Synthesis and anti-proliferative activity of substituted- anilinoquinazolines and its relation to EGFR inhibition

Article abstract of DOI:10.1055/s-0032-1312601

4-Anilinoquinazoline is a privileged scaffold in developing small molecule inhibitors of tyrosine kinases (TK) especially epidermal growth factor receptor (EGFR). 2 series belonging to 3'-substituted-4-anilinoquinazoline scaffold were synthesized and screened in vitro on isolated and a breast cancer cell line. The research aims at exploring the activity of compounds having diverse substituents at 3' position of the aniline moiety. Generally, the meta-substituted-anilinoquinazolines exhibited significant inhibitory activity against isolated enzyme as well as MCF-7 cancer cell line. For instance, compound 10b inhibited >99% of EGFR activities at 10 M concentration. 6 of the tested compounds exhibited range of anti-proliferative activity below 10 M potency. In particular, compounds 6e and 10b displayed the highest activity among the tested compounds with IC₅₀ values equal to 8.6 and 4.84 M, respectively. Structure-based tools were utilized to rationalize EGFR-TK binding of compound 10b since it is the most active compound in the enzyme inhibition test. Georg Thieme Verlag KG Stuttgart.New York.

Full text of DOI:10.1055/s-0032-1312601

360 Original Article
Synthesis and Anti-proliferative Activity of
Substituted-Anilinoquinazolines and Its Relation to
EGFR Inhibition
Authors
D. A. A El Ella1*, K. A. Saleh2, M. Hassan2, N. Hamdy3, M. E. El-Araby4, K. A. M. Abouzid1*
Affiliations
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Russian Egyptian University, Cairo, Egypt
3 Department of Biochemistry, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt
4 Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Helwan University, Helwan, Egypt
Key words
Abstract
tory activity against isolated enzyme as well as
▶
quinazoline
anilinoquinazoline
EGFR inhibitors
tyrosine kinase inhibitors
anti-cancer
structure-based design
●
MCF-7 cancer cell line. For instance, compound
10b inhibited >99% of EGFR activities at 10μM
concentration. 6 of the tested compounds exhib-
ited range of anti-proliferative activity below
10μM potency. In particular, compounds 6e and
10b displayed the highest activity among the
tested compounds with IC50 values equal to 8.6
and 4.84μM, respectively. Structure-based tools
were utilized to rationalize EGFR-TK binding of
compound 10b since it is the most active com-
pound in the enzyme inhibition test.
▼
▶
●
4-Anilinoquinazoline is a privileged scaffold in
developing small molecule inhibitors of tyro-
sine kinases (TK) especially epidermal growth
factor receptor (EGFR). 2 series belonging to
▶
●
▶
●
▶
●
▶
●
3'-substituted-4-anilinoquinazoline
scaffold
were synthesized and screened in vitro on iso-
lated and a breast cancer cell line. The research
aims at exploring the activity of compounds
having diverse substituents at 3' position of the
aniline moiety. Generally, the meta-substituted-
anilinoquinazolines exhibited significant inhibi-
received 17.02.2012
accepted 20.04.2012
Bibliography
DOI http://dx.doi.org/
10.1055/s-0032-1312601
Published online:
June 21, 2012
Arzneimittelforschung 2012;
62: 360–366
© Georg Thieme Verlag KG
Stuttgart · New York
ISSN 0004-4172
Introduction
▼
form of the enzyme [6]. Resistance to EGFR-TK
inhibitors have been attributed to specific muta-
tions which usually make receptors less sensitive
to their inhibitors. Common point mutations
include T790M (also reported as T766M), G719S,
L747S, and L858R [7,8]. The latter, also called
L834R in another sequence numbering conven-
tion, is the most discovered resistance source as
it represents 40% of detected mutations in some
tumors such as Non-Small Cell Lung Cancer
(NSCLC) [9,10]. Fortunately, both drugs seem to
be effective in single L858R patients as they pos-
sess higher affinity to this particular mutant
enzyme [11,12]. Lapatinib, a dual EGFR/erb-B2
inhibitor, seems to overcome the resistance
emerged due to the somatic mutations of T790M
in the tyrosine kinase domain of the EGFR gene
that occur in patients with breast cancer who
showed a poor response to the EGFR tyrosine
kinase inhibitors gefitinib and erlotinib [13].
Chemically, all EGFR inhibitors currently in clini-
cal use are functionalized on the meta position of
the aniline moiety with small hydrophobic halo
or hydrocarbon substituents. Nonetheless, we
found no enough research addressing the SAR of
this position with variety of bulkier groups
[14,15]. Therefore, we realized that this point of
Inappropriate or uncontrolled activation of pro-
tein tyrosine kinases (PTKs), either by over-
expression, constitutive activation, or mutation
has been shown to cause uncontrolled cell
growth [1]. The EGFR (erb-B1) PTK has been
identified as a major target for medicinal chemis-
try programs especially in cancer therapy such as
breast, ovarian, colon, and prostate [2,3] as it is
often associated with vascularity and poor prog-
nosis of patients [4]. Drug discovery efforts tar-
geting this aberrant kinase activity resulted in
introduction of several FDA approved small mol-
ecule anticancer agents such as gefitinib, erlo-
▶
Correspondence
Prof. K. A. M. Abouzid, PhD
Department of Pharmaceutical
Chemistry
Faculty of Pharmacy
Ain Shams University
Abassia
Cairo 11566
Egypt
Tel.: +20/2/2405 1150
Fax: +20/2/2508 0728
khaled.abouzid@pharm.asu.
edu.eg
tinib and lapatinib ( Fig. 1) which all belong to
●
4-anilinoquinazoline scaffold [5]. Thus, among
the great number of reported structural classes
of tyrosine kinase inhibitors, 4-anilinoquina-
zilines along with their congeners 4-anilinoqui-
nolines and pyrrolopyrimidines represent the
most researched classes of EGFR-TK inhibitors.
G e fitinib and erlotinib, the first generation
quinazoline EGFR-TK inhibitors, have similar
therapeutic profile since they target the activated
Prof. D. A. A El Ella, PhD
Department of Pharmaceutical
Chemistry
Faculty of Pharmacy
Ain Shams University
Abassia
Cairo 11566
Egypt
Tel.:+20/2/2405 1180
Fax:+20/2/2405 1107
dalal999@hotmail.com
*These authors contributed equally to this work.
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366

Original Article 361
Fig. 1 Quinazoline derivatives as EGFR TK
inhibitors.
