Design and Synthesis of some new 2,4,6-trisubstituted quinazoline EGFR inhibitors as targeted anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2020.103726

The present study describes the synthesis of 6-bromo-2-(pyridin-3-yl)-4-substituted quinazolines starting from 4-chloro derivative VI via the reaction with either phenolic compounds to obtain VIIa-f, IXa-d, 2-amino-6-(un)substituted benzothiazole to produce VIIIa-c or hydrazine hydrate to give X. Reaction of the hydrazino functionality of X with appropriate acid anhydride, acid chloride or aldehyde affords XIa-c, XIIa-c and XIVa-i, respectively. The target compounds were screened for their efficacy as EGFR inhibitors compared to gefitinib. Compounds eliciting superior EGFR inhibitory activity were further screened for their in vitro cytotoxicity against two human cancer cell lines namely: MCF7 (breast) and A549 (lung), in addition to normal fibroblast cell WI38 relative to gefitinib as a reference. Furthermore, compounds that showed potent inhibitory activity on wild-type EGFR were screened against mutant EGFR and assayed for their cytotoxicity against mutant EGFR-expressing cell lines PC9 and HCC827. The unsubstituted benzothiazol-2-amine VIIa showing superior EGFR inhibition (IC₅₀ = 0.096 μM) and anticancer activity against MCF-7 cell line (IC₅₀ = 2.49 μM) was subjected to cell cycle analysis and apoptotic assay. Moreover, a molecular docking study was performed to investigate the interaction of some representive compounds with the active site of EGFR- TK.

Full text of DOI:10.1016/j.bioorg.2020.103726

Journal Pre-proofs
Design and Synthesis of some new 2,4,6-trisubstituted quinazoline EGFR in-
hibitors as targeted anticancer agents
Heba Abdelrasheed Allam, Enayat E. Aly, Ahmed K.B.A.W. Farouk, Ahmed
M. El Kerdawy, Essam Rashwan, Safinaz E.S. Abbass
PII:
S0045-2068(20)30106-1
DOI:
https://doi.org/10.1016/j.bioorg.2020.103726
YBIOO 103726
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
14 January 2020
20 February 2020
4 March 2020
Please cite this article as: H. Abdelrasheed Allam, E.E. Aly, A.K.B. Farouk, A.M. El Kerdawy, E. Rashwan,
S.E.S. Abbass, Design and Synthesis of some new 2,4,6-trisubstituted quinazoline EGFR inhibitors as targeted
anticancer agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103726
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Design and Synthesis of some new 2,4,6-trisubstituted quinazoline EGFR
inhibitors as targeted anticancer agents
1
1
1
Heba Abdelrasheed Allam , Enayat E. Aly , Ahmed K. B. A. W. Farouk , Ahmed M. El
1,2
3
1
Kerdawy , Essam Rashwan , Safinaz E. S. Abbass
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box, 11562, Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, Newgiza,
km 22 Cairo–Alexandria Desert Road, Cairo, Egypt
3
Head of Confirmatory Diagnostic Unit, VACSERA, Cairo, Egypt
*Corresponding author: Heba Abdelrasheed Allam
E-mail: hebaallam80@hotmail.com ; Tel: +201006377655.

Design and Synthesis of some new 2,4,6-trisubstituted quinazoline EGFR
inhibitors as targeted anticancer agents
1
1
1
Heba Abdelrasheed Allam , Enayat E. Aly , Ahmed K. B. A. W. Farouk , Ahmed M. El
1,2
3
1
Kerdawy , Essam Rashwan , Safinaz E. S. Abbass
1
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Cairo University, Kasr El-Aini
Street, Cairo, P.O. Box, 11562, Egypt
2
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, New Giza University, Newgiza,
km 22 Cairo–Alexandria Desert Road, Cairo, Egypt
3
Confirmatory Diagnostic Unit, VACSERA, Cairo, Egypt
Abstract
The present study describes the synthesis of 6-bromo-2-(pyridin-3-yl)-4-substituted
quinazolines starting from 4-chloro derivative VI via the reaction with either phenolic
compounds to obtain VIIa-f, IXa-d, 2-amino-6-(un)substituted benzothiazole to produce
VIIIa-c or hydrazine hydrate to give X. Reaction of the hydrazino functionality of X with
appropriate acid anhydride, acid chloride or aldehyde affords XIa-c, XIIa-c and XIVa-i,
respectively. The target compounds were screened for their efficacy as EGFR inhibitors
compared to gefitinib. Compounds eliciting superior EGFR inhibitory activity were further
screened for their in vitro cytotoxicity against two human cancer cell lines namely: MCF7
(breast) and A549 (lung), in addition to normal fibroblast cell WI38 relative to gefitinib
as a reference. Furthermore, compounds that showed potent inhibitory activity on wild-type
EGFR were screened against mutant EGFR and assayed for their cytotoxicity against
mutant EGFR-expressing cell lines PC9 and HCC827. The unsubstituted benzothiazol-
2-amine VIIa showing superior EGFR inhibition (IC50 = 0.096 µM) and anticancer
activity against MCF-7 cell line (IC50 = 2.49 µM) was subjected to cell cycle analysis
and apoptotic assay. Moreover, a molecular docking study was performed to investigate
the interaction of some representive compounds with the active site of EGFR- TK.
Keywords: Quinazoline, EGFR, MCF-7, A549, WI38, Apoptosis
*Corresponding author: Heba Abdelrasheed Allam
E-mail: hebaallam80@hotmail.com ; Tel: +201006377655.

1
. Introduction
Cancer is a malignant life-threatening disease which stands next to cardiovascular
diseases in terms of morbidity and mortality and is projected to be the primary cause of
death worldwide in the future.[1] The discovery of effective safe new anticancer agent is
still a serious research field due to the side effects of the conventional non-selective
cytotoxic chemotherapies, their systemic toxicity and resistance.[2,3] Targeted therapies
selectively aim cancer cells or the tumor microenvironment that supports cancer growth
with higher effectiveness and slight off-target side effects on normal cells.[3] Targeted
chemotherapies specifically attack signaling pathways regulating tumor cell cycle and its
microenvironment prompting cell apoptosis, delaying tumor cell proliferation and/or
obstructing tumor mass growth.[4,5] Epidermal growth factor receptor (EGFR) signaling
pathway has been extensively investigated for its significant role in the progression of
different types of malignant tumors, where development of small molecules targeting
EGFR is a well-known strategy for design of antitumor agents.[6] Many reports referred
to the pivotal role of quinazoline derivatives as selective and potent EGFR inhibitors
along with their remarkable antitumor activity.[7–9] Reviewing the literature revealed
that
chloroanilino)quinazolines 1 and 2 exert a significant EGFR inhibition.[10,11] Also, the
disubstituted quinazolines: 4-(7-amino-2-aryl)-2-(4-fluorophenyl)-5-
certain
monosubstituted
quinazolines,
namely:
4-(3-bromo
/
bromoindolequinazolines 3a,b show good cytotoxicity against A549, MCF-7 and HeLa
cell lines, in addition to their nanomolar EGFR inhibitory activity.[12] Furthermore,
2,4,6-trisubstituted quinazoline 4, displays a significant anticancer activity against
different subpanel tumor cell lines with GI50 value 16.9 µM.[13] (Figure 1)
Accordingly, we design a series of novel 2,4,6-trisubstituted quinazoline
derivative by keeping the disubstituted 4-amino quinazoline scaffold in 3a,b and
bioisosteric replacement of their indolyl and 2-phenyl moieties with benzothiazolyl and
pyridin-3-yl ones, respectively. In addition, introduction of bromo group at position 6 is
carried out to obtain compounds VIIa-c. Moreover, guided by the significant anticancer
activity of the trisubstituted quinazoline derivative 4 compounds XIa-c were designed via
retaining NH linker at position 4 and replacing of methyl benzene sulfonamide moiety by
amido moieties, bioisosteric replacement of thien-2-yl with pyridin-3-yl and substitution

