel promising analogs via
scaffold hopping. Some synthesized analogs (6i and 6n-6p) showed
promising in vitro biological activity in terms of cytotoxicity, dual
inhibition whether upstream (EGFR) or downstream (ERK) via
western blotting, followed by validation using ELISA. Furthermore
in-silico molecular modeling studies were carried out to suggest the
binding mode of the synthesized analogs toward ATP catalytic
binding pocket of EGFR kinase. The synthesized analogs can offer
potential hits for further scaffold optimization.
were measured on a Helwett Packard 5988 spectrometers at 70 e.V.
High-resolution mass spectra were obtained using a LTQ Orbitrap
XL instrument. Elemental analyses was carried out at the Regional
Center for Microbiology and Biotechnology, Al-Azhar University,
Cairo, Egypt. Reaction courses and product mixtures were routinely
monitored by thin layer chromatography (TLC) on silica gel pre-
coated F254 Merck plates. Unless otherwise noted, all solvents and
reagents were commercially available and used without further
purification. General procedures for preparation of compounds 2,
3a-f and 4a-f are provided in the supplementary materials.
5. Experimental
5.1.2. Synthesis of N0-(2-Cyano-4-nitrophenyl)-N,N-
dimethylimidoformamide 2
5.1. Chemistry
5.1.1. General
Compound 2 was prepared according to reported procedures
[27] (yield 96%; mp 153e155 ꢁC, as reported).
Melting points were measured with a Stuart melting point
apparatus and were uncorrected. The NMR spectra were recorded
by a Varian Gemini-300 at 300 MHz (Varian Inc., Palo Alto, CA) and
Bruker spectrophotometer at 400 MHz. 1H NMR spectra were run at
300 or 400 MHz, while 13C NMR spectra were run at 75 or 100 MHz
5.1.3. Synthesis of 6-nitro-4-arylaminoquinazoline 3a-f
Compounds 3a-f was prepared according to reported proced-
ures [28,29].
in deuterated dimethylsulphoxide (DMSO-d6). Chemical shifts (dH
)
are reported relative to TMS as internal standard. All coupling
constant (J) values are given in hertz. Chemical shifts (dC) are re-
ported relative to DMSO-d6 as internal standards. IR spectra were
recorded as potassium bromide discs with a Bruker FT-IR spectro-
photometer and expressed in wave number (cmꢀ1). Mass spectra
5.1.4. Synthesis of 6-amino-4-arylaminoquinazolines 4a-f
Compounds 4a-f was prepared according to reported proced-
ures [28e30].

790
R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
5.1.5. General procedure for preparation of N-(4-((substituted
phenyl)amino)quinazolin-6-yl)-2-chloroacetamides 5a-f
J ¼ 8.7 Hz), 8.04e8.11 (2H, m, H-60 and H-8), 8.29 (1H, s, H-2’), 8.89
(1H, s, H-5), 9.00 (1H, s, H-2), 11.09 (1H, s, NH, D2O exchangeable),
11.20 (1H, s, NH, D2O exchangeable); HRMS m/z 415.0335 Mþþ1,
calcd for C17H11Cl2F3N4O 415.0340.
To a stirred ice cooled suspension or solution of intermediates
4a-f (2 mmol) in dioxane (10 mL), chloroacetyl chloride (2.2 mmol)
was added dropwise, then the mixture was stirred for 30 min at
0 ꢁC. The formed solid was filtered off, washed with ice water, dried
and crystallized from ethanol to give compounds 5a-f.
5.1.6. General procedure for preparation of target compounds 6a-x
To a solution of 5a-f (2 mmol) in acetonitrile, anhydrous po-
tassium carbonate (5.5 g, 4 mmol) and catalytic amount of KI were
added. The appropriate secondary amine (2.5 mmol) was then
added drop wise and the reaction mixture was refluxed for 6e8 h.
After the reaction was completed as judged by TLC, the mixture was
poured onto ice-water, acidified with 1 N HCl and left to settle. The
formed precipitate was filtered off, washed by ether, air-dried and
purified by thin layer plate chromatography using (CH2Cl2: meth-
anol 9:1) as eluent to afford the pure target compounds 6a-x.
5.1.5.1. N-(4-(((3-triflouromethyl)phenyl)amino)quinazolin-6-yl)-2-
chloroacetamide 5a. Yellow crystals (yield: 456 mg, 60%),
m.p. > 300 ꢁC; IR (KBr, cmꢀ1): 1692 (CO), 2953 (CH aliphatic), 3229
n
(NH anilino), 3368 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
4.39 (2H, s, CH2CO), 7.60e7.69 (2H, m, H-40 and H-50), 7.88 (1H, d, H-
7, J ¼ 8.7 Hz), 7.97 (1H, d, H-8, J ¼ 9.0 Hz), 8.10 (1H, d, H-60,
J ¼ 9.0 Hz), 8.16 (1H, s, H-20), 8.79 (1H, s, H-5), 8.92 (1H, s, H-2),
10.85 (1H, s, NH, D2O exchangeable), 11.05 (1H, s, NH, D2O
exchangeable).
5.1.6.1. 2-(Pyrrolidin-1-yl)-N-(4-((3-(trifluoromethyl)phenyl)amino)
quinazolin-6-yl)acetamide 6a. Yellow powder (yield: 622 mg, 75%),
5.1.5.2. N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-2-
chloroacetamide 5b. Yellow crystals (yield: 277 mg, 40%),
m.p. 140e142 ꢁC. IR (KBr, cmꢀ1): 1705 (CO), 2949 (CH aliphatic),
n
m.p. > 300 ꢁC; IR (KBr,
n
cmꢀ1): 1679 (CO), 2925 (CH aliphatic), 3176
3130 (NH anilino), 3351 (NH amide); 1H NMR (300 MHz, DMSO-d6)
: 1.79 (4H, s. br, H-300and H-400), 2.66 (4H, s br., H-200and H-500), 3.33
(NH anilino), 3368 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
d
4.40 (2H, s, CH2CO), 7.29 (1H, d, H-40, J ¼ 8.1 Hz), 7.45 (1H, t, H-50,
J ¼ 8.1 Hz), 7.70 (1H, d, H-6’, J ¼ 7.8 Hz), 7.91e7.95 (2H, m, H-7 and
H-20), 8.02 (1H, d, H-8, J ¼ 8.4 Hz), 8.80 (1H, s, H-5), 8.93 (1H, s, H-2),
10.98 (1H, s, NH, D2O exchangeable), 11.10 (1H, s, NH, D2O
exchangeable); HRMS m/z 347.0462 Mþþ1, calcd for C16H12Cl2N4O
347.0466.
(2H, s, CH2CO), 7.43 (1H, d, H-40, J ¼ 7.5 Hz), 7.59 (1H, t, H-50,
J ¼ 7.8 Hz), 7.78 (1H, d, H-7, J ¼ 8.7 Hz), 8.08 (1H, d, H-8, J ¼ 9 Hz),
8.20 (1H, d, H-60, J ¼ 7.8 Hz), 8.27 (1H, s, H-20), 8.59 (1H, s, H-5), 8.65
(1H, s, H-2), 9.94 (2H, s, NH, D2O exchangeable); MS (M.Wt.:
415.42):m/z (rel.int %), 415.10 (Mþ.,0.42); Anal. Calcd. For
C21H20F3N5O: C, 60.72; H, 4.85; N, 16.86. Found: C, 60.88; H, 4.90; N,
17.04.
