Design, synthesis and in vitro anti-proliferative activity of 4,6-quinazolinediamines as potent EGFR-TK inhibitors

Article abstract of DOI:10.1016/j.ejmech.2012.10.017

4-Anilino-6-substituted-quinazolines were designed, synthesized and evaluated for EGFR-TK and tumor growth inhibitory activities. The target compounds were designed with enamine ester or urea moieties appended at the C-6 of quinazoline as additional hydrogen bond acceptor functions. Most of the synthesized compounds displayed potent EGFR-TK inhibitory activity at 10 μM and the 6-ureido-anilinoquinazoline derivative 7a showed IC₅₀ value of 0.061 μM. Moreover, six compounds were tested by National Cancer Institute (NCI), USA for their anti-proliferative activity at 10 μM in full NCI 60 cell panel. Compound 7a was further assayed for five dose molar ranges in full NCI 60 cell panel and exhibited remarkable growth inhibitory activity pattern against Non-Small Cell Lung Cancer EKVX (GI₅₀ = 0.37 μM), NCI-H322M (GI₅₀ = 0.36 μM), Renal Cancer A498 (GI₅₀ = 0.46 μM), TK-10 (GI₅₀ = 0.99 μM) and Breast Cancer MDA-MB-468 (GI₅₀ = 1.096 μM) which are of high EGFR expression. Docking study was performed for the active compounds into ATP binding site of EGFR-TK which showed similar binding mode to gefitinib and additional binding with Cys-773 at the gatekeeper of EGFR-TK enzyme.

Full text of DOI:10.1016/j.ejmech.2012.10.017

European Journal of Medicinal Chemistry 61 (2013) 132e145
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and in vitro anti-proliferative activity of
4,6-quinazolinediamines as potent EGFR-TK inhibitors
,
Samar Mowafy a, Nahla A. Farag a, Khaled A.M. Abouzid b
*
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Misr International University, Abbassia, 11566 Cairo, Egypt
b Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University, Abbassia, 11566 Cairo, Egypt
a r t i c l e i n f o
a b s t r a c t
Article history:
4-Anilino-6-substituted-quinazolines were designed, synthesized and evaluated for EGFR-TK and tumor
growth inhibitory activities. The target compounds were designed with enamine ester or urea moieties
appended at the C-6 of quinazoline as additional hydrogen bond acceptor functions. Most of the
Received 2 February 2012
Received in revised form
24 September 2012
Accepted 11 October 2012
Available online 27 October 2012
synthesized compounds displayed potent EGFR-TK inhibitory activity at 10
noquinazoline derivative 7a showed IC50 value of 0.061 M. Moreover, six compounds were tested by
National Cancer Institute (NCI), USA for their anti-proliferative activity at 10 M in full NCI 60 cell panel.
mM and the 6-ureido-anili-
m
m
Compound 7a was further assayed for five dose molar ranges in full NCI 60 cell panel and exhibited
remarkable growth inhibitory activity pattern against Non-Small Cell Lung Cancer EKVX
Keywords:
4-Anilinoquinazoline
EGFR-TK inhibitors
Docking study
(GI50
(GI50 ¼ 0.99
¼
0.37
m
M), NCI-H322M (GI50
¼
0.36
m
M), Renal Cancer A498 (GI50
¼
0.46 mM), TK-10
m
M) and Breast Cancer MDA-MB-468 (GI50 ¼ 1.096
m
M) which are of high EGFR expres-
sion. Docking study was performed for the active compounds into ATP binding site of EGFR-TK which
showed similar binding mode to gefitinib and additional binding with Cys-773 at the gatekeeper of
EGFR-TK enzyme.
Anti-proliferative activity
Ó 2012 Elsevier Masson SAS. All rights reserved.
1. Introduction
the FDA for the treatment of advanced Non-Small Cell Lung Cancer
[13], Fig. 1. These agents belong to the 4-anilinoquinazoline class of
The epidermal growth factor receptor (EGFR) protein belongs to
the ErbB family of receptor tyrosine kinases (RTKs) which plays an
important role in the regulation of cell growth, differentiation, and
survival [1]. EGFR is a glycoprotein that contains an extracellular
ligand-binding domain, a transmembrane region, and an intracel-
lular domain with kinase activity [2]. EGFR is over expressed in
several human tumors including Non-Small Cell Lung Cancer
(NSCLC), prostate, breast and ovarian cancers [3e10] and correlates
with a poor prognosis in many cancer patients [11]. Thus, EGFR is an
attractive target for the design and development of compounds that
can specifically bind to the receptor and inhibit its tyrosine kinase
(TK) activity and its signal transduction pathway in cancer cells.
Inhibition of EGFR has been achieved through two main approaches:
by blocking ligand binding to the extracellular domain with
monoclonal antibodies and by using small-molecule inhibitors that
interact at the ATP-binding site [12]. A well-studied class of these
latter inhibitors is represented by 4-anilinoquinazolines, exempli-
fied by the lead compound gefitinib, erlotinibwhich are approved by
inhibitors and the key features between the receptor and this
template have been revealed as follows [14,15]. (1) The quinazoline
core fits intothe ATP binding pocketof the kinase domain, where the
N-1 nitrogen of the quinazoline nucleus interacts with the backbone
NH of Met-769 via a hydrogen bond, and water mediated hydrogen
bonding is observed between the N-3 of the quinazoline and the
Thr-766 side chain. (2) The aniline ring fills an adjacent lipophilic
pocket. (3) The solubilizing side chains at C-6 and/or C-7 of the
quinazoline core act to improve physical properties and confer
a more favorable pharmacokinetic profile.
In the past decade, the SAR study of quinazoline as EGFR
inhibitors was directed toward the modification of the C-4 aryla-
mino group [16], by varying the aniline substitution pattern, fine
tuning of the kinase selectivity profile can be achieved [17]. Also,
the substitution at C-6 position of quinazoline has received
increasing attention in the development of more potent & selective
inhibition [9,17]. Almost all substituents in this position were
designed to bind with Cys-773 in EGFR through hydrogen bonding
or covalent bonding [16,18,19]. Since 6-position of quinazoline
points toward the outside of the protein & theoretical studies have
indicated that bulky substituents are tolerated at the 6-position.
Moreover the 6-position substitution allows optimum distance for
* Corresponding author. Tel.: þ20 1222165624; fax: þ20 225080728.
E-mail address: Khaled.abouzid@pharm.asu.edu.eg (K.A.M. Abouzid).
0223-5234/$ e see front matter Ó 2012 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2012.10.017

S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
133
Erlotinib
Gefitinib
Fig. 1. Examples of EGFR receptor tyrosine kinase inhibitors and target compounds.
interaction with unique Cys-773 [20,21] as suggested by molecular
modeling [22], and may also improve physical properties and
impart a more favorable pharmacokinetics profile [17].
Encouraged by all these findings, we present three new series of 6-
substituted 4-anilinoquinazolines as promising selective candidates
for EGFR inhibition. Our strategyisdirected toward designingavariety
of ligands with diverse chemical properties hypothesizing that the
potency and selectivity of these molecules might be enhanced by
attaching new moieties at C-6 of substituted 4-anilinoquinazolines
and/or by varying the aniline substitution pattern.
in Schemes 1 and 2. The key intermediate 6-amino-4-
anilinoquinazolines 4aeh, were prepared following the litera-
ture methods [27e29] illustrated in Scheme 1. 6-Nitro-4-
phenylaminoquinazoline derivatives 3aeh were synthesized in
good yield, through the reaction of 5-nitroanthranilo nitrile 1
with dimethylformamide dimethyl acetal affording the corre-
sponding formamidine 2 followed by its reaction with appropriate
anilines in acetic acid. Compounds 3aeh were reduced to their
amino derivatives using sodium dithionite in ammonia yielded
the 6-aminoquinazolines 4aeh.
(Z)-Ethyl 3-[4-(arylamino)quinazolin-6-ylamino]-2-cyanoacry-
lates 5aeh were obtained as sole product, which was confirmed as
Z isomer by means of low field signal of the NH-proton, where intra-
molecular H-bond between amino group and carbonyl group is
accompanied by this deshielding effect [30]. Second, the HeNeCH
coupling constants in this (Z) series are higher & less variable [31]
with 1H NMR of NH peak at 6-position of quinazoline appeared as
2. Rational and design
The design of our ligands was based on earlier work with
quinazoline-based inhibitors of EGFR [9,17,23,24] and SAR studies on
type I inhibitors of active EGFR [25]. Type I inhibitors are ATP
competitivewhichbind tothehingeregionof theenzymaticallyactive
kinase domain and represent the prototypical kinase inhibitor. In this
study, three series of novel 6-substituted-quinazolines were designed
to selectively inhibit EGFR, while preserving stereo electronic contacts
responsible for binding to EGFR-TK. Accordingly, all the designed
compounds incorporated the 4-anilinoquinazoline motif and derive
selectivity through C-6 substitution with amino-2-cyanoacrylate 5,
aminomethylene malonate 6, ureido 7 moieties. Where the conjuga-
tion in the enamine ester system (series A and B) and urea system
(series C) directs the negative potential toward the carbonyl oxygen,
which could lead to the formation of stronger hydrogen bonds and
better affinity of these inhibitors in the active site, Also, different
substitution pattern have been introduced at aniline in a fashion
similar to gefitinib or erlotinib and finally phenolic hydroxyl group.
In this way, such substitution pattern could target additional
regions of the ATP-binding site of the protein kinase domain to
create differentially selective molecules. In addition, Genetic Opti-
mization for Ligand Docking (GOLD) was used as flexible docking
program [26] to predict the binding affinities and orientation of the
target compounds 4b, 5aeh, 6aef, 7a,b at the active site of the ATP
binding site of EGFR-TK.
doublet at
with proton of (NHeCH]C) with J ¼ 13.5 Hz, moreover on adding
D2O (NHeCH]C) appeared as single peak at 8.44 ppm (1H), (Z)-
d
11.00e11.04 ppm (1H) with J ¼ 13.5 Hz, that is coupled
d
ethyl 3-[4-(arylamino)quinazolin-6-ylamino]-2-cyanoacrylate 5ae
h were prepared by refluxing 4aeh with (E)-ethyl 2-cyano-3-
ethoxyacrylate in absolute ethanol. Also, their reaction with diethyl
2-(ethoxymethylene)malonate yielded diethyl 2-[(4-arylamino)
quinazolin-6-ylaminomethylene]malonate 6aef. Finally, the reac-
tion of the amino derivatives 4aeh with potassium cyanate in acetic
acid gave the corresponding 4-(substituted amino)-6-ureidoquin-
azoline 7a,b Scheme 2.
4. Results and discussion
4.1. Pharmacology
4.1.1. In vitro EGFR tyrosine kinase activity
4.1.1.1. The initial screening effect. The EGFR tyrosine kinase assays
were performed at BPS Bioscience (www.bpsbioscience.com). In
the initial screening effect; the seventeen selected compounds
indicated in Fig. 2 were tested over EGFR tyrosine kinase at a single
3. Chemistry
dose concentration of 10 mM. At this concentration, compounds 5c,
5d, 5f, 5g, 6b, 6d, 6e, 6g, 7a, and 7b have demonstrated a significant
inhibition of 100% for the activity EGFR tyrosine kinase, neverthe-
less the inhibition exerted by 4b, 5a, 5b, 5e, 5h and 6c were above
The route adopted for the synthesis of the new 6-substituted
amino-4-anilinoquinazoline derivatives in this study is depicted

