Enhancement of EGFR tyrosine kinase inhibition by C-C multiple bonds-containing anilinoquinazolines

Article abstract of DOI:10.1016/j.bmc.2009.11.035

A series of 4-anilinoquinazolines with C-C multiple bond substitutions at the 6-position were synthesized and investigated for their potential to inhibit epidermal growth factor receptor (EGFR) tyrosine kinase activity. Among the compounds synthesized, alkyne 6d and allenes 7d and 7f significantly inhibited EGFR tyrosine kinase activity. These compounds inhibited EGF-mediated phosphorylation of EGFR in A431 cells, resulting in cell-cycle arrest and apoptosis induction. The C-C multiple bonds substituted at the C-6 position of the anilinoquinazoline framework were essential for the significant inhibitory activity. Compounds with long carbon chains (n = 3-6), such as 6c-f, 7c-f, 11, and 12, displayed prolonged inhibitory activity.

Full text of DOI:10.1016/j.bmc.2009.11.035

Bioorganic & Medicinal Chemistry 18 (2010) 870–879
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhancement of EGFR tyrosine kinase inhibition by C–C multiple
bonds-containing anilinoquinazolines
*
Hyun Seung Ban, Yuko Tanaka, Wataru Nabeyama, Masako Hatori, Hiroyuki Nakamura
Department of Chemistry, Faculty of Science, Gakushuin University, Tokyo 171-8588, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 4-anilinoquinazolines with C–C multiple bond substitutions at the 6-position were synthe-
sized and investigated for their potential to inhibit epidermal growth factor receptor (EGFR) tyrosine
kinase activity. Among the compounds synthesized, alkyne 6d and allenes 7d and 7f significantly inhib-
ited EGFR tyrosine kinase activity. These compounds inhibited EGF-mediated phosphorylation of EGFR in
A431 cells, resulting in cell-cycle arrest and apoptosis induction. The C–C multiple bonds substituted at
the C-6 position of the anilinoquinazoline framework were essential for the significant inhibitory activity.
Compounds with long carbon chains (n = 3–6), such as 6c–f, 7c–f, 11, and 12, displayed prolonged inhib-
itory activity.
Received 21 October 2009
Revised 17 November 2009
Accepted 17 November 2009
Available online 22 November 2009
Keywords:
EGFR tyrosine kinase inhibitor
Anticancer agent
Quinazoline
Ó 2009 Elsevier Ltd. All rights reserved.
C–C multiple bonds
Allene
1. Introduction
We have been trying to uncover the efficiency of carbon–carbon
multiple bonds, including alkenes, alkynes, and allenes, in medici-
Epidermal growth factor receptor (EGFR) tyrosine kinase plays a
fundamental role in signal transduction pathways,1,2 and the
uncontrolled activation of this EGFR-mediated signaling may be
due to the overexpression of the receptors in numerous tumors,
including head and neck, lung, breast, bladder, prostate, and kidney
cancers; mutations resulting in their constitutive activation in
brain tumors; or the overexpression of the ligands, including EGF
nal chemistry.16,17 Allenes, in particular, have extraordinary prop-
erties, such as the potential for axial chirality and a higher
reactivity than alkenes.18 Therefore, there is much expectation that
the introduction of an allenic moiety to an existing molecular scaf-
fold would improve the biological and pharmacological properties
of the compound.19 Recently, we developed a simple protocol for
the introduction of an allene moiety into various electrophiles un-
der palladium-catalyzed conditions.20–22 Using this protocol, we
found that the introduction of allene into 4-anilinoquinazolines
selectively inhibited EGFR tyrosine kinase activity.17 In this paper,
we demonstrated the introduction of various C–C multiple bond
substituents into the 6-position of the anilinoquinazoline frame-
work and investigated their potential to inhibit EGFR tyrosine
kinase activity.
or TGF-a, in pancreatic, prostate, lung, ovarian, and colon can-
cers.3,4 Therefore, EGFR tyrosine kinase is an attractive target for
novel anticancer drug development.5 Gefitinib (Iressa™)6,7 and
erlotinib (Tarceva™)8 are reversible binding inhibitors of EGFR
kinase activity and have been approved for non-small-cell lung
cancer (NSCLC) therapy. Lapatinib is a dual reversible ErbB inhibi-
tor with potent activity toward EGFR and Her-2 kinases, and has
been approved for breast cancer therapy.9 Although the therapeu-
tic response to those inhibitors can persist for as long as 2–3 years,
most patients who show recurrence ultimately develop acquired
resistance to those agents. In particular, a mutation, T790M, within
the EGFR kinase domain was found in ꢀ50% of the patients.10 In
this regard, irreversible EGFR inhibitors have attracted attention
in recent years because of their potential to inhibit the activity of
EGFR tyrosine kinase with the T790M mutation.11 EKB-569,12
HKI-272,13 and CI-103314,15 have been investigated as irreversible
inhibitors and phase I/II clinical trials are under way (Fig. 1).
2. Chemistry
6-Alkoxyanilinoquinazoline derivatives
5 were synthesized
from 6,7-dimethoxyquinazoline 2 via selective deprotection of
the methyl group from the 6-methoxy group of 2 according to
the procedure in the literature, with modification (Scheme 1).6
Briefly, 2 was treated with D,L-methionine in methanesulfonic acid
to give 6-hydroxy-7-methoxyquinazoline, and acetyl protection of
the phenolic hydroxyl group followed by chlorination with POCl3
gave 3 in 36% yield in three steps. Treatment of 4-chloroquinazo-
line 3 with 3-chloroaniline or 3-chloro-4-fluoroaniline in isopropyl
alcohol gave corresponding 4-anilinoquinazolines 4a and 4b in 69%
* Corresponding author. Tel.: +81 3 3986 0221; fax: +81 3 5992 1029.
E-mail address: hiroyuki.nakamura@gakushuin.ac.jp (H. Nakamura).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.11.035

H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
871
Reversible inhibitors
O
F
O
F
HN
HN
N
Cl
HN
HN
Cl
O
O
S
N
O
O
O
O
N
O
O
N
N
O
N
N
Iressa
Tarceva
Lapatinib
Irreversible inhibitors
F
F
N
O
O
O
HN
N
Cl
N
HN
Cl
O
HN
HN
CN
N
HN
Cl
N
HN
O
CN
N
O
O
N
O
N
CI-1033
EKB-569
HKI-272
Figure 1. Structures of EGFR tyrosine kinase inhibitors.
Table 1
O
N
Cl
Synthesis of alkynes from 6-hydroxyquinazolines 5
O
O
AcO
O
a
N
NH
R1
Cl
n
N
HN
X
3
2
O
n
K2CO3
N
R1
5a-b
DMF,12 h
O
N
HN
N
Cl
6a-f
AcO
O
b
N
N
Compound
R1
n
X
Yielda (%)
R1 = H (69%)
R1 = F (99%)
4a:
b:
6a
6b
6c
6d
6e
6f
H
F
H
F
F
F
1
1
3
3
4
5
Br
Br
I
I
Cl
I
88
>99
78
59
74
48
R1
Cl
HN
N
a
Isolated yields based on 5.
HO
O
c
R1 = H (50%)
R1 = F (quant)
5a:
b:
Scheme 1. Reagents and conditions: (a) (i)
D
,
L
-methionine, CH3SO3H, 120 °C, 12 h;
I
phenol,K 2CO3
(ii) Ac2O, DMAP, pyridine, 0 °C to rt, 12 h; (iii) POCl3, DIPEA, toluene, 120 °C, 14 h,
three steps, 36%; (b) 3-chloroaniline or 3-chloro-4-fluoroaniline, iPrOH, 90 °C, 2 h;
(c) 25% NH3 aq, MeOH, rt, 12 h.
DMF,65 oC,30min
quant.
Scheme 2. Reaction of 1-(4-iodo-1-butynyl)naphthalene with phenol in the
presence of K2CO3 as a base.
and 99% yields, respectively. The removal of the acetyl group with
25% aqueous ammonia in methanol afforded 4-anilino-6-hydroxy-
7-methoxyquinazoline derivatives 5a and 5b. As shown in Table 1,
5a and 5b reacted with various haloalkynes in the presence of
K2CO3 as a base to give corresponding alkynyl ether derivatives
6a–f (n = 1–5) in good to high yields. Interestingly, ether formation
was not observed whereas quantitative recovery of 5a (or 5b) was
observed in the case of the reaction with 4-iodo-1-butyne (n = 2),
although 4-iodo-1-butyne was consumed readily. Indeed, the ether
formation of alcohols upon reacting with b-halogenated alkynes
has not been reported so far. Therefore, we conducted a model
reaction as shown in Scheme 2 to examine the reaction in greater
detail. We synthesized 1-(4-iodo-1-butynyl)naphthalene and re-
acted it with phenol in the presence of K2CO3 as a base in DMF
at 65 °C. 1-(4-Iodo-1-butynyl)naphthalene was consumed in
30 min and the corresponding elimination (E2) product was
obtained quantitatively. The results indicate that b-halogenated
alkynes undergo not the SN2 reaction with alkoxy anions but the
E2 reaction to afford conjugated enynes.