5.1.5.3. N-(4-((4-chlorophenyl)amino)quinazolin-6-yl)-2-
chloroacetamide 5c. Yellow crystals (yield: 416 mg, 60%),
5.1.6.2. 2-(Piperidin-1-yl)-N-(4-((3-(trifluoromethyl)phenyl)amino)
quinazolin-6-yl)acetamide 6b. Yellow powder (yield: 602 mg, 70%),
m.p. > 300 ꢁC; IR (KBr, cmꢀ1): 1679 (CO), 2946 (CH aliphatic), 3205
n
(NH anilino), 3361 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
m.p. 98e100 ꢁC. IR (KBr, cmꢀ1): 1682 (CO), 2962 (CH aliphatic),
n
4.38 (2H, s, CH2CO), 7.47 (2H, d, H-30, H-50, J ¼ 8.4 Hz), 7.78e7.96
(4H, m, H-20, H-6’, H-7 and H-8), 8.69 (1H, s, H-5), 8.84 (1H, s, H-2),
10.84 (1H, s, NH, D2O exchangeable), 11.12 (1H, s, NH, D2O
exchangeable); HRMS m/z 347.0458 Mþþ1, calcd for C16H12Cl2N4O
347.0466.
3229 (NH anilino), 3400 (NH amide); 1H NMR (300 MHz, DMSO-d6)
: 1.43 (2H, s br., H-400), 1.61 (4H, s br., H-300 and H-500), 2.5 (4H, t, H-
d
200 and H-600), 3.16 (2H, s, CH2CO),7.43 (1H, d, H-40, J ¼ 8.1 Hz), 7.59
(1H, t, H-50, J ¼ 8.1 Hz), 7.78 (1H, d, H-7, J ¼ 9 Hz), 8.07 (1H, d, H-8,
J ¼ 9.0 Hz), 8.19 (1H, d, H-60, J ¼ 6.9 Hz), 8.27 (1H, s, H-20), 8.58 (1H,
s, H-5), 8.65 (1H, s, H-2), 9.94 (1H, s, NH, D2O exchangeable), 9.99
5.1.5.4. N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-2-
chloroacetamide 5d. Yellow crystals (yield: 422 mg, 54%),
m.p. > 300 ꢁC (as reported) [31].
(1H, s, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6)
d:
23.9, 25.9, 54.6, 63.0, 112.5, 115.8, 118.5, 119.9, 123.3, 126.0, 126.1,
127.8, 128.8, 129.4, 129.7, 130.0, 136.9, 153.4, 157.7, 169.4; MS
(M.Wt.: 429.45): m/z (rel.int.%), 429.27 (Mþ$,27.35); Anal. Calcd. For
5.1.5.5. N-(4-((4-bromophenyl)amino)quinazolin-6-yl)-2-
C22H22F3N5O: C, 61.53; H, 5.16; N, 16.31. Found: C, 61.74; H, 5.23; N,
chloroacetamide 5e. Yellow crystals (yield: 470 mg, 60%), m.p.
16.47; HRMS m/z 430.1843 Mþþ1, calcd for C22H22F3N5O 430.1855.
>300 ꢁC. IR (KBr, cmꢀ1): 1692 (CO), 2938 (CH aliphatic), 3209 (NH
n
anilino), 3358 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d: 4.39
5.1.6.3. 2-Morpholino-N-(4-((3-(trifluoromethyl)phenyl)amino)qui-
nazolin-6-yl)acetamide 6c. Yellow powder (yield: 605 mg, 70%), m.p.
(2H, s, CH2CO), 7.58 (2H, d, H-20 and H-60, J ¼ 8.4 Hz), 7.73 (2H, d, H-
30 and H-50, J ¼ 8.7 Hz), 7.85 (1H, d, H-7, J ¼ 8.7 Hz), 7.95 (1H, d, H-8,
J ¼ 8.4 Hz), 8.74 (1H, s, H-5), 8.84 (1H, s, H-2), 10.85 (1H, s, NH, D2O
exchangeable), 10.91 (1H, s, NH, D2O exchangeable).
100e112 ꢁC. IR (KBr,
n
cmꢀ1): 1683 (CO), 2962 (CH aliphatic), 3296
(NH anilino), 3525 (NH amide); 1H NMR (400 MHz, DMSO-d6)
d: 2.56
(4H, t, H-200 and H-600, J ¼ 4.36 Hz), 3.23 (2H, s, CH2CO), 3.67 (4H, t, H-
300 and H-500, J ¼ 4.44 Hz), 7.44 (1H, d, H-40, J ¼ 7.68 Hz), 7.61 (1H, t, H-
50, J ¼ 7.92 Hz), 7.80 (1H, d, H-7, J ¼ 8.92 Hz), 8.01 (1H, d, H-8,
J ¼ 8.96 Hz), 8.20 (1H, d, H-60, J ¼ 8.24 Hz), 8.28 (1H, s, H-20), 8.60
(1H, s, H-5), 8.69 (1H, s, H-2), 10.03 (1H, s, NH, D2O exchangeable),
10.05 (1H, s, NH, D2O exchangeable); 13C NMR (100 MHz, DMSO-d6)
5.1.5.6. N-(3-(triflouromethyl)4-chlorophenyl)quinazolin-6-yl)-2-
chloroacetamide 5f. Yellow crystals (yield: 500 mg, 60%),
m.p. > 300 ꢁC; IR (KBr, cmꢀ1): 1718 (CO), 2942 (CH aliphatic), 3230
n
(NH anilino), 3375 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
4.41 (2H, s, CH2CO), 7.82 (1H, d, H-50, J ¼ 8.1 Hz), 7.96 (1H, d, H-7,
d: 53.7, 62.4, 66.5, 112.7, 115.7, 119.0, 120.6, 126.1, 128.0, 128.8, 130.0,

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
791
136.8,140.7,147.1,153.5,157.7,168.8; MS (M.Wt.: 431.42): m/z (rel.int.
%), 431.23 (Mþ$, 75.78); Anal. Calcd. For C21H20F3N5O2: C, 58.47; H,
4.67; N,16.23. Found: C, 58.65; H, 4.72; N,16.49; HRMS m/z 432.1638
Mþþ1, calcd for C21H20F3N5O2 432.1647.
60 and H-7), 8.01e8.05 (2H, m, H-8 and H-20), 8.58 (1H, s, H-5), 8.64
(1H, s, H-2), 9.86 (1H, s, NH, D2O exchangeable), 10.00 (1H, s, NH,
D2O exchangeable); MS (M. Wt: 410.91): m/z (rel.int. %), 410 (Mþ$,
0.03), 412 (Mþþ2, 0.01); Anal. Calcd. For C21H23ClN6O: C, 61.38; H,
5.64; N, 20.45. Found: C, 61.49; H, 5.62; N, 20.78.
5.1.6.4. 2-(4-Methylpiperazin-1-yl)-N-(4-((3-(trifluoromethyl)
phenyl)amino)quinazolin-6-yl)acetamide 6d. Yellow powder (yield:
5.1.6.9. N-(4-((4-chlorophenyl)amino)quinazolin-6-yl)-2-(pyrroli-
605 mg, 68%), m.p. 128e130 ꢁC. IR (KBr,
n
cmꢀ1): 1677 (CO), 2938
din-1-yl)acetamide 6i. Yellow powder (yield: 470 mg, 62%), m.p.
