2,3-Disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines: A novel class of antitumor agents

Article abstract of DOI:10.1016/j.ejmech.2008.01.009

A series of 2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines were synthesized and evaluated for their cytotoxic activity in vitro against five human cancer cell lines (human lung carcinoma cell line: A549, human leukemia cell lines: K562 and Molt-4, human prostate cancer cell line: PC-3, human breast carcinoma cell line: MDA-MB-231). Most of these compounds show potent activity against these tumor cell lines, especially against the A549 cell line. The cell cycle analysis was also studied by flow cytometry measurement on A549 cell line.

Full text of DOI:10.1016/j.ejmech.2008.01.009

Available online at www.sciencedirect.com
European Journal of Medicinal Chemistry 44 (2009) 448e452
http://www.elsevier.com/locate/ejmech
Short communication
2,3-Disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines:
A novel class of antitumor agents
a,
b,**
, Yongping Yu
*
Yandong Zhang a, Zhe Chen b, Yijia Lou
a Institute of Material Medica, College of Pharmaceutical Science, Zhejiang University, Zijin Campus, Hangzhou 310058, PR China
b Institute of Pharmacology & Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Science, Zhejiang University, Zijin Campus,
Hangzhou 310058, PR China
Received 23 August 2007; received in revised form 20 December 2007; accepted 10 January 2008
Available online 25 January 2008
Abstract
A series of 2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines were synthesized and evaluated for their cytotoxic activity in vitro
against five human cancer cell lines (human lung carcinoma cell line: A549, human leukemia cell lines: K562 and Molt-4, human prostate cancer
cell line: PC-3, human breast carcinoma cell line: MDA-MB-231). Most of these compounds show potent activity against these tumor cell lines,
especially against the A549 cell line. The cell cycle analysis was also studied by flow cytometry measurement on A549 cell line.
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords: Imidazoquinazoline derivatives; Antitumor activity
1. Introduction
incorporating the imidazo moiety on its 6- and 7-positions
[15e23]. In this present work, we would like to report the
2,3-disubstituted-8-arylamino-3H-imidazo[4,5-g]quinazoline’s
cytotoxic activity in vitro against human lung cell lines
(Fig. 2). Several of these compounds showed promising anti-
proliferative effect against tumor cell lines.
Quinazoline compounds have been well-recognized for
their pharmacological properties, such as anticonvulsant
[1,2], sedative, antihypertensive [3,4], vasodilator [5], antiin-
flammatory [6], antibiosis [7], phosphodiesterase inhibitors
[8], and fibrinogen receptor antagonists [9]. Among them, 4-
anilinoquinazolines are verified as the most promising small
molecule EGFR tyrosine kinase inhibitors [10e14]. For exam-
ple, PD153035 (6,7-dimethoxy-4-(3-bromophenyl)amino-qui-
nazoline) was discovered as the potent inhibitor of EGFR
tyrosine kinase (IC50 0.025 nM) [14]. IRESSAÔ (gefitinib,
ZD1839), the novel drugs granted by FDA, was used for treat-
ment of non-small-cell lung cancer (Fig. 1).
Most of the SAR researches of 4-anilinoquinazolines fo-
cused on the variation of substitutions at its 6- and 7-positions
as well as the substitutions at the 4-anilino portion. However,
the activity of the 4-anilinoquinazoline family could be mod-
ulated by changing the quinazoline core structure through
2. Results and discussion
2.1. Chemistry
2.1.1. Synthesis of derivatives
The synthesis of a series of 2,3-disubstituted-8-arylamino-
3H-imidazo[4,5-g]quinazolines (Scheme 1) was accomplished
according to our procedure reported previously [24]. The
methodology of parallel solid-phase synthesis was employed
using arylamines as the first building blocks, alkylamines as
the second building blocks, alkylaldehydes as the third build-
ing blocks and 4-chloro-7-fluoro-6-nitroquinazoline as the
scaffold. The reaction sequence is illustrated in Scheme 1.
