Synthesis and anticancer activities of 5,6,7-trimethoxy-N-phenyl(ethyl)-4- aminoquinazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2013.05.043

A series of 5,6,7-trimethoxy-N-phenyl(ethyl)-4-aminoquinazoline compounds was prepared by microwave irradiation and conventional heating methods. Compounds 6p, 6q , and 6x strongly inhibited extracellular regulated kinas-1/2 (ERK1/2) phosphorylation induced by epidermal growth factor (EGF) at 1.28 μM in PC3 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that all compounds had certain anticancer activities, and the IC50 values of 6x were 6.2 ± 0.9, 3.2 ± 0.1, and 3.1 ± 0.1 μM against PC3, BGC823, and Bcap37 cells, respectively. Acridine orange/ethidium bromide staining, Hoechst 33258 staining, DNA ladder, and flow cytometry analyses revealed that 6x induced cell apoptosis in PC3 cells, with apoptosis ratios of 11.6% at 1 μM and 31.8% at 10 μM after 72 h.

Full text of DOI:10.1016/j.ejmech.2013.05.043

European Journal of Medicinal Chemistry 66 (2013) 335e344
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activities of 5,6,7-trimethoxy-N-
phenyl(ethyl)-4-aminoquinazoline derivatives
,
b
,
c
,
,
b
,
c
,
,
b
,
,
b
,
,b,c
Ying Zhang a 1, Linhong Jin a 1, Hongmei Xiang a c, Jian Wu a c, Peiyi Wang a
,
Deyu Hu a c, Wei Xue a c, Song Yang a
,
b
,
,
b,
,b,c,
*
a State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Guizhou University, Guiyang 550025, PR China
b Key Laboratory of Green Pesticide and Agricultural Bioengineering, Ministry of Education, Guizhou University, Guiyang 550025, PR China
c Center for R&D of Fine Chemicals of Guizhou Province, Guizhou University, Guiyang 550025, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 5,6,7-trimethoxy-N-phenyl(ethyl)-4-aminoquinazoline compounds was prepared by micro-
wave irradiation and conventional heating methods. Compounds 6p, 6q, and 6x strongly inhibited
extracellular regulated kinase1/2 (ERK1/2) phosphorylation induced by epidermal growth factor (EGF) at
Received 29 March 2013
Received in revised form
27 May 2013
Accepted 29 May 2013
Available online 7 June 2013
1.28
all compounds had certain anticancer activities, and the IC50 values of 6x were 6.2 ꢀ 0.9, 3.2 ꢀ 0.1, and
3.1 ꢀ 0.1 M against PC3, BGC823, and Bcap37 cells, respectively. Acridine orange/ethidium bromide
staining, Hoechst 33258 staining, DNA ladder, and flow cytometry analyses revealed that 6x induced cell
apoptosis in PC3 cells, with apoptosis ratios of 11.6% at 1 M and 31.8% at 10 M after 72 h.
Ó 2013 Elsevier Masson SAS. All rights reserved.
mM in PC3 cells. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed that
m
Keywords:
Quinazoline
Anticancer
m
m
ERK1/2
Antiphosphorylation
Cell apoptosis
1. Introduction
catalytic site of EGFR [5]. Fry et al. [6] first discovered that the 4-
anilinoquinazoline derivativePD153035 possesses specific inhibi-
tory activity against EGFR tyrosine kinase. Since then, various 4-
anilinoquinazoline derivatives have been synthesized based on
the 4-anilinoquinazoline framework [7e11]. Modification of the
quinazoline structure has been performed in many anticancer
studies, such as against NSCLC, PC3, BTC, and MCF-7 cells [12e20].
Gefitinib (Iressa, ZD-1839) [21,22] and erlotinib (OSI-774, Tarceva)
[23,24], which are first-generation EGFR-targeting 4-anilino
quinazoline chemotherapeutics, have been approved for the
treatment of non-small-cell lung cancer. These two small com-
pounds directly act on the ATP binding area of EGFR, interfering
with the binding of ATP to EGFR and inhibiting the activity of EGFR-
TK. The second-generation EGFR-targeting chemotherapeuticBIB
W2992, which is an excellent non-irreversible EGFR inhibitor [25],
has also been approved for Phase-II clinical trials against lung
cancer [26].