(CH aliphatic), 3248 (NH anilino), 3483 (NH amide); 1H NMR
(300 MHz, DMSO-d6)
: 2.18 (3H, s, CH3), 2.40 (4H, s br., H-200 and H-
111e113 ꢁC. IR (KBr, cmꢀ1): 1672 (CO), 2962 (CH aliphatic), 3271
n
d
(NH anilino), 3424 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
600), 2.57 (4H, s br., H-300and H-500), 3.19 (2H, s, CH2CO), 7.39 (1H, d,
H-40, J ¼ 8.1 Hz), 7.57 (1H, t, H-50, J ¼ 8.1 Hz), 7.74 (1H, d, H-7,
J ¼ 9.3 Hz), 8.02 (1H, d, H-8, J ¼ 9.9 Hz), 8.15 (1H, d, H-60, J ¼ 9.9 Hz),
8.24 (1H, s, H-20), 8.53 (1H, s, H-5), 8.66 (1H, s, H-2), 9.99 (2H, s, NH,
D2O exchangeable); MS (M.Wt.: 444.46): m/z (rel.int. %), 444.15
(Mþ$, 1.48); Anal. Calcd. For C22H23F3N6O: C, 59.45; H, 5.22; N,
18.91; O, 3.60. Found: C, 59.62; H, 5.31; N, 19.08; HRMS m/z
445.1958 Mþþ1, calcd for C22H23F3N6O 445.1964.
1.79 (4H, s br., H-300 and H-400), 2.69 (4H, s br., H-200 and H-500), 3.37
(2H, s, CH2CO), 7.42 (2H, d, H-30 and H-50, J ¼ 7.8 Hz), 7.76 (1H, d, H-
7, J ¼ 8.4 Hz), 7.87 (2H, d, H-20 and H-60, J ¼ 8.1 Hz), 8.06 (1H, d, H-8,
J ¼ 8.7 Hz), 8.54 (1H, s, H-5), 8.60 (1H, s, H-2), 9.82 (1H, s, NH, D2O
exchangeable), 9.97 (1H, s, NH, D2O exchangeable); MS (M. Wt:
381.86): m/z (rel.int. %), 381.10 (Mþ$, 0.66), 383.05 (Mþþ2, 0.21);
Anal. Calcd. For C20H20ClN5O: C, 62.91; H, 5.28; N, 18.34. Found: C,
63.14; H, 5.29; N, 18.51.
5.1.6.5. N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-2-(pyrroli-
5.1.6.10. N-(4-((4-chlorophenyl)amino)quinazolin-6-yl)-2-(piper-
din-1-yl)acetamide 6e. Buff powder (yield: 420 mg, 55%), m.p.
idin-1-yl)acetamide 6j. Yellow powder (yield: 475 mg, 60%), m.p.
112e115 ꢁC. IR (KBr,
n
cmꢀ1): 1681 (CO), 2958 (CH aliphatic), 3241
162e163 ꢁC. IR (KBr, cmꢀ1): 1702 (CO), 2934 (CH aliphatic), 3203
n
(NH anilino), 3411 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
(NH anilino), 3314 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
1.76 (4H, s br., H-300 and H-400), 2.63 (4H, s br., H-200and H-500), 3.33
(2H, s, CH2CO), 7.14 (1H, d, H-40, J ¼ 7.8 Hz), 7.41 (1H, t, H-50,
J ¼ 8.1 Hz), 7.77e7.83 (2H, m, H-60 and H-7), 8.06e8.10 (2H, m, H-8
and H-20), 8.58 (1H, s, H-5), 8.62 (1H, s, H-2), 9.85 (1H, s, NH, D2O
exchangeable), 9.95 (1H, s, NH, D2O exchangeable); Anal. Calcd. For
1.40 (2H, m, H-400), 1.82 (4H, m, H-300, H-500), 2.5 (overlapped, H-200,
H-600), 4.2 (2H, s, CH2CO), 7.48 (2H, d, H-30, H-50, J ¼ 8.7 Hz), 7.79
(1H, d, H-7, J ¼ 8.7 Hz), 7.87 (2H, d, H-20, H-60, J ¼ 9.3 Hz), 8.01 (1H,
d, H-8, J ¼ 9 Hz), 8.70 (1H, s, H-5), 8.76 (1H, s, H-2),10.60 (1H, s, NH),
11.01 (1H, s, NH); MS (M. Wt: 395.89): m/z (rel.int. %), 395.10 (Mþ$,
0.91), 397.10 (Mþþ2, 0.33); Anal. Calcd. For C21H22ClN5O: C, 63.71;
H, 5.60; N, 17.69. Found: C, 63.17; H, 5.37; N, 17.52; HRMS m/z
396.1584 Mþþ1, calcd for C21H22ClN5O 396.1591.
C
20H20ClN5O: C, 62.91; H, 5.28; N, 18.34. Found: C, 63.16; H, 5.37; N,
18.56;
5.1.6.6. N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-2-(piperidin-
1-yl)acetamide 6f. Yellow powder (yield: 435 mg, 55%),
5.1.6.11. N-(4-((4-chlorophenyl)amino)quinazolin-6-yl)-4-
m.p.120e121 ꢁC. IR (KBr,
n
cmꢀ1): 1685 (CO), 2935 (CH aliphatic),
morpholinylacetamide 6k. Yellow powder (yield: 515 mg, 65%), m.p.
3264 (NH anilino), 3549 (NH amide); 1H NMR (300 MHz, DMSO-d6)
: 1.43 (2H, m, H-400), 1.61 (4H, m, H-300 and H-500), 2.5 (4H, m, H-200
112e114 ꢁC. IR (KBr, cmꢀ1): 1677 (CO), 2961 (CH aliphatic), 3271
n
d
(NH anilino), 3454 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
and H-600), 3.15 (2H, s, CH2CO), 7.14 (1H, d, H-40, J ¼ 7.5 Hz),7.38 (1H,
t, H-50, J ¼ 7.8 Hz), 7.76e7.79 (2H, m, H-60 and H-7), 8.04e8.09 (2H,
m, H-8 and H-20), 8.57 (1H, s, H-5), 8.6 (1H, s, H-2), 9.89 (1H,s, NH,
D2O exchangeable), 9.91 (1H, s, NH, D2O exchangeable); MS (M.Wt.:
395.89): m/z (rel.int. %), 395.05 (Mþ$, 0.72), 397.05 (Mþþ2, 0.27);
Anal. Calcd. For C21H22ClN5O: C, 63.71; H, 5.60; N, 17.69. Found: C,
63.88; H, 5.67; N, 18.01; HRMS m/z 396.1584 Mþþ1, calcd for
2.50 (overlapped, H200, H-600), 3.25 (2H, s, CH2CO), 3.60 (4H, s br., H-
300, 500), 7.42 (2H, s.br, H-30, H-50), 7.74 (1H, s br., H-7), 7.9 (2H, s br.,
H-20, H-60), 8.33 (1H, s br., H-8), 8.50 (1H, s, H-5), 8.62 (1H, s, H-2),
8.69 (1H, s, NH), 9.26 (1H, s, NH); MS (M. Wt: 397.86): m/z (rel.int.
%), 397.05 (Mþ$, 0.95), 399.05 (Mþþ2, 0.33); Anal. Calcd. For
C
20H20ClN5O2: C, 60.38; H, 5.07; N, 17.60. Found: C, 60.72; H, 5.12;
N, 17.49; HRMS m/z 398.1376 Mþþ1, calcd for C20H20ClN5O2
C
21H22ClN5O 396.1591.
398.1384.
5.1.6.7. N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-2-
5.1.6.12. N-(4-((4-chlorophenyl)amino)quinazolin-6-yl)-2-(4-
morpholinoacetamide 6g. Yellow powder (yield: 460 mg, 58%), m.p.
methylpiperazin-1-yl)acetamide 6l. Yellow powder (yield: 517 mg,
130e133 ꢁC. IR (KBr,
n
cmꢀ1):1683 (CO), 2973 (CH aliphatic), 3172
63%), m.p. 115e117 ꢁC. IR (KBr, cmꢀ1): 1681 (CO), 2935 (CH
n
(NH anilino), 3531 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
aliphatic), 3223 (NH anilino), 3512 (NH amide); 1H NMR (300 MHz,
2.56 (4H, s, br, H-200 and H-600), 3.33 (2H, s, CH2CO), 3.66 (4H, s br.,
H-300 and H-500), 7.13 (1H, d, H-40, J ¼ 7.8 Hz), 7.37 (1H, t, H-50,
J ¼ 7.8 Hz), 7.76e7.81 (2H, m, H-60 and H-7), 8.0e8.03 (2H, m, H-8
and H-20), 8.57 (1H, s, H-5), 8.64 (1H, s, H-2), 9.89 (1H, s, NH, D2O
exchangeable), 10.03 (1H, s, NH, D2O exchangeable); Anal. Calcd.