of 6-iodo with 6-bromo aiming to obtain new derivatives with promising anticancer and
EGFR inhibitory activities. Further modification includes bioisosteric substitution of NH
at position 4 with O to give VIIIa-f in order to inspect the effect of this substitution on
the designed biological activity. The extended side chain of lapatinib 5 at position 4
directed our interest to synthesize series IXa-d hoping to improve the anticancer and
EGFR inhibitory activities of the newly prepared quinazolines.
Another approach deals with NH spacer elongation at position 4 to NH NH CO
and NH N=CH to produce 4-substituted benzohydrazides XIIa-c and 4-substituted
arylideneaminoquinazolines XIVa-i.

Br
N
HN
N
HN
Cl
HN
Br
HN
N
N
R
N
N
3
a,b
1
2
F
SO2NH2
R
S
N
R
HN
HN
N
N
HN
Br
Br
I
N
N
N
N
S
N
N
XIa-c
VIIa-c
4
O
F
R
O
N
N
HN
Cl
Br
O S
O
N
O
N
N
5
VIIIa-f
Lapatinib
R
H
N
O
N
NH
O
N
N
R
HN
N
Br
Br
N
N
N
IXa-d
XIIa-c
R
N
N
HN
N
Br
N
XIVa-i
Fig. 1: Reported quinazoline anticancer agents, and the scaffold of the target compounds

2
. Results and discussion
2
.1. Chemistry
The synthetic pathways implemented for the synthesis of the intermediates and
target quinazolines are shown in Schemes 1 - 4. Reaction of 2-aminobenzoic acid with
Br in refluxing glacial acetic acid afforded 5-bromoanthranilic acid which upon reaction
2
with freshly prepared nicotinoyl chloride gave the amidated intermediate III. Reflux of
III with acetic anhydride produced the benzoxazinone IV. Heating of IV under reflux
with formamide resulted in quinazolinone V, which upon reflux with POCl afforded 4-
3
chloro derivative VI. Reaction of VI under reflux with the appropriate 2-amino-6-
un/substituted benzothiazoles in dimethylformamide and in presence of anhydrous
potassium carbonate produced VIIa-c, while reaction of VI at room temperature with the
appropriate phenolic compounds in dimethylformamide and in presence of anhydrous
potassium carbonate gave VIIIa-f. Scheme 1. IR spectra of compounds VIIa-c showed
-1 1
the appearance of NH bands at 3394 – 3369 cm . H-NMR spectra of VIIa, VIIb and
VIIc exhibited D O exchangeable singlet signals at 8.76, 8.68 and 8.67 ppm,
respectively, corresponding to NH proton. In addition, a (triplet quartet) pattern was
2
1
3
observed at 1.38 and 4.00 ppm for compound VIIb corresponding to ethoxy protons. C-
NMR spectrum of VIIb revealed two aliphatic signals at 15.19 and 64.04 attributed to
CH
3
and CH
2
of ethyl carbons, respectively. IR spectrum of VIIId revealed three strong
-1
bands at 2819, 2792 and 1693 cm , assignable for the aldehydic H and C=O groups,
-1
respectively, while that of VIIIf elicited two strong bands at 3251 and 1670 cm , attributed
1
to the NH and C=O groups of respectively. H-NMR spectra revealed the presence
of a singlet signal at 2.4 ppm integrated for the three protons of the methyl group of
VIIIb, a singlet signal at 10.09 ppm due to the proton of the aldehydic group of VIIId
and a singlet signal at 2.1 ppm and a D
2
O exchangeable singlet signal at 10.11 ppm
integrated for the methyl group and NH protons of VIIIf, respectively.

Br
COOH
NH
Cl
O
N
COOH
NH2
NH2
ii
i
+
Br
COOH
O
N
III
2
-Aminobenzoic acid
I
II
acid)
(Anthranilic
iii
O
O
Br
iv
Br
O
NH
N
N
N
N
V
IV
v
Cl
Br
N
N
N
VI
vi
vii
R
R
S
N
O
N
HN
N
Br
N
Br
VIII
R
N
N
VIIa-c
N
VIIIa-f
a
H
b
c
CH3
F
VII
R
H
a
d
e
f
CHO
NO2
b
c
OC H5
2
NO2
NHCOCH3
Scheme 1: Synthesis of the intermediates I, II, III, IV, V, VI and the target compounds
VIIa-c and VIIIa-f; Reagents and conditions: (i) Br , glacial acetic acid, ice bath; (ii)
Methylene chloride, triethylamine, 24 h RT; (iii) Acetic anhydride, reflux 4.5 h; (iv)
Formamide, reflux 2.5 h; (v) POCl , reflux 2.5 h; (vi) appropriate 2-amino benzothiazole
derivatives, DMF, anhyd. K CO , reflux 9 h; (vii) (un)substituted phenol derivatives,
DMF, anhyd. K CO , stirr on cold 24 h.
2
3
2
3
2
3

Reaction of compound VIIId with the appropriate un/substituted phenyl hydrazines in
absolute ethanol and in presence of few drops of glacial acetic acid furnished the target
compounds IXa-d. Scheme 2. Structures assigned to the products were deduced from
concordant microelemental and spectral analyses. IR spectra of compounds IXa-d
-1
showed presence of a sharp band corresponding to NH groups at 3221 – 3313 cm . IR
-1
spectrum of compound IXd revealed a forked band at 3421 and 3331 cm corresponding
-1
to NH
2
group, in addition to two bands attributed to SO group at 1319 and 1141 cm .
2
1H-NMR spectra of compounds IXa-d showed an increase in the integration of the
aromatic protons indicating the presence of additional aromatic ring, in addition to the
absence of singlet signal of the aldehydic proton along with the appearance of a sharp
singlet signal around 7.91 – 8.05 ppm corresponding to azomethine proton together with
the appearance of a D
2
O exchangeable singlet signal around 10.30 – 10.90 ppm
corresponding to NH proton. Compound IXc, displayed a singlet signal at 3.70 ppm
attributed to OCH
3
protons. NH protons of compound IXd appeared as a singlet signal at
2
7
.09 ppm which disappeared upon deuterium exchange.
H
N
CHO
N
O
N
i
O
R
Br
Br
N
N
N
N
N
VIIId
IXa-d
I
R
X
H
a
Cl
b
c
OCH3
SO NH
2
2
d
Scheme 2: Synthesis of the target compounds IXa-d from the target compound VIIId;
Reagent and condition: (i) absolute ethanol, drops of glacial acetic acid,
phenylhydrazine/4-hydrazinylbenzenesulfonamide,
reflux
9.5-15
h.
4-
chlorophenylhydrazine/4-methoxyphenylhydrazine, stirr on cold 24 h.