Trimethoxyphenyl is a crucial pharmacophoric group for analogs
of the antitumor natural product CA-4((Z)-2-methoxy-5-[2-(3,4,5-
trimethoxyphenyl)ethenyl]phenol) [27]. In our study on PD153035
(the specific inhibitor for EGFR-TK) as the leading compound, we
have successfully produced 6,7,8-trimethoxy-N-aryl-4-aminoquina
zoline compounds that are potent inhibitors of PC3, Bcap37,
BGC823, and A431 cells. We found that methoxy-substituted
Protein kinases play diverse and important roles in regulating
cellular processes such as cell proliferation, cell cycle, metabolism,
survival, apoptosis, and DNA damage/repair. Thus, protein kinases
are extensively targeted for the discovery of inhibitors as potential
cancer-treatment drugs [1e4]. Epidermal growth factor receptor
(EGFR) protein tyrosine kinase is one of the most important kinases
that play a fundamental role in signal transduction pathways. The
tyrosine kinase activity of EGFR is activated by ligand binding to the
receptor, and an inhibitor of EGFR tyrosine kinase may compete
with a ligand for EGFR binding or may directly interfere with the
Abbreviations: AO/EB, acridine orange/ethidium bromide; 13C NMR, 13C nuclear
magnetic resonance; CH, conventional heating; DMF, N,N-dimethylformamide;
DMSO, dimethyl sulfoxide; EGF, epidermal growth factor; EGFR, epidermal growth
factor receptor; ERK1/2, extracellular regulated kinase1/2; ESI-MS, electrospray
ionization mass spectrometry; HCPT, 10-hydroxyl camptothecine; 1H NMR, proton
nuclear magnetic resonance; IR, infra-red; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide; MW, microwave irradiation.
* Corresponding author. Center for R&D of Fine Chemicals of Guizhou Province,
Guizhou University, Guiyang 550025, PR China. Tel.: þ86 (851)829 2171; fax: þ86
(851)829 2170.
E-mail addresses: jhzx.msm@gmail.com, yangsdqj2002@yahoo.com (S. Yang).
Both authors contributed equally to this work.
1
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.05.043

336
Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
Table 1
quinazoline compounds possess potent antitumor activity but not
antiphosphorylation inhibitory activity, suggesting that cytotoxicity
may not result from inhibiting EGFR [28]. Thus, a series of new
quinazoline compounds 6ae6z was designed and synthesized with
three methoxy (5,6,7-OCH3) groups on the quinazoline ring to study
further the effect of the substitution position of quinazoline methoxy
groups on anticancer and antiphosphorylation activities. Twenty-six
new 5,6,7-trimethoxy-N-aryl-4-aminoquinazoline derivatives were
synthesized from 2,3,4-trimethoxybenzoic acid by the synthesis
route shown in Scheme 1. The structures of the title compounds were
characterized by infrared (IR), 1H nuclear magnetic resonance
(NMR), 13C NMR, and elemental analyses. The microwave irradiation
(MW) synthesis conditions for the target compounds were also
optimized. The antiproliferation activities of the title compounds
against PC3, Bcap-37, andBGC823 cells in vitro were evaluated by
the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) colorimetric assay. The results showed that title compounds
6ae6z possessed weak to strong anticancer activities, and most of
the compounds exerted an inhibitory effect on EGF-induced ERK1/2
phosphorylation in PC3 cells.
Effects of reaction time and MW power on the yield of 6o.a
Entry
Reaction time
Power (Watt)
Yield (%)
1
2
3
4
5
6
7
8b
30 min.
30 min.
30 min.
30 min.
10 min.
20 min.
40 min
8 h
40
60
80
100
100
100
100
0
73.1
78.5
79.4
80.7
72.3
78.7
77.3
45.0
a
Reaction conditions: reactants molar ratio, 1:1; reflux temperature of iso-
propanol, 80 ꢁC.
b
Conventional heating.
compounds increased from 30.1% to 71.6% under CH to 80.1%e90.0%
under MW with decreased reaction time from 2 to 12 h to 30 min.
Interestingly, 6f and 6r were obtained only under CH method,
whereas 6g and 6m were obtained only under MW irradiation.