For C20H20ClN5O2: C, 60.38; H, 5.07; N, 17.60. Found: C, 60.59; H,
5.04; N, 17.88; HRMS m/z 398.1377 Mþþ1, calcd for C20H20ClN5O2
398.1384.
DMSO-d6)
d
: 2.41 (4H, s br., H-200, H-600, J ¼ 9 Hz), 3.17 (2H, s,
CH2CO), 2.57 (4H, s br., H-300, H-500, J ¼ 9 Hz), 2.19 (3H, s, CH3), 7.41
(2H, d, H-30, H-50, J ¼ 9.0 Hz), 7.75 (2H, d, H-20, H-600, J ¼ 9 Hz), 7.75
(1H, d, H-7), 8.02 (1H, d, H-8, J ¼ 9.0 Hz), 8.53 (1H, s, H-5), 8.59 (1H,
s, H-2), 9.80 (1H, s, NH), 9.91 (1H, s, NH); 13C NMR (75 MHz, DMSO-
d6) d: 45.7, 52.7, 54.4, 61.6, 112.2, 115.2, 123.4, 123.8, 126.6, 127.0,
128.3, 131.7, 132.2, 136.2, 138.3, 146.6, 153.1, 157.2, 168.5; MS (M.
Wt.: 410.91): m/z (rel.int.%), 410.23 (Mþ$, 2.23%), 412.24 (Mþþ2,
0.76%); Anal. Calcd. For C21H23ClN6O: C, 61.38; H, 5.64; N, 20.45.
Found: C, 61.71; H, 5.69; N, 20.67; HRMS m/z 411.1693 Mþþ1, calcd
for C21H23ClN6O 411.1700.
5.1.6.8. N-(4-((3-chlorophenyl)amino)quinazolin-6-yl)-2-(4-
methylpiperazin-1-yl)acetamide 6h. Yellow powder (yield: 450 mg,
55%), m.p. 120e122 ꢁC. IR (KBr,
n
cmꢀ1): 1682 (CO), 2854 (CH
aliphatic), 3115 (NH anilino), 3532 (NH amide); 1H NMR (300 MHz,
5.1.6.13. N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-2-(pyrroli-
DMSO-d6)
d
: 2.07 (3H, s, CH3), 2.57 (4H, t, H-200 and H-600, J ¼ 4.8 Hz),
din-1-yl)acetamide 6m. Yellow powder (yield: 570 mg, 67%), m.p.
3.29 (2H, s, CH2CO), 3.66 (4H, t, H-300 and H-500, J ¼ 5.1 Hz), 7.15 (1H,
109e111 ꢁC. IR (KBr, cmꢀ1): 1683 (CO), 2940 (CH aliphatic), 3286
n
d, H-40, J ¼ 7.5 Hz), 7.38 (1H, t, H-50, J ¼ 8.1 Hz), 7.77e7.82 (2H, m, H-
(NH anilino), 3419 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:

792
R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
1.78 (4H, s.br, H-300, H-400), 2.65 (4H, s.br, H-200, H-500), 3.32 (2H, s,
CH2CO), 7.27-7-37 (2H, m, H-40, H-50), 7.76 (1H, d, H-7, J ¼ 9.3 Hz),
7.86 (1H, d, H-60, J ¼ 8.1 Hz), 8.07 (1H, d, H-8, J ¼ 9.0 Hz), 8.17 (1H, s,
H-20), 8.58 (1H, s, H-5), 8.61 (1H, s, H-2), 9.82 (1H, s, NH), 9.93 (1H, s,
5.1.6.18. N-(4-((4-bromophenyl)amino)quinazolin-6-yl)-2-(piper-
idin-1-yl)acetamide 6r. Buff powder (yield: 633 mg, 72%), m.p.
125e127 ꢁC. IR (KBr,
n
cmꢀ1): 1685(CO), 2929 (CH aliphatic), 3224
(NH anilino), 3479 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
NH); 13C NMR (100 MHz, DMSO-d6)
d: 23.9, 54.2, 59.9, 112.6, 115.7,
1.43e1.45 (2H, m, H-400),1.61 (4H, t, H-300, H-500, J ¼ 4.8 Hz), 2.50 (4H,
t, H-200, H-6''(overlapped)), 3.14 (2H, s, CH2CO), 7.54 (2H, d, H-30, H-
50, J ¼ 8.4 Hz), 7.75 (1H, d, H-7, J ¼ 9.0 Hz), 7.81 (2H, d, H-20, H-60,
J ¼ 8.7 Hz), 8.07 (1H, d, H-8, J ¼ 8.7 Hz), 8.53 (1H, s, H-5), 8.58 (1H, s,
H-2), 9.81 (1H, s, NH), 9.89 (1H, s, NH); 13C NMR (100 MHz, DMSO-
121.2, 121.5, 124.6, 126.3, 127.8, 128.8, 130.7, 136.9, 144.5, 147.0,
153.5, 157.6, 169.5; MS (M. Wt.: 426.32): m/z (rel.int.%), 425.15 (Mþ$
,
28.10%), 427.18 (Mþþ2, 25.76%); Anal. Calcd. For C20H20BrN5O: C,
56.35; H, 4.73; N, 16.43. Found: C, 56.58; H, 4.66; N, 16.32.
d6) d: 23.9, 25.9, 54.6, 62.9, 112.6, 115.6, 115.8, 124.6, 127.6, 128.8,
131.6, 136.7, 139.2,147.0, 153.5,157.6, 169.3; MS (M. Wt.: 440.35): m/
z (rel.int.%), 439.10 (Mþ$, 2.23%), 441.21 (Mþþ2, 2.06%); Anal. Calcd.
For C21H22BrN5O: C, 57.28; H, 5.04; N, 15.90. Found: C, 57.41; H,
5.09; N, 16.14; HRMS m/z 440.1081 Mþþ1, calcd for C21H22BrN5O
440.1086.
5.1.6.14. N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-2-(piper-
idin-1-yl)acetamide 6n. Buff powder (yield: 615 mg, 70%), m.p.
115e117 ꢁC. IR (KBr,
n
cmꢀ1): 1682 (CO), 2932 (CH aliphatic), 3252
(NH anilino), 3511 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
1.42 (2H, d, H-400, J ¼ 5.4 Hz), 1.59 (4H, t, H-300, H-500, J ¼ 5.4 Hz), 2.5
(4H, m, H-200, H-600, overlapped), 3.15 (2H, s, CH2CO), 7.27e7.37 (2H,
m, H-40, H-50), 7.77 (1H, d, H-7, J ¼ 9.0 Hz), 7.86 (1H, d, H-60,
J ¼ 7.5 Hz), 8.06 (1H, d, H-8, J ¼ 9.0 Hz), 8.16 (1H, s, H-20), 8.57 (1H, s,
H-5), 8.60 (1H, s, H-2), 9.83 (1H, s, NH), 9.90 (1H, s, NH); MS (M. Wt:
440.35): m/z (rel.int. %), 439 (Mþ., 0.45), 441.05 (Mþþ2, 0.43); Anal.
Calcd. For C21H22BrN5O: C, 57.28; H, 5.04; N, 15.90. Found: C, 57.62;
H, 5.11; N, 16.07.
