Design, synthesis and biological evaluation of novel quinazoline derivatives as potential antitumor agents: Molecular docking study

Article abstract of DOI:10.1016/j.ejmech.2010.06.013

Novel derivatives of quinazoline (1-27) have been synthesized and tested for their antitumor activity against three tumor cell lines among these cell lines the human breast carcinoma cell line (MCF-7) in which EGFR is highly expressed. All tested compound

Full text of DOI:10.1016/j.ejmech.2010.06.013

European Journal of Medicinal Chemistry 45 (2010) 4188e4198
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and biological evaluation of novel quinazoline derivatives
as potential antitumor agents: Molecular docking study
,
a
a
b
Adel S. El-Azab a , Mohamed A. Al-Omar , Alaa A.-M. Abdel-Aziz , Naglaa I. Abdel-Aziz ,
*
Magda A.-A. El-Sayed c, Abdulaziz M. Aleisa d, Mohamed M. Sayed-Ahmed d, Sami G. Abdel-Hamide e
,
**
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University. Riyadh 11451, Saudi Arabia
b Department of Medicinal Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
c Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, University of Mansoura, Mansoura 35516, Egypt
d Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi Arabia
e Department of Pharmaceutical Chemistry, College of Pharmacy, Al-Kharj University, Al-Kharj, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Novel derivatives of quinazoline (1e27) have been synthesized and tested for their antitumor activity
against three tumor cell lines among these cell lines the human breast carcinoma cell line (MCF-7) in
which EGFR is highly expressed. All tested compounds showed potent and selective activity against
Received 2 May 2010
Received in revised form
7 June 2010
Accepted 9 June 2010
Available online 16 June 2010
breast cancer (MCF-7) with IC50 range of 3.35e6.81
compounds 5, 9, 15, 18 and 20 exploited potent antitumor against human liver cell line (HEPG2), human
breast cell line (MCF-7) and human cervix cell line (HELA) with IC50 range of 3.35e5.59 g/ml. Virtual
mg/ml. With regarding broad-spectrum activity
m
screening was carried out through docking the designed compounds into the ATP binding site of
epidermal growth factor receptor (EGFR) to predict if these compounds have analogous binding mode to
the EGFR inhibitors.
Keywords:
Synthesis
Quinazoline
Antitumor
Ó 2010 Elsevier Masson SAS. All rights reserved.
Drug-likeness
Molecular docking
1. Introduction
growth factor receptor (EGFR) is cellular trans-membrane tyrosine
kinases that is over-expressed in a significant number of human
Cancer is continuing to be a major health problem in developing
as well as undeveloped countries [1e9]. Surpassing heart diseases,
it is taking the position number one killer due to various worldwide
factors. Although major advances have been made in the chemo-
therapeutic management of some patients, the continued
commitment to the laborious task of discovering new anticancer
agents remains critically important. In the course of identifying
various chemical substances which may serve as leads for
designing novel antitumor agents, we are particularly interested in
the present work with quinazoline derivatives which have been
identified as a new class of cancer chemotherapeutic agents with
significant therapeutic efficacy against solid tumors [7e9]. It is well
known that quinazoline derivatives are potent inhibitors of
epidermal growth factor receptor (EGFR) [10e18]. The epidermal
tumors (e.g., breast, ovarian, colon, and prostate), their expression
levels often correlate with vascularity, and is associated with poor
prognosis in patients [19e22]. Inhibitors of the EGFR PTK are
therefore expected to have great therapeutic potential in the
treatment of malignant and nonmalignant epithelial diseases. A
number of small molecule EGFR kinase inhibitors have been eval-
uated in cancer clinical trials [10e22]. For example (Fig. 1), anili-
noquinazoline-containing compounds Gefitinib (IressaÔ) [10e12],
Erlotinib (TarcevaÔ) [13], Lapatinib (TykerbÔ, also known as GW-
572016) and Vandetanib (ZactimaÔ) were recently approved for
the treatment of HER2-positive metastatic breast cancer [14e18].
Many more compounds are still under evaluation in clinical trials
for the treatment of cancer [20e22].
In view of the previous rationale and in continuation of an
ongoing program aiming at finding new structure leads with
potential chemotherapeutic activities [7e9], new series of
4-substituted 6-chloro-2-p-tolylquinazolin (1e27) have been
synthesized and screened for antitumor activity (Fig.1). These series
comprise the derived 4-substituted quinazoline pharmacophores
that are structurally related to Erlotinib and Lapatinib (Fig. 1). The
* Corresponding author. Tel.: þ966 50 2875710; fax: þ966 14091909.
** Corresponding author. Tel.: þ966 56 0921396.
E-mail addresses: adelazaba@yahoo.com (A.S. El-Azab), gabersami@yahoo.com
(S.G. Abdel-Hamide).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.06.013

A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
4189
Fig. 1. Reported and proposed antitumor quinazoline derivatives.
thrust of efforts in the derivatization of such type of compounds
focused mainly on the aryl moiety of the 4-substituted quinazoline.
In the present study, the substitution pattern at the 4-substituted
quinazoline pharmacophores was selected so as to confer different
electronic environment that would affect the lipophilicity, and
hence the activity of the target molecules. The objective of forming
these hybrids is an attempt to reach an active antitumor agent with
potentiated activity and selectivity toward cancerous cells. More-
over drug-likeness and molecular docking methodology were used
to identify the structural features required for the antitumor prop-
erties of these new series. However the results of this molecular
docking could support the postulation that our active compounds
may act on the same enzyme target where EGFR inhibitor acts
confirming the molecular design of the reported class of antitumor
agents.
benzoic acid (1), which was boiled with acetic anhydride to give
compound 2. Upon stirring of compound 2 with concentrated
ammonia solution at room temperature, the corresponding diamide
3 was obtained in relatively good yield. Moreover the treatment
of 2 with formamide or methylformamide produced 6-chloro-
2-p-tolylquinazolin-4(3H)-one (4) and 6-chloro-3-methyl-2-p-
tolylquinazolin-4(3H)-one (5) in 73% and 68% yields, respectively.
On the other hand, when compound 2 was reacted with hydrazine
hydrate gave the corresponding 3-amino-4(3H)quinazolinone (12)
in 88% yield. The reaction of compound 4 with P2S5 in anhydrous
xylene afforded 6-chloro-2-p-tolylquinazolin-4(3H)-thione (6). 4-
Substituted aminoquinazolines (7e9) were obtained by the reaction
of compound 6 with formamide, hydroxylamine hydrochloride
and/or hydrazine hydrate. N-(6-chloro-2-p-tolylquinazolin-4-yl)
benzamide (10) and O-benzoyl-N-(6-chloro-2-p-tolylquinazolin-
4-yl)hydroxylamine (11) were obtained in a relatively good yield
(69% and 71%, respectively) via the reaction of compound 7 or 8 with
benzoyl chloride in pyridine.
2. Results and discussion
2.1. Chemistry
2.1.2. Synthesis of compounds 13e27 (Scheme 2)
2.1.1. Synthesis of compounds 1e12 (Scheme 1)
Scheme 2 outlines the synthetic pathway used to obtain
compounds 13e27 (series-II). The reaction of 2 with P2S5 in
anhydrous xylene afforded 6-chloro-2-p-tolyl-4H-benzo[d][1,3]-
oxazin-4-thione (13) which was converted into compound 6 by
heating with formamide. Interaction of 6 with various haloge-
nated compounds in acetone containing K2CO3 at room
Scheme 1 outlines the synthetic pathway used to obtain
compounds 1e12 (series-I). The starting material 6-choro-2-p-
tolyl-4H-benzo[d][1,3]oxazin-4-one (2) was prepared by the
reaction of 5-chloroanthranilic acid with 4-methylbenzoyl chlo-
ride in pyridine to afford the 5-chloro-2-(4-methylbenzamido)-

4190
A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
O
O
O
Cl
Cl
OH
OH
Cl
+
pyridine
NH
NH2
H3C
CH3
O
1
Ac2O,reflux
O
O
O
N
R
Cl
Cl
N
Cl
NH2
O
N
NH
HCONH2 or
HCONMe
CH3
4:R=H
O
Conc.NH3
CH3
2
5:R=CH3
3
CH3
NH2NH2
P2S5,xylene
NH
S
N
O
N
Cl
Cl
NNH2
6
12
CH3
CH3
R
HN
N
R
Cl
NH
N
NH2COH,
NH2OH.HClor
NH2NH2
Cl
N
PhCOCl
pyridine
N
CH3
CH3
10:R=COPh
11:R=OCOPh
7:R=H
8:R=OH
9:R=NH2
Scheme 1. Synthesis of the proposed antitumor 4-quinazolone and 4-aminoquinazoline derivatives (series-I).