2.2. Inhibition activity of title compounds against ERK1/2
phosphorylation induced by EGF
2. Results and discussion
Western blot analysis demonstrated that the title compounds had
significant inhibitory activities against EGF-induced ERK1/2 phos-
phorylation in PC3 cells. As shown in Fig. 1, 6b, 6de6g (Fig. 1A), 6i,
6me6q (Fig. 1B), 6r (Fig. 1C), and 6x (Fig. 1D) inhibited ERK1/2
2.1. Chemistry
The starting material 2,3,4-trimethoxybenzoic acid was nitrated
with70% nitric acid, esterified with methanol in the presence of 98%
sulfuric acid, hydrogenated with Pd/C as catalyst in EtOH, cyclized
with formamide, and finally chlorinated with phosphorus oxy-
chloride to give the key intermediate 4-choloro-5,6,7-trimethoxy-
quinazoline. The target compounds 6ae6z were obtained by the
substitution reaction of 4-chloro-5,6,7-trimethoxyquinazoline with
amine to yield 5,6,7-trimethoxyl-N-aryl-4-aminequinazoline
(Scheme 1). The yields of the title compounds were relatively low
under conventional heating (CH) method. MW method was used to
improve the yields of the title compounds, and the reaction con-
ditions under MW were optimized using 6o as the model com-
pound. Table 1 shows that when the power of MW was optimized
from 40 W to 100 W, the yield of 6o increased from 73.1% to 80.7%
(Table 1, entries 1e4) within 30 min at 80 ꢁC. Then, the effect of
reaction time on yield was investigated. With increased reaction
time from 10 min to 30 min, the yield increased from 72.3% to 80.7%
within 30 min under an MW power 100 W (Table 1, entries 4e6).
However, the yield did not obviously increase when the reaction
time was 40 min (Table 1, entry 7). Thus, the reaction conditions for
MW irradiation were as follows: temperature, 80 ꢁC; reactant molar
ratio, 1:1; MW power, 100 W; and reaction time, 30 min. For
comparison, the yield of 6o was found to be only 45.0% after 8 h of
reaction (Table 1, entry 8).
phosphorylatin in PC3 cells induced by EGF at 10 mM. The 4-phenyl
ring containing a fluorine atom or trifluoromethyl group generally
had good antiphosphorylation activities (6e, 6f, 6i, 6m, 6oe6q, and
6x).
Further dose experiments were carried out on 6d, 6e, 6f, 6p, 6q,
and 6x. Fig. 2 shows that the inhibition activities of 6de6f against
ERK phosphorylation were weak to moderate at 5.12
at 10 M (Fig. 2AeC). However, 6p, 6q, and 6x showed strong in-
hibition activities against ERK phosphorylation using low concen-
tration of 1.28 M (Fig. 2DeF). Furthermore, the 4-aniline moiety
mM but strong
m
m
bearing a 3- or 4-trifluoromethyl group showed very strong inhi-
bition activities against ERK1/2 phosphorylation in PC3 cells.
Interestingly, 4-phenylethylamine substitution also showed very
strong inhibition activities against ERK phosphorylation in PC3 cells.
2.3. Antiproliferation activities of title compounds against PC3,
BGC823, and Bcap-37 cells
The antiproliferation activities of title compounds 6ae6z
were evaluated against PC3, BGC823, and Bcap-37 cells using
PD153035as a positive control. As shown in Table 3, the anti-
proliferation activity of 6z against PC3 cells at 10
61.75% ꢀ 12.5%, similar to that of PD153035. The antiproliferation
activities of 6l, 6v, 6w, and 6z against BGC823 cells at 10 M were
mM was
All title compounds were synthesized under both CH and MW
methods, and the results are listed in Table 2. The yields of the title
m
Scheme 1. Reagents and conditions of the synthesis of title compounds 6: (a) nitric acid, 0 ꢁC, 2 h; (b) CH3OH, 98% H2SO4, reflux, 12 h; (c) H2, Pd/C, 95% C2H5OH, reflux, 12 h; (d)
DMF, formamide, CH3OH, CH3ONa, 150 ꢁC, 24 h; (e) N2, toluene, POCl3, reflux, 3.5 h; (f) aryl amine, 2-propanol, MW, 100 W, 80 ꢁC, 30 min to obtain 6ae6z; aryl amine, 2-propanol,
reflux, 2e12 h to obtain 6ae6z.

Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
337
Table 2
Reaction conditions and yields of title compounds 6ae6z.a
Bcap37 cells were 6.2 ꢀ 0.9, 3.2 ꢀ 0.1, and 3.1 ꢀ 0.1
m
M, respectively,
which were higher than those of PD153035 (8.3 ꢀ 1.1, 14.4 ꢀ 1.5,
Microwave irradiationb
Reaction time (min)
Conventional heatingc
and 15.7 ꢀ 1.2 M, respectively).
m
Product
Yield (%)
Reaction time (h)
Yield (%)
2.4. Preliminary investigation on the apoptosis-inducing effect of
title compound 6x on PC3 cells
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
30
30
30
30
30
85.8
89.7
86.1
84.8
89.0
/
81.9
85.0
88.9
83.2
81.0
80.1
81.9
82.4
80.7
81.1
85.1
/
83.6
90.0
81.2
84.8
80.5
83.9
85.4
83.7
6
6
4
2
6
7
/
6
6
6
8
8
/
6
8
6
9
6
12
3
9
8
10
9
12
12
51.2
70.2
32.6
71.6
50.6
37.2
/
46.3
53.6
31.5
51.3
47.7
/
41.3
45.0
30.1
40.1
45.8
42.8
65.0
61.4
54.2
60.2
63.0
51.5
50.8
PD153035 reportedly exerts apoptosis-inducing effects [29,30].