Resultant terminal alkynes 6 were converted into correspond-
ing allenes 7 by treatment with paraformaldehyde and N,N-diiso-
propylamine in the presence of 2 equiv of copper bromide in
dioxane at 110 °C in 29–50% yields.23,24 Recently, we found that
the copper–bromide-catalyzed allene transformation was acceler-
ated by microwave irradiation.25 Therefore, we examined the
transformation of 6d into 7d under microwave condition. The reac-
tion was carried out in a sealed vial and the temperature of the
microwave apparatus was set at 150 °C. The reaction was com-
pleted in 5 min and 7d was obtained in 52% yield (Table 2).
Acetylene-conjugated anilinoquinazoline 10 was also synthe-
sized from 4-anilino-6-hydroxy-7-methoxyquinazoline derivative

872
H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
Table 2
F
Synthesis of allenes from alkynes 6
R1
HN
N
Cl
O
O
a
N
N
HN
N
Cl
5b
(CH2O)n, i-Pr2NH, CuBr
O
n
N
6a-f
dioxane,110oC
b
11
O
F
7a-f
HN
Cl
Compound
R1
n
Time (h)
Yielda (%)
O
O
7a
7b
7c
7d
7e
7f
H
F
H
F
F
F
1
1
3
3
4
5
4
4
4
50
38
22
N
4 (5 min)b
40 (52)b
39
12
4
5
Scheme 4. Reagents and conditions: (a) 6-chloro-1-hexene, K2CO3, KI, DMF, 60 °C,
12 h, 44%; (b) 1-chlorohexane, K2CO3, KI, DMF, 60 °C, 12 h, 52%.
29
a
Isolated yields based on 6.
b
The reaction was carried out in the microwave reactor and the reaction time
and yield were indicated in the parenthesis.
Table 3
In vitro inhibition of EGFR tyrosine kinase activitya
Compd
EGFR inhibition (IC50/nM)
5b, as shown in Scheme 3. Compound 5b was treated with trifluo-
romethanesulfonic anhydride to furnish triflate 8, and 8 under-
went the Sonogashira coupling reaction with ethynyltrimethyl
silane in the presence of Pd(PPh3)4 catalyst in THF under reflux
condition to give 9 in 68% yield. Removal of the TMS group with
K2CO3 in MeOH gave 10, quantitatively.
To compare the inhibitory effects of the C–C multiple bond moi-
eties on EGFR tyrosine kinase activity, hexenyloxy- and hexyloxy-
group-conjugated anilinoquinazolines 11 and 12 were synthesized
from 5b (Scheme 4).
6a
7a
6b
7b
6c
7c
6d
7d
6e
7e
6f
7f
25.9 1.49
16.9 0.82
14.8 5.15
23.8 1.61
17.7 4.85
28.5 0.86
2.53 0.58
3.16 1.33
87.1 1.30
65.5 2.81
32.7 1.55
4.45 2.15
>1000
10
3. Results and discussion
11
12
32.6 1.68
106 11.3
20.3 1.02
6.30 0.10
2.62 0.36
21.0 0.01
Tarceva
AG1476
PD168393
Iressa
3.1. Assay for in vitro kinase inhibitory activity
The inhibitory effect on EGFR tyrosine kinase activity was deter-
mined by measuring the extent of phosphorylation of the tyrosine-
kinase-specific peptide poly(Glu/Tyr) in vitro.26 Table 3 shows the
results of assay for the EGFR tyrosine kinase inhibitory activity of
the synthesized compounds and four EGFR tyrosine kinase inhibi-
tors, Tarceva, AG1476, PD168393, and Iressa. Compounds 6a–f and
7a–f inhibited EGFR tyrosine kinase activity with subnanomolar
IC50 values. Especially, compounds 6d (n = 3), 7d (n = 3), and 7f
a
The drug concentration required to inhibit the phosphorylation of the poly(Glu/
Tyr) substrate by 50% (IC50) was determined from semi-logarithmic dose–response
plots. The results represent mean SD of three independent experiments per-
formed in duplicate.
(n = 5) showed significant inhibition with IC50 values of 2.53,
3.16, and 4.45 nM, respectively. Their inhibitory activities are sim-
ilar to that of AG1476 and PD168393 (IC50 = 6.30 and 2.62 nM,
respectively), known EGFR tyrosine kinase inhibitors, and are high-
er than that of Tarceva and Iressa (IC50 = 20.3, 21.0 nM, respec-
tively). Compound 10, in which an acetylene group is directly
conjugated to the C-6 position of the quinazoline ring, did not dis-
play EGFR tyrosine kinase inhibitory activity (IC50 = >1000 nM). To
the contrary, it should be noted that an acetylene group directly
conjugated to the aniline ring such as Tarceva is essential for EGFR
tyrosine kinase inhibition. We also tested compound 11, which has
a terminal alkene group substitution at the C-6 position, and com-
pound 12, which has a hexyl group substitution at the C-6 position.
Compound 11 displayed higher EGFR tyrosine kinase inhibitory
activity than 6e but lower activity than 7d, although all these three
compounds have six carbon substituents at the C-6 positions of
their quinazoline rings. Meanwhile, compound 12 did not display
significant kinase inhibitory activity (IC50 = 106 nM). The results
indicate that alkyne and allene substitutions at the C-6 position
are essential to induce the EGFR tyrosine kinase inhibitory activity,
and alkene and alkane substituents are less effective although they
have the same carbon number.27
F
HN
N
Cl
a
b
TfO
O
N
c
5b
8
F
F
TMS
HN
Cl
HN
N
Cl
N
N
O
N
O
9
10
Scheme 3. Reagents and conditions: (a) Tf2O, DMAP, pyridine, 0 °C to rt, 12 h, 94%;
(b) ethynyltrimethylsilane, TEA, Pd(PPh3)4, CuI, THF, reflux, 12 h; (c) K2CO3, MeOH/
CH2Cl2, rt, 3 h, two steps, 74%.

H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
873
3.2. Inhibition of EGF-induced EGFR phosphorylation
To clarify the EGFR tyrosine kinase inhibitory activity of the
compounds at the cellular level, we examined the effects of com-
pounds 6a–f and 7a–f on EGF-induced EGFR autophosphorylation
in EGFR-overexpressing A431 cells. The immunoblot analysis in
Figure 2a revealed that all the synthesized compounds suppressed
the EGF-induced EGFR phosphorylation (Tyr1173) in a concentra-
tion-dependent manner and almost complete inhibition was ob-
served at 100 nM without affecting EGFR levels. AG1476, Tarceva,
PD168393, and Iressa also down-regulated EGFR phosphorylation
in a manner similar to the synthesized compounds. Interestingly,
the prolonged inhibitory activity was observed in an experiment
where A431 cells were treated with the compounds and washed
prior to the assay.28 After continuous incubation of A431 cells with
the synthesized compounds, AG1476, Tarceva, PD168393, and Ires-
sa at 10 and 100 nM for 1 h, followed by EGF stimulation, each
compound inhibited the autophosphorylation of EGFR in A431
cells, as shown by the immunoblot analysis (Fig. 2b). When the
cells were washed to remove unbound compounds from the cell
medium and incubated for an additional 5 h prior to EGF stimula-
tion, EGFR autophosphorylation activity was restored in the case of
compounds 6a–b, 7a–b, AG1476, and Tarceva. Meanwhile, for cells
treated with compounds 6c–f, 7c–f, and Iressa, the autophosphory-
lation activity was still inhibited in a manner similar to PD168393,
an irreversible EGFR tyrosine kinase inhibitor.29 These results sug-
gest that compounds 6c–f, 7c–f, and Iressa, showed prolonged
inhibition of EGFR tyrosine kinase activity, whereas other com-
pounds, such as compounds 6a–b, 7a–b, AG1476, and Tarceva,
reversibly interacted with EGFR tyrosine kinase. We speculated
that the prolonged inhibitory activity may be caused by the length
of the side chains substituted at the C-6 position of the quinazoline
rings. Therefore, we compared the prolonged inhibitory activities
of compounds 6e, 7d, 11, and 12, which have the same carbon
number at the C-6 position of the molecules, in a wash-out exper-
iment. As shown in Figure 3, EGFR autophosphorylation activity
was restored by compounds 6e, 7d, 11, and 12. As compounds
6a–b, 7a–b, AG1476, and Tarceva, which have short alkyl or hydro-
philic chains substituted at the C-6 position, did not show pro-
longed inhibition (Fig. 2b), the results indicate that the prolonged
inhibitory activities of compounds 6c–f, 7c–f, 11, and 12 are in-
duced by the hydrophobic interaction between the long alkyl
chains of the compounds and the target protein.
Figure 3. Prolonged inhibition of EGF-induced EGFR phosphorylation by com-
pounds 6e, 7d, 11, and 12. A431 cells were pre-incubated for 10 min with the
indicated concentrations of compounds 6e, 7d, 11, or 12, and then stimulated with
EGF (10 ng/ml) (a) or washed with fresh medium for 5 h prior to stimulation with
EGF (b). Levels of phosphorylated (P)-EGFR and EGFR were detected by immunoblot
analysis with specific antibody. Similar results were observed in three independent
experiments.