temperature furnished 6-chloro-4-(substitutedthio)-2-p-tolylqui-
nazolines (16e21) in 80e95% yield. The oxidation of 6-chloro-4-
(benzylthio)-2-p-tolylquinazoline (16) with KMnO4 in acetic acid
afforded 4-(benzylsulfonyl)-6-chloro-2-p-tolylquinazoline (22) in
a 63% yield. Moreover 2-(6-chloro-2-p-tolylquinazolin-4-ylthio)
substituted acetamides (23e27) were produced by heating of
compound 6 with various chloroacetanilides in acetone contain-
ing K2CO3 in 84e90% yield. Finally, the reaction of 13 with
hydrazine hydrate afforded 3-amino-6-chloro-2-p-tolylquinazo-
lin-4(3H)-thione (14) and N-(4-chloro-2-(hydrazinecarbono-
thioylphenyl)-4-methylbenzamide (15) in 46% and 43% yields
respectively.
causing a net 50% loss of initial cells at the end of the incubation
period (48 h). The antitumor drug discovery screen has been
designed to distinguish between broad-spectrum antitumor
compounds and tumor selective agents [24].
In present study, the active analogs showed a distinctive
potential pattern of selectivity as well as broad-spectrum anti-
tumor activity. With regard to selectivity against individual cell
lines, most of the compounds showed effectiveness against cell
line human breast cancer MCF-7 with IC50 values range of
3.35e6.81 mg/ml comparative to DOX IC50 (3.76 mg/ml) (Table 1).
HEPG2 human liver cell line proved to be sensitive toward
compounds 5, 9, 15, 18, 20 and 25 with IC50 concentration range
of 4.17e5.99 mg/ml comparative to DOX IC50 (4.57 mg/ml).
2.2. Biological activity
Regarding HELA cervix cell line, higher sensitivity was observed
with compounds 5, 6, 7, 9, 11, 15, 18, 20 and 23 with IC50
2.2.1. In vitro antitumor evaluation
concentration range of 3.56e5.39
(3.64 g/ml). With regard to broad-spectrum antitumor activity,
compounds 5, 9, 15, 18 and 20 showed IC50 concentration range
of 3.35e5.59 g/ml against the three cell lines (Table 1). On the
other hand compounds 6, 7, 11 and 23 showed higher cytotoxic
activity against MCF-7 cell line and HELA cervix cell line with low
activity against HEPG2 human liver cell line. Moreover
compounds 1, 2, 10, 14 and 27 proved to be ineffective against the
three cell lines.
mg/ml comparative to DOX IC50
The antitumor activity of all compounds against HEPG2 human
liver cell line, MCF-7 cell line and HELA cervix cell line was deter-
mined using Sulphorhodamine-B assay and Doxorubicin (DOX) as
a reference drug control [23]. Each cell line was incubated with four
m
m
concentrations (6e62 mg/ml) for each compound and was used to
create compound concentration versus survival fraction curves. The
response parameter (IC50) was calculated for each cell line (Table 1).
The IC50 value corresponds to the compound’s concentration

A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
4191
CH2CONHR
N
S
N
S
Cl
Cl
O
NH2COH
P2S5
RNHCOCH2Cl
2
6
N
(13)
23-R=H
CH3
CH3
24-R=Ph
RCl
25-R=4-EtO-Ph
26-R=4-CH3-Ph
27-R=4-F-Ph
NH2NH2
O
O
R
N
CH2Ph
S
S
S
Cl
Cl
Cl
KMnO4
CH3
NNH2
N
N
N
N
14
CH3
CH3
22
+
16: R=CH3
17: R=CH2Ph
S
NH2
18
: R=CH2CN
Cl
19: R=allyl
N
H
20: R=3-NO2-2-pyridine
21: R=CH2CH2OH
CH3
NH
O
15
Scheme 2. Synthesis of the proposed antitumor quinazoline-4-thione and 4-substituted mercaptoquinazoline derivatives (series-II).
2.2.2. Structuraleactivity relationship, Lipinski rule of five and
drug-likeness profile
2.2.2.1. Structuraleactivity relationship. The activity of the tested
compounds could be correlated to structure variation and modifi-
cations. By investigating the variation in selectivity of the tested
compounds over the three cell lines, it was revealed that nearly all
of the compounds belonging to series-I and series-II (Schemes 1
and 2) showed significant inhibition for the breast cancer cell line
(MCF-7). This great inhibition at the mentioned concentration
indicates a great potency for the compound with a strong lethal
effect over (MCF-7) breast cancer cells. Moreover the IC50 of
cyctotoxic activity for HEPG2 human liver cell line and HELA cervix
Table 1
In vitro antitumor activity of the designed quinazoline deriviatives.
Compd no.
IC50 (m
g/ml)a
MCF-7b
HELAc
HEPG2d
1
2
3
4
5
6
7
8
14.7
10.5
5.39
4.98
3.35
3.35
3.76
6.81
3.76
16.1
5.59
5.39
5.18
9.25
3.35
8.64
9.66
3.35
8.03
3.56
3.76
3.96
5.59
4.78
15.8
5.79
14.7
3.76
18.9
8.23
8.23
10.1
4.37
4.57
3.56
12.7
3.98
16.2
5.18
12.1
13.3
14.01
4.98
14.7
11.3
3.76
9.66
5.39
9.25
7.01
4.98
11.7
10.1
12.3
10.5
3.64
15.95
22.5
21
9.86
4.17
7.01
14.5
23.1
4.17
17.5
12.1
18.4
11.1
17
5.59
13.3
18.4
4.37
23.9
5.59
9.04
7.21
9.45
14.3
5.99
18.8
18.4
4.57
cancer cell reached 3.56 mg/ml in a number of the tested derivatives
(Table 1). However, distinguished selectivity was observed between
the first series-I (compounds 1e12), and the second series-II
(compounds 13e27). Both series-I and series-II showed significant
and selective inhibition for (MCF-7) breast cancer (IC50 value of
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
DOX
3.35 mg/ml). On the other hand series-I were more selective for
HELA cervix cancer cell than HEPG2 human liver cell line as
observed in Table 1. The agreement between the two series in the
inhibition of (MCF-7) breast cancer cells could be correlated to
a similar inhibitory mechanism related to the common structural
feature in the two series (the quinazoline fragment), while the
variation in selectivity over HEPG2 human liver cell line and HELA
cervix cancer cell line is probably caused by the differences in the
substitution pattern in the two series. These variations could be
also correlated to the lipophilic difference substitution pattern of
the quinazoline cores of the two series.
Briefly the obtained screening results showed that, among the
tested compounds, compounds 5e7 and 9 from series-I and
compounds 15, 18, 20e22 from series-II are the most active
a
IC50, compound concentration required to inhibit tumor cell proliferation by
members with IC50 values of 3.35e3.96 mg/ml. Regarding two
50%.
series, upon cyclization of compound 1 afforded compound 2 with
little increase in activity while replacement of the carboxylic
moiety of compound 1 with amide group as in 3 this resulted in
b
c
Human breast cell line (MCF-7).
Human cervix cell line (HELA).