F
O
HN
N
CI
HN
N
3′ Br
3′
HN
N
3′
N
O
O
O
O
O
O
N
O
O
N
N
PD153035
Gefitinib (Iressa)
Erlotinib (Tarceva)
O
F
F
O
F
S
O
3′
3′
HN
CI
HN
NH
NH
O
O
O
N
N
O
N
N
Lapatinib (Tyverb)
EGFR IC50 = 0.49 μM
diversity is an intriguing opportunity to develop novel antipro-
liferative agents which may act through EGFR-TK inhibition and/
or another mechanism.
the C-4 chloro group without affecting the chlorine atom at C-2
position. At the following stage, 4-(3-carboxyanilino)-2-chloro-
6,7-dimethoxyquinazoline 4 was refluxed with thionyl chloride
to produce the unstable acyl chloride hydrochloride derivative
(5). Without isolation, the acid chloride 5 was reacted with
Design and Rationale
▼
amines at ambient temperature, gave the final compounds (6a–i).
▶
On the other hand, Fig. 3 describes the synthetic pathway of
●
Most of the precedent SAR studies of 4-anilinoquinazolines were
dedicated to substitutions on 4' position of the aniline moiety
while halo group or small alkyl groups were recruited to satisfy
a presumed small pocket around the 3' position [16,17]. In these
early works on quinazoline inhibitors of EGFR-TK, screening
results discouraged researchers of pursuing SAR studies that
cover a diverse molecular space around this position since meth-
oxy and methyl analogues exhibited lower potency than halo
compounds. Nevertheless, the success of erlotinib and availabil-
ity of several crystal structures of EGFR-TK bound to quinazoline
inhibitors brought us a renewed interest in this less explored
position and we decided to investigate it with more challenging
bulkier functionalities. The SAR around the 3' position was stud-
ied through the synthesis of 2 sets of compounds that have sub-
stituents linked through ether or amide linker. Moreover, the 2
compound series contained chloro group on the C2 of the core
quinazoline nucleus because it was reported in recent studies to
enhance the anticancer activity and blood brain barrier penetra-
tion [18]. We kept 2 methoxy groups at positions 6- and 7- of the
quinazoline as they have been established as compatible groups
to good EGFR-TK inhibition [19]. Accordingly, the proposed com-
pounds were synthesized and evaluated for their in vitro enzyme
inhibitory activity as well as antiproliferative potency on MCF-7
breast carcinoma cell line in which EGFR-TK is over expressed.
the other series of the 4-anilinoquinazoline compounds (10a–f).
The protected m-acetamidophenol (7) was reacted with the cor-
responding alkyl or alkyl halides to afford the corresponding
alkoxy derivatives (8a–e). After removal of the protecting acetyl
group using alkaline hydrolysis, the aminophenyl ethers (9a–e)
were reacted with the 2,4-dichloro-6,7-dimethoxyquinazoline
(3) in presence in basic conditions to furnish the final com-
pounds (10a–e). The structures of all the newly synthesized
compounds were elucidated with 1H NMR, EI-Mass, FT-IR and
elemental analyses.
In Vitro EGFR Inhibition and Antiproliferative Cell
Assay
▼
EGFR-TK inhibitory activities of the title compounds were deter-
mined using K-LISA (Kinase activity ELISA) technique. K-LISA
enabled us to measure kinase activity semi-quantitatively using
enzyme-linked immunosorbent assays (ELISA) with phospho-
specific antibodies utilizing colorimetric detection [22,23]. The
test compounds that showed >50% inhibition, were evaluated
for IC50 on the MCF-7 breast cancer cell lines where the EGFR-TK
▶
is over-expressed ( Table 1) [24,25].
●
Results and Discussion
Chemistry
▼
Most of the tested compounds exhibited significant inhibitory
activity against the isolated EGFR enzyme except compounds
6b, 6d, 6g, 6i and 10d. Generally the ether series (10a–e) showed
higher EGFR inhibition and cellular activities than the amide
series (6a–i). In particular, the allyloxy analogue 10b was
approximately as potent as the reference lapatinib. Also, the cel-
lular assay results are consistent with enzyme inhibition, impli-
cating that anticancer activities are partially or mostly related to
EGFR-TK inhibition.
▼
▶
●
General synthesis of target compounds are depicted in Fig. 2,
3 from the central intermediate 2,4-quinazolinedione (2) [20].
Chlorination of the dione with POCl3 in the presence N,N-
dimethylaniline gave the key starting material, 2,4-dichloro-6,7-
dimethoxyquinazoline 3 [21]. In the second figure, the synthesis
of the final compounds 4-anilinoquinazolines (6a–i) was
described where the C-3' bulky substituent was linked to the
aniline ring via amide linker. 4-(3-Carboxyanilino)-2-chloro-
6,7-dimethoxyquinazoline (4) was synthesized via the reaction
of (3) with m-aminobenzoic acid under carefully controlled con-
ditions to strictly direct the SNAr substitution reaction towards
To rationalize the EGFR-TK binding potency of 10b, a structure-
based modelling study was performed by docking this molecule
on the crystal structures of EGFR-TK (pdb codes: 1M17, 2ITY and
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366

362 Original Article
Fig. 2 Synthetic pathway of 4-anilinoquinazo-
lines (6a–i). Reagents and conditions a KCNO,
AcOH then NaOH; b POCl3, N,N-dimethylaniline;
c m-aminobenzoic acid, ethanol, rt, 24h; d SOCl2,
reflux; e Amine compound, dioxane, K2CO3, rt.
O
CI
N
O
H3CO
OH
H3CO
H3CO
H3CO
H3CO
a
b
NH
N
H3CO
NH2
CI
N
H
O
3
1
2
c
COOH
COCI
HN
N
NH
.HCI
H3CO
H3CO
H3CO
H3CO
d
N
N
e
CI
N
CI
4
5
6a; R = 4- Morpholinyl
6b; R = Cyclohexylamino
6c; R = Benzylamino
R
HN
N
6d; R = Ethyl 4-piperazinyl-1-carboxylate
6e; R = 1-Piperidinyl
O
H3CO
H3CO
N
6f; R = Phenylethylamino
6g; R = i-Propylamino
CI
6h; R = t-Butylamino
6i; R = 4-Phenylpiperazin-1-yl
6a–i
Fig. 3 Synthesis of 4- alkyloxyanilinoquinazolines
(10a–f); Reagents and conditions a (CH3CO)2O,
CH3COOH, rt.; b Alkyl/Aralkyl halide, K2CO3, KI,
Acetone, reflux.; c NaOH, H2O, EtOH, reflux.;
d CH3COONa & EtOH, rt, 24h.