Starting from 4-(4-formyl-3-methoxyphenoxy)butyryl AM resin
1, in the presence of NaBH3CN in DMF, an arylamine was
attached to the resin by reductive amination. The resin-bound
* Corresponding author. Tel./fax: þ86 571 88208452.
** Corresponding author.
E-mail addresses: yijialou@zju.edu.cn (Y. Lou), yyu@zju.edu.cn (Y. Yu).
0223-5234/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.01.009

Y. Zhang et al. / European Journal of Medicinal Chemistry 44 (2009) 448e452
449
cytotoxicity on A549 cell line, such as compounds 7a
(IC50 ¼ 4.59 mM, R2 ¼ isopropyl), 7b (IC50 ¼ 2.81 mM,
R2 ¼ isobutyl) and 7c (IC50 ¼ 6.05 mM, R2 ¼ ethyl). How-
ever, long alkyl chain or butyl group on the R2 position is
detrimental to the compound’s cytotoxicity, such as com-
pounds 7e (IC50 ¼ 22.05 mM, R2 ¼ benzyl) and 7f
(IC50 ¼ 38.98 mM, R2 ¼ n-hexyl). Compared with the data
of 7a, 7gej, which have the identical substitutions on R1
and R2 positions, the effect of R3 group on the compound’s
cytotoxic activity is not apparent. All these compounds dis-
play the good cytotoxic activity with different substitutions
on R3 position, except a little loss of activity of compound
7j (R3 ¼ g-methoxypropyl, IC50 ¼ 15.76 mM). With identical
substitutions on R2 and R3 positions, 40-F (compound 7k,
IC50 ¼ 4.25 mM), and 30,40-di(OCH3) (compound 7l,
IC50 ¼ 5.58 mM) were found as promising substitutions on
the R1 position after the comparison of the data 7kem. In
addition, compound 7k was discovered as a promising and
potent inhibitor against most of the selected cell lines (with
IC50 ¼ 4.25 mM against A549 cell line, IC50 ¼ 5.55 mM
against K562 cell line, IC50 ¼ 9.74 mM against PC-3 cell
line, IC50 ¼ 11.43 mM against Molt-4 cell line).
Fig. 1. The structure of 4-anilinoquinazolines, PD153035 and IRESSA.
arylamine 3 was then reacted with 4-chloro-7-fluoro-6-nitro-
quinazoline scaffold 8 to yield the corresponding chemoselec-
tive resin-bound quinazoline 4, which was then treated with an
alkylamine to give resin-bound compound 5. The imidazo ring
of resin-bound compound 6 was formed through the reduction
of nitro group of resin-bound compound 5 with tin chloride
and intramolecular cyclization with an alkylaldehyde in one
step at 50 ꢀC. Then the desired 2,3-disubstituted 8-aryla-
mino-3H-imidazo[4,5-g]quinazoline 7 was obtained in good
yield and purity after the cleavage of resin-bound compound
6 by using TFA/DCM (1:1). The products were characterized
Next, flow cytometry was used to evaluate the effects of the
most active compounds (7a, 7b and 7k) on the growth and di-
vision of A549 cells, by measuring the DNA content of eu-
karyotic cells. The results show the proportion of cells
emitting a given level of fluorescent proportional to the
DNA content. Unlike antimitotic agents which accumulate
their DNA content in the G2/M phase of the cell cycle, the re-
sults in Table 2 show that compounds (7a, 7b and 7k) cause
significant arrest of the cell cycle at the G0/G1 phase. As
shown in Table 2, compounds 7a, 7b and 7k caused significant
arrest of the cell cycle at the G0/G1 phase, with a correspond-
ing decrease in the proportion of cells in S phase in compari-
son with control cultures. This result is consistent with the
behavior of an EGFR tyrosine kinase inhibitor, IRESSA
(ZD1839).