Human liver cell line (HEPG2).
d

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A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
Table 2
thione (13) (IC50 value; 5.18 mg/ml) into 3-aminoquinazoline-4-
Solubility and calculated Lipinski's rule of five for the most active compounds over
breast cancer (MCF-7) cell line.
thione (14) resulted in two fold decrease in activity (IC50 value;
9.25 g/ml). The ring opening of 3-aminoquinazoline-4-thione (14)
resulted in the formation of benzothiohydrazide (15) with broad-
spectrum antitumor agent with IC50 values of 3.35 g/ml, 4.49 g/
ml and 5.59 g/ml against MCF-77, HELA and HEPG2 cell lines,
m
log Sb
Parameter
a
Compd no.
IC50
log Pc
PSAd
MWe
nONf
nOHNHg
m
m
m
5
6
7
9
15
18
20
21
22
3.35
3.35
3.76
3.76
3.35
3.35
3.56
3.76
3.96
ꢀ3.92
ꢀ4.22
ꢀ5.58
ꢀ5.66
ꢀ4.50
ꢀ6.33
ꢀ6.37
ꢀ5.67
ꢀ7.10
3.58
4.16
4.73
4.27
2.66
4.92
5.46
4.42
4.92
34.89
28.68
51.81
63.83
67.15
49.57
84.50
46.01
59.92
284.75
286.78
269.73
284.75
319.82
325.82
408.87
330.84
408.91
3
2
3
4
4
3
6
3
4
0
1
2
3
4
0
0
1
0
respectively. More interestingly, S-substituted compound 6 resul-
ted in derivative with variant activity against the three cell line such
as compounds 18 and 20 were broad-spectrum antitumor agents
with IC50 range of 3.35e5.59
cancer cell line of such two compounds 18 (3.35
(3.56 g/ml) over similar analogs 16 (8.64 g/ml), 17 (9.66
and 19 (8.03 g/ml) may be attributed to the occurrence of CN and
m
g/ml. The high activity over breast
g/ml) and 20
g/ml)
m
m
m
m
m
a
NO2 moieties which may be an important fragments for hydrogen
bonding formation at the receptor site as described in docking
section.
Data taken from Table 1.
Solubility parameter.
Calculated lipophilicity.
Polar surface area (A ).
Molecular weight.
b
c
d
e
f
2
ꢀ
2.2.2.2. Lipinski rule of five and drug-likeness profile. In this work,
we submitted the most active compounds over MCF-7 cell line to
the analysis of Lipinski rule of five that indicates if a chemical
compound could be an orally active drug in humans [25]. Our
results showed that all active compounds (5e7, 9,15,18 and 20e22)
Number of hydrogen bond acceptor.
Number of hydrogen bond donor.
g
a remarkable increase in the magnitude of antitumor activity
against MCF-7-and HELA cell lines with IC50 value 5.39 and 8.23 g/
ml respectively. On the other hand, introduction of NH or N-CH3
moieties at position-3 of compound 2, afforded 4 and 5 with
a remarkable increase in the antitumor activity against breast
cancer, IC50 values, 4.98 and 3.25
compound 4 showed moderate activity toward HELA and HEPG2
cancer cell lines with IC50 values, 10.1 and 9.86 g/ml respectively.
Upon thiation of compound 4 to the corresponding quinazoline-
thione, compound 6 was obtained with a dramatic broad-spectrum
antitumor activity. In case of compound 2, the presence of NH2
moiety at position-3 favoured the antitumor activity against breast
cancer MCF-7 and retains the activity against HELA and HEPG2
cancer cell lines. Interestingly the addition of such NH2 group at
position-4 rather than the position-3 resulted in an increase in
antitumor activity against both breast cancer and HELA cell lines,
while replacement of such group with NHOH moiety lead to
decrease in cytotoxic activity against all cell lines. On the other
hand insertion of eNHNH2 fragment at the same position-4
resulted in sharp increase in antitumor activity with broad-spec-
trum effect. In addition, an increase in antitumor activity over
breast cancer was observed when the benzoxazine-4-one (2) (IC50
fulfilled
this
rule
(molecular
weight ¼ 269.73e408.91,
m
c log P ¼ 2.66e4.92, nON ¼ 2e6, and nOHNH ¼ 0e4) (Table 2),
similarly to clinically used drugs (i.e. Tarceva, Iressa, Vandetanib
and Lapatinib) (data not shown). Currently there are many
approaches that assess a compound drug-likeness based on topo-
logical descriptors, fingerprints of molecular drug-likeness struc-
ture keys or other properties such as c log P and molecular weight
[26]. In the Osiris program (http://www.organic-chemistry.org/
prog/peo) the occurrence frequency of each fragment is deter-
mined within the collection created by shreddering 3300 traded
drugs as well as 15,000 commercially available chemicals (Fluka)
yielding a complete list of all available fragments. In this work, we
used the Osiris program for calculating the fragment based drug-
likeness of the active compounds also comparing them with
Tarceva, Iressa, Vandetanib and Lapatinib (Fig. 2). Interestingly, the
derivatives 5e7, 9, 15, 18 and 20e21 presented better drug-likeness
values (from 2.22 to ꢀ5.54) than Tarceva (ꢀ6.73) and similar results
to Iressa, Vandetanib and Lapatinib (ꢀ2.62, 3.51 and ꢀ4.8 respec-
tively). In this study we also verified the drugscore [27]. Our
theoretical data showed that compounds 5e7, 9, 15, 18 and 20e21
presented values once again higher than Lapatinib (Fig. 2). More-
over, we used the Osiris program (Fig. 2) to predict the overall
toxicity of the most active derivatives as it may point to the pres-
ence of some fragments generally responsible for the irritant,
mutagenic, tumorigenic, or reproductive effects in these molecules.
mg/ml respectively. Moreover, also
m
value; 10.5
(IC50 value; 5.18
10.5 g/ml) to 12 (IC50 value; 5.39
m
g/ml) was converted to benzoxazine-4-thione (13)
g/ml). On contrary to conversion of 2 (IC50 value;
g/ml) with increased in anti-
m
m
m
tumor activity over breast cancer, conversion of benzoxazine-4-
Toxicity effects
Compd No.
Drug score
M
T
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
Low
I
Low
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
Low
R
Low
Low
Low
Low
Low
Low
Medium
Low
Low
Low
Low
5
6
7
9
15
18
20
21
0.72
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Medium
Medium
Low
Medium
Medium
Medium
Medium
Low
0.51
0.46
0.39
0.35
0.26
0.24
0.22
0.2
0.17
0.15
0.1
0.06
22
Erlotinib
Gefitinib
Vandetanib
Lapatinib
Low
Low
Low
Medium
Low
Low
Low
Fig. 2. In silico toxicity risks (left panel) and drugscore (right panel) of the reported and the most active antitumor quinazoline derivatives over breast cancer (M, mutagenic;
T, tumorigenic; I, irritant; R, reproductive).

A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
4193
Fig. 3. Lowest energy (green color) and bioactive conformers (red color) of the most active compound 18 as representative example and their superposition (left panel).
Interestingly, most of the active derivatives presented a low in silico
toxicity risk profile, similar to Tarceva, Iressa, Vandetanib and
Lapatinib (Fig. 2). These theoretical data reinforced the cytotoxicity
experimental data described in this work pointing these
compounds as lead compounds with low toxicity risk profile.
molecular orbital calculation [30]. The global minimum was
confirmed as true minimum not saddle point by the absence of
negative eigen value of the Hessian through frequency calculation
[3,31]. The calculation results showed that the lowest energy-
minimized structures of the compounds under investigation
exhibited a common arrangement of the 2-phenyl moiety, such
group arranged in a coplanar form to the quinazoline core. On the
other hand the bioactive conformation resulted from conforma-
tional analysis in the vicinity of receptor site showed that 2-phenyl
moiety attached to quinazoline core was out of the plane of qui-
nazoline ring by 30.7e51.9ꢁ (Fig. 3).
2.3. Molecular modeling study
Modeling studies are required in order to construct molecular
models that incorporate all experimental evidences reported. These
models are necessary to obtain a consistent and more precise
picture of the biological active molecules at the atomic level and
furthermore, provide new insights that can be used to design novel
therapeutic agents.