O
O
b
N
H
OR
H3C
N
H3C
OH
H
7
8a–f)
c
CI
OR
HN
N
H2N
OR
H3CO
H3CO
H3CO
H3CO
N
9a–f
N
CI
N
d
CI
3
10a–f
8a, 9a, 10a; R = Benzyl
8b, 9b, 10b; R = Allyl
8c, 9c, 10c; R = 3,4-Dichlorobenzyl
8d, 9d, 10d; R = 4-Methylbenzyl
8e, 9e, 10e; R = 2-(Morpholin-4-yl)ethyl
8f, 9f, 10f; R = 3-Fluorobenzyl
2ITZ) after modifying each ligand to the required structure while
keeping the rest of the structural features as given [26,27]. The
new structure was then re-docked into the ATP site. Concerning
the alkoxy side chain, the least energy conformer (all anti-con-
formation) was used. Also, 2 docking experiments were per-
formed due to difference in orientation of the allyl with respect
Table 1 EGFR-TK inhibition and MCF cell line inhibition activity of test com-
pounds. Note that IC50 >10μM is considered to be out of range. IC50 of refer-
ence lead compound (Lapatinib) was 7.70nM. ND means Not Determined.
Compound No.
inhibition % at 10μM
Cytotoxicity (IC50) μM
6a
6b
84
17.9
N D
0
to quinazoline ring as it either attains cis or trans conformation
6c
69.7
0
11.9
N D
▶
(
Fig. 4). In the process, it was worthy to compare the orienta-
●
6d
tion and contacts of this allyloxy group with erlotinib’s alkynyl
group in its crystal structure (PDB Code: 1M17) which is proved
to acquire cis conformation in its binding with the activated con-
formation of EGFR. The compound 10b with extended confor-
mation of allyloxy group (least energy conformer) rested
comfortably with good VdW radii in this medium size pocket
6e
65.4
33
8.6
6f
ND
6g
0
N D
6h
48
ND
6i
2.4
0
ND
10a
10b
10c
10d
10e
N D
99.5
53
4.8
composed of hydrophobic side chains of Leu764, Thr766,
out of range
ND
▶
Met742 and Lys721 ( Fig. 4b). Additionally, the ether oxygen
●
22.2
70.5
established a hydrogen bond with Thr766 terminal hydroxyl
group. On the other hand, the chloro group approached and
14.3
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366

Original Article 363
Fig. 4 a Docking of cis-conformation of 10b on
a
b
crystal structure of wild type EGFR-TK (PDB Code
1M17). b Docking of trans-conformation of 10b on
mutant L834R EGFR-TK (PDB Code: 2ITZ) shown
in dark grey. Same amino acid residues from wild
type are shown in light grey sticks.
PHE832
LYS721
LEU764
ASP31
MET742
LEU763
THR766
GLN767
MET769
backbone carbonyl of Gln767 in unsuitable short distance. Con-
formational search about the 2 bonds between the C$ atom of
the quinazoline and the phenyl ring suggested that rotating of
the 2 dihedral angles between the 2 rings resulted in relaxation
of inter-atomic distances between the chloro group and atoms
around without affecting other interactions such as the critical
bridged H-bond between quinazoline N-3 and Thr766 (hinge
region interaction). This way, the chloro group had contacts
with a small pocket in the hinge loop composed of residues
Gln767-Met769. Similar results were obtained from docking of
10b cis conformation on the wild-type EGFR crystal structure
complexed with gefitinib (PDB Code: 2ITY).
In this modelling study, the possibility of the allyloxy group to
attain trans conformation with respect to the quinazoline moi-
ety was investigated according to findings of Yun et al. about
binding mode of gefitinib with mutated EGFR (L834R) [27].The
authors reported an inverted (trans) positioning of the chloro
group as they formed a halogen bond with Asp831. Docking of
trans conformation of compound 10b on crystal structure L834R
EGFR crystal structure (PDB Code: 2ITZ) resulted in a convincing
short hydrogen bond with Asp831. In addition, the allyl group
lyser (Perkin-Elmer, Norwalk, CT, USA) at the microanalytical
laboratories of the Faculty of Science, Cairo University. All analy-
ses of the new compounds were within ±0.4% of the theoretical
values. The IR spectra (KBr) were measured on Shimadzu IR 110
spectrophotometer (Shimadzu, Koyoto, Japan), 1H-NMR spectra
were obtained on a Bruker proton NMR-Avance 300 (300MHz)
(Bruker, Munich, Germany), in DMSO-d6 as a solvent, using
tetramethylsilane (TMS) as internal standard. The electron
impact (EI) mass spectra were recorded on Finnigan Mat SSQ
7000 (70 ev) mass spectrometer. All reactions were monitored
by thin layer chromatograph (TLC) using precoated Aluminium
sheets Silica gel Merck 60 F254 and were visualized by UV lamp
(Merck, Darmstadt, Germany). All reagents and solvents were
purified and dried using standard techniques. Intermediates 8a,
8b, 8c were synthesized according to reported methods [28–30].
4-(3-Carboxyanilino)-2-chloro-6,7-dimethoxyquinazoline (4)
2,4-Dichloro-6,7-dimethoxyquinazoline (3) (0.5g, 1.93mmol)
was added portion-wise to a mixture of 3-aminobenzoic acid
(0.265g, 1.93mmol) and sodium acetate (0.2g, 2mmol) in abso-
lute ethanol (20mL). The mixture was stirred at room tempera-
ture overnight. The resulting faint green precipitate was
collected and dissolved in saturated solution of sodium carbon-
ate. The solution was filtered, acidified using glacial acetic acid.
The produced precipitate was collected, washed with water and
dried then crystallized from acetone. Yield: 77%; m.p.: >250°C;
IR (ν, cm−1): 3500–3078 (NH, OH acidic), 1692 (C=O), 1642
(C=N). 1H NMR (DMSO-d6) δ: 3.92 (s, 3H, OCH3), 3.96 (s, 3H,
OCH3), 7.16 (s, 1H, C5-H of quinazoline), 7.51-8.29 (m, 4H, Ar-H),
7.92 (s, 1H, C8-H of quinazoline), 10.02 (s, 1H, NH, D2O exchange-
able), 10.71 (s, 1H, OH, D2O exchangeable). EIMS, m/z: 359 (M+,
100%), 361 (M+2, 32.2%).
attained a bent conformation make suitable hydrophobic con-
▶
tact with the aromatic ring of Phe832 ( Fig. 4b) .