134
S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
N
N
O N
O N
2
2
i
N
N
NH
2
1
2
ii
5
5
2
4
4
3
6
6
R
R
3
HN
N
HN
2
iii
O N
H N
2
2
N
N
N
3a-h
4a-h
a:
e
R = 2 -Cl, 4 -Cl
: R = 3 -Ethynyl
b: R = 2 - Cl
f: R = 3 -OH
c:
g:
R = 4 - Cl
R = 4 -OH
d: R = 3 - Br
h: R = 2 -C H
2 5
Scheme 1. Synthesis of 6-amino-4-anilinoquinazolines. Reagents and conditions: (i) dimethylformamide dimethyl acetal, 100 ꢁC, 90 min; (ii) substituted aniline, glacial acetic acid,
reflux, 60 min; (iii) sodium dithionite/ammonia, reflux.
80%. The mean percentage inhibition of the compounds on EGFR at
the testing concentration (10 M) was shown on Table 1, Fig. 2.
4.1.2. Pharmacological (in vitro anticancer activity)
m
The structures of the final 4-anilino-quinazoline based products
were submitted to National Cancer Institute “NCI” (www.dtp.nci.
nih.gov), Bethesda, Maryland, USA, and six compounds were
selected on the basis of degree of structure variation and computer
modeling techniques for evaluation of their antineoplastic activity.
The tumor growth inhibition properties of the six compounds 5a,
5b, 5c, 5e, 7a, and 7b with the NCI codes NSC D-757174/1, NSC
D757173/1, D-757176/1, D-757175/1, D-757177/1, D-757178/1,
selected among 5aeh, 6aef, 7a,b by the National Cancer Institute
(NCI), USA, were screened on human tumor cell lines at 10ꢀ5 M,
under the drug discovery program of the NCI. Among the selected
six compounds, compound 7a (NSC D-757177/1) was further
screened for 5-log dose molar range due to its selective prominent
cell growth inhibition on renal cell lines at 10ꢀ5 M concentration
against verity of cell lines.
Structureeactivity correlation of the newly synthesized 4-
anilino-6-substituted aminoquinazolines derivatives revealed that
the N substitution of the 6-amino group in the 4 series increased
the inhibition percent in all the prepared final series (5,6 and 7).
Combining these results together with preliminary gold docking
scores, it was generally concluded that substitution at 6-position
with ureido moiety (7 series) results into the most active series
followed by amino-methylene malonate moiety (6 series), then
the amino-2-cyanoacrylate moiety (5 series). Moreover, for the
4-anilino ring substitution, we have introduced electron-
withdrawing as halogen(s) and ethynyl group and electron-
donating as ethyl, hydroxy function(s) to the 4-anilino ring. It was
revealed that the 3-bromo (5d and 6d), 3-hydroxy (5f and 7b),
4-hydroxy (5g and 6f), and 4-chloro (5c and 7a) derivatives resul-
ted into the highest inhibition percent for all the derivatives
synthesized (100% inhibitions), However, the inhibition percent of
the 4-chloro substituted derivative of amino-methylene malonate
series (6c, 82%) revealed less inhibition percent than the corre-
sponding 5 and 7 series derivatives (5c and 7a). As for the rest of
derivatives, 3-ethynyl (5e, 6e), 2-chloro (5b, 6d) and 2,4-dichloro
(5a) showed inhibition percent range from (90e95%) and finally
2-ethyl derivative (5h) which was less potent than other
compounds tested (84%). This study showed that the position and
types of the substitutions affect the activity of the synthesized
derivatives.
4.1.2.1. Primary single high dose (10ꢀ5 M) full NCI 60 cell panel
in vitro assay. Primary in vitro one dose anticancer assay was per-
formed in full NCI 60 cell panel. Results for each compound were
reported as a mean graph of the percent growth of the treated cells
when compared to the untreated control cells. Analysis of historical
Development Therapeutics Programme (DTP) was performed and
compound 7a which satisfied predetermined threshold inhibition
criteria was selected for NCI full panel 5 dose assays. The tested 4-
anilino-quinazoline based inhibitors showed a distinctive pattern
of selectivity & sensitivity against different NCI cell panel Table 2.
Compound 7a showed remarkably lowest cell growth promotion
against breast cancer MDA-MB-468 cell line with cell growth
promotion (7%). Compound 7a exhibited broad spectrum cell
growth inhibition against Non-Small Cell Lung Cancer NCI-H322M
(cell growth promotion 18.02%, inhibition 81.98%), EKVX (cell
growth promotion 22.36%, inhibition 77.64%), Ovarian SK-OV-3
(cell growth promotion 30.22%, inhibition 69.78%), Prostate
4.1.1.2. Measurement of potential enzyme inhibitory activity (IC50).
The profiling data for compound 7a against EGFR showed increased
inhibition of EGFR activity with increasing concentration of
compound 7a Fig. 3. The compound inhibited the EGFR activity by
99% at 10
mM, the IC50 value for 7a was determined to be 0.061
mM,
compared to that of gefitinib which is 0.033
mM.

S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
135
Scheme 2. Synthesis of 6-substituted amino-4-anilinoquinazolines. Reagents and conditions: (i) ethyl 2-cyano-3-ethoxyacrylate, abs. EtOH, reflux; (ii) diethyl 2-(ethoxy-
methylene)-malonate, abs. EtOH, reflux; (iii) KCNO, glacial acetic acid, stir on cold.
Cancer DU-145 (cell growth promotion 51.96%, inhibition 48.04%),
Renal Cancer TK-10 (cell growth promotion 26.52%, inhibition
73.48%), CAKI-1 (cell growth promotion 40.11%, inhibition 59.98%),
A498 (cell growth promotion 43.42%, inhibition 56.58%), ACHN (cell
growth promotion 47.43%, inhibition 52.57%).
The second most active compound 5c was found to be active
against Non-Small Cell Lung Cancer EKVX (cell growth promotion
20.64%, inhibition 79.36%), NCI-H322M (cell growth promotion
22.61%, inhibition 77.39%), Renal Cancer CAKI-1 (cell growth
Table 1
The EGFR tyrosine kinase assays of the seventeen
compounds over EGFR tyrosine kinase at a single dose
concentration of 10
mM.
Compound
% Inhibition
4b
5a
5b
5c
5d
5e
5f
5g
5h
6a
6b
6c
90a
92a
94a
100a
100a
95a
100a
100a
84a
90a
100a
82a
6d
6e
6f
7a
100a
100a
100a
100a
100a
100a
59b
7b
Gefitinib
Staurosporine
Staurosporine
96c
a
The inhibition % measured at 10
The inhibition % measured at 0.5
The inhibition % measured at 1 m
m
m
M.
M.
M.
b
c
Fig. 2. The EGFR tyrosine kinase assay of synthesized compounds at a single dose
concentration of 10
mM.