5.1.6.19. N-(4-((4-bromophenyl)amino)quinazolin-6-yl)-4-
morpholinylacetamide 6s. Buff powder (yield: 575 mg, 65%), m.p.
128e130 ꢁC. IR (KBr,
n
cmꢀ1): 1675 (CO), 2960 (CH aliphatic), 3267
(NH anilino), 3442 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
2.55 (4H, t, H-200,H-600, J ¼ 4.2 Hz), 3.21 (2H, s, CH2CO), 3.674 (4H, t,
H-300, H-500, J ¼ 4.2 Hz), 7.54 (2H, 0d, H-50, H-30, J ¼ 9.0 Hz), 7.75 (1H, d,
H-7, J ¼ 9.0 Hz), 7.80 (2H, d, H-6 , H-20, J ¼ 8.4 Hz), 8.00 (1H, d, H-8,
J ¼ 9.0 Hz), 8.52 (1H, s, H-5), 8.63 (1H, s, H-2), 9.86 (1H, s, NH), 10.01
(1H, s, NH); Anal. Calcd. For C20H20BrN5O2: C, 54.31; H, 4.56; N,
15.83. Found: C, 54.47; H, 4.53; N, 15.75; HRMS m/z 442.0869
Mþþ1, calcd for C20H20BrN5O2 442.0879.
5.1.6.15. N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-4-
morpholinylacetamide 6o. Buff powder (yield: 530 mg, 60%), m.p.
112e113 ꢁC. IR (KBr,
n
cmꢀ1): 1683 (CO), 2968 (CH aliphatic), 3172
(NH anilino), 3529 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
2.57 (4H, t, H-200,H-600, J ¼ 4.20 Hz), 3.29 (2H, s, CH2CO), 3.67 (4H, t,
H-300, H-500, J ¼ 4.50 Hz), 7.27e7.37 (2H, m, H-40, H-50), 7.76 (1H, d,
H-7, J ¼ 9.0 Hz), 7.84 (1H, d, H-60, J ¼ 7.50 Hz), 8.01 (1H, d, H-8,
J ¼ 9.0 Hz), 8.15 (1H, s, H-20), 8.56 (1H, s, H-5), 8.63 (1H, s, H-2), 9.86
(1H, s, NH), 9.99 (1H, s, NH); MS (M. Wt: 442.32): m/z (rel.int. %),
441 (Mþ$, 0.88), 443 (Mþþ2, 0.87); Anal. Calcd. For C20H20BrN5O2:
C, 54.31; H, 4.56; N, 15.83. Found: C, 54.57; H, 4.54; N, 16.02; HRMS
m/z 442.0873 Mþþ1, calcd for C20H20BrN5O2 442.0879.
5.1.6.20. N-(4-((4-bromophenyl)amino)quinazolin-6-yl)-2-(4-
methylpiperazin-1-yl)acetamide 6t. Buff powder (yield: 665 mg,
73%), m.p. 120e121 ꢁC. IR (KBr,
n
cmꢀ1):1675(CO), 2934 (CH
aliphatic), 3198 (NH anilino), 3250 (NH amide); 1H NMR (300 MHz,
DMSO-d6)
: 2.17 (3H, s, CH3), 2.49 (4H, s br., H-300, H-500), 2.56 (4H, s
d
br., H-200, H-600), 3.18 (2H, s, CH2CO), 7.53 (2H, d, H-30, H-50,
J ¼ 8.7 Hz), 7.74 (1H, d, H-7, J ¼ 9.0 Hz), 7.80 (2H, d, H-20, H-60,
J ¼ 9.0 Hz), 8.02 (1H, d, H-8, J ¼ 9 Hz), 8.52 (1H, s, H-5), 8.61 (1H, s,
H-2), 9.95 (2H, s, NH); MS (M.Wt: 455.36): m/z (rel.int.%), 454.10
(Mþ$, 0.98%), 456.10 (Mþþ2, 0.96%); Anal. Calcd. For C21H23BrN6O:
C, 55.39; H, 5.09; N, 18.46. Found: C, 55.53; H, 5.13; N, 18.63.
5.1.6.16. N-(4-((3-bromophenyl)amino)quinazolin-6-yl)-2-(4-
methylpiperazin-1-yl)acetamide 6p. Buff powder (yield: 528 mg,
58%), m.p. 140e142 ꢁC. IR (KBr,
n
cmꢀ1): 1674 (CO), 2961 (CH
5.1.6.21. N-(4-((4-chloro-3-(trifluoromethyl)phenyl)amino)quinazo-
lin-6-yl)-2-(pyrrolidin-1-yl)acetamide 6u. Brown powder (yield:
aliphatic), 3262 (NH anilino), 3474 (NH amide); 1H NMR (300 MHz,
DMSO-d6) d
: 2.18 (3H, s, CH3), 2.40 (4H, s br., H-300, H-400), 2.56 (4H, s
585 mg, 65%), m.p. 160e162 ꢁC. IR (KBr,
n
cmꢀ1): 1693 (CO), 2962
br., H-200, H-600), 3.18 (2H, s, CH2CO), 7.24e7.35 (2H, m, H-40,H-50),
7.73 (1H, d, H-7, J ¼ 9 Hz), 7.81 (1H, d, H-60, J ¼ 8.4 Hz), 8.01 (1H, d,
H-8, J ¼ 9.0 Hz), 8.14 (1H, s, H-20), 8.53 (1H, s, H-5), 8.61 (1H, s, H-2),
(CH aliphatic), 3187 (NH anilino), 3278 (NH amide); 1H NMR
(300 MHz, DMSO-d6)
: 1.77 (4H, m, H-300, H-400), 2.65 (4H, s br., H-
d
200, H-500), 3.32 (2H, s, CH2CO), 7.69 (1H, d, H-50, J ¼ 8.4 Hz), 7.76 (1H,
d, H-7, J ¼ 9.0 Hz), 8.07 (1H, d, H-60, J ¼ 8.7 Hz), 8.25 (1H, d, H-8,
J ¼ 8.7 Hz), 8.40 (1H, s, H-20), 8.58 (1H, s, H-5), 8.67 (1H, s, H-2), 9.80
(1H, s, NH), 10.01 (1H, s, NH); MS (M.Wt: 449.86): m/z (rel.int.%),
449.05 (Mþ$, 0.51%), 450.05 (Mþþ1, 0.17%), 451.05 (Mþþ2, 0.18%);
Anal. Calcd. For C21H19ClF3N5O: C, 56.07; H, 4.26; N, 15.57. Found: C,
56.28; H, 4.34; N, 15.71; HRMS m/z 450.1301 Mþþ1, calcd for
9.94 (2H, s, NH); MS (M. Wt: 455.36): m/z (rel.int. %), 454.05 (Mþ$
,
1.45), 456.05 (Mþþ2, 1.41); Anal. Calcd. For C21H23BrN6O: C, 55.39;
H, 5.09; N, 18.46. Found: C, 55.48; H, 5.13; N, 18.61; HRMS m/z
455.1182 Mþþ1, calcd for C21H23BrN6O 455.1195.
5.1.6.17. N-(4-((4-bromophenyl)amino)quinazolin-6-yl)-2-(pyrroli-
din-1-yl)acetamide 6q. Buff powder (yield: 510 mg, 60%), m.p.
C21H19ClF3N5O 450.1308.