2.3.2. Electrostatic and hydrophobic mappings [9,31]
In an attempt to understand the lowest activity of compound 17
and the highest antitumor activity of 18 as a representative
example of the same series, electrostatic and hydrophobic
mappings have been carried out for the lowest energy conformers,
to examine the similarity and dissimilarity in electronic, electro-
static binding characteristics of the surface of the molecules and
conformational properties (Fig. 4; left panel). Comparison of the
electrostatic mappings of 17 and 18 show that compound 18
possesses an increased negative charge regions located on the CN
groups representing hydrogen bond regions (in red). In contrast,
2.3.1. Conformational analysis
As an attempt to gain a better insight into the molecular
structures of the most active compounds 5e7, 9, 15, 18 and 20e22,
conformational analysis has been performed by use of the MMþ
force-field [28] (calculations in vacuo, bond dipole option for
electrostatics, PolakeRibiere algorithm, and RMS gradient of
ꢀ
0.01 kcal/A mol) as implemented in HyperChem 5.1 [29]. The most
stable conformer was fully optimized with AM1 semi-empirical
Fig. 4. Electrostatic maps (left panels) and hydrophobic maps (right panels) of the lowest energy conformers for the most active compound 18 (upper panel) and the least active
compound 17 (lower panel); maps are color coded: red for a hydrogen bond and a hydrophilic region, green for a medium polar region, and blue or cyan for a hydrophobic region.

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A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
Fig. 5. Docking of compounds 17 (upper right panel), 18 (lower left panel) and 20 (lower right panel) into the active site of epidermal growth factor receptor. Upper left panel
showed the Erlotinib inhibitor/EGFR complex. Hydrogen bonds are shown in green.
the lowest activity of compound 17 maybe attribute to the differ-
ence in electrostatic mapping with lack of the red hydrogen bond
area on such site which may be important for receptor interaction
(Fig. 4; lower left panel).
Compounds 18 (3.35
mg/ml; MCF-7), 17 (9.66 mg/ml; MCF-7) and 20
(3.56 g/ml; MCF-7) were used for docking study as representative
m
examples of the most active and moderate active compounds of the
same series. All the calculations were performed using MOE
2008.10 software [32] installed on 2.0G Core 2 Duo. The crystal
structure of epidermal growth factor receptor with Erlotinib
(TarcevaÔ) (1M17) was obtained from protein data bank PDB
(Fig. 5, upper left panel) [33e35]. The automated docking program
of MOE 2008.10 was used to dock compounds 17, 18 and 20 into ATP
binding site of EGFR. The complexes were energy-minimized with
a MMFF94 force-field [36] till the gradient convergence 0.01 kcal/
mol was reached. The binding energies of ꢀ15.30, ꢀ25.66 and
ꢀ23.79 kcal/mol were obtained for 17, 18 and 20, respectively
(Fig. 5). These docking studies have revealed that the quinazoline
ring binds to a narrow hydrophobic pocket in the N-terminal
domain of EGFR-TK where N-1 of the quinazoline ring interacts
with the backbone NH of Met-769 via a hydrogen bond, and simi-
larly, a water (HOH-10) molecule-mediated hydrogen bonding
interaction is observed between the N-3 of the quinazoline ring and
the Thr-830 side chain. These interactions revealed the importance
of both nitrogen atoms for binding and the subsequent inhibitory
capacity.
Similarly, hydrophobic mappings of the most active compound
18 showed that the hydrophobic region in cyan was distributed on
both side of 2-phenyl fragment and the quinazoline core while the
hydrophilic region in red was located mainly on CN fragments at 4
position of quinazoline core (Fig. 4; upper right panel). On the other
hand the hydrophobic distributions of the compound 17 occupy
additional hydrophobic benzyl fragment at position-4 of quinazo-
line core and lacking of hydrophilic region at such position indi-
cating dissimilarity of such compound (Fig. 4; lower right panel).
It is clear that the related charge distribution, electrostatic and
hydrophobic mappings suggest a similar activity of the molecules.
As a result, we find that compounds 5e7, 9, 15, 18 and 20e22 all can
adopt conformations that have identical distances and orientations
and that show sufficient agreement of the overall structure and of
the electrostatic potential pattern.
2.3.3. Molecular docking studies
The level of antitumor activities of the proposed compounds
over MCF-7 breast cancer cell, in which EGFR is highly expressed,
prompted us to perform molecular docking into the ATP binding
site of epidermal growth factor receptor (EGFR) to predict if these
compounds have analogous binding mode to the EGFR inhibitors.
Compounds 18 and 20 complex with EGFR-TK showed the
occurrence of four hydrogen bonds with Met-769 (2.82 A, 3.07 A
ꢀ
ꢀ
ꢀ
ꢀ
ꢀ
respectively), Thr-766 (3.37 A, 3.22 A respectively), Lys-721 (3.21 A,
ꢀ
3.25 A respectively) and HOH-10 mediated hydrogen bonding

A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
4195
ꢀ
ꢀ
interaction with Thr-830 side chain (2.71 A, 2.66 A respectively).
The lower interaction energy observed for 17 (Fig. 5, upper right
panel) rationalizes the insufficient binding into the EGFR-TK active
site than that of the compounds 18 and 20 (Fig. 5, lower panels).
The insufficient binding can be explained in terms of the occur-
rence of only two weak hydrogen bonds between the N-1 and N-3
4.1.1. 5-Chloro2-(4-methylbenzamido)benzoic acid (1)
5-Chloroanthranilic acid (20 mmol, 3.43 g) and 4-methyl-
benzoyl chloride (22 mmol, 3.40 g) were stirred at room tempera-
ture in pyridine (80 ml) for 6 h. The solvent was removed under
reduced pressure; the obtained residue was washed with acidu-
lated water, filtered, washed with water, dried, recrystallized from
ethanol, mp 284e286 ꢁC in 94% yield.
ꢀ
of quinazoline ring system and Met-769 (2.83 A) and HOH-10
mediated hydrogen bonding interaction with Thr-766 side chain
(3.10 A). In short, Fig. 5 demonstrates binding models of quinazo-
1H NMR (DMSO-d6):
d 12.07 (s, 1H, NHCO), 8.71 (s, 1H, Ar.), 7.95
ꢀ
(d, 1H, J ¼ 2.0 Hz), 7.82 (d, 2H, J ¼ 7.5 Hz), 7.68e7.61 (m, 2H), 7.34
line in the ATP binding site. In each case, the N-1 atom of the qui-
nazoline is hydrogen bonded to the backbone nitrogen of Met-769,
while the N-3 atom is hydrogen bonded to the side chain hydroxyl
of Thr-830 via a water molecule, and in case of compounds 18 and
20 we noticed that an additional hydrogen bond is formed between
N-3 and the side chain hydroxyl of Thr-766 and hydrogen bond
between CN of compound 18 and NO2 of compound 20 with the
backbone nitrogen of Lys-721. The results of this molecular docking
could support the postulation that our active compounds may act
on the same enzyme target where EGFR inhibitor acts confirming
the molecular design of the reported class of antitumor agents
[7e9,11e16,37].
(d, 2H, J ¼ 8.0 Hz), 2.35 (s, 3H, CH3). 13C NMR (DMSO-d6):
d 169.2,
165.0, 143.4, 140.5, 134.4, 131.9, 130.8, 130.0, 127.5, 126.8, 122.1,
118.8, 21.4. MS: (M ꢀ 1, 288, 6%).
Anal. calcd. for C15H12ClNO3 (%): C, 62.19; H, 4.17; N, 4.83. Found:
C, 62.43; H, 4.12; N, 4.53.
4.1.2. 6-Chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-one (2)
A benzoic acid derivative 1 (20 mmol, 5.79 g) was heated under
reflux in acetic anhydride (100 ml) for 3 h. The solid obtained was
filtered while hot and dried, mp 199e201 ꢁC in 89% yield.
IR (KBr, cmꢀ1) : 1763 (CO); 1H NMR (CDCl3):
n d 8.47 (s, 1H, Ar.),
8.23 (d, 1H, J ¼ 7.5 Hz), 8.06 (s, 1H), 7.71e7.60 (m, 3H), 7.40e7.38 (m,
1H), 2.53 (s, 3H, CH3). MS: M (271, 30.84%), M þ 2 (273, 10.34%).