136
S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
Table 2
Sixty human tumor cell line anticancer screening data at single dose assay (10ꢀ5 M
concentration) as percent cell growth promotion of compounds 5a, 5b, 5c, 5e, 7a, 7b.
5a 5b 5c 5e 7a 7b
Leukemia
CCRF-CEM
HL-60 (TB)
K-562
MOLT-4
RPMI-8226
70.92
93.13
50.08 103.42
96.01 107.99
99.87 105.04
71.44 103.44
107.69
59.67
58.75
79.47
95.26
88.59
86.23
84.29
76.22
61.38
50.14
97.46
96.37
87.49
71.25
98.78
62.86 100.71 101.14 111.77
Non-small cell lung cancer
A549/ATCC
EKVX
HOP-62
72.08
82.40
39.02
99.08 106.94
66.13
20.64
85.06
31.89
99.94
90.30
89.77
72.48
22.36
86.15
74.20
93.49
82.62
92.77
77.99
41.70
100.94
56.46
99.82 116.37
99.50 103.48
75.65
84.78
95.79 107.01
HOP-92
70.36
NT
Fig. 3. Graph of 7a Concentration against activity of EGFR; to determine IC50 value for
7a to be 0.061 mM.
NCI-H226
NCI-H23
NCI-H322M
NCI-H460
NCI-H522
Colon cancer
COLO 205
HCC-2998
HCT-116
HCT-15
99.31
92.49
22.61
70.02 100.45
79.75 86.31
99.77 101.95
98.23 100.15 100.38
52.30 18.02 83.53
46.95
98.74
87.95 108.26
66.20 102.48
promotion 32.57%, inhibition 67.43%), UO-3 (cell growth promotion
42.95%, inhibition 57.05%), Breast Cancer MDA-MB-468 (cell
growth promotion 26.64%, inhibition 73.36%), Prostate Cancer DU-
145 (cell growth promotion 49.16%, inhibition 50.84%), Leukemia
CCRF-CEM (cell growth promotion 50.08%, inhibition 49.92%),
MOLT-4 (cell growth promotion 50.14%, inhibition 49.86%) and
Ovarian Cancer OVCAR-3 cell line (cell growth promotion 50.23%,
inhibition 49.77%). Contrary compound 7b has not shown signifi-
cant cell growth inhibition, while 5a showed significant cell growth
inhibition against Renal Cancer cell lines CAKI-1 (cell growth
promotion 39.03%, inhibition 60.97%), UO-31 (cell growth promo-
tion 42.02%, inhibition 57.98%), TK-10 (cell growth promotion
44.17%, inhibition 55.83%), A498 (cell growth promotion 48.72%,
inhibition 51.28%), and against Non-Small Cell Lung EKVX Cancer
cell line (cell growth promotion 41.70%, inhibition 58.3%).
Compound 5b is another active candidate which showed cell
growth inhibition against Non-Small Lung EKVX (cell growth
promotion 39.02%, inhibition 60.98%), Renal CAKI-1 (cell growth
promotion 44.98%, inhibition 55.02%), Breast MDA-MB-468 cancer
cell line (cell growth promotion 34.71%, inhibition 65.29%) and also
showed up to 38% cell growth inhibition against Prostate DU-145
Cancer cell line. Compound 5e emerged as active which cell
growth inhibition against Non-Small Cell Lung EKVX (cell growth
promotion was 31.89%, inhibition was 68.11%) and Breast MDA-MB-
468 Cancer cell line (cell growth promotion was 40.41%, inhibition
was 59.59%), moreover it also exhibited up to 44% cell growth
inhibition against Renal CAKI-1 Cancer cell line at single dose assay
(10ꢀ5 M concentration) as shown in Table 2.
107.97 110.19 102.45 103.55 101.41
94.71
118.94 116.56 100.56 101.67
92.91 113.15 84.49 103.35
92.73 99.00
79.44 100.15
79.14 88.55
97.94 101.92
96.36 109.43
93.12 108.18
91.64 106.15
98.53
99.11
84.92
86.27
74.03
94.18
97.97
98.12
HT29
KM12
SW-620
96.12
99.10
100.49 115.97
97.35 105.31
CNS cancer
SF-268
SF-295
SF-539
SNB-19
72.98 105.80
82.15 101.83
92.67 110.66 102.51
76.13
74.26
94.38
95.57
97.85
85.14
92.46
85.41
79.74
90.72
95.73 109.12
78.59 93.11
94.97 101.85
95.72 87.85
85.36
98.11
86.36
91.27
81.35
94.54
87.19
96.35
87.99
86.33
84.68
SNB-75
U251
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
PC-3
DU-145
87.45
93.03
87.52 103.36
95.55 101.45
140.24 156.12 141.92 119.27 117.44 120.67
105.56 119.42 105.88 114.26 106.65 102.48
75.74 100.26
109.86 105.66 105.34 123.28 127.38
109.03 119.39 104.64 103.09 106.02 114.29
78.35 88.32 71.99 89.38 89.20 100.77
97.16 120.30 107.96 99.77 100.15
90.03 107.31 102.89 103.87
70.46
92.03
97.83 101.34
NT
99.82
87.90 105.04
55.03
67.63
79.74
95.27
52.40
50.23
89.00
92.75
83.33
77.41
78.68
94.05
89.52
94.82
61.01 107.40
74.21
91.88 112.27
83.64
89.54
88.73
30.22
82.83
95.07 124.09
82.20 90.06
87.03 104.89
85.41 99.58
96.98 106.58
98.14
98.87
96.75
87.46
72.01 100.23
84.98
96.99
4.1.2.2. In vitro 5 dose full NCI 60 cell panel assay. All the sixty cell
lines, representing nine tumor subpanels, were incubated at five
different concentrations (0.01, 0.1, 1, 10 &100 mM). The outcomes
82.59
48.72
62.89
39.03
77.05
89.55
44.17
42.02
91.28
63.73
68.74
44.98
91.72
98.85
53.02
66.65
83.13
53.12
51.20
32.57
83.18
87.04
51.34
42.95
91.68
82.55
63.06
56.14
92.86
97.07
65.47
58.64
85.89
43.42
92.60
77.40
47.43 103.19
40.11 84.79
93.70 110.60
81.90 94.91
26.52 103.01
58.03 80.07
were used to create log concentration vs % growth inhibition curves
and three response parameters (GI50, TGI and LC50) were calculated
for each cell line, Table 3. The GI50 value (growth inhibitory activity)
corresponds to the concentration of the compound causing 50%
decrease in net cell growth, the TGI value (cytostatic activity) is the
concentration of the compound resulting in total growth inhibition
and LC50 value (cytotoxic activity) is the concentration of the
compound causing net 50% loss of initial cells at the end of the
incubation period of 48 h. Compound under investigation 7a (NSC
D-757177/1) exhibited remarkable anticancer activity against most
of the tested cell lines representing nine different subpanels with
59.33
55.92
98.75
62.12
60.60
49.16
91.43
69.86
79.04 102.31
51.96
90.36
Breast cancer
MCF7
MDA-MB-231/ATCC
HS 578T
BT-549
87.61 110.09 111.39
96.77
97.17
86.45
97.32
85.19
75.49
86.93
91.04
93.12
89.34 103.45
76.06
72.71
26.64
89.32 104.27
87.77 104.80
92.36 108.27
61.62
ꢀ7.15
89.56 112.43
99.56
73.36
40.41
T-47D
MDA-MB-468
58.64
56.36
77.56
34.71
76.56
67.92
GI50 values between “0.36e26.7
Cancer subpanel namely COLO 205, HCC-2998, KM12 and SW-620
showed GI50 up to concentration of 27.5, 37.6, 28.4 and 39.7
m
M”, except four cell lines of Colon
NT ¼ Not tested, O 30e40% growth inhibition, O 40e50% growth inhibition, O 50e
70% growth inhibition, O 70e90% growth inhibition, O 90e100% growth inhibition,
O highly potent compound.
mM
respectively Table 3, note that compounds which were found to be
insensitive at the highest tested concentration i.e. 100 mM

S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
137
Table 3
NCI in vitro testing result of compound 7a (NSC D-757177/1) at five dose level in
m
M.
Panel
Cell line
GI50
TGI
LC50
Concentration
per cell line
Subpanel
MIDb
Selectivity ratio
(MIDa:MIDb)
Leukemia
CCRF-CEM
HL-60 (TB)
MOLT-4
RPMI-8226
SR
16.4
24.4
18.1
20.5
19.1
19.2
0.37
21.1
22.3
0.36
22.4
2.40
27.5
37.6
24.9
17.8
24.0
28.4
39.7
17.1
14.8
25.5
9.02
3.36
23.9
19.3
22.0
25.0
16.9
15.1
26.7
13.5
23.5
13.5
6.85
8.36
9.14
15.2
21.7
19.5
3.52
20.7
0.45
1.47
1.33
15.1
11.1
0.98
4.22
16.7
2.72
26.2
8.92
19.7
0.53
>100
69.8
87.1
78.9
75.6
>100
4.46
84.4
>100
6.64
>100
20.9
>100
>100
>100
>100
>100
>100
>100
>100
45.5
>100
>100
47.0
>100
68.7
60.7
76.0
47.6
42.7
>100
30.7
75.2
32.2
27.6
69.5
>100
90.3
>100
>100
20.5
88.9
13.0
35.8
21.5
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
70.1
Non-small cell
lung cancer
A549/ATCC
EKVX
12.59
0.83
HOP-62
NCI-H226
NCI-H322M
NCI-H460
NCI-H522
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM12
SW-620
SF-268
SF-295
SF-539
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
IGROV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
786-0
A498
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
PC-3
DU-145
MCF7
Colon cancer
28.56
0.367
CNS cancer
Melanoma
15.61
19.5
0.67
0.54
>100
77.0
90.1
Ovarian cancer
Renal cancer
12.04
6.91
0.87
1.51
>100
>100
>100
>100
>100
>100
>100
64.8
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
75.9
50.0
16.2
43.0
Prostate cancer
Breast cancer
9.71
1.08
0.83
92.5
>100
>100
37.7
12.61
MDA-MB-231/
ATCC
HS 578T
BT-549
T-47D
14.8
18.2
6.47
1.09
10.47
55.9
79.3
72.3
3.95
>100
>100
>100
>100
MDA-MB-468
MIDa
a
MID ¼ Average sensitivity of all cell line in
m
M.
b
MID ¼ Average sensitivity of all cell line of a particular subpanel in
mM.
therefore a sign of “>” is used as prefix to the concentration. With
regard to the sensitivity against some individual cell lines Table 3,
the compound showed high activity against Non-Small Cell Lung
data revealed an obvious sensitivity profile toward renal subpanel
(GI50 value ranging from 0.46 to 20.7 M), least for A498 and
maximum for 786-0 cell line. Note that EGFR is over expressed in
cancer cells of Non-Small Cell Lung Cancer, renal tumors, breast and
hormone refractory prostate cancer [3e10].
m
Cancer EKVX (GI50 equal to 0.37
0.36 M), Renal Cancer A498 (GI50 equal to 0.46
equal to 0.99 M) and Breast Cancer MDA-MB-468 (GI50 equal to
1.096 M) in which EGFR is over expressed [16,33,34]. Obtained
mM), NCI-H322M (GI50 equal to
m
mM), TK-10 (GI50
m
The criterion for selectivity of a compound depends upon the
ratio obtained by dividing the full panel MID (the average
m

138
S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
Fig. 6. Docking of compound 7a in the ATP binding site of EGFR TK (PDB code 2J5F) in
3D style; showing key hydrogen bonding (dotted lines) between N-1 of quinazoline
hydrogen bonds with Met769 NH and the N-3 interacts with side chain of Thr766
through a water bridge.
Fig. 4. Docking of compound 5c (yellow carbons) in the ATP binding site of EGFR TK
(PDB code 2J5F) in 3D style; showing key hydrogen bonding (dotted lines) between
N-1 of quinazoline hydrogen bonds with Met769 NH and the N3 interacts with side
chain of Thr766 through a water bridge. (For interpretation of the references to colour
in this figure legend, the reader is referred to the web version of this article.)
compounds. Realization that compounds of either related or
unrelated structures and matched by mean-graph patterns
frequently shared the same or related biochemical mechanisms of
action. Compound 7a was chosen as a “seed” to search the NCI
database for compounds of known mechanism with similar
patterns of sensitivity, or for molecular targets, whose pattern of
expression correlates with sensitivity to compound 7a, thus
generating hypotheses as to the mechanism of action of the novel
compound that can be tested in the laboratory. Interestingly
COMPARE analysis illustrates a high correlation between GI50 mean
graph of compound 7a with that of gefitinib & erlotinib to be 0.76 &
0.819, respectively which were reported as selective EGFR inhibi-
tors [35], moreover the GI50 mean graph for renal & prostate cancer
panel of relative high sensitivity against tested compound 7a, when
sensitivity of all cell lines toward the test agent) by their individual
subpanel MID (the average sensitivity of all cell lines of a particular
subpanel toward the test agent). As per this criterion, compound 7a
in the study was found to be most selective toward renal cancer
subpanel, Table 3. Levels of EGFR mRNA and TGF-a (a ligand for
EGFR) mRNA were highest in cell lines derived from renal cancers
[33]. Thus on the basis of cytotoxicity patterns of these compounds
we were able to support the original EGFR targeting strategy.
In addition to that, COMPARE analysis was performed;
COMPARE is a computerized, pattern recognition algorithm which
has considerable utility in the evaluation and exploitation of data
generated by the NCI screen. In essence, COMPARE is a method of
determining and expressing the degree of similarity, or lack thereof,
of mean-graph profiles generated on the same or different
Fig. 5. Docking of compound 6d in the ATP binding site of EGFR TK (PDB code 2J5F) in
3D style; showing key hydrogen bonding (dotted lines) between N-1 of quinazoline
hydrogen bonds with Met769 NH and the N-3 interacts with side chain of Thr766
through a water bridge.
Fig. 7. Docking of gefitinib in the ATP binding site of EGFR TK (PDB code 2J5F) in 3D
style; showing key hydrogen bonding (dotted lines) between N-1 of quinazoline
hydrogen bonds with Met769 NH and the N-3 interacts with side chain of Thr766
through a water bridge.