3.3. Suppression of cell growth
As EGFR expression is associated with increased tumor cell
growth, the inhibition of EGFR expression is expected to lead to a
reduction in cell growth. Therefore, we next examined the cell
growth inhibitory effect of compounds 6d, 7d, and 7f by the MTT
assay. As shown in Table 4, treatment of A431 cells with com-
pounds 6d, 7d, and 7f resulted in decreased growth rate, and their
GI50 values were 0.221, 0.268, and 0.586 lM, respectively. EGFR
tyrosine kinase inhibitors Tarceva, AG1476, and PD168393 showed
cell growth inhibitory activity with GI50 values of 0.915, 0.302, and
0.021 lM, respectively. These results support the findings of EGFR
tyrosine kinase activity (Table 3) and EGF-induced EGFR phosphor-
ylation (Figure 2). It was reported that the lack of autophosphory-
lation sites resulted in the reduction of internalization and
degradation rates of EGFR.30 Therefore, the prolonged inhibition
of EGFR autophosphorylation by 6d, 7d, and PD168393 induces
Figure 2. Inhibition of EGF-induced EGFR phosphorylation by synthesized compounds. A431 cells were pre-incubated for 10 min with the indicated concentrations of the
synthesized compounds, AG1476 (AG), Tarceva (Tar), or PD168393 (PD), and then stimulated with EGF (10 ng/ml) (a) or washed with fresh medium for 5 h prior to
stimulation with EGF (b). Levels of phosphorylated (P)-EGFR and EGFR were detected by immunoblot analysis with specific antibody. Similar results were observed in three
independent experiments.

874
H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
Table 4
(n = 3–6), such as compounds 6c–f, 7c–f, 11, 12, and Iressa, played
Growth inhibition of A431 cells by 6d, 7d, and 7fa
an important role to display their prolonged inhibitory activity.
Although the irreversible inhibitor PD168393, which is
equipped with a Michael acceptor functional group, is more potent
than 6d, 7d, and Tarceva for the growth inhibition of A431 cells,
the findings presented herein show the potential of introducing
C–C multiple bonds, including alkynes and allenes, into pharmaco-
logically active molecular scaffolds for drug development.
Compound
Growth inhibition (GI50/lM)
6d
7d
7f
Tarceva
AG1476
PD168393
Iressa
0.221 0.023
0.268 0.053
0.586 0.083
0.915 0.171
0.302 0.083
0.021 0.001
0.514 0.045
5. Experimental
5.1. Chemistry
a
A431 cells were incubated for 72 h with various concentrations (1 nM to
lM) of compounds, and then the rates of viable cells were determined by MTT
10
assay. The drug concentration required to inhibit cell growth by 50% (GI50) was
determined from semi-logarithmic dose-response plots. The results represent
mean SD of triplicate samples.
1H NMR and 13C NMR spectra were measured on JEOL JNM-AL
300 (300 MHz) and Varian Unity-Inova 400 (400 MHz) spectrome-
ters. 1H NMR and 13C NMR chemical shifts are expressed in d
(ppm), and coupling constant are expressed in Hz. IR spectra were
measured on a Shimadzu FTIR-8200A spectrometer. Analytical TLC
was performed on glass plates (Merck Kieselgel 60 F254 or RP-18
F254, layer thickness: 0.2 mm). Samples were visualized by UV light
(254 nm), I2, or KMnO4. Column chromatography was performed
on silica gel (Merck Kieselgel 70–230 mesh). All reactions were car-
ried out under argon atmosphere using standard Schlenk tech-
niques. All the chemicals were of analytical grade and used
without further purification.
the extended reduction of internalization and degradation rates of
EGFR, which may lead to the prolonged reduction of cell growth.
3.4. Induction of cell-cycle arrest and apoptosis
It has been established that the inhibition of EGFR by tyrosine
kinase inhibitors induced G1 cell-cycle arrest and apoptosis in hu-
man carcinoma cells.31 To determine whether the cell growth
inhibitory activity of compounds 6d and 7d is mediated by the
induction of cell-cycle arrest and apoptosis or not, biparametric
cytofluorimetric analysis was performed using FITC-labeled annex-
in V and propidium iodide (PI), which, respectively, stain phospha-
tidylserine residues and DNA.32 As shown in Figure 4, compounds
6d and 7d induced cell-cycle arrest in the G1 phase in a concentra-
tion-dependent manner. Furthermore, the population of early
apoptotic cells (annexin V positive and PI negative, B4 area) in-
creased to 13.5% and 13.9% on treatment with compounds 6d
and 7d, respectively (Fig. 5). PD168393 also showed similar activity
for G1 cell-cycle arrest and apoptosis induction. These results indi-
cate that the inhibition of cell growth by compounds 6d and 7d is
due to the induction of cell-cycle arrest and apoptosis in A431 cells.
5.1.1. 6-Acetoxy-4-(30-chloroanilino)-7-methoxyquinazoline 4a
To a solution of 3 (239 mg, 0.95 mmol) in isopropanol (10 ml)
was added 3-chloroaniline (0.11 ml, 1.95 mmol) and the mixture
was refluxed for 2 h with stirring. After cooling to room tempera-
ture, the precipitated solids were filtered and washed with isopro-
panol to give 4a as a white solid (291 mg, 0.85 mmol, 89% yield). 1H
NMR (400 MHz, CD3OD) d 8.85 (s, 3H), 8.39 (s, 1H), 7.91 (s, 1H),
7.66–7.69 (d, J = 8.0 Hz, 1H), 7.49–7.53 (t, J = 8.0 Hz, 1H), 7.39–
7.42 (m, 1H), 7.38 (s, 1H), 7.13 (s, 3H), 2.42 ppm (s, 3H). 13C NMR
(75 MHz, CD3OD) d 170.1, 160.9, 160.3, 151.8, 143.2, 140.6,
139.1, 135.5, 131.3, 128.0, 125.7, 123.8, 119.0, 108.5, 101.8, 57.7,
20.2. IR (KBr) 3359, 3024, 2943, 2715, 1774, 1635, 1574, 1535,
1512, 1443, 1365, 1300, 1234, 1157, 1064, 999, 868 cmꢁ1; MS
(ESI) m/z 344 ([M+H]+). Anal. Calcd for C17H14ClN3O3: C, 59.49; H,
4.10; N, 12.22. Found: C, 59.23; H, 3.95; N, 12.05.
4. Conclusion
We have succeeded in the synthesis of various anilinoquinazo-
lines with C–C multiple bond substitutions at the 6-position and
investigated their potential to inhibit EGFR tyrosine kinase activity.
Among the compounds synthesized, alkyne 6d and allenes 7d and
7f displayed significant inhibitory effects on EGFR tyrosine kinase
activity. These compounds inhibited EGF-mediated phosphoryla-
tion of EGFR in A431 cells, resulting in cell-cycle arrest and apop-
tosis induction. The C–C multiple bonds substituted at the C-6
position of the anilinoquinazoline framework were essential for
the significant kinase inhibitory activity. Long carbon chains
5.1.2. 6-Acetoxy-4-(30-chloro-40-fluoroanilino)-7-methoxyquina
zoline 4b
Compound 4b was prepared from 3 (251 mg, 1.0 mmol) and 3-
chloro-4-fluoroaniline (175 mg, 1.2 mmol) in isopropanol (12 ml)
by the procedure described for 4a to give 4b as a white solid
(374 mg, 1.03 mmol, >99%). 1H NMR (400 MHz, CD3OD) d 8.83 (s,
1H), 8.34 (s, 1H), 7.98–8.00 (m, 1H), 7.67–7.70 (m, 1H), 7.39–7.43
Figure 4. Induction of G1 cell-cycle arrest by compounds 6d and 7d. A431 cells were incubated for 24 h with the indicated concentrations of 6d, 7d, or PD168393 (PD). The
cells were harvested and stained with propidium iodide, and the cell-cycle phases were determined by a flow cytometer. The results represent the mean SD of triplicate
samples.

H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
875
Figure 5. Apoptosis induction by 6d and 7d. A431 cells were incubated for 12 h with the indicated concentrations of 6d, 7d, or PD168393 (PD). The cells were harvested and
stained with annexin V-FITC and propidium iodide (PI), and the percentage of stained cells was determined by a flow cytometer. The results represent the mean SD of
triplicate samples.