1
by electrospray LCeMS and H NMR.
2.2. Biological activities
All compounds were evaluated for their cytotoxic activity
in vitro against five human cancer cell lines (human lung car-
cinoma cell line: A549, human leukemia cell lines: K562 and
Molt-4, human prostate cancer cell line: PC-3, human breast
carcinoma cell line: MDA-MB-231) using IRESSA as the ref-
erence drug. The results are summarized in Table 1.
As shown in Table 1, most of compounds exhibited inhi-
bitions on the growth of selected tumor cell lines, especially
on A549 cell line and K562 cell line. Preliminary structuree
activity relationship of this series of compounds’ cytotoxicity
against A549 cell line was investigated. The data of com-
pound 7aef in Table 1 summarize the effect of different
R2 group on their activity, with identical substitutions on
R1 and R3 positions of these compounds. In general, short chain
or little steric hindrance of R2 substitutions were beneficial for
2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazoline’s
3. Conclusion
The antiproliferative activity of 2,3-disubstituted 8-aryla-
mino-3H-imidazo[4,5-g]quinazolines against a variety of can-
cer cell lines was evaluated. Most of them displayed potent
cytotoxic activity in the micromolar range, especially against
the A549 cell line and K562 cell line. The preliminary SAR
of this series of compounds’ cytotoxic activity against A549
cell line was investigated. The cell cycle analysis on A549
cell line indicated that these compounds cause significant ar-
rest of the cell cycle at the G0/G1 phase.
4. Experimental protocols
1H NMR and spectra were recorded on a Bruker AM 400
instrument at 400 and 500 MHz (chemical shifts are expressed
as d values relative to TMS as internal standard). LCeMS
(ESI) spectra were recorded a Finnigan Mat LCQ mass
Fig. 2. The structure and positional designation of 2,3-disubstituted 8-aryla-
mino-3H-imidazo[4,5-g]quinazolines.

450
Y. Zhang et al. / European Journal of Medicinal Chemistry 44 (2009) 448e452
Scheme 1. Solid-phase synthesis of 2,3-disubstituted 8-arylamino-3H-imidazo[4,5-g]quinazolines 7. Reagents and conditions: (a) NaBH3CN (10 equiv, 0.1 M) in
DMF/AcOH (99:1), rt, 24 h, (b) 4-chloro-7-fluoro-6-nitroquinazoline 8 in THF (10 equiv, 0.1 M), Et3N (10 equiv, 0.1 M), 24 h, repeat, (c) R3NH2 (20 equiv, 0.2 M)
in DCM, 24 h, (d) R2CHO (10 equiv, 0.1 M), SnCl2$2H2O (2 M) in DMF, 50 ꢀC, 1 h and (e) TFA/DCM ¼ 1:1, 1 h.
˚
Spectrophotometer 214 nm using a Betasil C18 (3 mm, 100 A,
3 ꢁ 50 mm) column.
e2.05 (2H, m), 1.40e1.41 (6H, d, J ¼ 6.8 Hz), 1.13e1.17
(3H, t, J ¼ 7.0 Hz).
4.1. Synthesis
4.1.1.2.
2-Ethyl-3-(3-ethoxypropyl)-8-(4-methoxyphenyla-
mino)-3H-imidazo[4,5-g]quinazoline (7c). Yield: 88% LCe
MS (ESI) m/z 406.6 (M þ Hþ). 1H NMR (400 MHz, d6-
DMSO) d 9.61 (1H, br s), 8.83 (1H, s), 8.46 (1H, s), 7.82
(1H, s), 7.77e7.79 (2H, m), 6.97e6.99 (2H, m), 4.33e4.36
(2H, t, J ¼ 6.8 Hz), 3.78 (3H, s), 3.32e3.40 (4H, m), 2.96e
3.01 (2H, q, J ¼ 7.4 Hz), 2.00e2.03 (2H, m), 1.40e1.43
(3H, t, J ¼ 7.4 Hz), 1.12e1.15 (3H, t, J ¼ 6.8 Hz).