Anal. calcd. for C15H10ClNO2 (%): C, 66.31; H, 3.71; N, 5.16. Found: C,
66.70; H, 4.02; N, 5.32.
3. Conclusion
4.1.3. 5-Chloro-2-(4-methylbenzamido)benzamide (3)
The present work led to the development of novel antitumor
molecules containing 4-substituted quinazoline pharmacophore.
Three cell lines including human liver cell line (HEPG2), human
breast cell line (MCF-7) and human cervix cell line (HELA) were
used to measure cytotoxic activity of the proposed quinazoline
derivatives. Compounds 5, 9, 15, 18 and 20 exploited broad-spec-
trum and potent antitumor activity with IC50 range of
Benzoxazine (2) (2 mmol, 543 mg) was stirred at room
temperature in conc. ammonia solution (10 mL) for 6 h. The solid
obtained was filtered, washed with water, dried and recrystallized
from ethanol, mp 229e231 ꢁC in 78% yield.
1H NMR (DMSO-d6):
d 12.77 (s, 1H, NHCO), 8.67 (d, 2H,
J ¼ 6.5 Hz), 7.93e7.59 (m, 5H), 7.34 (s, 2H, CONH2), 2.35 (s, 3H, CH3).
MS: (M ꢀ 1, 287). Anal. calcd. for C15H13ClN2O2 (%): C, 62.40; H,
4.54; N, 9.70. Found: C, 62.67; H, 4.32; N, 9.47.
3.35e5.59
tive activity against breast cancer (MCF-7) with IC50 range of
3.35e6.81 g/ml. Since breast cells are known to overexpress EGFR,
mg/ml. All tested compounds showed potent and selec-
m
4.1.4. 6-Chloro-2-p-tolylquinazolin-4(3H)-one (4)
which leads to continuous activation of the EGFR pathway involved
in cell proliferation, therefore, molecular docking studies further
helps in understanding the antitumor selectivity over MCF-7 cell
line and thereby helps to design novel potent inhibitors. Molecular
docking studies further supported the strong inhibitory activity of
18 and 20 compared to 17 of the same series and further help
understanding the various interactions between the ligands and
enzyme active sites in detail and thereby help to design novel
potent inhibitors.
Benzoxazine (2) (10 mmol, 2.71 g) was heated under reflux in
formamide (50 ml) for 3 h. The solid obtained was filtered while
hot and dried, mp 300e302 ꢁC in 73% yield.
IR (KBr, cmꢀ1) : 3179 (NH), 1668 (CO); 1H NMR (DMSO-d6):
n
d
12.41 (s, 1H, NHCO), 8.11 (s, 1H, Ar.), 8.04 (d, 1H, J ¼ 1.5 Hz), 7.82
(d, 2H, J ¼ 8.0 Hz), 7.68 (d, 1H, J ¼ 8.5 Hz), 7.37e7.22 (m, 2H), 2.33
(s, 3H, CH3). 13C NMR (DMSO-d6):
167.7, 160.2, 147.9, 146.3, 143.4,
d
134.9, 129.9, 129.8, 128.2, 124.3, 125.3, 127.5, 21.6. MS: (M ꢀ 28,
252). Anal. calcd. for C15H11ClN2O (%): C, 66.55; H, 4.10; N, 10.35.
Found: C, 66.65; H, 4.34; N, 9.98.
4. Experimental
4.1.5. 6-Chloro-3-methyl-2-p-tolylquinazolin-4(3H)-one (5)
Benzoxazine (2) (10 mmol, 2.71 g) was heated under reflux in N-
methylformamide (50 ml) for 3 h. The solid obtained was filtered
while hot and dried, mp 146e148 ꢁC in 68% yield.
4.1. Chemistry
Melting points (uncorrected) were recorded on Barnstead 9100
Electrothermal melting apparatus. IR spectra were recorded on
a PerkineElmer spectrometer. 1H NMR and 13C NMR was recorded
in DMSO-d6 and/or CDCl3 on a Jeol 500 MHz instrument using TMS
1H NMR (DMSO-d6):
d
8.56 (d, 1H, J ¼ 8.5 Hz), 8.44 (d, 1H,
J ¼ 8.5 Hz), 8.23 (d, 1H, J ¼ 8.5 Hz), 7.48e7.42 (m, 2H), 7.18 (d, 2H,
J ¼ 7.5 Hz), 2.35 (s, 3H, CH3), 2.16 (s, 3H, NCH3). 13C NMR (DMSO-d6):
d
167.7, 160.2, 147.9, 146.3, 143.4, 134.9, 129.9, 129.8, 128.2, 124.3,
as internal standard (chemical shifts in
d
ppm). Microanalytical
125.3, 127.5, 24.8, 21.6, MS: (M, 284). Anal. calcd. for C16H13ClN2O
(%): C, 67.49; H, 4.60; N, 9.84. Found: C, 67.65; H, 4.36; N, 9.88.
data (C, H, and N) were performed on PerkineElmer 240B analyzer
and they agreed with proposed structures within ꢂ0.4% of the
calculated values. Mass spectra were recorded on a Shimadzu PQ-
5000 GCeMS apparatus. Solvent evaporation was performed under
reduced pressure using Buchan Rotatory Evaporator unless other-
wise stated. T.L.C. was performed on precoated silica gel plates (60-
F254, 0.2 mm), manufactured by E.M. Sciences, Inc, and shortwave
UV (254) nm was used to detect the U.V. absorbing compounds
(CH2Cl2, EtOH 10:1).
4.1.6. 6-Chloro-2-p-tolylquinazolin-4(3H)-thione (6)
Method-A
A
mixture of 6-chloro-2-p-tolylquinazolin-4(3H)-one (4)
(10 mmol, 2.70 g) and phosphorous pentasulfide (11 mmol, 2.43 g)
was heated under reflux in anhydrous xylene (100 ml) for 12 h. The

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A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
reaction mixture was filtered while hot, the solvent was evapo-
rated, and the residue was triturated with dimethylsulfoxide
(10 ml) and filtered. The clear filtrate was poured into ice water and
stirred; the solid obtained was filtered, washed with water, dried
and recrystallized from ethanol, mp 258e260 ꢁC in 77% yield.
IR (KBr, cmꢀ1) : 3170 (NH), 1705 (CO); 1H NMR (DMSO-d6):
n
d
8.14 (s, 1H, NHCO), 8.04 (d, 1H, J ¼ 2.0 Hz), 7.92e7.89 (m, 2H),
7.83e7.81 (m, 1H), 7.62e7.55 (m, 1H), 7.54e7.47 (m, 4H), 7.42 (d, 1H,
J ¼ 7.5 Hz), 7.30e7.25 (m, 2H), 2.35 (s, 3H, CH3). 13C NMR (DMSO-
d6): d 161.9, 141.9,135.9,135.2, 134.0,133.9,133.0,132.8,131.2, 129.9,
129.7, 129.2, 128.8, 128.5, 127.5, 126.1, 124.1, 21.5. MS: (M, 373). Anal.
calcd. for C22H16ClN3O (%): C, 70.68; H, 4.31; N, 11.24. Found: C,
70.58; H, 4.68; N, 11.34.
Method-B
6-Chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-thione (13) (5 mmol,
1.44 g) was heated under reflux in formamide (25 ml) for 3 h. The solid
obtained was filtered while hot and dried (75% yield).
4.1.11. O-Benzoyl-N-(6-chloro-2-p-tolylquinazolin-4-yl)
hydroxylamine (11)
IR (KBr, cmꢀ1)
n
: 3188 (NH), 1206 (CS); 1H NMR (DMSO-d6):
A mixture of N-(6-chloro-2-p-tolylquinazolin-4-yl)hydroxyl-
d
13.98 (s, 1H, Ar.), 8.49 (d, 1H, J ¼ 1.5 Hz), 8.06 (d, 2H, J ¼ 7.5 Hz),
7.87e7.83 (m, 1H), 7.75e7.69 (m, 1H), 7.33 (d, 2H, J ¼ 7.5 Hz), 2.38 (s,
3H, CH3). 13C NMR (DMSO-d6):
187.2, 161.8, 152.3, 147.9, 143.6,
amine (8) (2.0 mmol, 571 mg) and benzoyl chloride (2.1 mmol,
295 mg) in pyridine (10 ml) was heated under reflux for 12 h. The
reaction mixture was cooled and the solvent was removed under
reduced pressure; the residue was triturated with water and
filtered. The solid obtained was dried and recrystallized from
ethanol, mp 196e198 ꢁC in 71% yield.
d
135.7,132.6, 129.5, 128.9, 128.3, 126.4, 122.6, 21.5. MS: (M, 286).