(t, J = 8.8 Hz, 1H), 7.37 (s, 1H), 4.12 (s, 3H), 2.42 (s, 3H). 13C NMR
(75 MHz, CD3OD) d 169.9, 161.0, 160.4, 159.5, 156.2, 151.8,
143.2, 140.3, 134.7, 134.6, 128.2, 126.2, 126.1, 122.0, 121.8,
119.0, 118.0, 117.7, 108.4, 101.6, 57.7, 20.2. IR (KBr) 3020, 2623,
1952, 1778, 1639, 1612, 1574, 1501, 1435, 1362, 1319, 1258,
1215, 1153, 1053, 1003, 934, 853 cmꢁ1
. MS (ESI) m/z 320
([M+H]+). Anal. Calcd for C15H11ClFN3O2: C, 56.35; H, 3.47; N,
13.14. Found: C, 56.40; H, 3.21; N, 13.06.
5.1.5. 4-(30-Chloroanilino)-7-methoxy-6-(prop-2-ynyloxy)quin
azoline (6a)
To a solution of 5a (76.5 mg, 0.25 mmol) in DMF (3 ml) were
added propargylbromide (28 ll, 0.38 mmol) and K2CO3 (41.5 mg,
1223, 1177, 1150, 1069, 995, 879 cmꢁ1
; MS (ESI) m/z 362
([M+H]+). Anal. Calcd for C17H13ClFN3O3: C, 56.44; H, 3.62, N
11.62. Found: C, 56.13; H, 3.56; N, 11.50.
0.30 mmol) and the mixture was stirred for 12 h at room temper-
ature. The reaction was quenched with water and the mixture was
extracted with ethyl acetate, dried over anhydrous MgSO4, and
concentrated. Purification by column chromatography on silica
gel with dichloromethane/methanol (20/1) gave 6a as a yellow
solid (74.4 mg, 0.22 mmol, 88% yield). 1H NMR (400 MHz, CD3OD)
d 8.71 (s, 1H), 7.89 (s, 1H), 7.57–7.59 (d, J = 8.0 Hz, 1H), 7.32–7.36
(t, J = 8.4 Hz, 1H), 7.31 (s, 1H), 7.13–7.16 (d, J = 9.2, 1H), 7.10 (s,
1H), 4.94–4.95 (d, J = 2.4 Hz, 2H), 4.04 (s, 3H), 2.63 (s, 1H). 13C
NMR (75 MHz, CD3OD) d 156.1, 155.4, 153.9, 148.2, 147.2, 139.8,
134.7, 130.0, 124.2, 121.6, 119.5, 108.8, 108.5, 102.7, 77.6, 76.6,
57.3, 56.3. IR (KBr) 3449, 1219, 2931, 2124, 1904, 1622, 1597,
1574, 1504, 1483, 1431, 1385, 1230, 1205, 1140, 1099, 1076,
1030, 997, 929, 887, 856, 775, 707, 677, 592, 480 cmꢁ1. MS (ESI)
m/z 340 ([M+H]+). Anal. Calcd for C18H14ClN3O2: C, 63.63; H,
4.15; N, 12.37. Found: C, 63.83; H, 4.06; N, 12.10.
5.1.3. 4-(30-Chloroanilino)-6-hydroxy-7-methoxyquinazoline
(5a)
To a solution of 4a (291 mg, 0.85 mmol) in methanol (7 ml) was
added aqueous ammonia (25% in H2O, 0.85 ml) and the mixture
was stirred for 13 h at room temperature. Solvents were removed
under reduced pressure and the resulting solid was washed with
dichloromethane to give 5a as a white solid (229 mg, 0.76 mmol,
89% yield). 1H NMR (400 MHz, CD3OD) d 8.48 (s, 1H), 7.99 (s,
1H), 7.73 (s, 1H), 7.69–7.71 (d, J = 8.0 Hz, 1H), 7.37–7.41 (t,
J = 8.0 Hz, 1H), 7.23 (s, 1H), 7.16–7.19 (d, J = 10.0 Hz, 1H), 4.09 (s,
3H). 13C NMR (75 MHz, DMSO) d 155.9, 154.0, 151.9, 146.8,
146.2, 141.5, 132.7, 130.0, 122.3, 120.7, 119.7, 109.7, 107.1,
105.4, 55.9. IR (KBr) 3375, 3005, 2496, 1770, 1620, 1582, 1524,
1404, 1277, 1246, 1207, 1150, 1006, 933, 898, 849 cmꢁ1; MS
(ESI) m/z 302 ([M+H]+). Anal. Calcd for C15H12ClN3O2: C, 59.71; H,
4.01; N, 13.93. Found: C, 59.66; H, 4.21; N, 13.87.
5.1.6. 4-(30-Chloro-40-fluoroanilino)-6-(prop-2-ynyloxy)-7-met
hoxyquinazoline (6b)
5.1.4. 4-(30-Chloro-40-fluoroanilino)- 6-hydroxy-7-methoxyqui
nazoline (5b)
Compound 6d was prepared from 5b (120 mg, 0.38 mmol),
Compound 5b was prepared from 4b (371 mg, 1.03 mmol) and
aqueous ammonia (25%, 1 ml) in methanol (8 ml) by the procedure
described for 5a to give 5b as a white solid (341 mg, 1.07 mmol,
>99%). 1H NMR (400 MHz, CD3OD) d 8.45 (s, 1H), 8.03–8.06 (m,
1H), 7.70 (s, 2H), 7.26–7.31 (t, J = 10.0, 1H), 7.23 (s, 1H), 4.09 (s,
3H). 13C NMR (75 MHz, DMSO) d 155.9, 154.5, 153.9, 151.9,
151.3, 146.8, 146.2, 137.2, 137.1, 122.8, 121.7, 121.6, 118.8,
118.5, 116.6, 116.3, 109.5, 107.2, 105.3, 55.9. IR (KBr) d 3386,
2936, 2835, 2484, 1848, 1612, 1585, 1516, 1470, 1339, 1246,
propargylbromide (43 ll, 0.57 mmol), and K2CO3 (79 mg,
0.57 mmol) in DMF (6 ml) by the procedure described for 6a to give
6b as a white solid (136 mg, 0.38 mmol, >99%). 1H NMR (400 MHz,
CD3OD) d 8.67 (s, 1H), 7.86–7.88 (dd, J = 2.8, 6.4 Hz, 1H), 7.50–7.54
(m, 1H), 7.27 (s, 2H), 7.14–7.19 (t, J = 8.8 Hz, 1H), 4.90–4.91 (d,
J = 2.4 Hz, 2H), 4.01 (s, 3H), 2.60–2.62 (t, J = 2.4 Hz, 1H). 13C NMR
(75 MHz, CD3OD) d 156.2, 155.4, 153.8, 148.1, 147.2, 135.1,
124.2, 121.8, 121.7, 116.8, 116.5, 108.6, 108.5, 102.5, 76.6, 74.6,
57.2, 56.3. IR (KBr) 3271, 2131, 1674, 1628, 1583, 1531, 1502,

876
H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
1475, 1431, 1342, 1234, 1145, 997, 856, 661, 561 cmꢁ1. MS (ESI) m/
z 358 ([M+H]+). Anal. Calcd for C18H13ClFN3O2: C, 60.43; H, 3.66; N,
11.75. Found: C, 60.20; H, 3.72; N, 11.90.
5.1.10. 4-(30-Chloro-40-fluoroanilino)-6-(hept-6-ynyloxy)-7-met
hoxyquinazoline (6f)
Compound 6f was prepared from 5b (140 mg, 0.44 mmol), 7-
iodoheptyne (137 mg, 0.66 mmol), and K2CO3 (122 mg, 0.88 mmol)
in DMF (5 ml) by the procedure described for 6c to give 6f as a pale
yellow solid (88 mg, 0.21 mmol, 48%). 1H NMR (400 MHz, CD3OD) d
8.66 (s, 1H), 7.87–7.89 (dd, J = 2.8, 6.4 Hz, 1H), 7.52–7.56 (m, 1H),
7.16–7.20 (t, J = 8.8 Hz, 1H), 7.06 (s, 1H), 7.00 (s, 1H), 4.12–4.16
(t, J = 6.4 Hz, 2H), 4.02 (s, 3H), 2.26–2.27 (q, J = 2.8 Hz, 2H), 1.96–
1.98 (quint, J = 2.8 Hz, 3H), 1.64–1.67 (quint, J = 3.6 Hz, 4H). 13C
NMR (75 MHz, CD3OD) d 156.4, 156.1, 155.3, 153.1, 149.2, 147.5,
135.3, 135.2, 124.1, 121.7, 121.6, 121.2, 121.0, 116.8, 116.5,
108.8, 107.9, 100.3, 84.3, 69.3, 68.5, 56.2, 28.5, 28.0, 25.1, 18.3. IR
(KBr) 3649, 3287, 3255, 2943, 2341, 1626, 1578, 1501, 1473,
1425, 1398, 1354, 1246, 1217, 1146, 1070, 997, 927, 862, 813,
690, 658, 546 cmꢁ1. MS (ESI) m/z 414 ([M+H]+). Anal. Calcd for
C22H21ClFN3O2: C, 63.84; H, 5.11; N, 10.15. Found: C, 63.68; H,
5.19; N, 10.24.