4.1.1. Synthesis of derivatives [24]
To the 4-(4-formyl-3-methoxyphenoxy)butyryl AM resin 1
(220 mg, 0.2 mmol sealed within a polypropylene mesh packet)
was added an arylamine (10 equiv, 0.1 M), NaBH3CN (10 equiv,
0.1 M) in anhydrous DMF/HOAC (99:1). The mixture was
shaken for 24 h at room temperature. The resin was then washed
with DMF (three times), DCM (three times), and MeOH (three
times). The resulting resin-bound compound 3 was coupled with
4-chloro-7-fluoro-6-nitroquinazoline 8 (10 equiv, 0.1 M) using
triethylamine (10 equiv, 0.1 M) in anhydrous THF at room tem-
perature for 24 h. The resin was washed with DMF (three times),
DCM (three times), and MeOH (three times). This procedure
was repeated. Resin-bound compound 4 was reacted with an al-
kylamine (20 equiv, 0.2 M) in DCM for 24 h at room tempera-
ture. The resin was washed with DMF (three times), DCM
(three times), and MeOH (three times) to afford resin-bound
compound 5. Reduction and cyclization reaction of resin-bound
compound 5 were carried out using an alkylaldehyde (10 equiv,
0.1 M) and SnCl2$2H2O (2 M) in DMF to afford resin-bound
compound 6. After being washed with DMF (three times),
DCM (three times), and MeOH (three times), the resin-bound
tricycle quinazoline 6 was treated with TFA/DCM ¼ 1:1 at
room temperature for 1 h and the solvent was removed under
the reduced pressure. The pure product 7 was obtained after
a flash column purification with eluant EtOAc/EtOH ¼ 97:3.
4.1.1.3.
2-Propyl-3-(3-ethoxypropyl)-8-(4-methoxyphenyla-
mino)-3H-imidazo[4,5-g]quinazoline (7d). Yield: 82% LCe
MS (ESI) m/z 420.6 (M þ Hþ). 1H NMR (400 MHz, d6-
DMSO) d 9.62 (1H, br s), 8.83 (1H, s), 8.47 (1H, s), 7.83
(1H, s), 7.78e7.80 (2H, m), 6.97e6.99 (2H, m), 4.34e4.38
(2H, t, J ¼ 7.0 Hz), 3.82 (3H, s), 3.33e3.42 (4H, m), 2.93e
2.97 (2H, t, J ¼ 7.4 Hz), 2.00e2.04 (2H, m), 1.89e1.95 (2H,
m), 1.13e1.17 (3H, t, J ¼ 7.0 Hz), 1.05e1.09 (3H, t,
J ¼ 7.4 Hz).
4.1.1.4.
2-Benzyl-3-(3-ethoxypropyl)-8-(4-methoxyphenyla-
mino)-3H-imidazo[4,5-g]quinazoline (7e). Yield: 82% LCe
MS (ESI) m/z 468.6 (M þ Hþ). 1H NMR (400 MHz, d6-
DMSO) d 9.63 (1H, br s), 8.85 (1H, s), 8.45 (1H, s), 7.82
(1H, s), 7.77e7.79 (2H, m), 7.27e7.29 (2H, m), 7.24e7.26
(1H, m), 7.21e7.23 (2H, m), 6.96e6.98 (2H, m), 4.31e4.34
(2H, t, J ¼ 6.8 Hz), 3.77 (3H, s), 3.78 (2H, s), 3.28e3.39
(4H, m), 1.84e1.91 (2H, m), 1.12e1.15 (3H, t, J ¼ 7.0 Hz).