Additionally, 4-hydrazino derivative X was produced by reflux of quinazolinone
VI with hydrazine hydrate. Heating of X under reflux with appropriate acid anhydride
gave the corresponding compounds XIa-c, whereas treatment of X with appropriate p-
substituted benzoyl chlorides in methylene chloride and in presence of trimethylamine at
room temperature resulted in compounds XIIa-c as shown in Scheme 3. The structure X
was supported by analytical and spectral data. IR spectrum displayed the appearance of
the characteristic stretching vibrations of the hydrazinyl moiety at 3300, 3294 and 3209
-1
1
cm . H-NMR spectrum showed two singlet signals at 5.02 and 9.86 ppm, which
disappeared on deuteration, corresponding to the NH and NH, respectively. IR spectra of
compounds XIa-c showed two bands attributed to the two carbonyl groups at 1732 –
2
-
1 1
1
714 cm . H-NMR spectra of XIa-c lacked the NH
2
protons signal at 5.02 ppm along
-CH -
with the appearance of aliphatic signal at 3.03 ppm corresponding to the -CH
2
2
protons of XIa, in addition to an increase in the integration of aromatic protons of
compound XIc, while XIb spectrum indicated the presence of a singlet signal at 2.45
13
ppm corresponding to the methyl protons of the diacetyl moiety. C-NMR spectra of
XIa,b,c showed a signal at 185.20, 172.49 and 166.42 ppm attributed to the two additional
equivalent carbonyl groups, respectively. IR spectra of XIIa-c displayed the
disappearance of the characteristic stretching vibrations of the NH moiety of X and
2
-1
revealed the appearance of new band at 1643, 1645 and 1658 cm related to the new
1
carbonyl group of the benzohydrazides; XIIa, XIIb and XIIc, respectively. H-NMR
spectra of XIIa-c lacked the NH
2
protons signal at 5.02 ppm and showed D O
2
exchangeable protons at 10.68 - 11.13 ppm corresponding to the NH protons, in addition
to an increase in the aromatic integration. Compound XIIb revealed an extra aliphatic
singlet signal at 3.88 ppm related to the CH protons.
3

NH2
N
Cl
N
HN
N
Br
Br
N
i
N
N
VI
X
i
iii
R
R
N
O
HN
N
Br
NH
N
HN
N
Br
XIa-c
XI
R
N
N
O
XIIa-c
a
N
O
XII
R
a
b
c
O
CN
b
c
N
OCH3
Cl
O
O
N
O
Scheme 3: Synthesis of the new intermediate compound X, the target compounds XIa-c
and XIIa-c; Reagents and conditions: (i) NH NH 99%, reflux 8.5 h; (ii) acid anhydride,
2
2
glacial acetic acid, reflux 4-13 h; (iii) appropriate 4-substitutedbenzoylchloride, methylene
chloride, triethylamine, stirr on cold 10-72 h.
Finally, the target compounds XIVa-i were gained via condensation of 4-
hydrazino derivative X with appropriate aldehydes in absolute ethanol under reflux as
displayed in Scheme 4. The expected structures of compounds XIVa-i were confirmed
by IR spectra that displayed the presence of the characteristic stretching vibration of NH
-1 1
group in the range of 3448 - 3394 cm . H-NMR spectra of compounds XIVa-i revealed
an increase in the integration of the aromatic protons indicating the presence of additional

aromatic ring, in addition to the appearance of a singlet signal around 11.70 - 12.20 ppm
corresponding to the NH proton which disappeared upon deuterium exchange.
Additionally, compound XIVc showed a singlet signal at 2.70 ppm corresponding to
OCH
.77 ppm. Compound XIVh displayed a singlet signal at 2.23 ppm assigned to CH
piperazinyl protons along with two triplet signals at 2.46 and 3.28 ppm of the piperazinyl
3
protons. The morpholinyl protons of XIVg appeared as two triplets at 3.25 and
3
3
13
protons. C-NMR spectra of XIVc showed a signal at 55.84 ppm attributed to the OCH
3
carbon.
i
XIII
a
R
HN
R
CHO
F
CHO +
N
b
c
d
N
O
N
4
-fluorobenzaldehyde
appropriate 2ry amine
XIIIa-d
N
N
N
R
XIV
R
NH2
HN
N
N
HN
N
Br
ii
N
Br
a
b
N
N
N
c
OCH3
Cl
X
XIVa-i
d
e
NO2
N
f
g
N
O
h
i
N
N
N
N
Scheme 4: Synthesis of reported aldehydic reagents XIIIa-d and the target compounds
XIVa-i; Reagents and conditions: (i) DMF, anhyd. K
2
CO , reflux 6-7 h; (ii) appropriate
3
aldehyde, absolute ethanol, reflux 10-17 h.

2
.2. Biological evaluation
All the newly synthesized compounds were subjected to EGFR-TK inhibitory
assay and most active ones were screened for their in vitro cytotoxicity against two
cancer cell lines, namely; the non-small cell lung cancer cell line A549 and breast cancer
cell line MCF-7, in addition to normal fibroblast cell line WI38. Compounds showed
promising inhibitory activity against wild-type EGFR were evaluated for their inhibitory
activity on mutant EGFR and their in vitro cytotoxicity against two cancer cell lines
expressing mutant EGFR, namely, PC9 and HCC827. Furthermore, a representative
compound eliciting superior EGFR inhibition was subjected to cell cycle analysis and
apoptotic assay to investigate its effect on the cell cycle progression and the apoptosis
percentage induced by this compound.
2
.2.1.EGFR - TK inhibitory assay:
All the newly target compounds VIIa-c, VIIIa-f, IXa-d, XIa-c, XIIa-c and XIVa-i
were inspected in vitro for their EGFR inhibitory activity. Table 1 shows the inhibition
data (IC50 values) of the examined compounds and Gefitinib, as a reference standard,
towards EGFR. As presented in Table 1, all the tested compounds showed EGFR
inhibitory activity with IC50 values ranging from sub micromolar to single-digit
micromolar concentration (0.096 – 2.962 µM).
The obtained results showed that twenty-four compounds out of twenty-eight
exhibited superior activities at sub-micromolar level (IC50= 0.096 - 0.818 µM) as EGFR
inhibitors compared to Gefitinib (IC50= 0.166 µM). Three compounds: 4-(6-
unsubstituted benzothiazole) VIIa, 4-acetamido phenoxy VIIIf and 4-nitrobenzylidene
XIVe quinazoline derivatives demonstrated comparable enzyme inhibitory activity (IC50
=
0
.096, 0.149 and 0.141 µM, respectively) to that expressed by Gefitinib (IC50= 0.166
µM). The spacer variation at position 4 with NH, O, NH-N=CH or NH-NH-C=O gave
compounds with promising EGFR inhibitory activity at sub-micromolar level. This
indicated that alteration of the spacer does not greatly affect the EGFR inhibition. These
three compounds were further tested against mutant EGFR kinases (T790M and L858R)
and their results are shown in Table 2. The results showed that compound VIIa and