●
Conclusion
▼
In this work, we present compounds with low micromolar level
of cell growth inhibition on human breast carcinoma cell line
(MCF-7). The exact mechanism of its cytotoxic activity of our
compounds especially those belonging to amide series could not
be confirmed because the enzyme inhibition is not as high as the
cell line inhibition.
The SAR also indicates that the 3′ position is less tolerant to
bulkier groups than 4' position because the allyloxy group gave
better results in the isolated EGFR-TK inhibition tests. Nonethe-
less, the results reveal that EGFR inhibition is not highly sensi-
tive to group size at 3' position as previously implicated.
Therefore, this diversity point can be utilized to introduce novel
TK inhibitors with versatile biological and/or pharmacokinetic
properties.
2-Chloro-6,7-dimethoxy-4-{[3-(morpholin-4-yl)
carbonyl]anilino}quinazoline (6a)
A mixture of 4 (3.59g, 10mmol) and thionyl chloride (15mL,
excess) was refluxed for 4h. Excess thionyl chloride was distilled
under vacuum. The residue obtained was washed with dry ether
(Na2SO4) and dried. The freshly prepared 3-(2-chloro-6,7-
dimethoxyquinazolin-4-ylamino)benzoyl chloride hydrochlo-
ride (5) (0.2g, 0.53mmol) was mixed with morpholine (0.2g,
excess) and anhydrous K2CO3 (0.2g) in dioxane (1.5mL) and
cooled in ice bath for 30min then stirred at room temperature
overnight. The mixture was poured on warm freshly prepared
concentrated solution of K2CO3 (10–15mL) then stirred for
30min. The formed precipitate was filtered, washed with water,
dried and crystallized from the ethanol/methanol mixture.
Yield: 77%; m.p.: 270–272°C; IR (ν, cm−1): 3317 (NH), 1619
Experimental
▼
Melting points are uncorrected and were determined on a Stuart
melting point apparatus (Stuart Scientific, Redhill, UK). Elemen-
tal analyses (C, H, N) were performed on Perkin-Elmer 2400 ana-
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366

364 Original Article
1
(C=O), 1606 (C=N). H NMR (DMSO-d6) δ: 3.93 (s, 3H, OCH3),
3.95 (s, 3H, OCH3), 4.33 (t, 4H, CH2-O-CH2 of morpholine), 4.07
(t, 4H, CH2-N-CH2 of morpholine), 7.16 (s,1H, C5-H of quinazo-
line), 7.21–7.79 (m, 4H, Ar-H), 7.86 (s, 1H, C8-H of quinazoline),
9.931 (s,1H, NH, D2O exchangeable). EIMS, m/z: 428 (M+, 63.2%),
430 (M+2, 17.2%). Anal. Calcd. for C21H21ClN4O4.0.5H2O: C,
57.56; H, 5.02; N, 12.78. Found: C, 57.60; H, 5.34; N, 12.50.
2-Chloro-6,7-dimethoxy-4-[3-(2-phenylethylcarbamoyl)
anilino]quinazoline (6f)
Compound 6f was prepared as described for 6a from 4 and
2-phenylethylamine. The product was recrystallized from meth-
anol. Yield: 72%; m.p.: 122–125 °C; IR (ν, cm−1): 3301–3500
(NH, NH amidic), 1629 (C=O), 1581 (C=N). 1H NMR (DMSO-d6)
δ: 2.86 (t, 2H, Ph-CH2- CH2-NH), 3.50 (t, 2H, Ph-CH2- CH2-NH),
3.93(s, 3H, OCH3), 3.95 (s, 3H, OCH3), 7.16 (s, 1H, C5-H of quina-
zoline), 7.91 (s, 1H, C8-H of quinazoline), 7.12–8.07 (m, 9H,
Ar-H), 8.59 (t, 1H, NH amidic, D2O exchangeable), 10.02 (s,1H,
NH, D2O exchangeable). EIMS, m/z: 462 (M+, 26.4%), 464 (M+2,
7.1%). Anal. Calcd. For C25H23ClN4O3: C, 64.86; H, 5.01; N, 12.10.
Found: C, 65.03; H, 4.98; N, 12.43.
2-Chloro-4-[3-(cyclohexylcarbamoyl)anilino]-6,7-
dimethoxyquinazoline (6b)
Compound 6b was prepared as described for 6a from 4 and
cyclohexylamine then the product was recrystallized from ace-
tonitrile. Yield: 63%; m.p.: 248–250 °C; IR (ν, cm−1): 3378 (NH
amidic), 3354 (NH), 1628 (C=O), 1574cm−1 (C=N). 1H NMR
(DMSO-d6) δ: 1.03–1.83 (m,10H, cyclohexyl), 3.93 (s, 3H, OCH3),
3.96 (s, 3H, OCH3), 3.34–3.49 (m, 1H, NH-CH of cyclohexyl), 7.18
(s, 1H, C5-H of quinazoline), 8.09 (s, 1H, C8-H of quinazoline),
7.47–8.04 (m, 4H, Ar-H), 8.23 (d, 1H, NH amidic, D2O exchange-
able), 9.94 (s, 1H, NH, D2O exchangeable). MS, m/z: 440 (M+,
100%), 442 (M+2, 30.1%). Anal. Calcd. for C23H25ClN4O3.0.5H2O:
C, 62.65; H, 5.71; N, 12.71. Found: C, 62.76; H, 5.75; N, 12.79.
2-Chloro-4-[ 3-(isopropylcarbamoyl)anilino]-6,7-
dimethoxyquinazoline (6g)
Compound 6g was prepared as described for 6a from 4 and
i-propylamine then the product was recrystallized from metha-
nol. Yield: 55%; m.p.: 170–171 °C; IR (ν, cm−1): 3312–3700
(NH, NH amidic), 1628 (C=O), 1575 (C=N). 1H NMR (DMSO-d6)
δ: 1.18 (d, 6H, NHCH(CH3) CH3), 3.936 (s, 3H, OCH3), 3.965 (s, 3H,
OCH3), 4.140 (septet, 1H, NHCH(CH3) CH3), 7.187 (s, 1H, C5-H of
quinazoline), 7.92 (s, 1H, C8-H of quinazoline), 7.48–8.10 (m, 4H,
Ar-H), 8.24 (d, 1H, NH amidic, D2O exchangeable), 9.96 (s, 1H,
NH, D2O exchangeable). EIMS, m/z: 400 (M+, 100%), 402 (M+2,
35.5%). Anal. Calcd. For C20H21ClN4O3: C, 59.92; H, 5.28; N, 13.98.