4.1.1.1. 2-Isopropyl-3-(3-ethoxypropyl)-8-(4-methoxyphenyla-
mino)-3H-imidazo[4,5-g]quinazoline (7a). Yield: 85% LCe
MS (ESI) m/z 420.6 (M þ Hþ). 1H NMR (400 MHz, d6-
DMSO) d 9.61 (1H, br s), 8.84 (1H, s), 8.47 (1H, s), 7.85
(1H, s), 7.78e7.80 (2H, m), 6.98e7.00 (2H, m), 4.37e4.41
(2H, t, J ¼ 6.4 Hz), 3.79 (3H, s), 3.37e3.42 (5H, m), 2.02
4.1.1.5.
2-Hexyl-3-(3-ethoxypropyl)-8-(4-methoxyphenyla-
mino)-3H-imidazo[4,5-g]quinazoline (7f). Yield: 87% LCe
MS (ESI) m/z 462.7 (M þ Hþ). 1H NMR (400 MHz, d6-
DMSO) d 9.60 (1H, br s), 8.81 (1H, s), 8.45 (1H, s), 7.81
(1H, s), 7.77e7.79 (2H, m), 6.96e6.98 (2H, m), 4.32e4.36
(2H, t, J ¼ 6.6 Hz), 3.77 (3H, s), 3.31e3.40 (4H, m), 2.92e

Y. Zhang et al. / European Journal of Medicinal Chemistry 44 (2009) 448e452
451
Table 1
Cytotoxicity of the target compounds against five human cancer lines in vitro
Compd
R1
R2
R3
Cytotoxicity (IC50, mM)a
A549
K562
PC-3
Molt-4
MDA-MB-231
IRESSA
7a
7b
7c
e
e
eb
13.59 ꢃ 2.05
4.59 ꢃ 1.44
2.81 ꢃ 2.46
6.05 ꢃ 0.49
18.33 ꢃ 5.45
22.05 ꢃ 5.17
38.98 ꢃ 3.99
7.31 ꢃ 3.63
6.26 ꢃ 3.25
8.29 ꢃ 0.97
15.76 ꢃ 1.69
4.25 ꢃ 1.59
5.58 ꢃ 0.76
12.14 ꢃ 6.06
11.46 ꢃ 4.36
22.05 ꢃ 4.34
9.36 ꢃ 1.13
5.18 ꢃ 1.22
14.40 ꢃ 0.15
>100
15.10 ꢃ 3.78
>100
>100
15.02 ꢃ 0.32
>100
>100
36.61 ꢃ 6.08
>100
>100
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-CH3O
40-F
(CH3)2CH
(CH3)2CHCH2
CH3CH2
C2H5O(CH2)2CH2
C2H5O(CH2)2CH2
C2H5O(CH2)2CH2
C2H5O(CH2)2CH2
C2H5O(CH2)2CH2
C2H5O(CH2)2CH2
Cyclo-C6H11
>100
>100
>100
>100
>100
>100
7d
7e
CH3CH2CH2
C6H5CH2
>100
12.88 ꢃ 0.73
40.88 ꢃ 6.61
19.31 ꢃ 0.76
>100
>100
>100
>100
>100
15.49 ꢃ 2.61
>100
7f
CH3(CH2)4CH2
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
(CH3)2CH
CH3CH2CH2
(CH3)2CH
7g
7h
7i
22.97 ꢃ 4.74
>100
7.69 ꢃ 1.39
>100
21.08 ꢃ 3.37
35.73 ꢃ 5.93
>100
(CH3)2CH
CH3(CH2)2CH2
CH3O(CH2)2CH2
CH3(CH2)2CH2
CH3(CH2)2CH2
CH3(CH2)2CH2
CH3O(CH2)2CH2
Cyclo-C6H11
>100
>100
18.89 ꢃ 5.05
>100
11.46 ꢃ 2.55
>100
7j
>100
7k
7l
5.55 ꢃ 0.34
18.51 ꢃ 0.10
>100
9.74 ꢃ 2.09
>100
11.43 ꢃ 3.39
6.63 ꢃ 2.60
>100
20.00 ꢃ 6.32
19.97 ꢃ 3.28
10.65 ꢃ 1.62
20.63 ꢃ 3.64
>100
30,40-Di(CH3O)
40-CH3
30,40-Di(CH3O)
7m
7n
7o
a
24.82 ꢃ 5.65
24.59 ꢃ 1.28
24.73 ꢃ 3.07
14.33 ꢃ 0.32
10.93 ꢃ 4.53
2.83 ꢃ 1.45
H
17.35 ꢃ 0.16
Each experiment was independently performed three times and expressed as means ꢃ SD.