XIVe have comparable activity to Gefitinib against the mutated type T790M, and
compound VIIIf was comparable to Gefitinib against the mutated type L858R.
Table 1: EGFR inhibitory activity of the target compounds compared to Gefitinib as a
reference standard.
Cpd.
No.
EGFR
Cpd.
No.
EGFR
IC50
Cpd. No.
EGFR
IC50
IC50
(µM)
(µM)
(µM)
VIIa
VIIb
VIIc
0.096 ± 0.00278
0.454 ± 0.01309
0.339 ± 0.01303
0.483 ± 0.01391
0.818 ± 0.02356
1.983 ± 0.07618
0.728 ± 0.0279
0.660 ± 0.01902
0.149 ± 0.00429
IXb
IXc
0.431±0.01241
0.303±0.00874
0.231±0.0087
XIVb
XIVc
XIVd
XIVe
XIVf
XIVg
XIVh
XIVi
0.258 ± 0.00992
0.573 ± 0.01651
0.296 ± 0.01138
0.141 ± 0.00408
0.183 ± 0.00527
0.289 ± 0.01111
1.246 ± 0.04785
0.282 ± 0.00812
IXd
XIa
XIb
XIc
VIIIa
VIIIb
VIIIc
VIIId
VIIIe
VIIIf
IXa
1.748 ± 0.06714
0.461 ± 0.01771
0.331 ± 0.01274
0.303 ± 0.00874
0.292 ± 0.00843
XIIa
XIIb
XIIc
0.286 ± 0.01099 Gefitinib 0.166 ± 0.00638
2.962 ± 0.11374 XIVa 0.796 ± 0.03058
Table 2: Mutant EGFR inhibitory activity of the target compounds compared to
Gefitinib as a reference standard.
IC50 (uM)
Cpd. No.
EGFRL858R
EGFRT790M
VIIa
0
0
0
.028±0.00069
.048±0.00118
.025±0.00061
0.055±0.00136
0.012±0.00029
0.042±0.00103
0.018±0.00045
VIIIf
XIVe
Gefitinib
0.023±0.00057

2
.2.2. In vitro cytotoxic activity:
After being investigated for their EGFR inhibitory activity, Fifteen compounds
VIIa,c, VIIIf, IXc,d, XIc, XIIa-c and XIVb,d-g,i out of the twenty eight newly
synthesized compounds that elicited superior to good EGFR inhibitory activity (0.096-
0
.339 µM) were selected to be evaluated for their in vitro cytotoxicity against two
EGFR-overexpressing cell lines, viz, the non-small cell lung carcinoma cell line A549
which is well-known to overexpress wild-type EGFR [14,15] and the breast cancer cell
line MCF-7 which is well-known to overexpress various growth factor receptors
including wild-type EGFR [16–21] to demonstrate their cellular efficacy. Furthermore,
they were evaluated using normal fibroblast cells WI38 for their cytotoxicity to evaluate
the compounds’ selectivity towards tumor cells. Additionally, three quinazoline
derivatives VIIa, VIIIf and XIVe that revealed comparable enzyme inhibitory activity
(IC50= 0.096, 0.149 and 0.141 µM, respectively) to that expressed by Gefitinib (IC50=
0
.166 µM) were selected to be evaluated using mutant EGFR-expressing cell lines
including PC9 and HCC827 (both cell lines carry a Glu746-Ala750 deletion mutation in
exon 19) using MTT assay [22]. In this in vitro testing, Gefitinib was used as a reference
standard. The in vitro cytotoxic data of the tested compounds are shown in Table 3.
Table 3: Cytotoxicity of the tested compounds and Gefitinib against A549, MCF-7,
WI38, PC9 and HCC827 cell lines and the selectivity index (SI*) for the tested compounds
and Gefitinib relative to normal cell line.
Selectivity index
IC50 (uM)
(SI)
Cpd. No.
A549
MCF-7
WI38
PC9
HCC827
A549
MCF-7
1
78.34 ±
1.05±0.02
3.43±0.066
VIIa
VIIc
VIIIf
IXc
2.49 ± 0.12 82.8 ± 4.14
0.464
33.253
84.131
3.033
8
.9
2
4.55 ±
.22
3.195 ±
0.15
268.8 ±
11.6
NA
NA
10.949
1.979
7.158
1
2
9.16 ±
.45
19.03 ±
0.95
57.72 ±
2.88
4.02±0.077 1.21±0.023
1
4
5.53 ±
.28
NA
NA
6.36 ± 0.21 1.89 ± 0.03
24.089
1

5
.774 ±
.28
9.996 ±
0.44
56.80 ±
2.71
NA
NA
NA
NA
IXd
XIc
9.837
1.909
1.498
1.995
3.478
4.393
0.435
18.480
5.139
12.590
8.469
7.963
5.682
5.310
7.395
2.614
14.529
3.384
28.964
3.158
1.119
1.543
2.484
7.029
0
2
8.206 ±
.41
10.142 ±
0.46
53.86 ± 2.6
1
3
3.961 ±
.69
6.881 ±
0.34
50.89 ±
2.21
NA
NA
XIIa
1
1
0.77 ±
.52
21.49 ±
0.92
NA
NA
XIIb
8.22 ± 0.17
0
1
0.514 ±
.52
2.517 ±
0.12
36.57 ±
1.61
NA
NA
XIIc
0
9
.151 ±
.45
11.879 ±
0.51
40.20 ±
1.84
NA
NA
XIVb
XIVd
XIVe
XIVf
XIVg
XIVi
Gefitinib
0
6
3.572 ±
.17
0.956±
0.04
27.69 ±
1.33
NA
NA
3
2
0.48 ±
.02
64.68 ±
3.23
3.66±0.071
NA
5.49±0.11
NA
3.50 ± 0.17
1
1
5
2.31 ±
.61
56.50 ±
2.82
63.26 ±
3.16
0
.585 ±
.27
45.559 ±
2.14
70.32 ±
3.34
NA
NA
0
2
0.92 ±
.77
NA
NA
2.47 ± 0.06 8.42 ± 0.24
0
4
.389 ±
.21
4.972 ±
0.24
34.95 ±
1.72
0
1.36±0.02
3.99±0.07
SI* = activity of the tested compounds (IC50) against normal cell line (WI38)/activity of
the tested compounds (IC50) against cancer cell line.
NA = not determined
The results presented in Table 3 revealed that, most of the tested compounds showed a
selective anticancer activity against MCF-7 rather than A549 cell line. Two compounds
XIVe and XIVi elicited promising anticancer activity against A549 with IC50 of 3.50 and
2
.47 µM, respectively, relative to Gefitinib (IC50= 4.39 µM). Five compounds VIIa,
VIIc, IXc, XIIc and XIVd displayed a superior anticancer activity against MCF-7 with
IC50 of 0.096 – 3.19 µM) compared to Gefitinib (IC50= 4.97 μM). 4-(4-Phenylpiperazin-
1-yl)benzylidenehydrazinyl XIVi was the most potent quinazoline derivative against