However, studies on the apoptosis-inducing activity of 4-
phenylethylaminoquinazoline compounds are limited. Given that
6x had excellent antiproliferation activities against the three cell
lines, further investigation was carried out to evaluate its
apoptosis-inducing effect on PC3 cells.
c
/
30
30
30
30
30
30
30
30
30
30
30
/
30
30
30
30
30
30
30
30
2.4.1. Fluorescence staining
Changes in the morphology of PC3 cells were investigated using
acridine orange (AO)/ethidium bromide (EB) and Hoechst 33258
staining under fluorescence microscopy to determine whether the
growtheinhibitory activity of the selected compounds was related
to apoptosis induction.
6s
6t
6u
6v
6w
6x
6y
6z
2.4.1.1. AO/EB staining. AO is a vital green dye that can stain nuclear
DNA across an intact cell membrane, whereas EB can stain only cells
with lost membrane integrity. Thus, after simultaneous treatment
with AO and EB, live cells were uniformly stained green, early
apoptotic cells were densely stained as green yellow or show green
yellow fragments, late apoptotic cells were densely stained orange
or displayed orange fragments, and necrotic cells were stained or-
a
Reaction conditions: reactants molar ratio 1:1, at 80 ꢁC in isopropanol.
b
c
MW power ¼ 100 W.
Not obtained.
ange without condensed chromatin. The cytotoxicity of 6x at 10 mM
against PC3 cells from 24 h to 72 h was detected by AO/EB staining,
with 10-hydroxyl camptothecine (HCPT) used as a positive control
at 10 mM against PC3 cells for 72 h. The results are shown in Fig. S1,
60.7% ꢀ 5.4%, 68.4% ꢀ 10.7%, 51.9% ꢀ 7.2%, and 58.9% ꢀ 6.1%,
respectively, which were higher than that of PD153035
(32.6% ꢀ 6.5%). Compounds 6h, 6u, and 6y displayed higher inhi-
which shows that the cells treated with 6x from 24 h to 72 h
changed in PC3 cells. Nuclei were stained as yellow green or orange,
and the morphology showed pycnosis, membrane blebbing, and
cell budding. These phenomena were associated with cell
apoptosis.
Green live PC3 cells with normal morphology were observed in
the negative control group. Green yellow or orange dots were
detected in HCPT after 48 h. Cells treated with 6x displayed yellow
dots and cell budding in PC3 cells at 48 h and were stained green
72 h after treatment. The emerging orange dots at 72 h also showed
typical late apoptotic cells.
bition activities against Bcap37 cells at 10 mM, with inhibition rates
of 50.0% ꢀ 7.8%, 69.3% ꢀ 10.1%, and 53.7% ꢀ 8.1%, respectively,
which were higher than that of PD153035 (43.4% ꢀ 5.7%). Inter-
estingly, among all title compounds, 6x showed the strongest in-
hibition and antiproliferation activities against all three cell lines
(PC3, BGC823, and PC3 cells), with inhibition rates of 90.6% ꢀ 9.1%,
88.2% ꢀ 6.1%, and 90.4% ꢀ 2.8% at 10
mM, respectively. These values
were relatively higher than that of PD153035.
IC50 assays were further carried out on 6x and PD153035. As
shown in Table 4, the IC50 values of 6x against PC3, BGC823, and
Fig. 1. Effects of title compounds (10 mM, 60 min treatment) on ERK1/2 phosphorylation induced by EGF (10 ng/mL, 10 min treatment).

338
Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
Fig. 2. Dose effect of six title compounds (different concentrations, 60 min treatment) on their inhibition activities against ERK1/2 phosphorylation in PC3 cells induced by EGF
(10 ng/mL, 10 min treatment).