b
The structure of IRESSA was listed in Fig. 1.
2.96 (2H, t, J ¼ 7.6 Hz), 1.99e2.02 (2H, m), 1.83e1.88 (2H,
1.47 (6H, d, J ¼ 7.0 Hz), 1.43e1.46 (2H, m), 0.98e1.01
(3H, t, J ¼ 7.4 Hz).
m), 1.42e1.47 (2H, m), 1.29e1.36 (4H, m), 1.12e1.15 (3H,
t, J ¼ 7.0 Hz), 0.87e0.90 (3H, t, J ¼ 6.8 Hz).
4.1.1.9.
2-Isopropyl-3-cyclohexyl-8-phenylamino-3H-imi-
4.1.1.6. 2-Isopropyl-3-isopropyl-8-(4-methoxyphenylamino)-
dazo[4,5-g]quinazoline (7o). Yield: 88% LCeMS (ESI) m/z
1
3H-imidazo[4,5-g]quinazoline (7h). Yield: 90% LCeMS
386.6 (M þ Hþ) H NMR(400 MHz, CDCl3) d 8.72 (1H, s),
1
(ESI) m/z 376.6 (M þ Hþ). H NMR (400 MHz, d6-DMSO)
8.28 (1H, s), 8.07 (1H, s), 7.79e7.81 (2H, m), 7.66 (1H, br
s), 7.41e7.45 (2H, m), 7.15e7.19 (1H, m), 4.29e4.31 (1H,
m), 3.28e3.31 (1H, m), 1.93e2.04 (4H, m), 1.64e1.87 (4H,
m), 1.49e1.50 (6H, d, J ¼ 6.8 Hz), 1.34e1.46 (2H, m).
Physicochemical data for compounds 7b, 7g, 7i, 7k, 7l and
7n were reported in Ref. [24].
d 9.62 (1H, br s), 8.81 (1H, s), 8.43 (1H, s), 7.82 (1H, s),
7.76e7.78 (2H, m), 6.96e6.98 (2H, m), 4.81e4.84 (1H, q,
J ¼ 6.9 Hz), 3.26e3.30 (1H, q, J ¼ 6.7 Hz), 1.74e1.75 (6H,
d, J ¼ 6.9 Hz), 1.51e1.52 (6H, d, J ¼ 6.7 Hz).
4.1.1.7. 2-Isopropyl-3-(3-methoxypropyl)-8-(4-methoxypheny-
lamino)-3H-imidazo[4,5-g]quinazoline (7j). Yield: 87% LCe
MS (ESI) m/z 406.6 (M þ Hþ). 1H NMR (400 MHz, d6-
DMSO) d 8.94 (1H, br s), 8.68 (1H, s), 8.57 (1H, s), 7.81
(1H, s), 7.71e7.73 (2H, m), 6.95e6.96 (2H, m), 4.32e4.35
(2H, t, J ¼ 6.9 Hz), 3.79 (3H, s), 3.31e3.36 (6H, m), 2.09e
2.14 (2H, m), 1.48e1.49 (6H, d, J ¼ 6.6 Hz).