5.1.7. 4-(30-Chloroanilino)-7-methoxy-6-(pent-4-
ynyloxy)quinazoline (6c)
To a solution of 5a (179 mg, 0.59 mmol) in DMF (2 ml) were
added 5-iodo-1-pentyne (230 mg, 1.2 mmol) and K2CO3
(163 mg, 1.2 mmol) and the mixture was stirred for 12 h at
60 °C. The reaction was quenched with water and the mixture
was extracted with ethyl acetate, dried over anhydrous MgSO4,
and concentrated. Purification by column chromatography on sil-
ica gel eluted with hexane/ethyl acetate (1/1) gave the crude
product. Further purification was carried out by recrystallization
from dichloromethane and hexane to afford 6c as a white solid
(161 mg, 0.46 mmol, 78% yield). 1H NMR (400 MHz, CD3OD) d
8.66 (s, 1H), 7.87–7.89 (m, 1H), 7.57–7.59 (m, 1H), 7.31–7.35
(t, J = 8.0 Hz, 1H), 7.12–7.14 (dd, J = 6.0 Hz, 2H), 7.08 (s, 1H),
4.28–4.32 (t, J = 6.4 Hz, 2H), 4.02 (s, 3H), 2.49–2.53 (m, 2H),
2.13–2.19 (quint, J = 6.4 Hz, 2H), 2.04–2.05 (t, J = 2.4 Hz, 1H).
13C NMR (75 MHz, CD3OD) d 155.9, 155.3, 153.5, 149.1, 147.7,
139.9, 134.7, 130.0, 124.1, 121.5, 119.4, 109.0, 108.2, 100.5,
83.5, 69.3, 67.7, 56.2, 27.8, 15.2. IR (KBr) 3292, 3076, 2928,
2120, 1923, 1626, 1599, 1574, 1506, 1473, 1429, 1393, 1285,
1240, 1209, 1148, 1097, 1069, 1043, 1005, 935, 862, 777, 682,
5.1.11. 6-(Buta-2,3-dienyloxy)-4-(30-chlorophenylamino)-7-me
thoxyquinazoline (7a)
To
0.11 mmol), and paraformaldehyde (17 mg, 0.55 mmol) in dioxane
(2 ml) was added diisopropylamine (62 l, 0.44 mmol) under ar-
a mixture of 6a (74.4 mg, 0.22 mmol), CuBr (16 mg,
l
gon atmosphere and the mixture was stirred under refluxed condi-
tion for 4 h. The reaction mixture was filtered by a short silica gel
column chromatography to remove the copper catalysts. Purifica-
tion by column chromatography on silica gel with hexane/ethyl
acetate (2/1) gave 7a as a yellow solid (31 mg, 0.11 mmol, 50%
yield). 1H NMR (400 MHz, CD3OD) d 8.70 (s, 1H), 7.88–7.89 (t,
J = 2.4 Hz, 1H), 7.55–7.57 (d, J = 8.0 Hz, 1H), 7.30–7.34 (t,
J = 8.0 Hz, 1H), 7.20 (s, 1H), 7.13 (s, 1H), 7.12 (s, 1H), 5.47–5.51
(quint, J = 6.4, 1H), 4.92–4.95 (m, 2H), 4.78–4.81 (m, 2H), 4.02 (s,
3H). 13C NMR (75 MHz, CD3OD) d 210.2, 156.1, 155.2, 153.5,
148.1, 147.5, 139.9, 134.6, 129.9, 124.0, 121.4, 119.3, 109.0,
107.9, 101.6, 86.3, 76.6, 67.2, 56.2. IR (KBr) 3557, 2926, 1954,
1626, 1578, 1597, 1514, 1429, 1396, 1354, 1292, 1234, 1144,
1067, 929, 856, 773, 681, 660, 556 cmꢁ1. MS (ESI) m/z 354
([M+H]+). Anal. Calcd for C19H16ClN3O2: C, 64.50; H, 4.56; N,
11.88. Found: C, 64.63; H, 4.62; N, 11.62.
640, 590 cmꢁ1 MS (ESI) m/z 368 ([M+H]+). Anal. Calcd for
.
C20H18ClN3O2: C, 65.31; H, 4.97; N, 11.42. Found: C, 65.27; H,
4.83; N, 11.38.
5.1.8. 4-(30-Chloro-40-fluoroanilino)-7-methoxy-6-(pent-4-
ynyloxy)quinazoline (6d)
Compound 6d was prepared from 5b (197 mg, 0.62 mmol), 5-
iodo-1-pentyne (170 mg, 1.23 mmol), and K2CO3 (170 mg,
1.23 mmol) in DMF (3 ml) by the procedure described for 6c to
give 6d as a yellow solid (138 mg, 0.36 mmol, 59%). 1H NMR
(400 MHz, CD3OD) d 8.66 (s, 1H), 7.88–7.90 (m, 1H), 7.51–7.55
(m, 1H), 7.16–7.20 (t, J = 8.8 Hz, 1H), 7.07 (s, 1H), 7.04 (s, 1H),
4.28–4.31 (t, J = 6.4 Hz, 2H), 4.02 (s, 3H), 2.49–2.53 (m, 2H),
2.15–2.18 (m, 2H), 2.03–2.05 (t, J = 2.8 Hz, 1H). 13C NMR
(75 MHz, CD3OD) d 156.0, 155.3, 153.4, 149.1, 147.6, 135.2,
124.1, 121.7, 121.6, 116.8, 116.5, 108.8, 108.1, 100.4, 83.5, 69.3,
67.6, 56.2, 27.7, 15.2. IR (KBr) 3854, 3649, 3425, 2934, 2345,
1626, 1583, 1500, 1499, 1474, 1429, 1339, 1217, 1146, 1053,
1016, 954, 935, 853 cmꢁ1. MS (ESI) m/z 386 ([M+H]+). Anal. Calcd
for C20H17ClFN3O2: C, 62.26; H, 4.44; N, 10.89. Found: C, 62.26;
H, 4.34; N, 10.80.
5.1.12. 6-(Buta-2,3-dienyloxy)-4-(30-chloro-40-fluoroanilino)-7-
methoxyquinazoline (7b)
Compound 7b was prepared from 6b (132 mg, 0.37 mmol), CuBr
(27 mg, 0.19 mmol), paraformaldehyde (28 mg, 0.93 mmol), and
diisopropylamine (0.11 ml, 0.74 mmol) in dioxane (4 ml) by the
procedure described for 7a to give 7b as a pale yellow solid
(50 mg, 0.14 mmol, 38% yield). 1H NMR (400 MHz, CD3OD) d 8.67
(s, 1H), 7.88–7.91 (dd, J = 2.4, 6.4 Hz, 1H), 7.50–7.52 (m, 1H),
7.14–7.19 (t, J = 8.8 Hz, 1H), 7.13 (s, 1H), 5.47–5.50 (quint,
J = 6.8 Hz, 1H), 4.90–4.93 (m, 2H), 4.75–4.78 (m, 2H), 4.01 (s, 3H).
13C NMR (75 MHz, CD3OD) d 210.0, 156.3, 155.2, 153.4, 153.1,
148.2, 135.3, 124.0, 121.6, 121.5, 121.1, 116.7, 116.4, 107.8,
101.7, 86.2, 67.1, 56.1, 50.6. IR (KBr) 3402, 3080, 2932, 2216,
1955, 1625, 1579, 1504, 1472, 1425, 1377, 1336, 1215, 1142,
1167, 987, 927, 858, 816, 816, 792, 783, 579, 441 cmꢁ1. MS (ESI)
m/z 372 ([M+H]+). Anal. Calcd for C19H15ClFN3O2: C, 61.38; H,
4.07; N, 11.30. Found: C, 61.30; H, 3.90; N, 11.21.
5.1.9. 4-(30-Chloro-40-fluoroanilino)-6-(hex-5-ynyloxy)-7-met
hoxyquinazoline (6e)
Compound 6e was prepared from 5b (197 mg, 0.62 mmol), 6-
chloro-1-hexyne (0.11 ml, 0.93 mmol), KI (10 mg, 0.06 mmol),
and K2CO3 (171 mg, 1.24 mmol) in DMF (3 ml) by the procedure
described for 6c to give 6e as a pale yellow solid (183 mg,
0.46 mmol, 74%). 1H NMR (400 MHz, CD3OD) d 8.66 (s, 1H),
7.88–7.91 (m, 1H), 7.52–7.56 (m, 1H), 7.16–7.20 (t, J = 8.8 Hz,
1H), 7.00 (s, 2H), 4.17–4.20 (t, J = 6.4 Hz, 2H), 7.02 (s, 3H),
2.33–2.37 (m, 2H), 2.05–2.12 (quint, J = 6.4 Hz, 2H), 1.99–2.00
(t, J = 2.4 hz, 1H), 1.76–1.84 (quint, J = 7.6 Hz, 2H). 13C NMR
(75 MHz, CD3OD) d 167.4, 155.4, 155.0, 153.4, 149.2, 147.6,
135.4, 124.0, 121.5, 116.8, 116.5, 108.2, 100.1, 97.5, 84.0, 69.0,
68.9, 56.3, 27.9, 24.9, 18.1. IR (KBr) 3414, 3283, 2955, 2340,
1620, 1578, 1501, 1427, 1394, 1227, 1142, 1005, 929, 845, 816,
640, 552, 515 cmꢁ1. MS (ESI) m/z 400 ([M+H]+). Anal. Calcd for
C21H19ClFN3O2: C, 63.08; H, 4.79; N, 10.51. Found: C, 62.94; H,
4.53; N, 10.26.