Anal. calcd. for C15H11ClN2S (%): C, 62.82; H, 3.87; N, 9.77. Found: C,
62.91; H, 3.60; N, 9.55.
1H NMR (DMSO-d6):
d
9.43 (s, 1H, NHCO), 8.42 (d, 1H, J ¼ 8.5 Hz),
4.1.7. 6-Chloro-2-p-tolylquinazolin-4-amine (7)
8.03 (dd, 2H, J ¼ 3.0 Hz), 7.89 (d, 1H, J ¼ 2.0 Hz), 7.86e7.63 (m, 3H),
7.64 (dd, 1H, J ¼ 2.0 Hz), 7.44e7.39 (m, 3H), 7.20 (d, 1H, J ¼ 7.5 Hz),
2.23 (s, 3H, CH3). 13C NMR (DMSO-d6): 166.6, 161.1, 155.2,148.3,
137.4, 136.8, 135.4, 134.7, 131.7, 131.6 129.7, 128.1, 127.9, 124.3, 124.2,
124.0, 119.9, 21.9. MS: (M, 391). Anal. calcd. for C22H16ClN3O2 (%): C,
67.78; H, 4.14; N, 10.78. Found: C, 67.97; H, 4.42; N, 11.02.
6-Chloro-2-p-tolylquinazolin-4(3H)-thione (6) (2 mmol, 773 mg)
was heated under reflux in formamide (15 ml) for 3 h. The solid
obtained was filtered while hot, dried and recrystallized from
ethanol, mp 193e195 ꢁC in 68% yield.
IR (KBr, cmꢀ1) : 3413, 3287 (NH); 1H NMR (DMSO-d6):
n d 8.39 (s,
1H, Ar.), 8.34 (d, 2H, J ¼ 8.0 Hz), 7.84 (s, 2H, D2O exchangeable, NH2),
7.78e7.73 (m, 2H), 7.29 (d, 2H, J ¼ 8.0 Hz), 2.36 (s, 3H, CH3). 13C NMR
4.1.12. 3-Amino-6-chloro-2-p-tolylquinazolin-4(3H)-one (12)
Benzoxazine (2) (2 mmol, 543 mg) was heated under reflux in
hydrazine hydrate (5 ml) for 3 h. The solid obtained was filtered
while hot and dried, mp 208e210 ꢁC in 88% yield.
(DMSO-d6):d161.8,160.7,149.7,140.3,136.0,133.7,130.2,129.7,129.3,
128.4,123.4,114.5, 21.4. MS: (M, 269). Anal. calcd. for C15H12ClN3 (%):
C, 66.79; H, 4.48; N, 15.58. Found: C, 67.02; H, 4.50; N, 15.56.
IR (KBr, cmꢀ1)
d6):
(m, 3H), 7.29 (d, 2H, J ¼ 8.0 Hz), 5.70 (s, 2H, NH2), 2.39 (s, 3H, CH3).
13C NMR (DMSO-d6):
160.7, 156.6, 145.9, 139.9, 134.8, 132.1, 131.2,
n
: 3266, 3121 (NH), 1680 (CO); 1H NMR (DMSO-
4.1.8. N-(6-chloro-2-p-tolylquinazolin-4-yl)hydroxylamine (8)
6-Chloro-2-p-tolylquinazolin-4(3H)-thione (6) (2 mmol, 773 mg)
and hydroxylamine hydrochloride (2.1 mmol, 146 mg) was heated
under reflux for 12 h in pyridine (15 ml). The reaction mixture
was cooled and the solvent was removed under reduced pressure;
the residue was triturated with water and filtered. The solid obtained
was dried and recrystallized from ethanol, mp 227e229 ꢁC in 72%
yield.
d
8.10 (d, 1H, J ¼ 2.5 Hz), 7.84 (dd, 1H, J ¼ 2.5 Hz), 7.74e7.71
d
130.2, 130.1, 128.4, 125.3, 121.6, 21.4. MS: M (285, 60%), M þ 2 (287,
20%). Anal. calcd. for C15H12ClN3O (%): C, 63.05; H, 4.23; N, 14.71.
Found: C, 63.41; H, 4.22; N, 14.77.
4.1.13. 6-Chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-thione (13)
6-Chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-one (2) (5 mmol,
1.36 g) and phosphorous pentasulfide (5.5 mmol, 1.22 g) was
heated under reflux in anhydrous xylene (150 ml) for 12 h. The
reaction mixture was filtered while hot, the solvent was evapo-
rated, and the residue was triturated with dimethylsulfoxide
(10 ml) followed by filtration. The clear filtrate was poured into ice
water and stirred; the solid obtained was filtered, washed with
water and dried, mp 188e190 ꢁC in 93% yield.
IR (KBr, cmꢀ1)
n
: 3429 (NH), 3104 (OH); 1H NMR (DMSO-d6):
d
10.51 (s, 1H, OH), 7.98 (m, 2H), 7.76 (s, 1H, Ar.), 7.53 (d, 1H,
J ¼ 8.0 Hz), 7.44 (d, 1H, J ¼ 8.0 Hz), 7.34 (d, 2H, J ¼ 8.0 Hz), 2.39 (s,
3H, CH3). MS: (M, 285). Anal. calcd. for C15H12ClN3O (%): C, 63.05; H,
4.23; N, 14.71. Found: C, 63.08; H, 4.41; N, 14.47.
4.1.9. 6-Chloro-4-hydrazino-2-p-tolylquinazoline (9)
6-Chloro-2-p-tolylquinazolin-4(3H)-thione(6) (2 mmol, 773 mg)
was heated under reflux in (5 ml) hydrazine hydrate for 3 h The
solid obtained was filtered while hot, dried and recrystallized
from ethanol, mp 273e275 ꢁC in 76% yield.
IR (KBr, cmꢀ1) : 1251 (CS); 1H NMR (DMSO-d6):
n d 7.81 (d, 1H,
J ¼ 8.5), 7.44 (s, 1H), 7.41e7.30 (m, 3H), 6.82e6.75 (m, 2H), 2.11
(s, 3H). MS: M (287, 31%, M þ 2, 289, 10%). Anal. calcd. for
C15H10ClNOS (%): C, 62.61; H, 3.50; N, 4.87. Found: C, 62.53; H, 3.36;
N, 4.88.
1H NMR (DMSO-d6):
d
9.69 (s, 1H), 8.45 (d, 2H, J ¼ 8.0 Hz), 8.32
(s, 1H), 7.74 (d, 2H, J ¼ 11.0 Hz), 7.29 (d, 2H, J ¼ 8.0 Hz), 4.92 (s, 2H),
2.37 (s, 3H). 13C NMR (DMSO-d6):
160.2, 159.5, 148.8, 140.4, 136.0,
d
133.3, 130.1, 129.4, 129.2, 128.6, 122.2, 114.0, 21.5. MS: (M þ 1, 285).
Anal. calcd. for C15H13ClN4 (%): C, 63.27; H, 4.60; N, 19.68. Found: C,
63.27; H, 4.39; N, 19.45.