Found: C, 60.01; H, 5.63; N, 13.71.
4-[3-(N-Benzylcarbamoyl)anilino]-2-chloro-6,7-
dimethoxyquinazoline (6c)
Compound 6c was prepared as described for 6a from 4 and ben-
zylamine. The product was recrystallized from methanol. Yield:
76%; m.p.: 170–173°C; IR (ν, cm−1): 3346 (NH amidic), 3289
(NH), 1626 (C=O), 1578 cm−1 (C=N). 1H NMR (DMSO-d6) δ: 3.93
(s, 3H, OCH3), 3.96 (s, 3H, OCH3), 4.50 (s, 2H, NH-CH2-Ph), 7.18
(s, 1H, C5-H of quinazoline), 7.33–8.18 (m, 9H, 2 Ar-H), 7.90 (s,
1H, C8-H of quinazoline), 9.06 (t, 1H, NH amidic, D2O exchange-
able), 9.96 (s, 1H, NH, D2O exchangeable). MS, m/z: 448 (M+,
41.9%), 450 (M+2, 14.4%). Anal. Calcd. for C24H21ClN4O3: C,
64.21; H, 4.72; N, 12.28. Found: C, 63.88; H, 5.11; N, 11.98.
4-[3-(t-Butylcarbamoyl)anilino]-2-chloro-6,7-
dimethoxyquinazoline (6h)
Compound 6h was prepared as described for 6a from 4 and
t-butylamine then the product was recrystallized from metha-
nol. Yield, 63%; m.p. 200–202 °C; IR (ν, cm−1): 3374–3600 (NH,
NH amidic), 1630 (C=O), 1575 (C=N). 1H NMR (DMSO-d6) δ:
1.39 (s, 9H, NHC(CH3) 3), 3.93 (s, 3H, OCH3), 3.96 (s, 3H, OCH3),
7.19 (s, 1H, C5-H of quinazoline), 7.91 (s, 1H, C8-H of quinazo-
line), 7.41(m, 4H, Ar-H), 8.03 (s, 1H, NH amidic, D2O exchangea-
ble), 9.945 (s,1H, NH, D2O exchangeable). EIMS, m/z: 414 (M+,
73.6%), 416 (M+2, 32.4%). Anal. Calcd. for C21H23ClN4O3: C,
60.79; H, 5.59; N, 13.50. Found: C, 61.10; H, 5.93; N, 13.17.
4-{3-[(4-Carbethoxypiperazinyl)carbonyl]anilino}-2-
chloro-6,7-dimethoxyquinazoline (6d)
Compound 6d was prepared as described for 6a from 4 and ethyl
piperazinyl-1-carboxylate. The product was recrystallized from
ethanol. Yield: 80%; m.p. 255–257 °C; IR (ν, cm−1): 3501 (NH),
1686 (C=O ester), 1623 (C=O amidic), 1579 (C=N). 1H NMR
(DMSO-d6) δ: 1.12 (t, 3H, COOCH2CH3), 3.46 (t, 4H, 2-CH2 of pip-
erazine), 3.56 (t, 4H, 2-CH2 of piperazine), 3.94(s, 3H, OCH3),
3.96 (s, 3H, OCH3), 4.07 (q, 2H, COOCH2C H 3), 7.18 (s, 1H, C5-H of
quinazoline), 7.80 (s, 1H, C8-H of quinazoline), 7.16–7.84 (m, 4H,
Ar-H), 9.94 (s,1H, NH, D2O exchangeable). EIMS, m/z: 499 (M+,
100%), 501 (M+2, 38.1%). Anal. Calcd. for C24H26ClN5O5.0.65
H2O: C, 56.33; H, 5.32; N, 13.65. Found: C, 56.07; H, 4.94; N,
13.42.
2-Chloro-6,7-dimethoxy-4-[3-(4-phenylpiperazin-1-yl)
anilino]quinazoline (6i)
Compound 6i was prepared as described for 6a from 4 and
1-phenylpiperazine then the product was recrystallized from
methanol. Yield: 75%; m.p.: 198–200 °C; IR (ν, cm−1): 3322
1
(NH), 1630 (C=O), 1594 (C=N). H NMR (DMSO-d6) δ: 3.592–
3.801 (m, 8H, piperazine-H), 3.93 (s, 3H, OCH3), 3.96 (s, 3H,
OCH3), 6.78–7.86 (m, 9H, Ar-H), 7.17 (s, 1H, C5-H of quinazo-
line), 7.83 (s, 1H, C8-H of quinazoline), 9.87 (s, 1H, NH, D2O
exchangeable). EIMS, m/z: 504 (M+, 82.0%), 506 (M+2, 20.8%).
Anal. Calcd. For C27H26ClN5O3.H2O: C, 62.12; H, 5.36; N, 13.42.
Found: C, 61.54; H, 5.61; N, 12.98.
2-Chloro-6,7-dimethoxy-4-{3-[(piperidin-1-yl)carbonyl]
anilino}quinazoline (6e)
Compound 6e was prepared as described for 6a from 4 and pip-
eridine then the product was recrystallized from ethanol. Yield:
75%; m.p.: 240–241 °C; IR (ν, cm−1): 3246 (NH), 1676 (C=O),
3-(3-Methylbenzyloxy)acetanilide (8d)