S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
139
compared with mean graph based on expression pattern of mRNA
of EGFR in the cell lines of National Cancer Institute [33]. Both mean
graphs are visually similar and the profile of cell line response was
consistent with expected expression of EGFR; showing bars pro-
jecting to the right for both drug sensitivity and mRNA for the EGFR
target.
showed highest selectivity ratio among all tested subpanels.
Moreover, the selectivity profile was further confirmed through
COMPARE analysis results. All these findings support EGFR target-
ing strategy and present 7a as promising EGFR-TK inhibitor that can
be further investigated to be used for a well-characterized tumor
type that is highly expressing of targeted kinase.
According to these results, we can conclude that 4-chloro
derivatives 7a, 5c appear to be the most interesting compounds
among the newly synthesized and seem potentially attractive as
antitumor agents. These preliminary encouraging results of bio-
logical screening done on the synthesized compounds could offer
an excellent framework in this field that may lead to discovery of
potent and selective antitumor agent.
4.2. Docking studies
Docking study using GOLD 4.1 [26] was performed. GOLD is
a genetic algorithm based docking program that allows a wide
range of flexibility for the ligand and the protein [36]. It is used to
predict the binding modes and orientation of the synthesized
compounds at the active site of the ATP binding site of EGFR-TK.
The coordinates of the EGFR-TK structure were obtained from
the crystal structure of EGFR with its inhibitor (PDB code 2J5F),
which was chosen because of the interaction between the
6-substitution of the irreversible inhibitor to unique cys-793 of
EGFR. The root mean square difference (RMSD) between the top
6. Experimental protocols
6.1. Chemistry
Starting materials and reagents were purchased from Sigmae
Aldrich. Melting points were recorded on Stuart Scientific appa-
ratus and were uncorrected. FT-IR spectra were recorded on a Per-
kineElmer spectrophotometer. 1H NMR spectra were recorded in
ꢀ
docking pose and original crystallographic geometry was 1.45 A.
Nevertheless, further validation of docking algorithm was achieved
by docking of the selected lead compound, gefitinib in the active
site of EGFR kinase PDB (2J5F), this was found to retrieve the re-
ported binding mode of gefitinib in the X-ray crystal structure of
the active site of EGFR kinase PDB (2ITY), Fig. 7, where the N-1
atom of quinazoline interacts via H-bond with backbone NH of
Met769 [37].The binding mode of the 4-anilinoquinazoline based
inhibitors 5aeh, 6aeg, 7a, b were in consistency with the
proposed design and rational. One of the best-scored poses of
compounds in 5, 6, 7 series is presented in Figs. 4e6, where the
main interactions between the ligand 5c, 6d, 7a respectively and
the receptor can be observed and identified as following: (i) the N-
1 atom of quinazoline interacted via H-bond with backbone NH of
Met-769 (ii) the N-3 made water mediated H-bond to the side
chain of Thr-766 (iii) the substituted 4-anilinoquinazoline lied in
a deep pocket at the back of the ATP binding site. Moreover the
carbonyl bond of ester (or urea) group made extra hydrogen
bonding with cys-793 which was absent in reference compound
gefitinib Fig. 7, which could enhance the binding affinity and fix
the ligands in its position.
d
scale given in ppm on a GEMINI-200 MHz spectrophotometer, or
Varian Mercury VX-300 NMR spectrometer. Coupling patterns are
described as follows: s, singlet; d, doublet, dd, doubled doublet; t,
triplet; m, multiplet; and 1H, 2H, 3H, etc. J describes a coupling
constant. The coupling constants were rounded off to one decimal
place. MS spectra mass were recorded on Shimadzu GCMS-QP
5050A gas chromatograph mass spectrometer (70 eV). Elemental
analyzes were performed at the Microanalytical Center, Cairo
University.
6.1.1. (E)-N0-(2-Cyano-4-nitrophenyl)-N,N-dimethylimidoformamide
(2)
5-Nitroanthranilonitrile 1 (5.00 g, 30.7 mmol) was refluxed in
20 ml dimethylformamide dimethyl acetal. After 90 min, the
mixture was allowed to cool at room temperature. The solid was
filtered off, washed with several portions of diethyl ether and dried
to yield 2 as yellow crystals (6.50 g, 96.9%), mp 153e155 ꢁC as re-
ported [27,38].
5. Conclusion
6.1.2. Generalmethodforpreparationof6-nitro-4-arylaminoquinazol-
ine (3aeh)
Compound 7a showed remarkably lowest cell growth promo-
tion against breast cancer MDA-MB-468 cell line and exhibited
broad spectrum cell growth inhibition followed by compound 5c
which is the most active one in the 5 series showing highest
percent mean growth inhibition over the whole cell panels tested
and 100% EGFR-TK inhibition then 5a, 5e, 5b and finally, 7b which
although it showed 100% enzyme inhibition but it was the least for
cell line inhibition. Although it was reported that the presence of
electron withdrawing lipophilic groups at the meta-position of
aniline is favored for EGFR activity [32], both 5f, 7b have shown
high interaction energy in docking studies which is further
confirmed by 100% EGFR enzyme inhibition. As for compound 7b
which showed the least cell line inhibition, it may be because
of the hydrophilic substitution of the 3-hydroxy group at
4-anilinoquinazoline scaffold which decreases log P (partition
coefficient) of the compound decreasing penetration across cell
membrane and hence its inhibition percent. Furthermore,
compound 7a was found to be the most selective candidate of the
tested compounds at 5 dose level screening against cell lines which
are of high EGFR expression [16,33,34], and obvious sensitivity
profile toward renal subpanel, which has the highest expression
levels of EGFR mRNA and TGF-a mRNA [33], also, renal subpanel
A mixture of N0-(2-cyano-4-nitrophenyl)-N,N-dimethylforma-
mide (3.0 g, 13.7 mmol) and appropriate aniline (15.1 mmol) in
glacial acetic acid was stirred and refluxed for about 2 h, reactions
were monitored with TLC. After the reaction was completed the
mixture was filtered while hot. The solid was washed with diethyl
ether and dried to yield 3aeh [27,38].
6.1.2.1. 4-(2,4-Dichlorophenylamino)-6-nitroquinazoline
ld 97% as yellow crystals, mp 256e258 ꢁC. IR: (nmax, cmꢀ1): 3406 (NH),
1618 (NO2) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
: 7.53e7.77 (m, 3H,
(3a). Yie-
d
anilino Hs), 7.941 (d, 1H, quinazoline H8), 8.56e8.59 (m, 2H, quina-
zoline H2, H7), 9.58 (s, 1H, quinazoline H5), 10.63 (s, 1H, anilinoeNH
exchangeable by D2O). MS: m/z (%): 334 (Mþ ꢀ 1, 11.98%), 335 (Mþ,
4.72%), 336 (Mþ þ 1, 7.93%), Anal. Calcd. for C14H8Cl2N4O2: C 50.17, H
2.41, N 16.72; found: C 50.42, H 2.47, N 16.92.
6.1.2.2. 4-(2-Chlorophenylamino)-6-nitroquinazoline (3b). Yield 95%
as yellow crystals, mp 180e182 ꢁC. IR: (nmax, cmꢀ1): 3359 (NH), 1617
(NO2) cmꢀ1 1H NMR (300 MHz) (DMSO-d6)
. d: 7.38e7.62 (m, 4H,
anilino H’s), 7.93 (d, 1H, quinazoline H8), 8.54e8.58 (m, 2H, quina-
zoline H2, H7), 9.59 (s, 1H, quinazoline H5), 10.61 (s, 1H, anilinoeNH
exchangeable by D2O). MS: m/z (%): 300.1 (Mþ, 10.61%), 302