4.2. Biology
4.2.1. Cytotoxic assay
The tumor cell lines panel consisted of A549, K562, PC-3,
Molt-4 and MDA-MB-231. Cultured cancer cells were grown
in the presence of the putative anticancer agents in 96-well
plates. After 96 h treatment, the cell growth rates were deter-
mined by MTT assay and the IC50 values were calculated with
LOGIT method. Assays were performed in triplicate on three
independent experiments.
4.1.1.8. 2-Isopropyl-3-butyl-8-(4-methylphenylamino)-3H-imi-
dazo[4,5-g]quinazoline (7m). Yield: 91% LCeMS (ESI) m/z
1
374.6 (M þ Hþ). H NMR (400 MHz, d6-DMSO) 9.62 (1H,
br s), 8.83 (1H, s), 8.50 (1H, s), 7.83 (1H, s), 7.79e7.81
(2H, m), 7.39e7.40 (2H, m), 4.32e4.35 (2H, t, J ¼ 7.0 Hz),
3.25e3.28 (1H, m), 2.31 (3H, s), 1.86e1.89 (2H, m), 1.45e
4.2.2. Cell cycle analysis
Test substances were dissolved in DMSO and diluted to
10 mmol/l. A549 cells (5 ꢁ 104 cells/ml, 5 ml) were cultured
in five flasks for 24 h with test compounds (for each test com-
pound, one flask was used) or without any addition. The cells
were harvested, washed with PBS and centrifuged. The fixa-
tion of cells was realized through the addition of 4 ml ethanol
(70% ice-cold) and then keeping cells at ꢂ20 ꢀC overnight un-
til DNA staining. The fixed cells were treated with 100 mg/ml
Rnase A in PBS for 1 h, followed by staining with 50 mg/ml
propidium iodide in PBS in the dark. The DNA content of eu-
karyotic cells was then measured with flow cytometery.
Table 2
Cell cycle distribution by flow cytometry in A549 cells treated for 24 h
Compound
G0/G1 (%)
S (%)
G2/M (%)
Control
IRESSA (ZD1839)
61.26 ꢃ 7.63
76.12 ꢃ 2.47
80.38 ꢃ 6.63
81.47 ꢃ 5.67
86.28 ꢃ 1.90
29.52 ꢃ 5.98
19.24 ꢃ 4.51
16.22 ꢃ 5.18
15.41 ꢃ 4.81
10.73 ꢃ 1.78
9.22 ꢃ 1.79
4.64 ꢃ 4.02
3.40 ꢃ 2.07
3.12 ꢃ 2.75
2.99 ꢃ 2.68
7a
7b
7k

452
Y. Zhang et al. / European Journal of Medicinal Chemistry 44 (2009) 448e452
[10] G.W. Rewcastle, W.A. Denny, A.J. Bridges, H. Zhou, D.R. Cody,
Acknowledgements
A. McMichael, D.W. Fry, J. Med. Chem. 38 (1995) 3482e3487.
[11] A.J. Barker, Eur. Patent 0,566,226 (1993).
Yongping Yu thanks for the National Natural Science Foun-
dation of China (NSFC 20572098), Zhejiang Provincial Natu-
ral Science Foundation of China (ZJNSF Z405037), Zhejiang
Science and Technology Programme (2005c3401), Program
for New Century Excellent Talents in University (NCET-05-
0523). Yijia Lou thanks for National Natural Science Founda-
tion of China (NSFC 30672564).
[12] W.H.J. Ward, P.N. Cook, A.M. Slater, D.H. Davies, G.A. Holdgate,
L.R. Green, Biochem. Pharmacol. 48 (1994) 659e666.
[13] A.J. Bridges, H. Zhou, D.R. Cody, G.W. Rewcastle, A. Mc-Michael,
H.D. Hollis Showalter, D.W. Fry, A.J. Kraker, W.A. Denny, J. Med.
Chem. 39 (1996) 267e276.