4.1.14. 3-Amino-6-chloro-2-p-tolylquinazolin-4(3H)-thione (14)
and N-(4-chloro-2-(hydrazinecarbonothioylphenyl)-4-
methylbenzamide (15)
6-Chloro-2-p-tolyl-4H-benzo[d][1,3]oxazin-4-thione
(13)
4.1.10. N-(6-chloro-2-p-tolylquinazolin-4-yl)benzamide (10)
A mixture 6-chloro-2-p-tolylquinazolin-4-amine (7) (2 mmol,
539 mg) and benzoyl chloride (2.1 mmol, 295 mg) in pyridine
(10 ml) was heated under reflux for 12 h. The reaction mixture was
cooled and the solvent was removed under reduced pressure; the
residue was triturated with water and filtered. The solid obtained
was dried and recrystallized from ethanol, mp 191e193 ꢁC in 69%
yield.
(3 mmol, 863 mg) was heated under reflux in hydrazine hydrate
(10 ml) for 5 h. The reaction mixture was cooled and the solvent
was removed under reduced pressure; the solid obtained was dried
and chromatographed with chloroform as fluent.
Compound 14: yield 46%, mp 218e220 ꢁC. IR (KBr, cmꢀ1) n: 3455,
3339 (NH), 1220 (CS); 1H NMR (DMSO-d6):
7.85e7.81 (m,1H), 7.74e7.70 (m, 3H), 7.29 (d, 2H, J ¼ 8.5 Hz), 5.70 (s,
d
8.10 (d, 1H, J ¼ 7.5 Hz),
2H, NH2), 2.38 (s, 3H, CH3). MS: (M, 301). Anal. calcd. for

A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
4197
C15H12ClN3S (%): C, 59.70; H, 4.01; N,13.92. Found: C, 60.04; H, 4.01;
N, 14.21.
for C17H15ClN2OS (%): C, 61.72; H, 4.57; N, 8.47. Found: C, 61.76; H,
4.87; N, 8.74.
Compound 15: yield 43%, mp 140e142 ꢁC. 1H NMR (DMSO-d6):
d
13.11 (s, 1H, SH), 8.71 (s, 1H, NHCO), 7.95 (d, 1H, J ¼ 2 Hz), 7.82 (d,
4.1.16. 4-(Benzylsulfonyl)-6-chloro-2-p-tolylquinazoline (22)
6-Chloro-4-(benzylthio)-2-p-tolylquinazoline (17) (1 mmol,
376 mg) was stirred with KMnO4 (1.5 mmol, 235 mg) in acetic acid
(5 ml) at room temperature for 2 h. The solvent was evaporated
under reduced pressure; the solid obtained was washed with
water, filtered, dried and recrystallized from acetic acid, mp
234e236 ꢁC in quantitative yield.
2H, J ¼ 7.5 Hz), 7.62 (d, 1H, J ¼ 2.0 Hz), 7.34 (s, 2H, NH2), 7.31e7.28
(m, 3H), 2.33 (s, 3H, CH3). MS: (M ꢀ 35) 284. Anal. calcd. for
C15H14ClN3OS (%): C, 56.33; H, 4.41; N, 13.14. Found: C, 56.68; H,
4.26; N, 12.88.
4.1.15. 6-Chloro-4-(substitutedthio)-2-p-tolylquinazolines (16e21)
A
mixture of 6-chloro-2-p-tolylquinazolin-4(3H)-thione (6)
1H NMR (CDCl3):
7.72 (d, 2H, J ¼ 8.0 Hz), 7.41e7.32 (m, 4H), 7.18 (d, 2H, J ¼ 7.5 Hz),
5.48 (s, 2H, CH2Ph), 2.29 (s, 3H, CH3). 13C NMR (CDCl3):
161.4,
d
8.26 (d, 2H, J ¼ 8.5 Hz), 7.92 (d, 2H, J ¼ 8.0 Hz),
(2 mmol, 773 mg) and the appropriate alkyl, allyl, aryl or aralkyl
halides derivatives (2.1 mmol) in pyridine (10 mL) was heated
under reflux for 10e12 h. The reaction mixture was cooled and the
solvent was removed under reduced pressure; the residue was
triturated with water and filtered. The solid obtained was dried and
recrystallized.
d
154.0, 152.6, 147.6, 144.3, 138.9, 136.8, 135.3, 131.6, 129.6, 127.5,
126.6, 133.9, 122.3, 120.0, 117.2, 70.2, 24.3. MS: (M þ 1, 409). Anal.
calcd. for C22H17ClN2O2S (%): C, 64.62; H, 4.19; N, 6.85. Found: C,
64.87; H, 4.60; N, 6.73.
4.1.15.1. 6-Chloro-4-(methylthio)-2-p-tolylquinazoline (16). Yield 86%,
4.1.17. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)substituted
acetamides (23e27)
ꢁ
1
mp 207e209 C (AcOH). H NMR (DMSO-d6):
d
8.30e8.28 (m, 2H),
8.16 (dd, 1H, J ¼ 7.5 Hz), 7.49e7.46 (m, 2H), 7.41e7.40 (m, 2H), 2.35 (s,
A mixture of 6-chloro-2-p-tolylquinazolin-4(3H)-thione (6)
3H, CH3), 2.11 (s, 3H, SCH3).
(2 mmol, 773 mg) and the appropriate chloroacetanilide
(2.1 mmol) in acetone (15 ml) containing anhydrous K2CO3
(2.5 mmol, 345 mg) was heated under reflux for 10e12 h. The
reaction mixture was filtered while hot, the solvent was removed
under reduced pressure; the solid obtained was dried and
recrystallized.
13C NMR (DMSO-d6):
d 161.6, 159.6, 151.9, 148.1, 136.6, 134.9,
131.6, 130.9, 129.7, 127.9, 124.1, 119.9, 21.4, 17.3. MS: (M, 300). Anal.
calcd. for C16H13ClN2S (%): C, 63.89; H, 4.36; N, 9.31. Found: C, 63.88;
H, 4.57; N, 9.60.
4.1.15.2. 4-(Benzylthio)-6-chloro-2-p-tolylquinazoline (17). Yield 95%,
mp 152e154 ꢁC (EtOH).1H NMR (CDCl3):
(m, 4H), 7.52e7.33 (m, 7H), 4.82 (s, 2H, CH2Ph), 2.40 (s, 3H, CH3). MS:
(M, 376). Anal. calcd. for C22H17ClN2S (%): C, 70.11; H, 4.55; N, 7.43.
Found: C, 70.47; H, 4.55; N, 7.12.
d
8.46 (s,1H, Ar.), 8.05e7.98
4.1.17.1. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)acetamide (23).
Yield 90%, mp 266e268 ꢁC (AcOH). IR (KBr, cmꢀ1)
n
: 3366, 3180
(NH), 1639 (CO); 1H NMR (CDCl3):
d 8.02 (s, 1H, Ar.), 7.90 (d, 1H,
J ¼ 8.0 Hz), 7.44e7.38 (m, 4H), 7.30e7.16 (d, 1H, J ¼ 7.5 Hz), 6.39
(s, 1H, NHCO), 4.72 (s, 2H, CH2CO), 2.65 (s, 1H, NHCO), 2.21 (s, 3H,
CH3). 13C NMR (CDCl3): 24.0, 70.1, 119.2, 120.9, 122.2, 127.0, 127.5,
129.8, 131.6, 135.5, 136.5, 151.7, 154.5, 161.1, 172.6. MS: (M, 343).
Anal. calcd. for C17H14ClN3OS (%): C, 59.38; H, 4.10; N, 12.22. Found:
C, 59.00; H, 4.47; N, 12.46.
4.1.15.3. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)acetonitrile (18).
Yield 92%, mp 196e198 ꢁC (EtOH). 1H NMR (DMSO-d6):
d 8.41 (d,1H,
J ¼ 8.0 Hz), 8.17e8.07 (m,1H), 8.00e7.95 (m, 2H), 7.73e7.55 (m,1H),
7.42e7.25 (m, 2H), 5.62 (s, 2H, CH2CN), 2.36 (s, 3H, CH3). 13C NMR
(DMSO-d6):
d 164.2, 159.4, 150.6, 141.8, `135.7, 130.3, 129.8, 128.7,
128.2, 125.3, 122.4, 116.6, 115.0, 52.2, 21.5. MS: (M-2, 323). Anal.
calcd. for C17H12ClN3S (%): C, 62.67; H, 3.71; N, 12.90. Found: C,
62.41; H, 4.10; N, 12.13.