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S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
(Mþ þ 2, 4.21%), Anal. Calcd. for C14H9ClN4O2: C 55.92, H 3.02, N
6.1.3.3. N4-(4-Chlorophenyl)quinazoline-4,6-diamine (4c). Yield 88%
as yellow crystals, mp 237e239 ꢁC, (as reported) [38].
18.63; found: C 56.31, H 3.15, N 18.85.
6.1.2.3. 4-(4-Chlorophenylamino)-6-nitroquinazoline (3c). Yield 87%
6.1.3.4. N4-(3-Bromophenyl)quinazoline-4,6-diamine (4d). Yield 78%,
mp 267e270 ꢁC, (as reported) [27,28,38].
as yellow crystals, mp 264 ꢁC, (as reported) [27].
6.1.2.4. 4-(3-Bromophenylamino)-6-nitroquinazoline (3d). Yield 76.5%
as yellow crystals, mp 267e270 ꢁC, (as reported) [27,28,38].
6.1.3.5. N4-(3-Ethynylphenyl)quinazoline-4,6-diamine (4e). Yield 62%,
mp 110e111 ꢁC, (as reported) [29].
6.1.2.5. 4-(3-Ethynylphenylamino)-6-nitroquinazoline (3e). Yield 95%
as yellow crystals, mp 271e272 ꢁC, (as reported) [29].
6.1.3.6. N4-(3-Hydroxyphenyl)quinazoline-4,6-diamine
80%, mp 197e199 ꢁC. IR: (nmax, cmꢀ1): 3520, 3357 (2 NH), 3224 (OH)
cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
: 5.6 (s, 2H, quinazolineeNH2),
(4f). Yield
d
6.1.2.6. 4-(3-Hydroxyphenylamino)-6-nitroquinazoline(3f). Yield 89%,
mp 306e308 ꢁC. IR: (nmax, cmꢀ1): 3737 (OH), 3381 (NH), 1540 (NO2)
5.9e5.9 (dd, 1H, quinazoline H7, J ¼ 9 Hz, J ¼ 2.4 Hz), 5.97e6.00
(m, 1H, quinazoline H5, J ¼ 2.4 Hz), 6.47e6.51 (m, 1H, anilino H2),
7.15e7.26 (m, 2H, anilino H4, H6), 7.36e7.37 (m,1H, anilino H5), 7.50e
7.53 (d,1H, quinazoline H8, J ¼ 9 Hz), 8.34 (s, 1H, quinazoline H2), 8.80
(s, 1H, phenoleOH, exchangeable by D2O), 9.35 (s, 1H, anilinoeNH,
exchangeable by D2O). MS: m/z (%): 251.3 (Mþ ꢀ 1, 35.35%), 252.5
(Mþ, 18.72%) & base peak at 78 (100%), Anal. Calcd. for C14H12N4O: C
66.65, H 4.79, N 22.2; found: C 66.71, H 4.82, N 22.31.
cmꢀ1
.
1H NMR (300 MHz) (DMSO-d6)
d
: 7.17e7.28 (m, 3H, anilino
H2, H4, H6), 7.37e7.38 (m, 1H, anilino H5), 7.92e7.95 (d, 1H, quina-
zoline H8, 9.3 Hz), 8.54e8.57 (dd, 1H, quinazoline H7,
J
¼
J ¼ 9.3 Hz, J ¼ 2.4 Hz), 8.71 (s, 1H, quinazoline H2), 9.50 (s, 1H, OH
exchangeable by D2O), 9.67e9.68 (d, 1H, quinazoline H5, J ¼ 2.4 Hz),
10.32 (s, 1H, anilinoeNH, exchangeable by D2O). MS: m/z (%): 281.3
(Mþ ꢀ 1, 46.85%), 282.4 (Mþ, 37%), 283.6 (Mþ þ 1, 8.68%), Anal. Calcd.
for C14H10N4O3: C 59.57, H 3.57, N 19.85; found: C 59.88, H 3.61, N
20.11.
6.1.3.7. N4-(4-Hydroxyphenyl)quinazoline-4,6-diamine
83%, mp 192e194 ꢁC. IR: (nmax, cmꢀ1): 3402, 3350 (2 NH), 3226 (OH)
cmꢀ1 1H NMR (300 MHz) (DMSO-d6)
: 5.61 (s, 2H, quinazolinee
(4g). Yield
.
d
6.1.2.7. 4-(3-Hydroxyphenylamino)-6-nitroquinazoline(3g). Yield 84%,
NH2), 5.90e5.94 (dd, 1H, quinazoline H7, J ¼ 9 Hz, J ¼ 2.4 Hz),
5.86e5.97 (m, 1H, quinazoline H5, J ¼ 2.4 Hz), 6.72e6.77 (m, 2H,
anilino H2, H6), 7.35e7.57 (m, 2H, anilino H3, H5), 7.63e7.66 (d, 1H,
quinazoline H8, J ¼ 9 Hz), 8.34 (s, 1H, quinazoline H2), 8.56 (s, 1H,
phenoleOH, exchangeable by D2O), 9.42 (s, 1H, anilinoeNH,
exchangeable by D2O). MS: m/z (%): 251.3 (Mþ ꢀ 1, 32.5%), 252.5
(Mþ, 13.72%) & base peak at 77.9 (100%), Anal. Calcd. for C14H12N4O: C
66.65, H 4.79, N 22.2; found: C 66.8, H 4.84, N 22.4.
mp 314 ꢁC, (as reported) [39].
6.1.2.8. 4-(2-Ethylphenylamino)-6-nitroquinazoline (3h). Yield 92%,
mp 178e80 ꢁC. IR: (nmax, cmꢀ1): 3332 (NH), 2959 (CH aliphatic),
1563 (NO2) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d: 1.07e1.12 (t, 3H,
ePheCH2eCH3), 2.54e2.61 (q, 2H, PheCH2eCH3), 7.29e7.39 (m,
4H, anilino Hs), 7.88e7.90 (d, 1H, quinazoline H8, J ¼ 9.3 Hz), 8.50e
8.54 (dd, 2H, quinazoline H2, H7, J ¼ 9.3 Hz, J ¼ 2.4 Hz), 9.59e9.60 (d,
1H, quinazoline H5,
J
¼
2.4 Hz), 10.37 (s, 1H, anilinoeNH,
6.1.3.8. N4-(2-Ethylphenyl)quinazoline-4,6-diamine (4h). Yield 66%,
mp 264e265 ꢁC. IR: (nmax, cmꢀ1): 3428, 3321 (2 NH), 2964 (CH)
exchangeable by D2O). MS: m/z (%): 294.4 (Mþ, 31.21%), 296.7
(Mþ þ 2, 0.51%) Anal. Calcd. for C16H14N4O2: C 65.30, H 4.79, N
19.04; found: C 65.62, H 4.82, N 19.12.
cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d: 1.07e1.12 (t, 3H, CH2eCH3),
2.54e2.61 (q, 2H, CH2eCH3), 5.49 (s, 2H, quinazolineeNH2,
exchanged by D2O), 7.20e7.33 (m, 6H, anilino H3, H4, Hs, H6, qui-
nazoline H5, H7), 7.47e7.50 (m, 1H, quinazoline H8), 8.10 (s, 1H,
quinazoline H2), 9.08 (s, 1H, anilinoeNH, exchangeable by D2O).
MS: m/z (%): 264.2 (Mþ, 20.47%), 265.6 (Mþ þ 1, 2.84%) & base peak
at 89.85 (100%). Anal. Calcd. for C16H16N4: C 72.7, H 6.1, N 21.2;
found: C 72.89, H 6.18, N 21.43.
6.1.3. General method for preparation of 6-amino-4-
arylaminoquinazoline (4aeh)
To a solution of compound 3 (3.42 g, 0.01 mol) in NH4OH (20 ml,
30%), a solution of sodium dithionite (7 g, 0.04 mol) in water (30 ml)
was quickly added, the reaction mixture was refluxed for 30 min.
After cooling, the crude product was filtered off, washed and
crystallized from ethanol [40].
6.1.4. General method for preparation of (Z)-ethyl 3-[4-(arylamino)
quinazolin-6-ylamino]-2-cyanoacrylate (5aeh)
To a solution (0.01 mol) of appropriate amine in absolute ethanol,
(Z)-ethyl 2-cyano-3-ethoxyacrylate (1.7 g, 0.01 mol) was added, the
reaction mixture was refluxed about 3 h. After cooling, the crude
product was filtered off, washed and crystallized from ethanol.
6.1.3.1. N4-(2,4-Dichlorophenyl)-4,6-quinazolinediamine
80%, mp 244e246 ꢁC. IR: (nmax, cmꢀ1): 3415, 3385 (2 NH) cmꢀ1. H
NMR (300 MHz) (DMSO-d6) : 5.62 (s, 2H, quinazolineeNH2), 6.89e
(4a). Yield
1
d
7.54 (m, 5H, anilino H3, H5, H6, quinazoline H5, H7), 7.72e7.75 (m, 1H,
quinazoline H8), 8.27 (s, 1H, quinazoline H2), 9.31 (s, 1H, anilinoeNH,
exchangeable by D2O). MS: m/z (%): 303.9 (Mþ ꢀ 1, 22.6%), 304.95
(Mþ, 8.97%), 305.9 (Mþ þ 1, 13.97%), 306.9 (Mþ þ 2, 3.87%) & base peak
at 268.5 (100%) Anal. Calcd. for C14H10Cl2N4: C 55.10, H 3.30, N 18.36;
found: C 55.23, H 3.35, N 18.45.
6.1.4.1. (Z)-Ethyl 3-[4-(2,4-dichlorophenylamino)quinazolin-6-
ylamino]-2-cyanoacrylate (5a). Yield 85%, mp 155e156 ꢁC. IR:
(
nmax, cmꢀ1): 3402, 3294 (2 NH), 2218 (CN), 3194 (aliphatic CH),
1678 (CO),1624 (vinyl C]C) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d:
1.245e1.32 (t, 3H, eOeCH2eCH3), 4.23e4.30 (q, 2H, eOeCH2e
CH3), 7.50e7.52 (m, 1H, anilino H6), 7.55e7.61 (m, 1H, anilino H5),
7.61e7.64 (m, 1H, anilino H3), 7.79 (d, 1H, quinazoline H8), 8.01 (d,
1H, quinazoline H7), 8.31 (d, 1H, quinazoline H5), 8.51e8.57 (m, 1H,
NHeCH]C), 8.63 (s, 1H, quinazoline H2), 9.73 (s, 1H, anilinoeNH,
exchangeable by D2O), 11.02 (d, 1H, quinazolineeNH, exchange-
able by D2O). MS: m/z (%): 426.9 (Mþ ꢀ 1, 27.43%), 427.9 (Mþ,
16.84%), 428.9 (Mþ þ 1, 9.62%), 430 (Mþ þ 2, 9.6%) & base peak at
346 (100%). Anal. Calcd. for C20H15Cl2N5O2: C 56.09, H 3.53, N 16.35;
found: C 56.18, H 3.63, N 16.39.
6.1.3.2. N4-(2-Chlorophenyl)quinazoline-4,6-diamine (4b). Yield 75%,
mp 269e271 ꢁC. IR: (nmax, cmꢀ1): 3415, 3854 (2 NH) cmꢀ1. 1H NMR
(300 MHz) (DMSO-d6) d: 5.62 (s, 2H, quinazolineeNH2), 7.27e7.59
(m, 6H, anilino H3, H4, H5, H6 & quinazoline H5, H7), 7.62e7.75
(m, 1H, quinazoline H8), 8.26 (s, 1H, quinazoline H2), 9.28 (s, 1H,
anilinoeNH, exchangeable by D2O). MS: m/z (%): 269.7 (Mþ ꢀ 1,
3.84%) 270.5 (Mþ, 11.97%), 272.6 (Mþ þ 2, 4.87%) & base peak at 51.5
(100%), Anal. Calcd. for C14H11ClN4: C 62.11, H 4.1, N 20.7; found: C
62.28, H 4.15, N 20.8.