5.1.13. 4-(30-Chlorophenylamino)-6-(hexa-4,5-dienyloxy)-7-me
thoxyquinazoline (7c)
Compound 7c was prepared from 6a (161 mg, 0.44 mmol), CuBr
(32 mg, 0.22 mmol), paraformaldehyde (33 mg, 1.1 mmol), and
diisopropylamine (0.12 ml, 0.88 mmol) in dioxane (4 ml) by the
procedure described for 7a to give 7c as a yellow solid (37 mg,
0.097 mmol, 22% yield). 1H NMR (400 MHz, CD3OD) d 8.70 (br,

H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
877
1H), 7.86 (s, 1H), 7.57–7.59 (d, J = 8.4 Hz, 1H), 7.30–7.34 (t,
J = 8.0 Hz, 1H), 7.19 (s, 1H), 7.12–7.14 (d, J = 8.0 Hz, 1H), 7.04 (s,
1H), 5.17–5.24, (quint, J = 6.4 Hz, 1H), 4.71–4.74 (dt, J = 3.6,
6.8 Hz, 2H), 4.15–4.19 (t, J = 6.8 Hz, 2H), 4.01 (s, 3H), 2.23–2.27
(m, 2H), 2.05–2.08 (quint, J = 6.8 Hz, 2H). 13C NMR (75 MHz,
CD3OD) d 208.5, 155.9, 155.3, 149.2, 139.9, 134.6, 130.0, 124.0,
121.6, 119.5, 107.9, 100.4, 89.1, 74.5, 68.6, 56.2, 28.1, 24.4. IR
(KBr) 2931, 1954, 1624, 1599, 1578, 1504, 1471, 1429, 1393,
548 cmꢁ1
.
MS (ESI) m/z 428 ([M+H]+). Anal. Calcd for
C23H23ClFN3O2: C, 64.56; H, 5.42; N, 9.28. Found: C, 64.38; H,
5.51; N, 9.57.
5.1.17. 4-(30-Chloro-40-fluorophenylamino)-7-methoxy-6-
trifluoromethanesulfonoylquinazoline (8)
To a solution of 5b (127 mg, 0.40 mmol) in pyridine (4 ml) was
added Tf2O (0.14 ml, 0.84 mmol) at 0 °C dropwise under argon
atmosphere and then the mixture was stirred at room temperature
for 5 h. After pyridine was removed under reduced pressure, the
residue was purified by column chromatography on silica gel
eluted with dichloromethane/methanol (30/1) to give 8 as a white
solid (170 mg, 0.38 mmol, 94%. 1H NMR (400 MHz, CDCl3) d 8.74 (s,
1H), 7.88–7.90 (dd, J = 2.8 Hz, 6.4 Hz, 1H), 7.73 (s, 1H), 7.49–7.53
(m, 1H), 7.42 (s, 1H), 7.21 (t, J = 8.8 Hz, 1H), 4.07 (s, 3H). 13C NMR
(75 MHz, CDCl3) d 157.4, 157.0, 156.0, 155.3, 153.7, 150.8, 138.2,
134.2, 127.9, 124.9, 122.4, 122.3, 121.4, 121.2, 120.8, 116.9,
116.6, 115.0, 109.6, 108.3, 56.7. IR (KBr) 3452, 3411, 3126, 3259,
1630, 1504, 1421, 1219, 1132, 895, 629, 498 cmꢁ1. MS (ESI) m/z
452 ([M+H]+). Anal. Calcd for C16H10ClF4N3O4S: C, 42.54; H, 2.23;
N, 9.30; S, 7.10. Found: C, 42.71; H, 2.12; N, 9.43; S, 6.96.
1335, 1240, 1209, 1146, 1069, 928, 849, 773, 679, 584, 556 cmꢁ1
.
MS (ESI) m/z 382 ([M+H]+). Anal. Calcd for C21H20ClN3O2: C,
66.05; H, 5.28; N, 11.00. Found: C, 66.06; H, 5.05; N, 11.00.
5.1.14. 4-(30-Chloro-40-fluorophenylamino)-6-(hexa-4,5-dieny
loxy)-7-methoxyquinazoline (7d)
Compound 7d was prepared from 6b (129 mg, 0.33 mmol), CuBr
(24 mg, 0.17 mmol), paraformaldehyde (15 mg, 0.83 mmol), and
diisopropylamine (0.093 ml, 0.66 mmol) in dioxane (2 ml) by the
procedure described for 7a to give 7c as a white solid (53 mg,
0.13 mmol, 40% yield). 1H NMR (400 MHz, CD3OD) d 8.67 (s, 1H),
7.87–7.89 (m, 1H), 7.51–7.55 (m, 1H), 7.16–7.20 (t, J = 8.8 Hz,
1H), 7.05 (s, 1H), 7.01 (s, 1H), 5.19–5.23 (quint, J = 6.8 Hz, 1H),
4.72–4.74 (quint, J = 3.6 Hz, 2H), 4.16–4.19 (t, J = 6.8 Hz, 2H), 4.01
(s, 3H), 2.23–2.29 (m, 2H), 2.04–2.11 (quint, J = 6.8 Hz, 2H). 13C
NMR (75 MHz, CD3OD) d 208.6, 156.1, 155.4, 153.4, 149.3, 147.5,
135.3, 124.2, 121.7, 116.3, 108.9, 108.0, 100.4, 89.1, 75.6, 68.6,
56.2, 28.2, 24.4. IR (KBr) 3425, 2945, 2345, 1949, 1603, 1578,
1500, 1394, 1329, 1285, 1194, 1136, 1057, 1013, 997, 844,
5.1.18. 4-(30-Chloro-40-fluorophenylamino)-6-ethynyl-7-meth
oxyquinazoline (10)
To a mixture of 8 (90 mg, 0.42 mmol), Pd(PPh3)4 (49 mg,
0.04 mmol), and CuI (16 mg, 0.08 mmol) in THF (5 ml) were added
ethynyltrimethylsilane (0.09 ml, 0.63 mmol) and triethylamine
(0.23 ml, 1.7 mmol) under argon atmosphere and the mixture
was stirred under reflux condition for 12 h. After the solvent was
removed under reduced pressure, the residue was filtered with
short column chromatography on silica gel eluted with dichloro-
methane/methanol (10/1) to remove the catalysts. The mixture
was again concentrated and dissolved in dichloromethane/metha-
nol (v/v 2/1, 3 ml). K2CO3 (59 mg, 0.43 mmol) was added and the
mixture was stirred for 3 h at room temperature. The reaction mix-
ture was filtered with Celite and concentrated. Purification by col-
umn chromatography on silica gel with dichloromethane/
methanol (30/1) gave 10 as a white solid (85 mg, 0.31 mmol, 74%
yield). 1H NMR (400 MHz, CD3OD) d 8.70 (s, 1H), 8.00 (s, 1H),
7.93–7.96 (dd, J = 2.8, 6.4 Hz, 1H), 7.49–7.53 (m, 1H), 7.16–7.21
(t, J = 8.4 Hz, 2H), 4.04 (s, 3H). 13C NMR (75 MHz, CD3OD): d
163.0, 153.5, 156.1, 152.2, 134.6, 126.7, 124.3, 121.7, 121.6,
116.9, 116.6, 113.3, 108.9, 107.3, 82.3, 78.8, 56.4. IR (KBr) 3587,
3449, 3288, 3179, 2100, 1624, 1562, 1535, 1500, 1472, 1420,
1383, 1337, 1231, 1132, 1001, 931, 850, 816, 789, 545 cmꢁ1. MS
(ESI) m/z 328 ([M+H]+). Anal. Calcd for C17H11ClFN3O: C, 62.30; H,
3.38; N, 12.82. Found: C, 62.53; H, 3.30; N, 12.68.
815 cmꢁ1
.
MS (ESI) m/z 400 ([M+H]+). Anal. Calcd for
C21H19ClFN3O2: C, 63.08; H, 4.79; N, 10.51. Found: C, 63.13; H,
4.51; N, 10.40.
5.1.15. 4-(30-chloro-40-fluorophenylamino)-6-(hepta-5,6-dieny
loxy)-7-methoxyquinazoline (7e)
Compound 7e was prepared from 6b (174 mg, 0.43 mmol), CuBr
(32 mg, 0.22 mmol), paraformaldehyde (32 mg, 1.1 mmol), and
diisopropylamine (0.12 ml, 0.86 mmol) in dioxane (3 ml) by the pro-
cedure described for 7b to give 7e as a pale yellow solid (70 mg,
0.17 mmol, 39% yield). 1H NMR (400 MHz, CD3OD) d 8.64 (s, 1H),
7.87–7.90 (dd, J = 2.8, 6.8 Hz, 1H), 7.52–7.57 (m, 1H), 7.16–7.20 (t,
J = 8.8 Hz, 2H), 7.00 (s, 1H), 5.13–5.17 (quint, J = 6.8 Hz, 1H), 4.68–
4.71 (quint, J = 2.8 Hz, 2H), 4.14–4.17 (t, J = 6.4 Hz, 2H), 4.01 (s,
3H), 2.12–2.14 (m, 2H), 1.98–2.02 (quint, J = 7.2 Hz, 2H), 1.65–1.69
(quint, J = 7.6 Hz, 2H). 13C NMR (75 MHz, CD3OD) d 208.6, 156.0,
155.4, 153.4, 149.4, 135.3, 124.1, 121.7, 121.6, 116.8, 116.5, 108.1,
100.1, 89.6, 75.1, 69.3, 56.3, 28.4, 27.9, 25.4. IR (KBr) 3649, 2945,
1953, 1624, 1580, 1502, 1429, 1398, 1145, 1057, 1028, 999, 935,
845, 816, 773, 691, 670, 554 cmꢁ1. MS (ESI) m/z 414 ([M+H]+). Anal.