1
1579 (C=N). H NMR (DMSO-d6) δ: 1.53–1.64 (m, 6H, piperid-
A mixture of m-hydroxyacetanilide (7) (1g, 6mmol), 3-methyl-
benzyl chloride (0.84g, 6mmol), anhydrous K2CO3 (0.2 g,
1.4mmol) and KI (0.2 g, 1.2mmol) in dry acetone (15mL) was
heat to reflux for 11h. The mixture was cooled to room tempera-
ture, solvent was evaporated and the residue was partitioned
between water and ether (3×30mL). The organic layer was
washed with 10% aqueous sodium hydroxide, brine and dried
ine- H), 3.50 (m, 4H, CH2-N-CH2 of piperidine), 3.93 (s, 3H,
OCH3), 3.96 (s, 3H, OCH3), 7.17 (s, 1H, C5-H of quinazoline),
7.15–7.86 (m, 4H, Ar-H), 7.77 (s, 1H, C8-H of quinazoline), 9.83
(s,1H, NH, D2O exchangeable). EIMS, m/z: 426 (M+, 46.2%), 428
(M+2, 14.1%). Anal. Calcd. for C22H23ClN4O3: C, 61.90; H, 5.43; N,
13.12. Found: C, 62.24; H, 5.52; N, 13.21.
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366

Original Article 365
(anhydrous potassium carbonate) and evaporated. The residue
was washed with dry n-hexane then crystallized from chloro-
form. Yield, 76%; m.p. 92–95 °C; IR (ν, cm−1): 3310 (NH amidic)
and 1668 (C=O). 1H NMR (DMSO-d6) δ: 2.03 (s, 3H, CH3CONHPh),
2.32 (s, 3H, PhOCH2PhCH3), 5.01 (s, 2H, PhOCH2P h C H 3), 6.66–
7.36 (m, 8 H, Ar-H), 9.89 (s, 1H, NH amidic, D2O exchangeable).
MS, m/z: 255 (M+, 5.7%).
7H, 2 Ar-H), 7.16 (s, 1H, C5-H of quinazoline), 7.91 (s, 1H, C8-H
of quinazoline), 9.87 (s, 1H, NH, D2O exchangeable). EIMS, m/z:
489(M+,45.5%),491(M+2,14.2%).Anal.Calcd.ForC23H18Cl3N3O3:
C, 56.29; H, 3.70; N, 8.56. Found: C, 56.38; H, 4.00; N, 8.65.
2-Chloro-6,7-dimethoxy-4-[3-(3-methylbenzyloxy)
anilino]quinazoline (10d)
Compound 10d was prepared as described for 10a from 8d
(0.795gm, 3.12mmol) and 3 then the product was recrystallized
from ethanol. Yield, 71.2%; m.p. 185–188°C; IR (ν, cm−1): 3111
(NH) and 1590 (C=N). 1H NMR (DMSO-d6) δ: 2.30 (s, 3H, CH3P h -
CH2O), 3.91(s, 3H, OCH3), 3.94 (s, 3H, OCH3), 4.99 (s, 2H, PhCH2O),
7.02–7.42 (m, 10H, Ar-H and quinazoline – H), 7.87 (s, 1H, NH,
D2O exchangeable). EIMS, m/z: 435 (M+, 7.4%), 437 (M+2, 3.9%).
Anal. Calcd. For C24H22ClN3O3: C, 66.13; H, 5.09; N, 9.64. Found:
C, 66.52; H, 5.15; N, 9.67.
3-[2-(Morpholin-4-yl)ethoxy]acetanilide (8e)
Compound 8e was prepared as described for 8d from 7 and
2-(4-morpholinyl)ethyl chloride (0.89g, 6mmol) and then crys-
tallized from chloroform. Yield: 73.5%; m.p. 67–70 °C; IR (ν,
cm−1): 3296.7 (NH amidic) and 1654.6 (C=O). 1H NMR (DMSO-
d6) δ: 2.03 (s, 3H, CH3CONHPh), 2.47 (t, 4H, CH2-N-CH2 of mor-
pholine), 2.68 (t, 2H, PhOCH2CH2-morpholine), 3.57 (t, 4H,
CH2- O - CH2- of morpholine), 4.03 (t, 2H, PhOCH2C H 2morpho-
line), 6.59-7.31 (m, 4H, Ar-H), 9.92 (s,1H, NH amidic, D2O
exchangeable). MS, m/z: 266 (M+, 7%).
2-Chloro-6,7-dimethoxy-4-{3-[2-(morpholin-4-yl)
ethoxy]anilino}quinazoline (10e)
4-(3-Benzyloxyanilino)-2-chloro-6,7-
dimethoxyquinazoline (10a)
Compound 10e was prepared as described for 10a from 8e
(0.823gm, 3.12mmol) and 3 then the product was recrystallized
from ethanol. Yield: 85.7%; m.p. 245–247 °C; IR (ν, cm−1): 3283
(NH) and 1572 (C=N). 1H NMR (DMSO-d6) δ: 1.91 (t, 4H, CH2-N-
CH2 of morpholine), 2.73 (t, 2H, PhOCH2CH2N of morpholine),
3.75 (t, 4H, CH2- O - CH2 of morpholine), 3.93(s, 3H, OCH3), 3.96
(s, 3H, OCH3), 4.32 (t, 2H, PhOCH2C H 2N of morpholine), 6.81–
7.51 (m, 4H, Ar-H), 7.18 (s, 1H, C5-H of quinazoline), 7.93 (s, 1H,
C8-H of quinazoline), 9.90 (s, 1H, NH, D2O exchangeable). EIMS,
m/z: 444 (M+, 1.2%) 446 (M+2, 1.0%). Anal. Calcd. for
C22H25ClN4O4: C, 59.39; H, 5.66; N, 12.59. Found: C, 59.43; H,
5.75; N, 12.77.
A mixture of 8a (0.75gm, 3.12mmol), NaOH (6.25g, 156mmol)
in ethanol (125mL) and water (30mL) was heated to reflux with
stirring for 7h. The mixture was cooled and the solvent was
removed under vacuum. The residue was partitioned between
chloroform (100mL) and water (100mL). The organic phase was
washed with water (2×100mL), dried (MgSO4), and evaporated
under vacuum to give 9a as oily residue that was mixed with
sodium acetate (0.2g, 2mmol) in absolute ethanol (20mL) was
warmed gently till complete dissolution. The 2,4-dichloro-6,7-
dimethoxyquinazoline (3) (0.5g, 1.93mmol) was added portion-
wise and stirred at room temperature overnight. Precipitate
formed was collected, washed with ethanol, dried and crystal-
lized from ethanol. Yield: 77%; m.p.: 165–167 °C; IR (ν, cm−1):
3371 (NH), 1573 (C=N). 1H NMR (DMSO-d6) δ: 3.92 (s, 3H,
OCH3), 3.96 (s, 3H, OCH3), 5.14 (s, 2H, PhCH2O), 6.85-7.93 (m,
9H, Ar-H), 7.16 (s, 1H, C5-H of quinazoline), 7.93 (s, 1H, C8-H of
quinazoline), 9.87 (s, 1H, NH, D2O exchangeable). EIMS, m/z: 421
(M+, 20.5%), 423 (M+2, 9.0%). Anal. Calcd. for C23H20ClN3O3: C,
65.48; H, 4.78; N, 9.96. Found: C, 65.55; H, 4.92; N, 10.05.