4.1.17.2. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)phenylꢀac1)etamide
(24). Yield 88%, mp 277e279 ꢁC (AcOH). IR (KBr, cm
n: 3260
(NH), 1656 (CO); 1H NMR (CDCl3):
d 9.74 (s, 1H, exchangeable,
NHCO), 7.95 (d, 1H, J ¼ 7.0 Hz), 7.61e7.38 (m, 7H), 7.34 (s, 1H, Ar.),
4.1.15.4. 4-(Allylthio)-6-chloro-2-p-tolylquinazoline (19). Yield 88%,
7.03e6.89 (m, 3H), 4.81 (s, 2H, CH2CO), 2.23 (s, 3H, CH3). 13C NMR
mp 147e149 ꢁC (AcOH). 1H NMR (DMSO-d6):
d
8.00 (d, 1H,
(CDCl3): d 24.3, 70.1, 115.5, 115.7, 120.1, 121.5, 122.3, 126.6, 127.8,
J ¼ 1.0 Hz), 7.52 (d, 2H, J ¼ 8.5), 7.41e7.36 (m, 3H), 7.17e7.15 (m, 2H),
6.87 (d, 2H, J ¼ 8.5 Hz), 4.81 (s, 2H), 2.36 (s, 3H, CH3).
MS: (326, 4.3%). Anal. calcd. for C18H15ClN2S (%): C, 66.15; H,
4.63; N, 8.57. Found: C, 66.15; H, 4.45; N, 8.59.
129.8, 131.8, 136.5, 139.1, 152.5, 154.8, 158.6, 160.3, 161.0, 165.8. MS:
(M þ 1, 420). Anal. calcd. for C23H18ClN3OS (%): C, 65.78; H, 4.32; N,
10.01. Found: C, 65.41; H, 4.49; N, 10.06.
4.1.17.3. Ethyl(6-chloro-2-p-tolylquinazolin-4-ylthio)ethyl-4-(2-acet-
4.1.15.5. 6-Chloro-4-(2-nitropyridylthio)-2-p-tolylquinazoline (20).
amido)benzoate (25). Yield 88%, mp 242e244 ꢁC (EtOH). 1H NMR
Yield 90%, mp 214e216 ꢁC (AcOH). 1H NMR (CDCl3):
d
8.19 (d, 1H,
(CDCl3):
d
9.84 (s, 1H, exchangeable, NHCO), 8.00 (d, 1H, J ¼ 7.5 Hz),
J ¼ 7.5 Hz), 7.97 (d, 2H, J ¼ 7.0 Hz), 7.77 (d, 1H, J ¼ 7.0 Hz), 7.72e7.70
7.52 (d, 2H, J ¼ 8.5 Hz), 7.46e7.37 (m, 5H), 7.16 (d, 1H, J ¼ 7.5 Hz),
6.86 (d, 2H, 8.5), 4.83 (s, 2H, CH2CO), 4.01 (q, 2H, J ¼ 7.0 Hz), 2.23 (s,
(m, 1H), 7.51e7.73 (m, 5H), 7.07 (d, 1H, J ¼ 7.0 Hz), 2.30 (s, 3H, CH3).
13C NMR (CDCl3):
d
160.6, 154.9, 143.9, 141.1, 136.4, 135.1, 133.9,
3H, CH3), 1.39 (t, 3H, J ¼ 7.0 Hz). 13C NMR (CDCl3):
d 14.81, 24.1, 63.7,
131.6,129.7,129.5,129.0,128.7,128.0,127.7,127.6,126.3,126.1,122.5,
23.8. MS: (M, 408). Anal. calcd. for C20H13ClN4O2S (%): C, 58.75; H,
3.20; N, 13.70. Found: C, 58.64; H, 3.56; N, 13.74.
71.1, 114.8, 121.2, 122.2, 122.3, 127.1, 127.8, 129.8, 130.4, 131.6, 135.2,
136.6, 139.1, 152.9, 154.7, 156.1, 161.0, 168.5. MS: (M ꢀ 1, 462). Anal.
calcd. for C25H22ClN3O2S (%): C, 64.72; H, 4.78; N, 9.06. Found: C,
64.78; H, 4.88; N, 9.06.
4.1.15.6. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)ethanol
Yield 80%, mp 286e288 ꢁC (AcOH). IR (KBr, cmꢀ1) n: 3179 (OH); 1H
NMR (DMSO-d6):
8.11(s, 1H), 8.04 (d, 1H, J ¼ 7.5 Hz), 7.82 (d, 2H,
(21).
4.1.17.4. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)-p-tolyacetamide
d
(26). Yield 84%, mp 262e264 ꢁC (AcOH). 1H NMR (CDCl3):
d 9.71 (s,
J ¼ 7.5 Hz), 7.68 (d, 1H, J ¼ 8.5 Hz), 7.38e7.27 (m, 2H), 4.07 (s, 1H,
1H, exchangeable, NHCO), 8.02 (d,1H, J ¼ 7.0 Hz), 7.51e7.40 (m, 7H),
7.19e7.14 (m, 3H), 4.86 (s, 2H, CH2CO), 2.33 (s, 3H, CH3), 2.26 (s, 3H,
CH3). MS: (M, 433). Anal. calcd. for C24H20ClN3OS (%): C, 66.43; H,
4.65; N, 9.68. Found: C, 66.56; H, 4.90; N, 9.41.
OH), 3.50 (t, 2H, J ¼ 5 Hz), 3.27 (t, 2H, J ¼ 5 Hz), 2.29 (s, 3H, CH3). 13
C
NMR (DMSO-d6): d 166.6, 1601, 59.6, 151.9, 148.0, 136.6, 134.9, 131.8,
129.7, 127.9, 124.1, 119.9, 60.3, 41.7, 21.5. MS: (M, 330). Anal. calcd.

4198
A.S. El-Azab et al. / European Journal of Medicinal Chemistry 45 (2010) 4188e4198
4.1.17.5. 2-(6-Chloro-2-p-tolylquinazolin-4-ylthio)-p-flor-
geometry of resulting complexes was studied using the MOE’s Pose
Viewer utility.
ophenylacetamide (27). Yield 90%, mp 266e228 ꢁC (AcOH). 1H NMR
(CDCl3):
d
9.91 (s, 1H, NHCO), 8.01 (d, 1H, J ¼ 7.0 Hz), 7.61e7.58
(m, 2H), 7.49e7.38 (m, 5H), 7.16 (d, 1H, J ¼ 7.5 Hz), 7.04e7.00
Acknowledgments
(m, 2H), 4.85 (s, 2H, CH2CO), 2.35 (s, 3H, CH3). 13C NMR (CDCl3):
d
166.8, 161.0, 158.7, 154.8, 152.8, 139.2, 136.5, 135.2, 133.4, 131.7,
The authors would like to express their gratitude and thanks to
the National Cancer Institute, Cairo, Egypt for doing the antitumor
testing of the new compounds. Our sincere acknowledgments to
Chemical Computing Group Inc, 1010 Sherbrooke Street West, Suite
910, Montreal, H3A 2R7, Canada, for its valuable agreement to use
the package of MOE 2008.10 software.
129.8, 127.8, 127.1, 122.3, 121.4, 120.3, 115.8, 71.1, 24.2. MS: (M, 437).
Anal. calcd. for C23H17ClFN3OS (%): C, 63.08; H, 3.91; N, 9.60. Found:
C, 63.41; H, 4.17; N, 9.29.
4.2. Biological evaluation
References
4.2.1. Materials
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tissue culture plasticware were purchased from Costar (Milan,
Italy). All other chemicals were obtained from Sigma Chemical
Company (St. Louis, MO, USA).
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Initial molecular structures of the most active compounds 5e7,
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4.3.2. Molecular docking methodology
Docking studies have been performed using MOE 2008.10. With
this purpose, crystal structure of Erlotinib (TarcevaÔ) was obtained
from Protein Data Bank (PDB codes: 1M17) in order to prepare
protein for docking studies. Docking procedure was followed using
the standard protocol implemented in MOE 2008.10 and the