Calcd for C22H21ClFN3O2: C, 63.84; H, 5.11; N, 10.15. Found: C, 63.58;
H, 4.99; N, 10.34.
5.1.19. 4-(30-Chloro-40-fluorophenylamino)-6-(hex-5-enyloxy)-
7-methoxyquinazoline (11)
5.1.16. 4-(3-Chloro-4-fluorophenylamino)-6-(octa-6,7-dienyl
oxy)-7-methoxyquinazoline (7f)
To a mixture of 5b (80 mg, 0.25 mmol), K2CO3 (53 mg,
0.38 mmol), and KI (4.2 mg, 0.03 mmol) in DMF (2 ml) was added
6-chloro-1-hexene (0.05 ml, 0.38 mmol) and the mixture was stir-
red at 60 °C for 12 h. The reaction was quenched with water and
the reaction mixture was extracted with ethyl acetate, dried over
MgSO4, and concentrated. Purification by column chromatography
on silica gel with dichloromethane/methanol (30/1) gave the crude
11. Further purification was carried out by recrystallization from
methanol to give 11 as a white solid (44 mg, 0.11 mmol, 44% yield).
1H NMR (400 MHz, CD3OD) d 8.64 (s, 1H), 7.87–7.90 (dd, J = 2.8,
6.4 Hz, 1H), 7.52–7.57 (m, 1H), 7.16–7.20 (t, J = 8.4 Hz, 2H), 7.00
(s, 1H), 5.80–5.89 (q, J = 7.2 Hz, 1H), 5.00–5.09 (m, 2H), 4.14–4.17
(t, J = 6.8 Hz, 2H), 4.01 (s, 3H), 2.17–2.19 (q, J = 7.6 Hz, 2H), 1.95–
1.99 (quint, J = 7.2 Hz, 2H), 1.65–1.67 (quint, J = 8.0 Hz, 2H). 13C
NMR (75 MHz, CD3OD) d 156.2, 155.3, 153.1, 153.0, 149.2, 146.9,
138.2, 135.2, 121.9, 121.8, 121.1, 120.8, 116.7, 116.4, 114.9,
Compound 7f was prepared from 6b (88 mg, 0.21 mmol), CuBr
(15 mg, 0.11 mmol), paraformaldehyde (16 mg, 0.53 mmol), and
diisopropylamine (0.06 ml, 0.42 mmol) in dioxane (3 ml) by the
procedure described for 7b to give 7f as a white solid (25 mg,
0.1706 mmol, 29% yield). 1H NMR (400 MHz, CD3OD) d 8.66 (s,
1H), 7.86–7.88 (dd, J = 2.4, 6.4 Hz, 1H), 7.51–7.54 (m, 1H), 7.15–
7.19 (t, J = 8.8 Hz, 2H), 7.01 (s, 1H), 5.10–5.13 (quint, J = 6.4 Hz,
1H), 4.65–4.68 (quint, J = 3.2 Hz, 2H), 4.09–4.13 (t, J = 6.4 Hz, 2H),
4.00 (s, 3H), 2.05–2.07 (m, 2H), 1.93–1.96 (quint, J = 6.8 Hz, 2H),
1.53–1.55 ppm (m, 4H). 13C NMR (75 MHz, CD3OD) d 208.5,
156.5, 155.3, 153.2, 149.4, 135.3, 124.2, 121.8, 121.0, 116.8,
100.3, 89.7, 74.8, 69.4, 56.2, 29.0, 28.8, 28.7, 28.1, 25.4. IR (KBr)
3649, 3281, 3080, 2936, 2339, 1954, 1626, 1578, 1499, 1473,
1429, 1396, 1339, 1244, 1217, 1146, 1070, 995, 849, 815, 691,

878
H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
108.9, 107.3, 100.6, 69.2, 56.1, 33.3, 28.3, 25.1. IR (KBr) 3649, 3283,
3078, 2941, 2361, 1626, 1578, 1502, 1474, 1427, 1398, 1354, 1219,
1146, 1070, 997, 927, 907, 849, 794, 691, 546 cmꢁ1. MS (ESI) m/z
402 ([M+H]+). Anal. Calcd for C21H21ClFN3O2: C, 62.76; H, 5.27; N,
10.46. Found: C, 62.53; H, 5.44; N, 10.20.
expression was visualized with a ChemiDoc XRS image analyzer
(Bio-Rad).
5.1.20.4. Cell-cycle analysis. After incubation of A431 cells with
the drugs, the cells were washed with PBS and fixed with 70% eth-
anol for 2 h at 4 °C. The cells were incubated for 30 min at 37 °C in
1 ml of RNase solution (0.25 mg/ml in PBS), and further incubated
for 30 min at 4 °C in propidium iodide (PI) staining solution
(0.05 mg/ml in PBS). The suspension was then passed through a
5.1.20. 4-(3-Chloro-4-fluorophenylamino)-6-hexyloxy-7-metho
xyquinazoline (12)
Compound 12 was synthesized from 5b (79 mg, 0.24 mmol)
with K2CO3 (51 mg, 0.37 mmol), KI (4.0 mg, 0.03 mmol), and 1-
chlorohexane (0.051 ml, 0.37 mmol) by the procedure, described
for 11 to give 12 as a white solid (51 mg, 0.13 mmol, 52% yield).
1H NMR (400 MHz, CD3OD) d 8.61 (s, 1H), 7.88–7.91 (dd, J = 2.8,
6.8 Hz, 1H), 7.56–7.58 (m, 1H), 7.25 (s, 1H), 7.15–7.20 (t,
J = 8.8 Hz, 1H), 7.07 (s, 1H), 4.13–4.16 (t, J = 6.8 Hz, 2H), 4.00 (s,
3H), 1.90–1.97 (quint, J = 7.2 Hz, 2H), 1.36–1.52 (m, 6H), 0.91–
nylon mesh filter (40 lm) and analyzed by a Cytomics FC500 flow
cytometer (Beckman Coulter).
5.1.20.5. Apoptosis detection. Phosphatidylserine externaliza-
tion was measured using an annexin V-FITC kit (Beckman Coulter)
according to the manufacturer’s instructions. After incubation of
A431 cells with the drugs, the cells were suspended in 100
binding buffer, and 5 l annexin V-FITC (5 g/ml) and 2.5
(250 g/ml) were added. The cell suspensions were incubated for
l
l
l 1ꢂ
0.94 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, CD3OD)
d
156.2,
l
l
l PI
155.5, 152.6, 149.4, 146.3, 135.1, 124.3, 121.9, 116.7, 116.4,
108.7, 107.0, 101.3, 100.5, 69.6, 56.2, 31.5, 28.9, 25.6, 22.5,
14.0. IR (KBr) 3275, 3084, 2934, 2332, 1636, 1578, 1501, 1474,
1427, 1339, 1246, 1219, 1146, 1070, 999, 862, 816, 690,
l
10 min at 4 °C and analyzed by a flow cytometer.
Acknowledgements
554 cmꢁ1
.
MS (ESI) m/z 404 ([M+H]+). Anal. Calcd for
C21H23ClFN3O2: C, 52.45; H, 5.74; N, 10.40. Found: C, 62.45; H,
5.74; N, 10.33.
This work was supported in part by a Grant-in-Aid for Scientific
Research on Priority Areas ‘Cancer Therapy’ from the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
5.1.20.1. Kinase assay. The kinase activity of various tyrosine
kinases was determined by ELISA.26 EIA/RIA strip-well plates
(Corning) were coated with poly(Glu/Tyr, 4:1) peptide (Sigma,
References and notes
50
phate-buffered saline (PBS, Ca/Mg-free). Kinase reaction was per-
formed in the plates by addition of 50 l kinase buffer (50 mM
HEPES, 125 mM NaCl, 10 mM MgCl2, pH 7.4) containing ATP,
10 ng recombinant EGFR (Invitrogen, catalytic domain), and inhib-
itors. After 20 min, the plates were washed three times with wash
buffer (0.1% Tween 20 in PBS) and incubated for 20 min with HRP
lg/ml, 100 ll per well) by incubation overnight at 4 °C in phos-
1. Aaronson, S. A. Science 1991, 254, 1146.
2. Ullrich, A.; Schlessinger, J. Cell 1990, 61, 203.
3. Blume-Jensen, P.; Hunter, T. Nature 2001, 411, 355.
4. Kim, H.; Muller, W. J. Exp. Cell Res. 1999, 253, 10.
l
5. Bridges, A. J. Chem. Rev. 2001, 101, 2541.
6. Barker, A. J.; Gibson, K. H.; Grundy, W.; Godfrey, A. A.; Barlow, J. J.; Healy, M. P.;
Woodburn, J. R.; Ashton, S. E.; Curry, B. J.; Scarlett, L.; Henthorn, L.; Richards, L.