Biological Testing
▼
The human tumor cell lines (MCF-7) were obtained as a gift from
NCI, MD, USA. All chemicals and solvents were purchased from
Sigma-Aldrich.
Assay for EGFR-TK activity inhibition using K-LISA
technique
4-(3-Allyloxyanilino)-2-chloro-6,7-
dimethoxyquinazoline (10b)
EGFR TK inhibitory activity was assayed according to reported
methods [22,23] using EGFR enzyme kit (Merck KGaA, Darm-
stadt, Germany). The assay was performed in 96-well plates pre-
coated with a substrate. In each well, 85μL of 8 μM ATP solution
and 100μL of the standards were added. Then 100μL of each test
compound solution were added at 10μM concentrations. A posi-
tive control for EGFR kinase was used. 100μl of sample diluents
were added to the blank well. After incubation for 1h at 37 °C on
a rotator set at 100rpm, the plate was washed 3 times with 250–
300μL PBS containing 1% Tween 20 (T-PBS) per well. After the
last wash, microwell strips were tapped on absorbent paper
towel to remove excess Wash Buffer. The microwell strips were
used immediately after washing or placing upside down on a
wet absorbent paper for not longer than 15min. Next, 100μL
HRP-conjugate anti-phosphotyrosine monoclonal antibody was
added to all wells. After 1h of incubation at 37°C, the plate was
washed 3 times as previously. TMB substrate solution (100μL)
diluted in T-PBS containing 5mg/mL BSA was added to all wells
including the blank well. The plate was re-incubated at room
temperature (18–25°C) for 15min on a rotator set at 100rpm.
Direct exposure to intense light was avoided. The point at which
Compound 10b was prepared as described for 10a from 8b
(0.595gm, 3.12mmol) and 3 then the product was recrystallized
from ethanol. Yield, 60%; m.p. 190–193 °C; IR (ν, cm−1): 3207
(NH), 1594 (C=N), 1548 (C=C). 1H NMR (DMSO-d6) δ: 3.93(s,
3H, OCH3), 3.96 (s, 3H, OCH3), 4.45 (d, 2H, OCH2CH=CH2), 5.25
(dd, 1H, OCH2CH=(CH)H), 5.36 (dd,1H,OCH2CH=(CH)H), 6.02
(m,1H, OCH2CH=CH2), 6.73–7.91 (m, 4H, Ar-H), 7.14 (s, 1H,
C5-H of quinazoline),8.05 (s, 1H, C8-H of quinazoline), 10.25
(s,1H, NH, D2O exchangeable). EIMS, m/z: 371 (M+, 16.7%). Anal.
Calcd. for C19H18ClN3O3: C, 61.38; H, 4.88; N, 11.30. Found: C,
61.39; H, 4.91; N, 11.33.
2-Chloro-4-[3-(3,4-dichlorobenzyloxy)anilino]-6,7-
dimethoxyquinazoline (10c)
Compound 6c was prepared as described for 10a from 8c
(0.964gm, 3.12mmol) and 3 then the product was recrystallized
from ethanol. Yield: 80.3%; m.p.: 200–202 °C; IR (ν, cm−1):
3421 (NH) and 1580 (C=N). 1H NMR (DMSO-d6) δ: 3.92 (s, 3H,
OCH3), 3.96 (s, 3H, OCH3), 5.16 (s, 2H, PhCH2O), 6.83–8.07 (m,
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366

366 Original Article
8 Chan SK, Gullick WJ, Hill ME. Mutations of the epidermal growth fac-
tor receptor in non-small cell lung cancer – search and destroy. Eur
J Cancer 2006; 42: 17–23
9 Gotoh N. Somatic mutations of the EGF receptor and their signal
transducers affect the efficacy of EGF receptor-specific tyrosine kinase
inhibitors. Int J Clin Exp Pathol 2011; 4: 403–409
10 Pao W, Miller VA, Politi KA et al. Acquired resistance of lung adenocar-
cinomas to gefitinib or erlotinib is associated with a second mutation
in the EGFR kinase domain. PLoS Med 2005; 2: e73
11 Pao W, Miller V, Zakowski M et al. EGF receptor gene mutations are
common in lung cancers from “never smokers” and are associated
with sensitivity of tumors to gefitinib and erlotinib. Proc Natl Acad
Sci USA 2004; 101: 13306–13311
12 Pao W, Chmielecki J. Rational, biologically based treatment of EGFR-
mutant non-small-cell lung cancer. Nat Rev Cancer 2010; 10: 760–774
13 Tevaarwerk AJ, Kolesar JM. Lapatinib: a small-molecule inhibitor of
epidermal growth factor receptor and human epidermal growth fac-
tor receptor-2 tyrosine kinases used in the treatment of breast cancer.
Clin Ther 2009; 31: 2332–2348
the substrate reaction should be stopped may be determined by
the ELISA reader being used. Finally, the reaction was terminated
by the addition of 100μL of 1 M H2SO4 as stop solution, and A492
was measured using an ELISA reader. Results should be read
immediately after addition of the Stop solution or within 1h if
the microwell strips are stored at 4°C in the dark. Absorbance of
each microwell was measured on a spectrophotometer at 450nm
(620nm served as the reference wave length; 610 nm to 650nm is
acceptable). The plate reader was blanked using the blank well.
The absorbance of the samples and the EGFR Standards were
determined. The inhibition rate (%) was calculated using the equa-
tion stated that the inhibition %=[1−(A492/A492 control)] ×100%.
In vitro anti-proliferative activity
Potential cytotoxic activity on human breast carcinoma cell line
(MCF-7) was performed using the Sulfo-Rhodamine-B stain
(SRB) assay according to the method of Skehan et al. [24]. Cells
were plated in 96-multiwell microtiter plate (104 cells/well) for
24h before treatment with the compound(s) to allow the attach-
ment of cell to the wall of the plate. Test compounds were dis-
solved in DMSO and diluted with saline to the appropriate
volume. Different concentrations of the compound under test
(0.1, 2.5, 5, and 10μM/mL) were added to the cell monolayer.
Triplicate wells were prepared for each individual dose. Monol-
ayer cells were incubated with the test compounds for 48h at
37 °C and in atmosphere of 5% CO2. After 48h, cells were fixed,
washed, and stained for 30min with 0.4% (wt/vol) with SRB dis-
solved in 1% acetic acid. Unbound dye was removed by 4 washes
with 1% acetic acid, and attached stain was recovered with Tris-
EDTA buffer. Color intensity was measured in an ELISA reader.