A549 lung carcinoma cell line with IC50 of 2.47 µM, whereas, the 4-
chlorobenzylidenehydrazinyl XIVd congener was the most active compound against
MCF-7 with IC50 of 0.956 μM. Twelve compounds VIIa, VIIc, VIIIf, IXc, IXd, XIc,
XIIa, XIIc, XIVb, XIVe, XIVf and XIVg out of fifteen displayed low cytotoxicity
against the normal fibroblast cells (WI38) with good selectivity index.
Among the selected compounds for evaluation against EGFR-dependent cell lines PC9
and HCC827, compound VIIa was better than Gefitinib against the two cell lines.
Whereas, Compound VIIIf was superior than Gefitinib on HCC827 cell line.
2
.2.3. Cell Cycle Analysis:
The unsubstituted benzothiazol-2-amine VIIa that showed a superior EGFR
inhibition and anticancer activity against MCF-7 cell line was subjected to cell cycle
analysis and apoptotic assay in order to investigate its effect on cell cycle progression and
the apoptosis percentage induced by the compound.
The results of the cell cycle analysis of compound VIIa are shown in Table 4 and Figure
2
.
Table 4: Cell cycle analysis results of compound VIIa after 24 h in MCF-7 cell line.
Cpd. No. %G0-G1
VIIa
%S
%G2/M %Pre-G1
Comment
Pre G1 apoptosis and
Cell growth arrest at
G2/M.
2
7.29
31.25
39.01
41.45
7.1
24.19
1.47
Control
53.89

Control/MCF-7
VIIa/MCF-7 after 24 h
Fig. 2. Effect of compound VIIa on the cell cycle of MCF-7 cells after 24 h.
The results obtained from Table 4 and Figure 2 revealed that compound VIIa led to pre
G1 apoptosis with cell growth arrest at G2/M phase in MCF-7 cells after 24 h which was
confirmed by an increase in the percentage of DNA content (41.46 %) after addition of
the compound compared to the control cells (7.1 %).
2
.2.4. Apoptotic Assay:
To investigate the apoptosis induction effect and to quantify the percentage of
apoptosis induced by compound VIIa in MCF-7 cells, Annexin V-FITC/propidium iodide
dual staining assay was carried out according to the reported method [23,24]. The Annexin
V assay offers the possibility of identifying early phases of apoptosis before the loss of
cell membrane integrity and permits measurements of the kinetics of apoptotic death in
relation to the cell cycle.
The effect of compound VIIa on apoptotic induction in MCF-7 cells are shown in Table
5
and Figure 3.
Table 5: Effect of compound VIIa on apoptotic induction compared to the control cells
after 24 h.
Apoptosis
Cpd. No.
Necrosis
Total
24.19 %
1.47 %
Early
6.27 %
0.87 %
Late
VIIa / MCF7
15.64 %
0.26 %
2.28 %
0.34 %
Control /MCF7

Control/MCF-7
VIIa/MCF-7 after 24 h
Fig. 3. Effect of compound VIIa on apoptosis induction of MCF-7 cells after 24h.
The results obtained from Table 6 and Figure 3 demonstrated that the percentage of the
total apoptotic cells in MCF-7 cell line increases after treatment with compound VIIa
(24.19 %) relative to control cells (1.47 %) which represents a prominent marker of
apoptosis. The quinazolin-4-yl-(6-unsubstituted benzothiazol-2-amine) VIIa revealed cell
growth arrest at G2/M phase, in addition to its apoptotic induction effect.
2
2
.3. Molecular Modeling Study
.3.1. Molecular docking study:
Molecular docking simulations were performed for compounds VIIa,c, VIIIf,
IXb, XIc, XIIb and XIVe eliciting significant EGFR inhibitory activity in order to
investigate their binding mode in the EGFR active site using the X-ray crystallographic
structure of EGFR co-crystallized with the 4-anilinoquinazoline derivative Lapatinib
(PDB ID: 1XKK). The molecular docking setup was first validated by carrying out self-
docking of Lapatinib in the EGFR active site. The re-docking validation step reproduced
the experimental binding mode of the co-crystallized ligand precisely demonstrating that
the used docking protocol is suitable for the intended docking study. This is shown by the
small RMSD between the docked pose and the co-crystallized ligand (1.63 Å); the energy
score (S) = −15.11 kcal/mol and the ability of the docking pose to reproduce all the key
interactions attained by the co-crystallized ligand with the hot spots in the active sites; H-

bonding with Met793 and through water mediated H-bonding with Thr854 as shown in
Figure 4 and 5. The validated setup was then used in expecting the ligands receptor
interactions at the binding site for the compounds of interest.
Fig. 4. 2D interaction diagram showing Lapatinib docking pose interactions with the key
amino acids in the EGFR binding site.
(a)
(b)
Fig. 5. 2D diagram (a) and 3D representation (b) of the superimposition of the co-
crystallized (red) and the docking pose (blue) of Lapatinib in the EGFR binding site with
RMSD of 1.63Å. (ligand hydrogen atoms were removed for clarity)

The experimentally used reference Gefitinib showed the same binding pattern as
Lapatinib as displayed in Figure 6.
(a)
(b)
Fig. 6. 2D diagram (a) and 3D representation (b) of Gefitinib in the EGFR binding site.
According to the performed docking study, the most active compounds VIIa,c, VIIIf,
IXb, XIc, XIIb and XIVe show a common expected binding pattern in the ATP binding
site as shown in Figure 7-9. They form a water-mediated hydrogen bond with the key
amino acid Thr854 backbone NH by their nitrogen atom at position 1 of the quinazoline
ring and H-bond with the key amino acid Met793 by their nitrogen atom in 2-pyridin-3-
yl. In addition to the interactions with the key amino acids, they achieve further
interactions with the ATP binding site, where they interact by their amino group at
−
position 4 of the quinazoline ring through hydrogen bonding with the COO moiety of
Asp855 and through hydrophobic interaction via the bromo group at position 6 of the
quinazoline ring with the hydrophobic side chains of the amino acids Met766, Leu788
and Leu858. Table 6 shows their docking scores. The binding pattern of these
compounds explains their superior EGFR inhibitory activity as revealed by their
experimental EGFR inhibitory assay results as presented in Table 1 and their binding
affinity (docking score) relative to the reference Gefitinib as displayed in Table 6.

(a)
(b)
Fig. 7. 2D diagram (a) and 3D representation (b) of compound VIIIf in the EGFR
binding site.
(a)
(b)
Fig. 8. 2D diagram (a) and 3D representation (b) of compound XIIb in the EGFR
binding site
The most potent compound VIIa shows a different expected binding pattern in the ATP
binding site as shown in Figure 9. It forms a H-bond with the key amino acid Met793 by
the nitrogen atom in the pyridyl ring which is attached to the quinazoline ring at position
2
. In addition, the amino group at position 4 of the quinazoline interacts with the COO−
moiety of Asp855 through hydrogen bonding. Uniquely, it inserts its benzothiazole
bicyclic scaffold in the hydrophobic sub-pocket surrounded by the hydrophobic side

chains of the amino acids Ala743, Met766, Leu777, Leu792 and Phe856. This unique
binding pattern rationalizes its higher activity. (For further details see SI)
(a)
(b)
Fig. 9. 2D diagram (a) and 3D representation (b) of compound VIIa in the EGFR
binding site.
Table 6: Docking energy scores (S) in kcal/mol, interacting amino acid, Distances in Å, H-
bond energies in kcal/mol of the tested compounds, Gefitinib and Lapatinib and their EGFR
inhibitory activity (IC50 µM).
H-bond
energies
Interacting Distances
Docking score
EGFR IC50
(µM)
Compound
VIIa
(S) (kcal/mol)
moiety
(Å)
(
kcal/mol)
Asp855
Met793
3.28
2.99
−1.6
−4.3
−12.74
0.096
Asp855
2.80
3.13
3.22
−5.7
−1.4
−3.7
VIIc
−12.34
−11.49
H
2
O
0.339
0.149
Met793
VIIIf
H
2
O
3.18
−1.2