[14] D.W. Fry, A.J. Kraker, A. McMichael, L.A. Ambroso, J.M. Nelson,
W.R. Leopold, R.W. Connors, A.J. Bridges, Science 265 (1994) 1093e
1095.
[15] G.W. Rewcastle, B.D. Palmer, A.J. Bridges, H.D. Hollis Showalter,
L. Sun, J. Nelson, A. McMichael, A.J. Kraker, D.W. Fry, W.A. Denny,
J. Med. Chem. 39 (1996) 918e928.
References
[16] H.D. Hollis Showalter, A.J. Bridges, H. Zhou, A.D. Sercel,
A. McMichael, D.W. Fry, J. Med. Chem. 42 (1999) 5464e5474.
[17] J.M. Rae, M.E. Lippman, Breast Cancer Res. Treat. 83 (2004) 99e107.
[18] A.J. Bridges, W.A. Denny, D.W. Fry, A. Kraker, R.F. Meyer, G.W. Re-
wcastle, A.M. Thompson, H.D. Hollis Showalter, U.S. Patent
5,679,683 (1994).
[1] D.G. Wenzel, J. Am. Pharm. Assoc. 44 (1955) 550e553.
[2] M. Hori, R. Iemura, H. Hara, A. Ozaki, T. Sukamoto, H. Ohtaka, Chem.
Pharm. Bull. 38 (1990) 681e687.
[3] S. Hayao, H.J. Havera, H.E.W.G. Stryeker, T.G. Leipzig, R.A. Kulp,
H.E. Hartzler, J. Med. Chem. 8 (1965) 807e815.
[4] Y. Nishikawa, T. Shindo, K. Ishii, H. Nakamura, T. Kona, H. Uno, J.
Med. Chem. 32 (1989) 583e593.
[19] Schnur, Rodneym Caughren, W.O. Patent 9,749,688 (1997).
[20] S.W. Wright, D.L. Hageman, L.D. McClure, A.A. Carlo, J.L. Treadway,
A.M. Mathiowetz, J.M. Withka, P.H. Bauer, Bio. Med. Chem. Lett. 11
(2001) 17e21.
[5] H.J. Havera, H.J. Vidrio, J. Med. Chem. 22 (1979) 1548e1550.
[6] Q. Chao, L. Deng, H. Shih, L.M. Leoni, D. Genini, D.A. Carson,
H.B. Cottam, J. Med. Chem. 42 (1999) 3860e3873.
[21] L. Toledo-Sherman, E. Deretey, J.J. Slon-Usakiewicz, W. Ng, J.R. Dai,
J.E. Foster, P.R. Redden, M.D. Uger, L.C. Liao, A. Pasternak, N. Reid,
J. Med. Chem. 48 (2005) 3221e3230.
[22] J.J. Slon-Usakiewicz, W. Ng, J.E. Foster, J.R. Dai, E. Deretey, L. Toledo-
Sherman, P.R. Redden, A. Pasternak, N. Reid, J. Med. Chem. 47 (2004)
5094e5100.
[7] P.P. Kung, M.D. Casper, K.L. Cook, L. Wilson-Lingardo, L.M. Risen,
T.A. Vickers, R. Ranken, L.B. Blyn, J.R. Wyatt, P.D. Cook, D.J. Ecker,
J. Med. Chem. 42 (1999) 4705e4713.
[8] T. Glaser, J. Traber, Agents Actions 15 (1984) 314e348.
[9] N.J. Liverton, D.J. Armstrong, D.A. Claremon, D.C. Remy, J.J. Bardwin,
R.J. Lynch, G. Zhang, R.J. Gould, Bioorg. Med. Chem. Lett. 8 (1998)
483e486.
[23] Y.B. Lee, C.H. Ahn, U.S. Patent 2005,187,231 (2005).
[24] Y. Zhang, C. Xu, R.A. Houghten, Y. Yu, J. Comb. Chem. 9 (2007) 9e11.