113e115 ꢁC. IR (KBr,
n
cmꢀ1): 1688 (CO), 2960 (CH aliphatic), 3191
(NH anilino), 3300 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
5.1.6.22. N-(4-((4-chloro-3-(trifluoromethyl)phenyl)amino)quinazo-
lin-6-yl)-2-(piperidin-1-yl)acetamide 6v. Brown powder (yield:
1.81 (4H, s br., H-300, H-400), 2.77 (4H, s br., H-200, H-500), 3.47 (2H, s,
CH2CO), 7.53 (2H, d, H-30, H-50, J ¼ 8.7 Hz), 7.75 (1H, d, H-7,
J ¼ 8.7 Hz), 7.82 (2H, d, H-20, H-60, J ¼ 8.7 Hz), 8.06 (1H, d, H-8,
J ¼ 8.7 Hz), 8.53 (1H, s, H-5), 8.62 (1H, s, H-2), 9.84 (1H, s, NH), 10.10
611 mg, 66%), m.p.112e114 ꢁC. IR (KBr, cmꢀ1): 1681 (CO), 2941 (CH
n
aliphatic), 3287 (NH anilino), 3496 (NH amide); 1H NMR (300 MHz,
DMSO-d6)
: 1.42 (2H, m, H-400), 1.60 (4H, m, H-300, H-500), 2.50 (4H,
d
(1H, s, NH); 13C NMR (100 MHz, DMSO-d6)
d
: 23.9, 54.2, 59.9, 113.0,
H-200, H-600, overlapped), 3.15 (2H, s,CH2CO), 7.70 (1H, d, H-50,
J ¼ 8.7 Hz), 7.79 (1H, d, H-7, J ¼ 8.1 Hz), 8.05 (1H, d, H-60, J ¼ 9.0 Hz),
8.26 (1H, d, H-8, J ¼ 8.1 Hz), 8.41 (1H, s, H-20), 8.60 (1H, s, H-5), 8.65
(1H, s, H-2), 9.94 (1H, s, NH), 10.06 (1H, s, NH); MS (M.Wt: 463.89):
m/z (rel.int.%), 463.05 (Mþ$, 0.18%), 464.05 (Mþþ1, 0.05%), 465.05
(Mþþ2, 0.06%); Anal. Calcd. For C22H21ClF3N5O: C, 56.96; H, 4.56; N,
115.5,115.8,124.5,128.7,131.6,136.8,139.2,147.0,153.5,157.6,169.5;
MS (M. Wt.: 426.32): m/z (rel.int.%), 425.08 (Mþ$, 1.45%), 427.07
(Mþþ2, 1.57%); Anal. Calcd. For C20H20BrN5O: C, 56.35; H, 4.73; N,
16.43. Found: C, 56.49; H, 4.81; N, 16.59; HRMS m/z 426.0923
Mþþ1, calcd for C20H20BrN5O 426.0929.

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
793
15.10. Found: C, 57.23; H, 4.62; N, 15.24; HRMS m/z 464.1461 Mþþ1,
(2 ꢂ 100 mL), dried, and evaporated under vacuum to give 9a, b as
oily residue which used in the next step without further
purification.
calcd for C22H21ClF3N5O 464.1465.
5.1.6.23. N-(4-((4-chloro-3-(trifluoromethyl)phenyl)amino)quinazo-
lin-6-yl)-4-morpholinylacetamide 6w. Brown powder (yield:
5.1.9. Preparation of compounds 10a, b
Following the same procedures descried above for the prepa-
ration of 6-nitroquinazoline 3a-f, using 4-(benzyloxy)anilines 9a, b.
679 mg, 73%), m.p.130e131 ꢁC. IR (KBr,
n
cmꢀ1): 1683 (CO), 2860 (CH
aliphatic), 3308 (NH anilino), 3495 (NH amide); 1H NMR (300 MHz,
DMSO-d6) d
: 2.57 (4H, s br., H-200, H-600), 3.21 (2H, s, CH2CO), 3.68 (4H,
S br., H-300, H-500), 7.70 (1H, d, H-50, J ¼ 9.0 Hz), 7.79 (1H, d, H-7,
J ¼ 9.0 Hz), 8.00 (1H, d, H-60, J ¼ 8.7 Hz), 8.25 (1H, d, H-8, J ¼ 8.7 Hz),
8.41 (1H, s, H-20), 8.60 (1H, s, H-5), 8.67 (1H, s, H-2),10.03 (1H, s, NH),
10.07 (1H, s, NH); 13C NMR (100 MHz, DMSO-d6)
d: 53.7, 62.4, 66.5,
112.6, 115.5, 115.7, 121.0, 125.2, 126.6, 127.1, 128.1, 128.9, 132.1, 136.9,
139.5, 147.2, 153.3, 157.5, 168.9; Anal. Calcd. For C21H19ClF3N5O2: C,
54.14; H, 4.11; N, 15.03. Found: C, 54.31; H, 4.09; N, 14.88.
5.1.6.24. N-(4-((4-chloro-3-(trifluoromethyl)phenyl)amino)quinazo-
lin-6-yl)-2-(4-methylpiperazin-1-yl)acetamide 6x. Brown powder
(yield: 575 mg, 60%), m.p. 160 ꢁC. IR (KBr, cmꢀ1): 1666 (CO), 2942
n
(CH aliphatic), 3113 (NH anilino), 3310 (NH amide); 1H NMR
(300 MHz, DMSO-d6)
: 2.18 (1H, s, CH3), 2.41 (4H, s br., H-200, H-600),
5.1.9.1. N-(4-(benzyloxy)phenyl)-6-nitroquinazolin-4-amine
10a.
d
Yellow crystals (yield: 700 mg, 60%), m.p. 246e248 ꢁC (as reported)
2.57 (4H, s br., H-300, H-500), 3.19 (2H, s, CH2CO), 7.68 (1H, d, H-50,
J ¼ 9.0 Hz), 7.75 (1H, d, H-7, J ¼ 9.0 Hz), 8.02 (1H, d, H-60, J ¼ 9.0 Hz),
8.22 (1H, d, H-8, J ¼ 9.0 Hz), 8.39 (1H, s, H-20), 8.55 (1H, s, H-5), 8.67
(1H, s, H-2), 10.00 (1H, s, NH), 13.82 (1H, s, NH); MS (M.Wt: 478.9):
m/z (rel.int.%), 478.10 (Mþ$, 1.55%), 479.10 (Mþþ1, 0.43%), 480.05
(Mþþ2, 0.55%); Anal. Calcd. For C22H22ClF3N6O: C, 55.18; H, 4.63; N,
17.55. Found: C, 55.34; H, 4.71; N, 17.73; HRMS m/z 479.1568 Mþþ1,
calcd for C22H22ClF3N6O 479.1574.
[33].
5.1.9.2. N-(4-((3-fluorobenzyl)oxy)phenyl-4-amino)-6-
nitroquinazoline 10b. Yellow powder (yield: 670 mg, 55%), m.p.
195e197 ꢁC. IR (KBr,
DMSO-d6)
n
cmꢀ1): 3065 (NH); 1H NMR (300 MHz,
d
: 5.16 (2H, s, CH2 benzylic), 7.06e7.45 (7H, m, 6 Ar-H and
D2O exchangeable anilino-NH), 7.68 (2H, d, H-20, H-6’, J ¼ 8.1 Hz),
7.87 (1H, d, H-8, J ¼ 9.6 Hz), 8.51 (1H, d, H-7, J ¼ 9.6 Hz), 8.63 (1H, s,
H-2), 9.62 (1H, s, H-5).
5.1.7. Preparation of N-(4-aralkyloxyphenyl)acetamide 8a, b
Benzyl chloride (0.76 g, 6 mmol) or 3-fluorobenzyl bromide
(1.13 g, 6 mmol) was added to a stirred mixture of paracetamol 7
(0.91 g, 6 mmol) and metallic sodium (0.28 g, 12 mol) in absolute
ethyl alcohol (20 mL). The reaction mixture was heated under
reflux for 1 h then cooled to room temperature and evaporated till
dryness under vacuum. Upon addition of water (30 mL), the
precipitated solid was filtered and washed with n-hexane then
crystallized from chloroform to afford compounds 8a and 8a,
respectively.