Met793
Met793
Asp855
3.41
2.80
−2.3
−3.8
IXb
XIc
−10.49
−13.00
0.431
0.331
2.70
3.14
3.31
−6.4
−1.4
−3.1
2
H O
Met793
Asp855
2.73
3.12
3.22
−6.2
−1.5
−3.8
XIIb
−12.44
−12.38
H
2
O
0.292
0.141
Met793
Asp855
2.78
3.14
3.34
−5.9
−1.4
−2.9
XIVe
2
H O
Met793
H
2
O
2.93
3.29
−1.1
−3.6
Gefitinib
−12.89
−15.12
0.166
NA*
Met793
H
2
O
2.79
2.96
−1.4
−5.4
Lapatinib
Met793
NA* = Not available.
2
.3.2. Physicochemical, ADME and pharmacokinetic properties prediction:
SwissADME online web tool provided by the Swiss Institute of Bioinformatics
SIB) is applied for the computation of the physicochemical properties in addition to the
(
prediction of the ADME parameters, pharmacokinetic properties and drug-like nature of
the selected compounds VIIa,c, VIIIf, IXb, XIc, XIIb and XIVe. This was carried out to
assure that they are not only hopeful candidates in terms of biological efficacy, but also
from the pharmacokinetic characteristics. The submitted compounds are predicted to
possess promising physicochemical and pharmacokinetic properties. They exhibit a
predicted wlogP in a range of 3.51 – 7.16, moderate water solubility, high GIT absorption
with no BBB permeability and so no predicted CNS adverse effects. Figure 10
demonstrates the BOILED-Egg graph of the WLOGP vs. TPSA (Topological Polar

Surface Area) for the submitted compounds [20]. All compounds except VIIc and IXb
are placed in the area of human intestinal absorption (HIA) with no BBB permeability.
This graph displays also that they are not P-glycoprotein substrates (PGP-), so they are
not liable to the efflux mechanism done by this transporter which is used by many tumor
cell lines as a drug-resistance mechanism [21]. Although compounds VIIc and IXb show
very promising kinase inhibitory activity, the lipophilicity of IXb or the high polar
surface area of VIIc impede their HIA and so their oral bioavailability. This increases
attention in designing kinase inhibitors to the pharmacokinetics of the compound and not
only the good activity attained. The anticipated high GIT absorption of these compounds
is due to their optimal physicochemical properties found in the suitable physicochemical
properties range for oral bioavailability.
Fig. 10. Predicted Boiled-Egg plot from SwissADME online web tool for compounds
VIIa,c, VIIIf, IXb, XIc, XIIb and XIVe.
SwissADME online web tool shows also that all the tested compounds except
IXb (due to its high molecular weight and logP) satisfy the drug-likeness characteristics
as defined by the major pharmaceutical companies; Lipinski's (Pfizer) [25], Ghose's
(Amgen) [26], Veber's (GSK) [27], Egan's (Pharmacia) [28] and Muegge's (Bayer) [29]
filters. However, they are not classified as lead-like as their molecular weights exceeded

3
50 and most of them is with predicted logPo/w from XLOGP3 [30] model that is higher
than 3.5. In summary, the computational study of the physicochemical and
pharmacokinetic properties of the newly synthesized compounds confirms that most of
them show promising biological efficiency with hopeful pharmacokinetic properties.
3
.Conclusion
Twenty-Eight new compounds were synthesized and evaluated for their EGFR inhibitory
activity. Twenty-four compounds exhibited significant wild-type EGFR inhibitory activity
at sub-micromolar level (IC50= 0.096 – 0.818 µM) compared to Gefitinib (IC50= 0.166
µM). Fifteen compounds VIIa,c, VIIIf, IXb,d, XIc, XIIa-c and XIVb,d-g,i that elicit a
significant enzyme inhibition were further screened for their anticancer activity against
MCF-7 and A549 cell lines, in addition to WI38 normal fibroblast cell line. The majority
of the tested compounds showed a selective anticancer activity against MCF-7 rather than
A549 cell line. Compounds showed promising inhibitory activity against wild-type EGFR
were screened for their inhibitory activity on mutant EGFR (EGFRT790M and EGFRL858R
)
and for their in vitro cytotoxicity against two cancer cell lines expressing mutant EGFR,
namely, PC9 and HCC827. The most active EGFR inhibitor VIIa (IC50 = 0.096 μM) was
subjected to cell cycle analysis and apoptotic assay. The results of molecular docking
study confirmed that the binding mode was consistent with the EGFR inhibitory activity of
the tested compounds. Most of the newly synthesized compounds are not only with
significant anticancer activity, but also possess promising pharmacokinetic properties.
4
. Experimental
4
.1.
Chemistry:
All chemicals were picked up from commercial suppliers and used without any
purification. Reactions were observed by TLC (Kieselgel 60 F254 precoated plates, E.
Merck, Germany) and the spots were visualized by exposure to UV lamp at λ 254 nm.
Determination of melting points was obtained using Electrothermal Stuart SMP
3
digital
and
1
13
melting point apparatus. H NMR and C NMR spectra were determined in DMSO-d
6
1
recorded on 400 MHz spectrophotometer for H NMR and 100 MHz spectrophotometer
for 13C NMR (Bruker AG, Switzerland) at Faculty of Pharmacy, Cairo University;
Chemical shift (δ) values are expressed in parts per million (ppm) and coupling constants