S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
141
6.1.4.2. (Z)-Ethyl 3-[4-(2-chlorophenylamino)quinazolin-6-ylamino]-
2-cyanoacrylate (5b). Yield 82%, mp 147e149 ꢁC. IR: (nmax, cmꢀ1):
3429, 3371 (2 NH), 2214 (CN), 2924, 2954 (aliphatic CH), 1678 (CO),
(
nmax, cmꢀ1): 3429, 3371 (2 NH), 3156 (OH) 2214 (CN), 2990
(aliphatic CH), 1678 (CO), 1635 (vinyl C]C) cmꢀ1 1H NMR
(300 MHz) (DMSO-d6) : 1.25e1.30 (t, 3H, eOeCH2eCH3), 4.22e
.
d
1643 (vinyl C]C) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d
: 1.24e1.30
4.29 (q, 2H, eOeCH2eCH3), 6.55e6.59 (m, 1H, anilino H2), 7.16e
7.26 (m, 2H, anilino H4, H6), 7.35e7.40 (m, 1H, anilino H5), 7.76e
7.80 (d, 1H, quinazoline H8, J ¼ 9 Hz), 7.89e8.05 (dd, 1H, quinazo-
line H7, J ¼ 9 Hz, J ¼ 2.4 Hz), 8.41e8.47 (d, 1H, quinazoline H5,
J ¼ 2.4 Hz), 8.54e8.58 (d, 1H, NHeCH]C, J ¼ 13.2 Hz), 8.60 (s, 1H,
quinazoline H2), 9.44 (s, 1H, OH exchangeable by D2O), 9.69 (s, 1H,
anilinoeNH exchangeable by D2O), 10.99e11.03 (d, 1H,
quinazolineeNH, exchangeable by D2O, J ¼ 13.2 Hz). MS: m/z (%):
375.3 (Mþ, 85.38%), 377.1 (Mþ þ 2, 7.01%) & base peak at 45.9
(100%), Anal. Calcd. for C20H17N5O3: C 63.99, H 4.56, N 18.66; found:
C 64.27, H 4.9, N 18.9.
(t, 3H, eOeCH2eCH3), 4.19e4.29 (q, 2H, eOeCH2eCH3), 7.34e7.48
(m, 2H, anilino H6, H4) 7.57e7.628 (m, 2H, anilino H3, H5), 7.77e7.82
(m, 1H, quinazoline H8, J ¼ 9 Hz), 7.91e8.07 (dd, 1H, quinazoline H7,
J ¼ 9 Hz, J ¼ 2.4 Hz), 8.40e8.42 (m, 1H, quinazoline H5, J ¼ 2.4 Hz),
8.46e8.60 (m, 2H, quinazoline H2, NHeCH]C), 9.69 (s,1H, anilinoe
NH exchangeable by D2O), 10.98e11.02 (d, 1H, quinazolineeNH,
exchangeable by D2O). MS: m/z (%): 393 (Mþ, 72.5%), 395
(Mþ þ 2, 43.75%) & base peak at 281 (100%), Anal. Calcd. for
C20H16ClN5O2: C 60.99, H 4.09, N 17.78; found: C 61.12, H 4.18, N
17.97.
6.1.4.3. (Z)-Ethyl 3-[4-(4-chlorophenylamino)quinazolin-6-ylamino]-
2-cyanoacrylate (5c). Yield 86%, mp 255e257 ꢁC. IR: (nmax, cmꢀ1):
3429, 3371 (2 NH), 2214 (CN), 2978 (aliphatic CH), 1678 (CO), 1628
6.1.4.7. (Z)-Ethyl 3-[4-(4-hydroxyphenylamino)quinazolin-6-
ylamino]-2-cyanoacrylate (5g). Yield 88%, mp 260e261.5 ꢁC. IR
(
nmax, cmꢀ1): 3429, 3371 (2 NH), 3267 (OH), 2214 (CN), 3000
(vinyl C]C) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d
: 1.25e1.32 (t,
(aliphatic CH), 1678 (CO), 1627 (vinyl C]C) cmꢀ1
.
1H NMR
3H, eOeCH2eCH3), 4.20e4.31 (q, 2H, eOeCH2eCH3), 7.45e7.48
(m, 2H, anilino H2, H6), 7.77e7.80 (m, 1H, quinazoline H8,
J ¼ 9 Hz), 7.86 (d, 2H, quinazoline H3, H5), 7.90e8.05 (dd, 1H, qui-
nazoline H7, J ¼ 9 Hz, J ¼ 2.4 Hz), 8.38e8.42 (d, 1H, quinazoline H5,
J ¼ 2.4 Hz), 8.50e8.60 (m, 2H, quinazoline H2, NHeCH]C), 9.71 (s,
1H, anilinoeNH exchangeable by D2O), 10.98e11.03 (d, 1H,
quinazolineeNH, exchangeable by D2O). MS: m/z (%): 393.5 (Mþ,
71.04%), 395.5 (Mþ þ 2, 29.8%), Anal. Calcd. for C20H16ClN5O2: C
60.99, H 4.09, N 17.78; found: C 61.42, H 4.14, N 18.12.
(300 MHz) (DMSO-d6) : 1.21e1.28 (t, 3H, eOeCH2eCH3), 4.18e
d
4.25 (q, 2H, eOeCH2eCH3), 6.79e6.82 (d, 2H, anilino H2, H6),
7.42e7.48 (m, 2H, anilino H3, H5), 7.71e7.74 (d, 1H, quinazoline H8,
J ¼ 9 Hz), 7.81e7.89 (dd, 1H, quinazoline H7, J ¼ 9 Hz, J ¼ 1.8 Hz),
8.27e8.33 (d, 1H, quinazoline H5, J ¼ 1.8 Hz), 8.38e8.44 (m, 1H,
NHeCH]C), 8.56 (s, 1H, quinazoline H2), 9.49 (s, 1H, phenoliceOH,
exchangeable by D2O), 9.66 (s, 1H, anilinoeNH exchangeable by
D2O), 10.95e11.01 (d, 1H, quinazolineeNH exchangeable by D2O).
MS: m/z (%): 375.5 (Mþ, 70.84%), 377 (Mþ þ 2, 7.84%), 378 (Mþ þ 3,
1.25%) & base peak at 328.5 (100%), Anal. Calcd. for C20H17N5O3: C
63.99, H 4.56, N 18.66; found: C 64.50, H 4.69, N 18.93.
6.1.4.4. (Z)-Ethyl 3-[4-(3-bromophenylamino)quinazolin-6-
ylamino]-2-cyanoacrylate (5d). Yield 70%, mp 188e190 ꢁC. IR:
(
nmax, cmꢀ1): 3549, 3325 (2 NH), 2214 (CN), 2989, 2939 (aliphatic
6.1.4.8. (Z)-Ethyl 3-[4-(2-ethylphenylamino)quinazolin-6-ylamino]-
CH), 1678 (CO), 1627 (vinyl C]CH) cmꢀ1
.
1H NMR (300 MHz)
2-cyanoacrylate (5h). Yield 84%, mp 198.5e200.5 ꢁC. IR (nmax
,
(DMSO-d6) : 1.28e1.33 (t, 3H, eOeCH2eCH3), 4.23e4.29 (q, 2H, e
d
cmꢀ1): 3429, 3371 (2 NH), 2214 (CN), 2970, 2933 (aliphatic CH),
1678 (CO),1629 (vinyl C]C) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
OeCH2eCH3), 7.31e7.41 (m, 2H, anilino H5, H6), 7.79e7.83 (d, 1H,
quinazoline H8, J ¼ 9 Hz), 7.85e7.90 (m, 1H, anilino H4), 7.92e8.08
(dd, 1H, quinazoline H7, J ¼ 9 Hz, J ¼ 2.4 Hz), 8.18e8.20 (d, 1H,
anilino H2), 8.40e8.43 (d, 1H, quinazoline H5, J ¼ 2.4 Hz), 8.55e8.59
(d, 1H, NHeCH]C, J ¼ 13.5 Hz), 8.61 (s, 1H, quinazoline H2), 9.70
(s, 1H, anilinoeNH exchangeable by D2O), 11.00e11.04 (d, 1H,
quinazolineeNH, exchangeable by D2O, J ¼ 13.5 Hz). 13C NMR
d:
1.07e1.12 (t, 3H, ePheCH2eCH3), 1.24e1.32 (t, 3H, eOeCH2eCH3),
2.49e2.51 (q, 2H, ePheCH2eCH3), 4.18e4.31 (q, 2H, eOeCH2e
CH3), 7.29e7.32 (m, 3H, anilino H4, H5, H6), 7.35e7.39 (m, 1H, ani-
lino H3), 7.74e7.78 (d, 1H, quinazoline H8, J ¼ 9 Hz), 7.96 (d, 1H,
quinazoline H7, J ¼ 9 Hz), 8.33e8.42 (m, 1H, quinazoline H5), 8.44e
8.45 (m, 1H, NHeCH]C), 8.61 (s, 1H, quinazoline H2), 9.73 (s, 1H,
anilinoeNH exchangeable by D2O) , 10.98 (d, 1H, quinazolineeNH,
exchangeable by D2O). MS: m/z (%): 388 (Mþ, 100%), 389 (Mþ þ 1,
21.7%), 390 (Mþ þ 2, 1.93%), Anal. Calcd. for C22H21N5O2: C 68.2, H
5.46, N 18.08; found: C, H, N (C 68.33, H 5.47, N 18.09).
d
(ppm): 166.72, 165.02, 153.7, 153.03, 147.44, 141.17, 138.38, 137.05,
130.82, 129.45, 126.65, 124.84, 121.33, 118.47, 115.92, 109.53, 108.93,
75.19, 61.11, 14.71, MS: m/z (%): 438 (Mþ, 8.8%), 439 (Mþ þ 1, 8.79%),
440 (Mþ þ 2, 82.36%), 441 (Mþ þ 3, 28.09%), Anal. Calcd. for
C20H16BrN5O2: C 54.8, H 3.68, N 15.98; found: C 55.02, H 3.72, N
15.68.
6.1.5. General method for preparation of diethyl 2-[(4-arylamino)
quinazolin-6-ylaminomethylene]malonate (6aef)
6.1.4.5. (E)-Ethyl 3-[4-(3-ethynylphenylamino)quinazolin-6-
A mixture of appropriate amine (0.01 mol) & diethyl 2-
(ethoxymethylene)malonate (2.16 g, 0.01 mol) in absolute ethanol
was refluxed about 2 h. After cooling, the crude product was filtered
off, washed and crystallized from ethanol.
ylamino]-2-cyanoacrylate (5e). Yield 91%, mp 383e384 ꢁC. IR:
(
nmax, cmꢀ1): 3549, 3271 (2 NH), 2214 (CN), 2985 (aliphatic CH),
1678 (CO),1635 (vinyl C]C) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d:
1.25e1.32 (t, 3H, eOeCH2eCH3), 4.21 (s, 1H, ethynyl CH), 4.22e4.32
(q, 2H, eOeCH2eCH3), 7.26e7.29 (m, 1H, anilino H6), 7.42e7.48 (m,
1H, anilino H2), 7.79e7.82 (d, 1H, quinazoline H8, J ¼ 9 Hz), 7.88e
7.95 (m, 1H, anilino H4), 7.97e8.00 (m, 1H, anilino H5), 8.04e8.11
(dd, 1H, quinazoline H7, J ¼ 9 Hz), 8.42e8.47 (m, 1H, quinazoline
H5), 8.58 (d, 1H, NHeCH]C), 8.63 (s, 1H, quinazoline H2), 9.81
(s, 1H, anilinoeNH, exchangeable by D2O), 11.02e11.06 (d, 1H,
quinazolineeNH, exchangeable by D2O). MS: m/z (%): 381 (Mþ ꢀ 2,
8.99%), 383 (Mþ, 7.96%), Anal. Calcd. for C22H17N5O2: C 68.9, H 4.47,
N 18.27; found: C 69.2, H 4.51, N 18.31.
6.1.5.1. Diethyl 2-[(2,4-dichlorophenylamino)quinazolin-6-
ylaminomethylene]malonate (6a). Yield 95%, mp 280.6e282.7 ꢁC.
IR: (nmax, cmꢀ1): 3412 cmꢀ1 (NH), 2923 cmꢀ1 (aliphatic CH), 1680
(CO), 1640 (vinyl C]C) cmꢀ1 1H NMR (300 MHz) (DMSO-d6)
. d:
1.24e1.31 (2t, 6H, 2ꢂ eOeCH2eCH3), 4.14e4.28 (2q, 4H, 2ꢂ eOe
CH2eCH3), 7.50e7.51 (m, 1H, anilino H6), 7.53e7.56 (m, 1H, ani-
lino H5), 7.61e7.64 (m,1H, anilino H3), 7.78e7.82 (d,1H, quinazoline
H8, J ¼ 9 Hz), 7.94e8.04 (d, 1H, quinazoline H7, J ¼ 9 Hz), 8.40 (d, 1H,
quinazoline H5), 8.57e8.61 (d, 1H, NHeCH]C, J ¼ 13.5 Hz), 8.42
(s, 1H, quinazoline H2), 9.89 (s, 1H, anilinoeNH exchangeable by
D2O), 10.95e10.99 (d, 1H, quinazolineeNH, exchangeable by D2O,
J ¼ 13.5 Hz). MS: m/z (%): 475 (Mþ, 10%), 476 (Mþ þ 1, 7.25%), 478.15
6.1.4.6. (Z)-Ethyl 3-[4-(3-hydroxyphenylamino)quinazolin-6-
ylamino]-2-cyanoacrylate (5f). Yield 77%, mp 271e272.5 ꢁC. IR:

142
S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
(Mþ þ 3, 1.31%) & base peak at 45.9 (100%), Anal. Calcd. for
C22H20Cl2N4O4: C 55.6, H 4.24, N 11.79; found: C 55.83, H 4.31, N
11.98.
MS: m/z (%): 430 (Mþ, 52%), 431 (Mþ þ 1, 19%), 432 (Mþ þ 2,
4.1%) & base peak at 383 (100%), Anal. Calcd. for C24H22N4O4: C
66.97, H 5.15, N 13.02; found: C 67.18, H 5.22, N 13.09.
6.1.5.2. Diethyl 2-[(2-chlorophenylamino) quinazolin-6-ylaminome-
6.1.5.6. Diethyl 2-[(4-hydroxyphenylamino) quinazolin-6-ylaminom-
thylene]malonate (6b). Yield 98%, mp 130e132 ꢁC. IR:
(
nmax
cmꢀ1): 3560 (NH), 2981 (aliphatic CH), 1698 (CO), 1603 (vinyl C]C)
cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
: 1.24e1.31 (2t, 6H, 2ꢂ eOe
,
ethylene]-malonate (6f). Yield 96%, mp 162e263.5 ꢁC. IR: (nmax
,
cmꢀ1): 3521, 3442 (2 NH), 3305 (OH), 2978 (aliphatic CH), 1642
(CO), 1641 (vinyl C]C) cmꢀ1 1H NMR (300 MHz) (DMSO-d6)
d:
d
.
CH2eCH3), 4.14e4.29 (2q, 4H, 2ꢂ eOeCH2eCH3), 7.36e7.47 (m, 2H,
anilino H6, H4), 7.57e7.62 (m, 2H, anilino H3, H5), 7.79e7.82 (d, 1H,
quinazoline H8, J ¼ 9 Hz), 7.92e8.96 (dd, 1H, quinazoline H7,
J ¼ 9 Hz, J ¼ 2.4 Hz), 8.39e8.40 (d, 1H, quinazoline H5, J ¼ 2.4 Hz),
8.42 (s, 1H, quinazoline H2), 8.58e8.62 (d, 1H, NHeCH]C,
J ¼ 13.5 Hz), 9.87 (s, 1H, anilinoeNH, exchangeable by D2O),
10.95e10.99 (d, 1H, quinazolineeNH, exchangeable by D2O,
J ¼ 13.5 Hz). MS: m/z (%): 440.8 (Mþ, 71.43%), 442.7 (Mþ þ 2,
33.94%), Anal. Calcd. for C22H21ClN4O4: C 59.93, H 4.8, N 12.71;
found: C 59.58, H 4.53, N 12.59.
1.21e1.28 (2t, 6H, 2ꢂ eOeCH2eCH3), 4.18e4.25 (2q, 4H, 2ꢂ eOe
CH2eCH3), 6.79e6.82 (d, 2H, anilino H2, H6), 7.42e7.48 (m, 2H,
anilino H3, H5), 7.71e7.74 (d, 1H, quinazoline H8, J ¼ 9 Hz), 7.81e7.89
(dd, 1H, quinazoline H7, J ¼ 9 Hz, J ¼ 1.8 Hz), 8.27e8.33 (d, 1H,
quinazoline H5, J ¼ 1.8 Hz), 8.56 (s, 1H, quinazoline H2), 8.57e8.61
(d, 1H, NHeCH]C, J ¼ 13.5 Hz), 9.34 (s, 1H, phenoliceOH
exchangeable by D2O), 9.68 (s, 1H, anilinoeNH exchangeable by
D2O), 10.97e11.01 (d, 1H, quinazoline NH, exchangeable by D2O,
J ¼ 13.5 Hz). MS: m/z (%): 423 (Mþ þ 1, 40%), 424 (Mþ þ 2, 14.3%) &
base peak at 376 (100%), Anal. Calcd. for C22H22N4O5: C 62.55, H
5.25, N 13.26; found: C 62.67, H 4.69, N 13.49.
6.1.5.3. Diethyl 2-[(4-dichlorophenylamino) quinazolin-6-ylaminome-
thylene]malonate (6c). Yield 97%, mp 147e149 ꢁC. IR: (nmax, cmꢀ1):
3452 (NH), 2985, 2935 (aliphatic CH), 1686 (CO), 1647 (vinyl C]C)
6.1.6. General methodforpreparation of 4-(arylamino)-6-ureidoquina-
zoline (7a, b)
A mixture of appropriate amine (0.01 mol) and potassium
cyanate (0.01 mol, 0.81 g) in glacial acetic acid was refluxed for
about 2 h. After cooling; the crude product was filtered off, washed
and crystallized from ethanol.
cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
d: 1.25e1.32 (2t, 6H, 2ꢂ eOe
CH2eCH3), 4.14e4.29 (2q, 4H, 2ꢂ eOeCH2eCH3), 7.45e7.48 (d, 2H,
anilino H2, H6), 7.79e7.82 (d, 1H, quinazoline H8, J ¼ 9 Hz), 7.89e7.92
(m, 2H, quinazoline H3, H5), 7.93e7.97 (dd, 1H, quinazoline H7,
J ¼ 9 Hz, J ¼ 2.4 Hz), 8.40e8.401 (d, 1H, quinazoline H5, J ¼ 2.4 Hz),
8.56 (s, 1H, quinazoline H2), 8.57e8.616 (d, 1H, NHeCH]C,
J ¼ 13.5 Hz), 9.83 (s, 1H, anilinoeNH exchangeable by D2O),
10.99e11.04 (d, 1H, quinazolineeNH, exchangeable by D2O,
J ¼ 13.5 Hz). MS: m/z (%): 440.8 (Mþ, 40.64%), 442.6 (Mþ þ 1,
20.25%), 444.1 (Mþ þ 3, 1.86%) & base peak at 394.65 (100%), Anal.
Calcd. for C22H21ClN4O4: C 59.93, H 4.8, N 12.71; found: C 59.6, H
4.77, N 12.52.
6.1.6.1. 4-(4-Chlorophenylamino)-6-ureidoquinazoline
76%, mp 147e149 ꢁC. IR: (nmax, cmꢀ1): 3363 (NH’s), 1686 (CO) cmꢀ1
1H NMR (300 MHz) (DMSO-d6)
6.11 (s, 2H, ureidoeNH2,
(7a). Yield
.
d:
exchangeable by D2O), 7.45e7.49 (m, 2H, anilino H2, H6), 7.73e7.84
(m, 3H, quinazoline H8 & anilino H3, H5), 7.89e7.92 (dd, 1H, quina-
zoline H7, J ¼ 9 Hz, J ¼ 2.4 Hz), 8.46e8.47 (d, 1H, quinazoline H5,
J ¼ 2.4 Hz), 8.46e8.57 (m, 1H, NH-CH]C), 8.60 (s, 1H, quinazoline
H2), 9.91 (s, 1H, anilinoeNH, exchangeable by D2O), 10.36 (s, 1H,
6.1.5.4. Diethyl 2-[(3-bromophenylamino) quinazolin-6-ylaminomethy
lene]malonate (6d). Yield 96%, mp 133e135 ꢁC. IR: (nmax, cmꢀ1):
3568 (NH), 3094 (aliphatic CH),1686 (CO),1640 (vinyl C]C) cmꢀ1. 1H
quinazolineeNH, exchangeable by D2O). 13C NMR
d (ppm): 169.22,
158.30, 152.59, 143.91, 140.13, 138.34, 129.04, 128.88, 128.43, 127.69,
126.89,124.89,115.61,112.32,110.04, MS: m/z (%): 313.75 (Mþ,19.5%),
312.75 (Mþ ꢀ 1, 49.25%), 311.75 (Mþ ꢀ 2, 22.15%) & base peak at 51.7
(100%), Anal. Calcd. for C15H12ClN5O: C 57.42, H 3.86, N 22.32; found:
C 57.71, H 3.90, N 15.38.
NMR (300 MHz) (DMSO-d6)
d: 1.25e1.38 (2t, 6H, 2ꢂ eOeCH2eCH3),
4.15e4.29 (2q, 4H, 2ꢂ eOeCH2eCH3), 7.30e7.34 (m, 1H, anilino H6),
7.36e7.41 (m, 1H, anilino H5), 7.81e7.84 (d, 1H, quinazoline H8,
J ¼ 9 Hz), 7.90 (d, 1H, anilino H4), 7.95e8.99 (dd, 1H, quinazoline H7,
J ¼ 9 Hz, J ¼ 2.4 Hz), 7.99e8.02 (m, 1H, anilino H2), 8.21e8.22 (d, 1H,
quinazoline H5, J ¼ 2.4 Hz), 8.41 (s,1H, quinazoline H2), 8.58e8.62 (d,
1H, NHeCH]C, J ¼ 13.5 Hz), 9.85 (s, 1H, anilinoeNH exchangeable
by D2O), 10.99e11.04 (d, 1H, quinazoline NH, exchangeable by D2O,
J ¼ 13.5 Hz). MS: m/z (%): 483 (Mþ ꢀ 2, 46.9%), 484 (Mþ ꢀ 1, 42%),
485 (Mþ, 35.8%), 486 (Mþ þ 1, 59.3%), Anal. Calcd. for C22H21BrN4O4:
C 54.44, H 4.36, N 11.54; found: C 54.14, H 4.27, N 11.25.
6.1.6.2. 4-(3-Hydroxyphenylamino)-6-ureidoquinazoline
86%, mp 271e272.5 ꢁC. IR: (nmax, cmꢀ1): 3423 (NH), 3313.11 (OH), 1701
(CO) cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
: 6.16 (s, 2H, ureidoeNH2,
(7b). Yield
d
exchangeable by D2O), 6.49e6.53 (m, 1H, anilino H2), 7.09e7.66 (m, 3H,
anilino H4, H5, H6), 7.70e7.73 (d, 1H, quinazoline H8, J ¼ 9 Hz), 7.89e
7.93 (dd, 1H, quinazoline H7, J ¼ 9 Hz, J ¼ 2.4 Hz), 8.46e8.47 (d, 1H,
quinazoline H5, J ¼ 2.4 Hz), 8.668 (s,1H, quinazoline H2), 9.11 (s, 1H, OH,
exchangeable by D2O), 9.64 (s, 1H, anilinoeNH, exchangeable by D2O),
10.34 (s, 1H, quinazolineeNH exchangeable by D2O). MS: m/z (%): 294
(Mþ ꢀ 1, 11.74%) & base peak at (108.85, 100%), Anal. Calcd. for
C15H13N5O2: C 61.01, H 4.44, N 23.72; found: C 61.37, H 4.56, N 23.79.
6.1.5.5. Diethyl 2-[(3-ethynylphenylamino) quinazolin-6-ylaminom
ethylene]malonate (6e). Yield 96%, mp 274e276 ꢁC. IR: (nmax
,
cmꢀ1): 3440 (NH), 3229 (aliphatic CH), 1678 (CO), 1636 (vinyl C]C)
cmꢀ1. 1H NMR (300 MHz) (DMSO-d6)
: 1.25e1.31 (2t, 6H, 2ꢂ eOe
d
CH2eCH3), 4.14 (s, 1H, ethynyl CH), 4.17e4.26 (2q, 4H, 2ꢂ eOeCH2e
CH3), 7.25e7.30 (m, 1H, anilino H6), 7.42e7.47 (m, 1H, anilino H2),
7.80 -7.83 (m, 1H, quinazoline H8), 7.94e7.97 (m, 1H anilino H4),
7.97e8.01 (m, 1H, anilino H5), 8.04e8.10 (dd, 1H, quinazoline H7,
J ¼ 9 Hz, J ¼ 2.4 Hz), 8.40e8.41 (d, 1H, quinazoline H5, J ¼ 2.4 Hz),
8.57 (s, 1H, quinazoline H2), 8.59e8.64 (d, 1H, NHeCH]C), 9.81 (s,
1H, anilinoeNH exchangeable by D2O), 11.02e11.05 (d, 1H,
6.2. Pharmacology
6.2.1. Materials and methods
6.2.1.1. In vitro EGFR tyrosine kinase activity. The EGFR tyrosine
kinase activity at single dose concentration of 10 mM was carried out
by BPS Bioscience (www.bpsbioscience.com) & EGFR (BPS#40187)
served as the enzyme source and Poly (Glu, Tyr) sodium salt, (4:1,
Glu:Tyr) (Sigma#P7244) served as the standardized substrate &
Kinase-Glo Plus Luminescence kinase assay kit (Promega#V3772).
The IC50 determination was carried out where quality control
testing is routinely performed on each of the targets to insure
quinazolineeNH, exchangeable by D2O). 13C NMR
d (ppm): 170.55,
158.57, 156.95, 152.18, 150.29, 139.84, 139.60, 129.83, 129.50, 125.67,
123.47, 124.84, 123.42, 122.36, 120.96, 109.38, 106.12, 84.00, 83.92,
81.21, 60.46, 56.18, 55.01, 14.82, 14.68.