Bioorg. Med. Chem. Lett. 2001, 11, 1911.
7. Vansteenkiste, J. F. Expert Rev. Anticancer Ther. 2004, 4, 5.
8. Shepherd, F. A.; Rodrigues, P. J.; Ciuleanu, T.; Tan, E. H.; Hirsh, V.; Thongprasert,
S.; Campos, D.; Maoleekoonpiroj, S.; Smylie, M.; Martins, R.; van Kooten, M.;
Dediu, M.; Findlay, B.; Tu, D.; Johnston, D.; Bezjak, A.; Clark, G.; Santabárbara,
P.; Seymour, L. N. Eng. J. Med. 2005, 353, 10.
9. Nelson, M. H.; Dolder, C. R. Ann. Pharmacother. 2006, 40, 261.
10. Paez, J. G.; Janne, P. A.; Lee, J. C.; Tracy, S.; Greulich, H.; Gabriel, S.; Herman, P.;
Kaye, F. J.; Lindeman, N.; Boggon, T. J.; Naoki, K.; Sasaki, H.; Fujii, Y.; Eck, M. J.;
Sellers, W. R.; Johnson, B. E.; Meyerson, M. Science 2004, 304, 1497.
11. Kwak, E. L.; Sordella, R.; Bell, D. W.; Godin-Heymann, N.; Okimoto, R. A.;
Brannigan, B. W.; Harris, P. L.; Driscoll, D. R.; Fidias, P.; Lynch, T. J.; Rabindran, S.
K.; McGinnis, J. P.; Wissner, A.; Sharma, S. V.; Isselbacher, K. J.; Settleman, J.;
Haber, D. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 7665.
12. Torrance, C. J.; Jackson, P. E.; Montgomery, E.; Kinzler, K. W.; Vogelstein, B.;
Wissner, A.; Nunes, M.; Frost, P.; Discafani, C. M. Nat. Med. 2000, 6, 1024.
13. Rabindran, S. K.; Discafani, C. M.; Rosfjord, E. C.; Baxter, M.; Floyd, M. B.; Golas,
J.; Hallett, W. A.; Johnson, B. D.; Nilakantan, R.; Overbeek, E.; Reich, M. F.; Shen,
R.; Shi, X.; Tsou, H.-R.; Wang, Y.-F.; Wissner, A. Cancer Res. 2004, 64, 3958.
14. Smaill, J. B.; Showalter, H. D. H.; Zhou, H.; Bridges, A. J.; McNamara, D. J.; Fry, D.
W.; Nelson, J. M.; Sherwood, V.; Vincent, P. W.; Roberts, B. J.; Elliott, W. L.;
Denny, W. A. J. Med. Chem. 2000, 44, 429.
conjugated anti-phosphotyrosine antibody (0.2
well, Santa Cruz). After two washes, the plates were developed
by the addition of tetramethylbenzidine (50 l per well, Sigma)
and stopped by addition of H2SO4 (2 N, 50 l per well). The absor-
lg/ml, 50 ll per
l
l
bance at 450 nm was measured by a 96-well plate reader (Tecan).
5.1.20.2. Cell growth assays. The human epidermoid carcinoma
cell line A431 was used for the cell growth assays. The cells
(5 ꢂ 103 cells per well of a 96-well plate) were incubated at
37 °C for 72 h in 100 ll RPMI-1640 medium containing various
concentrations of drugs. After incubation, MTT (10 ll, 5 mg/ml in
PBS; MTT [30-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide, Sigma] was added to each well, and the cells were further
incubated at 37 °C for 4 h. After removal of the medium, DMSO
(100 ll) was added, and the absorbance at 570 nm was determined
by a 96-well plate reader.
15. Tiseo, M.; Loprevite, M.; Ardizzoni, A. Curr. Med. Chem. Anti-Cancer Agents 2004,
4, 139.
16. Kim, H.; Williams, L. Curr. Opin. Drug Disc. Dev. 2008, 11, 870.
17. Ban, H. S.; Onagi, S.; Uno, M.; Nabeyama, W.; Nakamura, H. ChemMedChem
2008, 3, 1094.
18. Krause, N.; Hoffmann-Röder, A.. In Krause, N., Hashmi, S., Eds.; Modern Allene
Chemistry; Wiley-VCH: Weinheim, 2008; Vol. 2,. p 997.
19. Brzozowski, T.; Konturek, P. C.; Konturek, S. J.; Brzozowska, I.; Pawlik, T. J. Phys.
Pharmacol. 2005, 56, 33.
20. Nakamura, H.; Kamakura, T.; Ishikura, M.; Biellmann, J. F. J. Am. Chem. Soc.
2004, 126, 5958.
21. Nakamura, H.; Onagi, S.; Kamakura, T. J. Org. Chem. 2005, 70, 2357.
22. Nakamura, H.; Kamakura, T.; Onagi, S. Org. Lett. 2006, 8, 2095.
23. Searles, S.; Li, Y.; Nassim, B.; Lopes, M.-T. R.; Tran, P. T.; Crabbé, P. J. Chem. Soc.,
Perkin Trans. 1 1984, 747.
24. Crabbé, P.; Fillion, H.; André, D.; Luche, J.-L. J. Chem. Soc., Chem. Commun. 1979, 859.
25. Nakamura, H.; Sugiishi, T.; Tanaka, Y. Tetrahedron Lett. 2008, 49, 7230.
26. Barbacci, E. G.; Pustilnik, L. R.; Rossi, A. M.; Emerson, E.; Miller, P. E.; Boscoe, B.
P.; Cox, E. D.; Iwata, K. K.; Jani, J. P.; Provoncha, K.; Kath, J. C.; Liu, Z.; Moyer, J. D.
Cancer Res. 2003, 63, 4450.
5.1.20.3. Immunoblot analysis. After drug treatment for a speci-
fied period, the cells were washed three times with PBS, dipped in
100
ll ice-cold lysis buffer (20 mM HEPES, pH 7.4, 1% Triton X-100,
10% glycerol, 1 mM sodium vanadate, 5
lg/ml leupeptin, and
1 mMEDTA) for15 min, anddisruptedwitha HandySonicDisrupter,
and the lysate was boiled for 5 min in a sample buffer (50 mM Tris,
pH 7.4, 4% sodium dodecyl sulfate (SDS), 10% glycerol, 4% 2-thioeth-
anol, and 0.05 mg/ml bromophenol blue) at a ratio of 4:1. The
proteinswereseparatedby10%SDS–polyacrylamidegelelectropho-
resis and transferred onto a PVDF membrane (GE Healthcare). Anti-
bodies used for immunoblotting were phospho-EGFR (Tyr1173)
antibody and EGFR antibody (Santa Cruz). After incubation with
horseradish peroxidase-conjugated secondary antibody (Santa
Cruz), the blot was treated with ECL kit (GE Healthcare), and protein

H. S. Ban et al. / Bioorg. Med. Chem. 18 (2010) 870–879
879
27. We carried out the docking simulation of compounds 6d, 7d, and 10 using DS
modeling ver. 2.0 based on the X-ray analysis data of EGFR tyrosine kinase
(PDB code: 1M17). However, we could not obtain their reliable binding modes
except for compound 10. Therefore, we could not clarify the binding modes of
compounds for the current enhanced inhibitory activities in details.
28. Tsou, H.-R.; Mamuya, N.; Johnson, B. D.; Reich, M. F.; Gruber, B. C.; Ye, F.;
Nilakantan, R.; Shen, R.; Discafani, C.; DeBlanc, R.; Davis, R.; Koehn, F. E.;
Greenberger, L. M.; Wang, Y.-F.; Wissner, A. J. Med. Chem. 2001, 44, 2719.
29. Fry, D. W.; Bridges, A. J.; Denny, W. A.; Doherty, A.; Greis, K. D.; Hicks, J. L.;
Hook, K. E.; Keller, P. R.; Leopold, W. R.; Loo, J. A.; McNamara, D. J.; Nelson, J. M.;
Sherwood, V.; Smaill, J. B.; Trumpp-Kallmeyer, S.; Dobrusin, E. M. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 12022.
30. Decker, S. J.; Alexander, C.; Habib, T. J. Biol. Chem. 1992, 267, 1104.
31. Wu, X.; Fan, Z.; Masui, H.; Rosen, N.; Mendelsohn, J. J. Clin. Invest. 1995, 95, 1897.
32. Nakamura, H.; Kuroda, H.; Saito, H.; Suzuki, R.; Yamori, T.; Maruyama, K.; Haga,
T. ChemMedChem 2006, 1, 729.