5.1.10. General procedure for preparation of 6-aminoquinazolines
11a, b
Following the same procedures descried above for the prepa-
ration of compounds 4a-f.
5.1.10.1. N4-(4-(benzyloxy)phenyl)quinazoline-4,6-diamine
White powder (yield: 547 mg, 80%), m.p. 173e175 ꢁC (as reported)
[33].
11a.
5.1.10.2. N4-(4-((3-fluorobenzyl)oxy)phenyl)quinazoline-4,6-diamine
11b. Buff powder (yield: 432 mg, 60%), m.p.125e126 ꢁC. IR (KBr,
n
cmꢀ1): 3214, 3337, 3440 (NH and NH2). 1H NMR (300 MHz, DMSO-
d6) d: 5.13 (2H, s, CH2 benzyl), 5.49 (2H, s, NH2, exchangeable by
D2O), 6.99 (2H, d, H-30, H-50, J ¼ 9.0 Hz), 7.14e7.31 (3H, m, H-20, H-
60, D2O exchangeable anilino-NH), 7.40e7.50 (4H, m, H-200, H-400,H-
500, H-600), 7.68 (2H, d, H-7, H-8, J ¼ 9.0 Hz), 8.24 (1H, s, H-2), 9.20
(1H, s, H-5).
5.1.7.1. N-(4-(benzyloxy)phenyl)acetamide
8a. White
crystals
(yield: 1.23 g, 85%), m.p. 140e142 ꢁC (as reported) [32].
5.1.7.2. N-(4-((3-fluorobenzyl)oxy)phenyl)acetamide
8b. White
5.1.11. General procedure for preparation of 2-chloro-N-(4-
(phenylamino) quinazolin-6-yl)acetamide 12a, b
Following the same procedures descried above for the prepa-
ration of compounds 5a-f.
crystals (yield: 960 mg, 62%), m.p. 120e121 ꢁC. IR (KBr,
n
cmꢀ1):
1651 (CO), 3301 (NH); 1H NMR (300 MHz, DMSO-d6)
d: 2.06 (3H, s,
CH3), 5.08 (2H, s, benzylic CH2), 6.92e6.95 (2H, d, H-30, H-50,
J ¼ 9.0 Hz), 7.131 (1H, t, H-50, J ¼ 9.0 Hz), 7.22 (1H, s, H-20), 7.25e7.28
(1H, d, H-40, J ¼ 9.0 Hz), 7.38e7.41 (1H, d, H-6’, J ¼ 9.0 Hz), 7.45e7.48
(2H, d, H-2 and H-6, J ¼ 9.0 Hz), 9.72 (1H, s, NH, exchangeable by
D2O).
5.1.11.1. N-(4-((4-(benzyloxy)phenyl)amino)quinazolin-6-yl)-2-
chloroacetamide 12a. Yellow crystals (yield: 543 mg, 65%),
m.p. > 300 ꢁC. IR (nmax, cmꢀ1): 1687 (CO), 2926 (CH aliphatic), 3244
(NH anilino), 3350 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
5.1.8. Preparation of 4-(benzyloxy)anilines 9a, b
4.36 (2H, s, CH2CO), 5.13 (2H, s, CH2 benzylic), 7.05 (2H, d, H-3’, H-50,
J ¼ 9.0 Hz), 7.33e7.48 (5H, m, Ar-H), 7.60 (2H, d, Ar-H, J ¼ 8.4 Hz),
7.77 (1H, d, H-7, J ¼ 9.0 Hz), 7.87 (1H, d, H-8, J ¼ 9.3 Hz), 8.56 (1H, s,
H-5), 8.75 (1H, s, H-2), 10.31 (1H, s, NH, exchangeable by D2O),10.70
(1H, s, NH, exchangeable by D2O); HRMS m/z 419.1260 Mþþ1, calcd
for C23H19ClN4O2 419.1275.
A mixture of 8a, b (3.12 mmol), NaOH (6.25 g, 156 mmol) in
ethanol (125 mL) and water (30 mL) was heated to reflux for 7 h.
The mixture was cooled and the solvent was removed under vac-
uum. The residue was partitioned between chloroform (100 mL)
and water (100 mL). The organic phase was washed with water

794
R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
5.1.11.2. 2-Chloro-N-(4-((4-((3-fluorobenzyl)oxy)phenyl)amino)qui-
nazolin-6-yl)acetamide 12b. Yellow crystals (yield: 525 mg, 60%),
mp > 300 ꢁC. IR: (nmax, cmꢀ1): 1687 (CO), 3360 (NH amide),
15.02. MS (M. Wt.: 71.54): m/z (rel.int.%), 471.40 (Mþ$4.77%); HRMS
m/z 472.2144 Mþþ1, calcd for C27H26FN5O2 472.2149.
3262(NH anilino); 1H NMR (300 MHz, DMSO-d6)
d: 4.37 (2H, s,
5.1.12.4. N-(4-((4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-
yl)-4-morpholinylacetamide 13d. Brown powder (yield: 662 mg,
CH2CO),5.18 (2H, s, CH2 benzylic), 7.08e8.03 (10H, m, Ar-H), 8.80
(1H, s, H-5), 8.97 (1H, s, H-2),10.80 (1H,s,NH, exchangeable by D2O),
11.40 (1H,s,NH, exchangeable by D2O).
68%), m.p. 100e102 ꢁC. IR (KBr,
n
cmꢀ1): 1662 (CO), 2918 (CH
aliphatic), 3333 (NH anilino), 3394 (NH amide); 1H NMR (300 MHz,
DMSO-d6)
: 2.50 (4H, s br., H-2000, H-6000), 3.36 (2H, s, CH2CO), 3.61
d
(4H, S br., H-3000, H-5000), 5.18 (2H, s, CH2 benzylic), 7.07 (2H, s br., H-
30, H-50), 7.25 (1H, S br., H-500), 7.3 (2H, s br., H-200, H-400), 7.4 (1H, s
br., H-600), 7.6 (3H, m, H-20, H-60, H-7), 8.09 (1H, s br., H-8), 8.45 (1H,
s, H-5), 8.73 (1H, s, H-2), 9.60 (1H, s, NH),10.00 (1H, s, NH); 13C NMR
5.1.12. General procedure for preparation of target quinazolines
13a-d
Following the same procedures descried above for the prepa-
ration of target compounds 6a-x.
(100 MHz, DMSO-d6)
d
: 53.7, 62.3, 66.5, 68.9, 113.0, 114.5 (2JF-
¼ 22.1 Hz), 114.8 (2JF-C ¼ 8.4 Hz), 115.6, 123.9, 123.9, 124.0, 124.8,
C
127.6, 128.6, 130.8 (3JF-C ¼ 19.9 Hz), 132.9, 136.4, 140.6 (3JF-
¼ 7.6 Hz), 147.0, 153.9 (1JF-C ¼ 114.5 Hz), 158.0, 161.4, 163.8, 168.7;
C
Anal. Calcd. For C27H26FN5O3: C, 66.52; H, 5.38; N, 14.37. Found: C,
66.64; H, 5.34; N, 14.49. MS (M. Wt.: 487.54): m/z (rel.int. %), 487.23
(Mþ$ 3.71%).
5.2. Biological evaluation
5.2.1. In vitro cytotoxic activity
All of the synthesized compounds were soluble in 1% DMSO.