S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
143
compliance to acceptable standards. 33P-ATP was purchased from
a. Assay protocol
Perkin Elmer and ADP-GloÔ was purchased from Promega. All
other materials were of standard laboratory grade.
The human tumor cell lines of the cancer-screening panel are
grown in RPMI 1640 medium containing 5% fetal bovine serum and
2 mM L-glutamine. For a typical screening experiment, cells are
6.2.1.1.1. Assay protocols. The EGFR tyrosine kinase activity was
performed using Kinase-Glo Plus luminescence kinase assay kit
(Promega). It measures kinase activity by quantitating the amount
of ATP remaining in solution following a kinase reaction. The
luminescent signal from the assay is correlated with the amount of
ATP present and is inversely correlated with the amount of kinase
inoculated into 96 well microtiter plates in 100 ml at plating
densities ranging from 5000 to 40,000 cells/well depending on the
doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37 ꢁC, 5% CO2, 95% air and 100%
relative humidity for 24 h prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to
represent a measurement of the cell population for each cell line at
the time of drug addition (Tz). Experimental drugs are solubilized
in dimethyl sulfoxide at 400-fold the desired final maximum test
concentration and stored frozen prior to use. At the time of drug
addition, an aliquot of frozen concentrate is thawed and diluted to
twice the desired final maximum test concentration with complete
medium containing 50 mg/ml Gentamicin. Additional four, 10-fold
or ½log serial dilutions are made to provide a total of five drug
concentrations plus control. Aliquots of 100 ml of these different
drug dilutions are added to the appropriate microtiter wells already
containing 100 ml of medium, resulting in the required final drug
concentrations. Following drug addition, the plates are incubated
for an additional 48 h at 37 ꢁC, 5% CO2, 95% air, and 100% relative
humidity. For adherent cells, the assay is terminated by the addition
of cold TCA. Cells are fixed in situ by the gentle addition of 50 ml of
cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for
60 min at 4 ꢁC. The supernatant is discarded, and the plates are
washed five times with tap water and air dried. Sulforhodamine B
(SRB) solution (100 ml) at 0.4% (w/v) in 1% acetic acid is added to
each well, and plates are incubated for 10 min at room temperature.
After staining, unbound dye is removed by washing five times with
1% acetic acid and the plates are air dried. Bound stain is subse-
quently solubilized with 10 mM trizma base, and the absorbance is
read on an automated plate reader at a wavelength of 515 nm. For
suspension cells, the methodology is the same except that the assay
is terminated by fixing settled cells at the bottom of the wells by
gently adding 50 ml of 80% TCA (final concentration, 16% TCA)
activity. The compounds were diluted to 100
l of the dilution was added to a 50 l reaction so that the final
concentration of DMSO is 1% in all of reactions. All of the enzy-
matic reactions were conducted at 30 ꢁC for 40 min. The 50
reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2,
0.1 mg/ml BSA, 0.2 mg/ml Poly (Glu, Tyr) substrate, 10 M ATP and
EGFR. After the enzymatic reaction, 50 l of Kinase-Glo Plus
mM in 10% DMSO and
5
m
m
ml
m
m
Luminescence kinase assay solution (Promega) was added to each
reaction and incubate the plate for 5 min at room temperature.
Luminescence signal was measured using a Tecan Infinite M1000
microplate reader.
The protein kinase assays used to determine IC50 value were
performed using ADP-GloTM assay kit from Promega which
measures the generation of ADP by the protein kinase. Generation
of ADP by the protein kinase reaction leads to an increase in
luminescence signal in the presence of ADP-GloTM assay kit.
The assay was started by incubating the reaction mixture in
a 96-well plate at 30 ꢁC for 30 min. After the 30 min incubation
period, the assay was terminated by the addition of 25
GloTM Reagent (Promega). The 96-well plate was shaken and
then incubated for 40 min at ambient temperature. 50 l of
ml of ADP-
m
Kinase detection reagent was added, the 96-well reaction plate
was then read using the ADP-Glo Luminescences Protocol on
a GloMax plate reader (Promega: Catalog #E7031). Blank control
was set up that included all the assay components except the
addition of appropriate substrate (replace with equal volume of
kinase assay buffer). The corrected activity for each protein
kinase target was determined by removing the blank control
value.
6.2.1.1.2. Data analysis. EGFR activity assays were performed in
duplicate at each concentration. The luminescence data were
analyzed using the computer software, Graphpad Prism. The
difference between luminescence intensities in the absence of
EGFR (Lut) and in the presence of EGFR (Luc) was defined as 100%
activity (Lut ꢀ Luc). Using luminescence signal (Lu) in the presence
of the compound, % activity was calculated as:
% Activity ¼ {(Lut ꢀ Lu)/(Lut ꢀ Luc)} ꢂ 100%, where Lu ¼ the
luminescence intensity in the presence of the compound (all
percent activities below zero were set to 0%).
b. Data analysis
Using the seven absorbance measurements [time zero, (Tz),
control growth, (C), and test growth in the presence of drug at the
five concentration levels (Ti)], the percentage growth is calculated
at each of the drug concentrations levels. Percentage growth inhi-
bition is calculated as: [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 for concentrations
for which Ti>/ ¼ Tz and [(Ti ꢀ Tz)/Tz] ꢂ 100 for concentrations for
which Ti < Tz: three dose response parameters are calculated for
each experimental agent. Growth inhibition of 50% (GI50) is calcu-
lated from [(Ti ꢀ Tz)/(C ꢀ Tz)] ꢂ 100 ¼ 50, which is the drug
concentration resulting in a 50% reduction in the net protein
increase (as measured by SRB staining) in control cells during the
drug incubation. The drug concentration resulting in total growth
inhibition (TGI) is calculated from Ti ¼ Tz. The LC50 (concentration
of drug resulting in a 50% reduction in the measured protein at the
end of the drug treatment as compared to that at the beginning)
indicating a net loss of cells following treatment is calculated from
[(Ti ꢀ Tz)/Tz] ꢂ 100 ¼ ꢀ50. Values are calculated for each of these
three parameters if the level of activity is reached; however, if the
effect is not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum
concentration tested [41,42].
% Inhibition was calculated as: % inhibition ¼ 100 (%) ꢀ % activity.
IC50 determination for inhibitor against EGFR was estimated by
generating a graph of log inhibitor vs normalized response with
variable using the Prism software.
6.2.1.2. Evaluation for cytotoxic activity against a panel of sixty
human cancer cell lines. National Cancer Institute (NCI) for in vitro
anticancer assay evaluates different compounds for their anticancer
activity. The screening is a two-stage process, beginning with the
evaluation of all compounds against the full NCI 60 cell lines panel
representing leukemia, Non-Small Cell Lung Cancer, melanoma,
colon cancer, CNS cancer, breast cancer, ovarian cancer, renal cancer
and prostate cancer at a single dose of 10ꢀ5 M. The output from the
single dose screen is reported as a mean graph and is available for
analysis by the COMPARE program. Compounds which exhibit
significant growth inhibition are evaluated against the 60 cell panel
at five concentration levels.
Results for each compound were reported as a mean graph of
the percent growth of the treated cells when compared to the
untreated control cells. There after obtaining the results for one

144
S. Mowafy et al. / European Journal of Medicinal Chemistry 61 (2013) 132e145
growth factor receptor (EGF-R) could be predictive of early and long-term
dose assay, analysis of historical Development Therapeutics Pro-
gramme (DTP) was performed and compounds which satisfies
predetermined threshold inhibition criteria is selected for NCI full
panel 5 dose assay.
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All molecular modeling calculation and docking studies were
carried out using GOLD 4.1 [26]. The X-ray crystal structure of the
kinase domain of EGFR in complex with its inhibitor (PDB code
2J5F) was recovered RSCB protein data bank, which was chosen
because of the interaction between the 6-substitution of the
inhibitor to unique Cys-793 of EGFR. Docking engine was validated;
the co-crystallized ligand was extracted from the catalytic site and
redocked to calculate the root mean square difference (RMSD)
between the top docking pose and original crystallographic
geometry. The protein structure was prepared using protein prep-
aration protocol of Accelry’s discovery studio 2.5 [43]. The amino
acid residues were ionized using role based technique and the
missing residues were completed. The protein structure was typed
by CHARMM [44] force field then it was minimized using 500 step
of SMART minimizer of Discovery studio program (Minimizer adopt
hybrid of steepest descent [45] and conjugate gradient [46] mini-
mization algorithms). All the water molecules in the protein were
deleted except No. 10 because of its main role in ligand binding.
Based on the literature data of EGFR-TK inhibitors binding [14,15],
an interaction motif was added to Met-769 residue as hydrogen
bond donor. Kinase template was loaded in gold and the default
parameters of GOLD were used except that the number of opera-
tions was raised to 500,000 operations and the selection pressure
rose to 1.1. Chemscore kinase scoring function was used to score the
resulted docking poses [47].
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Acknowledgments
[22] D.W. Fry, A.J. Bridges, W.A. Denny, A. Doherty, K.D. Greis, J.L. Hicks, K.E. Hook,
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3-carbonitrile inhibitors of epidermal growth factor receptor kinase and their
bioisosteric relationship to the 4-anilino-6,7-dialkoxyquinazoline inhibitors,
J. Med. Chem. 43 (2000) 3244e3256.
The authors are grateful to The National Cancer Institute,
Maryland, USA for performing anticancer activity and also would
like also to thank BPS Bioscience for EGFR binding assay.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.ejmech.2012.10.017.
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potent adenosine 5’-triphosphate binding site inhibitors of the tyrosine kinase
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