(J) in Hertz (Hz). Elemental microanalyses were performed at the Regional Center for
Mycology and Biotechnology, Al-Azhar University, Egypt while mass spectra were carried
out using Shimadzu Qp-2010 plus Gas Chromatograph-Mass, at the MicroAnalytical
Center, Faculty of Science, Cairo University. Infrared Spectra were recorded on Shimadzu-
FTIR spectrophotometer, Faculty of Pharmacy, Cairo University, Egypt and expressed in
-1
wave number (cm ), using potassium bromide discs. Compounds I, II and XIIIa-d have
been synthesized as reported [31–34].
4
.1.1. 5-Bromo-2-(nicotinamido)benzoic acid (III)
Nicotinoyl chloride II (2.3 g, 16 mmol) was added to a solution of 5-
bromoanthranilic acid I (3.5 g, 16 mmol) in methylene chloride (30 ml) and triethylamine
2.26 ml, 16 mmol). The reaction mixture was stirred overnight at room temperature, the
obtained solid was filtered, dried and crystallized from ethanol.
(
-1
White powder, (yield 80%), m.p. 299-300°C; IR (KBr, νmax/cm ): 3450 - 3425 (OH
1
acid),3406 (NH), 1774 (C=O acid), 1678 (C=O amide), 1593, 1519 (C=C); H NMR
(
DMSO-d
H), 7.95 (s, 1H, D
m, 1H, Ar-H), 8.56 (d, J= 8.92 Hz, 1H, Ar-H), 8.83 (dd, J=1.16, 1.12 Hz, 1H, Ar-H),
6
)  ppm: 7.64 (t, J= 6.28 Hz, 1H, Ar-H), 7.86 (dd, J= 2.48, 2.48 Hz, 1H, Ar-
2
O exchangeable, -NH) , 8.13 (d, J= 2.44 Hz, 1H, Ar-H), 8.27 - 8.30
(
1
3
9
d
1
.12 (d, J=1.56 Hz, 1H, Ar-H), 12.17 (s, 1H, D O exchangeable, -OH); C NMR (DMSO-
2
6
)  ppm: 115.77, 119.25, 120.77, 123.41, 125.59, 131.41, 133.29, 136.58, 137.09,
+
39.68, 146.50, 162.97 (C=O amide), 168.77 (C=O acid); MS, m/z: 321 [M ], 323
+
[
M+2] ; Anal. Calcd. % for C13
H
9
BrN
2
O (321.13): C, 48.62; H, 2.83; N, 8.72; Found
3
%
C, 48.51; H, 3.04; N, 8.96.
4
.1.2. 6-Bromo-2-(pyridin-3-yl)-4H-benzo[d][1,3]oxazin-4-one (IV)
A mixture of the amidated anthranilic acid III (1.84 g, 5 mmol) in acetic anhydride
(15 ml) was heated under reflux for 5 h. Then, the reaction mixture was poured onto ice-
water. The obtained solid product was collected by filtration, dried and crystallized from
-1
methanol. Off white powder, (yield 65%), m.p. 170-173°C; IR (KBr, νmax/cm ): 1762
1
(
C=O), 1593, 1519 (C=C); H NMR (CDCl
3
) ppm: 7.50 (dd, J= 4.92,
4
.88 Hz, 1H, Ar-H), 7.63 (d, J= 8.60 Hz, 1H, Ar-H), 7.97 (dd, J= 2.28, 2.28 Hz, 1H, Ar-
H), 8.40 (d, J= 2.24 Hz, 1H, Ar-H), 8.54 - 8.57 (m, 1H, Ar-H), 8.83 (dd, J= 1.36, 1.44

13
Hz, 1H, Ar-H), 9.52 (d, J=1.56 Hz, 1H, Ar-H); C NMR (DMSO-d
6
)  ppm: 119.40,
1
1
21.48, 124.55, 126.45, 129.64, 130.49, 135.85, 140.06, 145.40, 149.09, 153.54, 155.89,
+
+
57.86 (C=O); MS, m/z: 303 [M ], 305 [M+2] ; Anal. Calcd. % for C13
H BrN O
7 2 2
(
303.12): C, 51.51; H, 2.33; N, 9.24; Found % C, 51.68; H, 2.45; N, 9.41.
.1.3. 6-Bromo-2-(pyridin-3-yl)quinazolin-4(3H)-one (V) [35]:
A mixture of the benzoxazinone IV (1 g, 3.3 mmol) in formamide (15 ml) was
4
heated under reflux for 4 h. Then, the reaction mixture was kept to cool at room
temperature. The obtained solid product was collected by filtration, washed with water,
dried at 120 °C and crystallized from ethanol.
-1
Buff crystals, (yield 80 %), m.p. 320-322°C; IR (KBr, νmax/cm ): 3325 (NH), 1670 (C=O
1
amide), 1597, 1562 (C=C); H NMR (DMSO-d
6
) ppm: 7.59 (dd, J= 4.84, 4.84 Hz, 1H,
Ar-H), 7.71 (d, J= 8.68 Hz, 1H, Ar-H), 8.00 (dd, J= 2.36, 2.36 Hz, 1H, Ar-H), 8.24 (d, J=
2
9
.24 Hz, 1H, Ar-H), 8.50 – 8.47 (m, 1H, Ar-H), 8.77 (dd, J= 1.28, 1.32 Hz, 1H, Ar-H),
.29 (d, J=1.96 Hz, 1H, Ar-H), 12.90 (s, 1H, D O exchangeable, -NH).
2
4
.1.4. 6-Bromo-4-chloro-2-(pyridin-3-yl)quinazoline (VI) [36,37]:
A mixture of quinazolin-4-one V (0.5 g, 1.65 mmol) in POCl
3
(4 ml) was heated
under reflux for 2.5 h. The reaction mixture was kept to cool at room temperature, poured
dropwisely onto crushed ice-water with vigorous stirring and neutralized with 20% NaOH.
The obtained solid product was filtered, washed with water, dried and crystallized from
ethanol.
-1
Olive green powder, (yield 85 %), m.p. 166-169°C; IR (KBr, νmax/cm ): 3082 (CH
1
aromatic), 1558, 1539 (C=C); H NMR (DMSO-d
6
) ppm: 7.64 (dd, J= 4.80, 4.80 Hz,
1H, Ar-H), 8.07 (d, J= 8.92 Hz, 1H, Ar-H), 8.25 (dd, J= 2.00, 2.04 Hz, 1H, Ar-H), 8.42
(d, J= 2.00 Hz, 1H, Ar-H), 8.72 - 8.78 (m, 2H, Ar-H), 9.56 (d, J=1.68 Hz, 1H, Ar-H).
4
.1.5. General procedure for the preparation of compounds VIIa-c:
A mixture of equimolar amounts of 4-chloro derivative VI and the appropriate
benzothiazole (10 mmol) was stirred under reflux in dry dimethylformamide (DMF) (10
ml) in presence of anhydrous potassium carbonate (K CO ) (0.4 g, 3 mmol) for 15 h. The
2
3
solid separated upon pouring the reaction mixture onto ice-water was filtered, washed
with water, dried and crystallized from ethanol.