HEPG2, MCF-7 cells were seeded in 96 well plates at 30,000 cells
per well and incubated for 24 h at 37 ꢁC and 5% CO2. The cells were
then treated with the drug (5-fold serial dilution) for 48 h at 37 ꢁC
and 5% CO2 in the incubator. The cells were treated with 5 mg/mL 3-
5.1.12.1. N-(4-((4-(benzyloxy)phenyl)amino)quinazolin-6-yl)-2-(pyr-
rolidin-1-yl)acetamide 13a. Yellow powder (yield: 525 mg, 58%),
m.p. 105e107 ꢁC. IR (KBr,
n
cmꢀ1): 1676 (CO), 2958 (CH aliphatic),
3258 (NH anilino), 3424 (NH amide); 1H NMR (300 MHz, DMSO-d6)
: 1.77 (4H, s br., H-3000, H-4000), 2.64 (4H, s br., H-2000, H-5000), 3.31 (2H,
d
s, CH2CO), 5.04 (2H, s, CH2 benzylic), 7.01 (2H, d, H-30, H-50,
J ¼ 9.0 Hz), 7.32e7.50 (5H, m, Ar-H), 7.65e7.73 (3H, m, H-20, H-60, H-
7), 8.04 (1H, d, H-8, J ¼ 9.0 Hz), 8.43 (1H, s, H-5), 8.58 (1H, s, H-2),
9.67 (1H, s, NH), 9.94 (1H, s, NH); Anal. Calcd. For C27H27N5O2: C,
71.50; H, 6.00; N, 15.44. Found: C, 71.69; H, 6.08; N, 15.62; HRMS m/
z 454.2277 Mþþ1, calcd for C27H27N5O2 454.2243.
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) in Phosphate Buffer Saline (PBS) for 4 h and lysed with 0.01 N
HCl containing 10% Sodium dodecyl sulfate (SDS). Plates were read
on a Biotech plate reader at 570 nm 4 h after lysing. The inhibitory
concentration at 50% (IC50) was determined from the exponential
curve of viability versus concentration. The viability was calculated,
as previously reported [34].
5.1.12.2. N-(4-((4-(benzyloxy)phenyl)amino)quinazolin-6-yl)-4-
5.2.2. ELISA assay
morpholinylacetamide 13b. Yellow powder (yield: 601 mg, 64%),
m.p. > 250 ꢁC. IR (KBr, cmꢀ1): 1668 (CO), 2954 (CH aliphatic), 3275
n
The kinase activity of EGFR was measured by use of an anti-
phosphotyrosine antibody with the Alpha Screen system (Perki-
nElmer, USA) according to manufacturer's instructions, as reported
earlier [24]. Percent inhibition was calculated by the comparison of
compounds treated to control incubations. The concentration of the
test compound causing 50% inhibition (IC50) was calculated from
the concentrationeinhibition response curve (triplicate de-
terminations) and the data were compared with Erlotinib as stan-
dard EGFR inhibitor. Moreover, the phosphorylated EGFR levels in
(NH anilino), 3444 (NH amide); 1H NMR (300 MHz, DMSO-d6)
d:
2.56 (4H, s br., H-2000, H-6000), 3.20 (2H, s, CH2CO), 3.67 (4H, t, H-3000,
H-5000, J ¼ 4.2 Hz), 5.11 (2H, s, CH2 benzylic), 7.01 (2H, d, H-30, H-50,
J ¼ 9.0 Hz), 7.32e7.48 (5H, m, Ar-H), 77.69e7.72 (3H, m, H-20, H-60,
H-7), 8.07 (1H, d, H-8, J ¼ 9.0 Hz), 8.43 (1H, s, H-5), 8.71 (1H, s, H-2),
9.78 (1H, s, NH), 10.20 (1H, s, NH); MS (M.Wt: 469.55): m/z
(rel.int.%), 469.20 (Mþ$, 6.31%); Anal. Calcd. For C27H27N5O3: C,
69.07; H, 5.80; N, 14.92. Found: C, 69.35; H, 5.89; N, 15.13; HRMS m/
z 470.2189 Mþþ1, calcd for C27H27N5O3 470.2192.
MCF-7 cell lysate, after treatment with 3 mM of compound 6n for 24
and 48 h, were measured using a Bio Vision Colorimetric 96-well
Phospho-EGFR (Tyr 1069) ELISA Kit (catalog no. K4245-100), ac-
cording to the manufacturer's instructions.
5.1.12.3. N-(4-((4-((3-fluorobenzyl)oxy)phenyl)amino)quinazolin-6-
yl)-2-(pyrrolidin-1-yl)acetamide 13c. Brown powder (yield:
630 mg, 67%), m.p. 115e117 ꢁC. IR (KBr,
n
cmꢀ1): 1683 (CO), 2960
(CH aliphatic), 3273 (NH anilino), 3419 (NH amide); 1H NMR
(300 MHz, DMSO-d6)
: 1.78 (4H, s br., H-3000,H-4000), 2.65 (4H, s br.,
5.2.3. Western blotting
d
MCF-7 cells were treated with 3 mM of 6n and incubated for 1, 2,
H-2000, H-5000), 3.31 (2H, s, CH2CO), 5.14 (2H, s, CH2 benzylic), 7.02
(2H, d, H-30, H-50, J ¼ 9.0 Hz), 7.15 (1H, t, H-500, J ¼ 8.4 Hz), 7.27e7.50
(4H, m, H-200, H-400, H-600), 7.65 (2H, d, H-20, H-60, J ¼ 8.7 Hz), 7.7 (1H,
d, H-7, J ¼ 8.7 Hz), 8.03 (1H, d, H-8, J ¼ 9.0 Hz), 8.43 (1H, s, H-5), 8.55
(1H, s, H-2), 9.63 (1H, s, NH), 9.88 (1H, s, NH); 13C NMR (100 MHz,
4, 6, 24, and 48 h. The cells were lysed by 1X RIPA buffer containing
protease and phosphatase inhibitors to quantify the protein con-
centration by BCA protein assay. 20
or 12% SDSPAGE and the protein was transferred into nitrocellulose
membrane 0.45 m for 90 min at 90 V and blocked with 5% BSA
mg/well was loaded on 8%, 10%,
m
DMSO-d6)
d
: 23.9, 54.2, 59.8, 68.9, 112.8, 114.52 (2JF-C ¼ 21.1 Hz),
after that was propped against Stat3 and its phosphorylated (10%
SDSPAGE), EGFR and its phosphorylated (8% SDSPAGE), and ERK 1/2
and its phosphorylated (12% SDSPAGE) overnight at 4 ꢁC. The
membrane was subjected to the corresponding IR-conjugated
114.86 (2JF-C ¼ 19.9 Hz), 115.65, 123.92, 123.94, 124.08, 124.73, 127.6,
128.5, 130.8 (3JF-C ¼ 8.4 Hz), 132.9, 136.5, 140.6 (3JF-C ¼ 7.6 Hz), 146.9,
153.9 (1JF-C ¼ 103.7 Hz), 157.9, 161.4, 163.8, 169.4; Anal. Calcd. For
C
27H26FN5O2: C, 68.77; H, 5.56; N,14.85. Found: C, 68.94; H, 5.63; N,
secondary antibodies. b-actin was used as a loading control.

R.S.M. Ismail et al. / European Journal of Medicinal Chemistry 155 (2018) 782e796
795
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Acknowledgments
Department of Pharmaceutical Chemistry, Faculty of Pharmacy,
Ain Shams University, Egypt, is highly appreciated for supporting
this research. Pharmaceutical Chemistry Department, Faculty of
Pharmacy, Cairo University, Egypt, is highly appreciated for funding
this research work, and for HRMS analyses, the authors would like
to extend their sincere appreciation to the Deanship of Scientific
Research and the Research Center, College of Pharmacy, King Saud
University for their funding of this research. The authors would like
to acknowledge OpenEye molecular modeling software for sup-
porting an academic license.
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