The relation between surviving fraction and drug concentration
is plotted to get the survival curve for breast tumor cell line after
the specified time.
14 Cruz-López O, Conejo-García A, Núñez MC et al. Novel substituted
quinazolines for potent EGFR tyrosine kinase inhibitors. Curr Med
Chem 2011; 18: 943–963
15 Cumming JG, McKenzie CL, Bowden SG et al. Novel, potent and selec-
tive anilinoquinazoline and anilinopyrimidine inhibitors of p38 MAP
kinase. Bioorg Med Chem Lett 2004; 14: 5389–5394
16 Rewcastle GW, Denny WA, Bridges AJ et al. Tyrosine kinase inhibitors.
5. Synthesis and structure-activity relationships for 4-[(phenylmethyl)
amino]- and 4-(phenylamino)quinazolines as potent adenosine 5'-tri-
phosphate binding site inhibitors of the tyrosine kinase domain of the
epidermal growth factor receptor. J Med Chem 1995; 38: 3482–3487
17 Thompson AM, Bridges AJ, Fry DW et al. Tyrosine kinase inhibitors.
7. 7-Amino-4-(phenylamino)- and 7-amino-4-[(phenylmethyl)amino]
pyrido[4,3-d]pyrimidines: a new class of inhibitors of the tyrosine
kinase activity of the epidermal growth factor receptor. J Med Chem
1995; 38: 3780–3788
18 Abouzid K, Shouman S. Design, synthesis and in vitro antitumor activ-
ity of 4-aminoquinoline and 4-aminoquinazoline derivatives targeting
EGFR tyrosine kinase. Bioorg Med Chem 2008; 16: 7543–7551
19 Kamath S, Buolamwini JK. Targeting EGFRand HER-2 receptor tyrosine
kinases for cancer drug discovery and development. Med Res Rev
2006; 26: 569–594
20 Heindel ND, Brodof TA, Kogelschatz JE. Cyclization of amine-acetylene
diester adducts. Modification of the Conrad-Limpach method. J Het-
erocycl Chem 1966; 3: 222–223
21 Althuis TH, Hess HJ. Synthesis and identification of the major metabolites
of prazosin formed in dog and rat. J Med Chem 1977; 20: 146–149
22 Jin Y, Li H-Y, Lin L-P et al. Synthesis and antitumor evaluation of novel
5-substituted-4-hydroxy-8-nitroquinazolines as EGFR signaling-tar-
geted inhibitors. Bioorg Med Chem 2005; 13: 5613–5622
23 Tsou H-R, Mamuya N, Johnson BD et al. 6-Substituted-4-(3-bromophe-
nylamino)quinazolines as Putative Irreversible Inhibitors of the Epi-
dermal Growth Factor Receptor (EGFR) and Human Epidermal Growth
Factor Receptor (HER-2) Tyrosine Kinases with Enhanced Antitumor
Activity. J Med Chem 2001; 44: 2719–2734
24 Skehan P, Storeng R, Scudiero D et al. New colorimetric cytotoxicity
assay for anticancer-drug screening. J. Natl. Cancer Inst 1990; 82:
1107–1112
25 Konecny GE, Pegram MD, Venkatesan N et al. Activity of the Dual
Kinase Inhibitor Lapatinib (GW572016) against HER-2-Overexpress-
ing and Trastuzumab-Treated Breast Cancer Cells. Cancer Res 2006;
66: 1630–1639
26 Stamos J, Sliwkowski MX, Eigenbrot C. Structure of the epidermal
growth factor receptor kinase domain alone and in complex with a
4-anilinoquinazoline inhibitor. J Biol Chem 2002; 277: 46265–46272
27 Yun CH, Boggon TJ, Li Y et al. Structures of lung cancer-derived EGFR
mutants and inhibitor complexes: mechanism of activation and
insights into differential inhibitor sensitivity. Cancer Cell 2007; 11:
217–227
28 Griera R, Armengo M, Reyes A et al. Synthesis and pharmacological
evaluation of new cysLT1 receptor antagonists. Eur J Med Chem 1997;
32: 547–570
Acknowledgement
▼
Authors thank Prof. Mohamed Ayman Al-Zahaby and Dr. Abdel-
sattar Omar, King Abdel-Aziz University, Jeddah, Saudi Arabia for
assisting the molecular modelling part of this work.
Conflict of Interest
▼
The authors report no conflict of interest.
References
1 Blume-Jensen P, Hunter T. Oncogenic kinase signaling. Nature 2001;
411: 355–365
2 Li R, Stafford JA. Kinase Inhibitor Drugs. 1st ed. Hoboken, New Jersey:
John Wiley & Sons, 2009
3 Yarden Y, Sliwkowski MX. Untangling the ErbB signalling network. Nat
Rev Mol Cell Biol 2001; 2: 127–134
4 Garcia R, Franklin RA, McCubrey JA. EGF induces cell motility and
multi-drug resistance gene expression in breast cancer cells. Cell
Cycle 2006; 5: 2820–2826
5 Zhang J, Yang PL, Gray NS. Targeting cancer with small molecule kinase
inhibitors. Nat Rev Cancer 2009; 9: 28–39
29 Brancaccio G, Lettieri G, Viterbo R. Orientation studies in the coumaran
series. Revised structure of the nitration product of 5-acetamido-2-
methylcoumaran via the elucidation of the Claisen rearrangement of
m-acetoamidophenyl allyl ether. Eur J Org Chem 1972; 38: 831–832
30 Dietrich SW, Smith RN, Fukunaga JY et al. Dihydrofolate Reductase
Inhibition by 2,4-Diaminotriazines: A Structure-Activity Study. Arch
biochem biophys 1979; 194: 600–611
6 Cataldo VD, Gibbons DL, Pérez-Soler R et al. Treatment of non-small-
cell lung cancer with erlotinib or gefitinib. N Engl J Med 2011; 364:
947–955
7 Nguyen KS, Kobayashi S, Costa DB. Acquired resistance to epidermal
growth factor receptor tyrosine kinase inhibitors in non-small-cell
lung cancers dependent on the epidermal growth factor receptor
pathway. Clin Lung Cancer 2009; 10: 281–289
Ella DAAE et al. Synthesis and Anti-proliferative Activity… Arzneimittelforschung 2012; 62: 360–366