4
.1.5.1. N-(6-bromo-2-(pyridin-3-yl)quinazolin-4-yl)benzo[d]thiazol-2-amine (VIIa)
-1
Dark brown powder, (yield 55%), m.p. 378-380°C; IR (KBr, νmax/cm ): 3369 (NH), 1608
1
(
NH bending), 1508 and 1435 (C=C); H NMR (DMSO-d
6
)  ppm: 7.35 (t, J= 7.54 Hz,
1H, Ar-H), 7.50 (t, J= 7.56 Hz, 1H, Ar-H), 7.63 – 7.67 (m, 2H, Ar-H), 7.88 (d, J= 6.34
Hz, 2H, Ar-H), 8.77 (d, J= 3.76 Hz, 1H, Ar-H), 8.92 (d, J= 7.69 Hz, 1H, Ar-H), 8.56 -
8
.67 (m, 2H, Ar-H), 8.97 (s, ½ H, D O exchangeable, -NH), 9.79 (s, 1H, Ar-H), 12.99 (s,
2
1
3
½
H, D
2
O exchangeable, -NH) ; C NMR (DMSO-d )  ppm: 119.82, 122.59, 123.80,
6
1
1
24.18, 124.70, 127.08, 128.00, 130.43, 133.48, 136.12, 137.17, 138.76, 149.60, 150.07,
+
+
51.66, 158.16, 171.28; MS, m/z: 434 [M ], 436 [M+2] ; Anal. Calcd. % for
S (434.32): C, 55.31; H, 2.79; N, 16.13; Found % C, 55.46; H, 2.84; N,
C
20
H
12BrN
5
1
6.45.
.1.5.2. N-(6-bromo-2-(pyridin-3-yl)quinazolin-4-yl)-6-ethoxybenzo[d] thiazol-2-amine
4
(VIIb)
-1
Dark brown powder, (yield 56%), m.p. 383-385°C; IR (KBr, νmax/cm ): 3394 (NH), 1604
1
(
NH bending), 1523 and 1458 (C=C); H NMR (DMSO-d
6
)  ppm: 1.38 (t, J= 6.92 Hz,
), 7.07 (dd, J= 2.48, 2.40 Hz, 1H, Ar-
H), 7.50 – 7.67 (m, 4H, Ar-H), 7.88 (d, J= 8.84, 1H, Ar-H), 8.06 (d, J= 8.92, 1H, Ar-H),
.79 (t, J= 7.68, 1H, Ar-H), 8.93 (d, J= 8.00 Hz, 1H, Ar-H), 9.02 (s, 1H, D
3
H, OCH
2
CH
3
), 4.12 (q, J= 6.86 Hz, 2H, OCH
2
CH
3
8
2
O
13
exchangeable, -NH), 9.79 (d, J= 1.52, 1H, Ar-H); C NMR (DMSO-d )  ppm:15.19
6
2 3 2 3
(OCH CH ), 64.04 (OCH CH ), 116.17, 117.54, 120.14, 124.24, 127.24, 130.51, 133.83,
+
1
36.19, 137.23, 139.90, 150.11, 151.73, 155.85, 158.27, 171.62; MS, m/z: 478 [M ], 480
+
[
M+2] ; Anal. Calcd. % for C22
C, 55.40; H, 3.49; N, 14.91.
.1.5.3. N-(6-bromo-2-(pyridin-3-yl)quinazolin-4-yl)-6-nitrobenzo[d]thiazol-2-amine
VIIc)
H
16BrN OS (478.37): C, 55.24; H, 3.37; N, 14.64; Found
5
%
4
(
-1
Red powder, (yield 63%), m.p. 378-380°C; IR (KBr, νmax/cm ): 3373 (NH), 1608 (NH
1
bending), 1508 and 1435 (C=C); H NMR (DMSO-d
6
)  ppm: 7.56 – 7.64 (m, 2H, Ar-H),
7
2
9
.72 (d, J= 8.76 Hz, 1H, Ar-H), 7.78 (dd, J= 2.28, 2.28 Hz, 1H, Ar-H), 8.15 (dd, J= 2.36,
.4 Hz, 1H, Ar-H), 8.67 (s, 1H, D O exchangeable, -NH), 8.73 – 8.79 (m, 3H, Ar-H),
.02 (d, J= 8.00 Hz, 1H, Ar-H), 9.84 (d, J= 1.32 Hz, 1H, Ar-H); C NMR (DMSO-d
2
13
6
) 
ppm: 117.84, 121.61, 122.65, 124.04, 127.91, 129.64, 134.41, 134.88, 135.39, 136.24,

+
1
40.28, 149.56, 150.27, 151.09, 157.57, 158.97, 161.62, 170.99; MS, m/z: 479 [M ], 481
+
[
M+2] ; Anal. Calcd. % for C20
H
11BrN
6
O S (479.31): C, 50.12; H, 2.31; N, 17.53;
2
Found % C, 50.31; H, 2.57; N, 17.75.
4
.1.6. General procedure for the preparation of compounds (VIIIa-f)
A mixture of equimolar amounts of 4-chloro derivative VI and the appropriate
phenol (10 mmol) was stirred at room temperature in dry DMF (10 ml) in presence of
anhydrous K CO for 24 h. The solid separated upon pouring the reaction mixture onto
ice-water was filtered, washed with water, dried and crystallized from ethanol.
2
3
4
.1.6.1. 6-Bromo-4-phenoxy-2-(pyridin-3-yl)quinazoline (VIIIa)
-1
Grey powder, (yield 75%), m.p. 169-172°C; IR (KBr, νmax/cm ): 3059 (CH aromatic
1
str.), 1566 and 1550 (C=C); H NMR (DMSO-d
6
)  ppm: 7.41 (t, J= 7.16 Hz, 1H, Ar-H),
7
8
.51 (t, J= 8.38Hz, 3H, Ar-H), 7.57 - 7.61 (m, 2H, Ar-H), 8.01 (d, J= 8.88Hz, 1H, Ar-H),
.19 (d, J= 8.68 Hz, 1H, Ar-H), 8.45 (d, J= 7.84 Hz, 1H, Ar-H), 8.53 (s, 1H, Ar-H), 8.68
13
(
d, J= 3.88 Hz, 1H, Ar-H), 9.27 (s, 1H, Ar-H); C NMR (DMSO-d
6
)  ppm: 116.45,
1
1
21.01, 122.45, 124.25, 126.25, 126.59, 130.18, 130.52, 132.61, 135.55, 138.53, 149.59,
+
+
50.95, 152.11, 152.43, 158.07, 166.18; MS, m/z: 378 [M ], 380 [M+2] ; Anal. Calcd. %
O (378.23): C, 51.02; H, 3.53; N, 17.50; Found % C, 51.30; H, 3.71; N,
for C19
H12BrN
3
1
7.69.
.1.6.2. 6-Bromo-2-(pyridin-3-yl)-4-(p-tolyloxy)quinazoline (VIIIb):
4
-1
Grey powder, (yield 70%), m.p. 170-173°C; IR (KBr, νmax/cm ): 2935 (CH aliphatic)
1
1
4
550 and 1504 (C=C); H NMR (DMSO-d
6
)  ppm: 2.42 (s, 3H, -CH ), 7.30 – 7.42 (m,
3
H, Ar-H), 7.52 (dd, J= 4.88, 4.80Hz, 1H, Ar-H), 8.01 (d, J= 8.84Hz, 1H, Ar-H), 8.19 (d,
J= 8.88 Hz, 1H, Ar-H), 8.48 (d, J= 7.84 Hz, 1H, Ar-H), 8.52 (s, 1H, Ar-H), 8.67 (d, J=
1
3
4
1
1
.48 Hz, 1H, Ar-H), 9.28 (s, 1H, Ar-H); C NMR (DMSO-d
6
)  ppm: 21.03 (CH
3
),
15.47, 120.96, 122.09, 124.27, 126.25, 130.15, 130.50, 132.67, 135.58, 138.49, 149.61,
+
+
50.21, 152.12; MS, m/z: 392 [M ], 394 [M+2] ; Anal. Calcd. % for C20
H14BrN O
3
(392.26): C, 54.76; H, 2.99; N, 15.96; Found % C, 54.97; H, 3.12; N, 16.14.
4
.1.6.3. 6-Bromo-4-(4-fluorophenoxy)-2-(pyridin-3-yl)quinazoline (VIIIc)
-1
Grey powder, (yield 73%), m.p. 193-195°C; IR (KBr, νmax/cm ): 3062 (CH aromatic),
1
1
550 and 1504 (C=C); H NMR (DMSO-d
6
)  ppm: 7.40 - 7.44 (m, 2H, Ar-H), 7.51 –