The results above indicated that the cells had apoptotic
morphology. The almost complete absence of red cells in 6x indi-
cated that it was associated with very low cytotoxicity. These
findings showed that 6x induced apoptosis with low cytotoxicity.
with uniformly light blue nuclei were observed under a fluores-
cence microscope after treatment with Hoechst 33258, whereas
apoptotic cells had bright blue nuclei because of karyopyknosis and
chromatin condensation. Meanwhile, the nuclei of dead cells were
not stained. PC3 cells treated with 6x at 1 and 10
mM for 72 h were
2.4.1.2. Hoechst 33258 staining. Hoechst 33258 is a membrane-
permeable blue fluorescent dye that stains cell nuclei. Live cells
stained with Hoechst 33258. HCPT was used as a positive control at
10
mM for 72 h. The results are shown in Fig. S2, which shows that
Table 3
Growth inhibitory ratios of selected cell lines.
Compdsa
Inhibitory ratio (%)a
PC3
BGC823
Bcap37
1
m
M
10
m
M
1
m
M
10
m
M
1
mM
10 mM
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
6n
6o
6p
6q
6r
6s
6t
6u
6v
6w
6x
7.9 ꢀ 7.8
9.3 ꢀ 6.3
24.9 ꢀ 6.7
24.6 ꢀ 7.4
25.5 ꢀ 6.2
45.6 ꢀ 7.2
9.1 ꢀ 6.9
NTb
NT
NT
NT
NT
NT
ꢂ12.0 ꢀ 10.0
ꢂ7.5 ꢀ 8.0
ꢂ8.3 ꢀ 7.6
7.3 ꢀ 8.8
7.8 ꢀ 4.0
7.0 ꢀ 2.5
14.1 ꢀ 7.9
31.6 ꢀ 5.4
NT
ꢂ16.3 ꢀ 11.6
3.3 ꢀ 6.0
15.4 ꢀ 4.3
11.1 ꢀ 11.0
10.8 ꢀ 6.2
13.5 ꢀ 3.1
28.8 ꢀ 3.9
50.0 ꢀ 7.8
NT
11.3 ꢀ 8.2
23.8 ꢀ 5.4
3.1 ꢀ 7.0
1.2 ꢀ 1.3
19.3 ꢀ 6.5
NT
NT
14.9 ꢀ 10.0
ꢂ4.5 ꢀ 24.6
14.5 ꢀ 4.9
0.9 ꢀ 4.6
38.9 ꢀ 7.9
11.9 ꢀ 19.6
26.4 ꢀ 9.5
24.0 ꢀ 5.5
19.8 ꢀ 7.7
49.7 ꢀ 4.8
34.7 ꢀ 2.7
21.5 ꢀ 10.4
22.2 ꢀ 4.4
29.2 ꢀ 5.4
29.0 ꢀ 8.0
46.6 ꢀ 4.1
28.8 ꢀ 7.9
25.3 ꢀ 6.4
31.3 ꢀ 7.1
42.7 ꢀ 5.6
43.0 ꢀ 9.5
22.6 ꢀ 7.1
90.6 ꢀ 9.1
40.4 ꢀ 10.7
61.7 ꢀ 8.5
62.8 ꢀ 4.7
NT
NT
NT
NT
9.0 ꢀ 5.2
16.3 ꢀ 5.3
NT
NT
9.7 ꢀ 7.4
0.6 ꢀ 9.2
NT
11.4 ꢀ 6.1
NT
NT
NT
ꢂ8.4 ꢀ 5.6
12.8 ꢀ 3.8
14.7 ꢀ 6.0
1.0 ꢀ 27.1
7.6 ꢀ 8.9
8.7 ꢀ 4.3
10.5 ꢀ 7.2
NT
10.4 ꢀ 5.8
14.4 ꢀ 6.4
8.6 ꢀ 3.3
21.0 ꢀ 8.6
11.0 ꢀ 8.6
NT
22.2 ꢀ 10.6
11.3 ꢀ 4.2
4.3 ꢀ 9.6
19.4 ꢀ 5.7
46.5 ꢀ 12.6
40.9 ꢀ 10.9
2.1 ꢀ 14.9
19.8 ꢀ 10.4
18.6 ꢀ 2.3
18.9 ꢀ 6.0
NT
26.1 ꢀ 3.7
27.6 ꢀ 6.2
32.3 ꢀ 2.2
23.5 ꢀ 9.6
17.5 ꢀ 4.7
NT
26.4 ꢀ 7.6
69.3 ꢀ 10.1
2.2 ꢀ 6.0
20.7 ꢀ 6.9
90.4 ꢀ 2.8
53.7 ꢀ 8.1
45.8 ꢀ 7.8
43.4 ꢀ 5.7
12.4 ꢀ 6.4
60.7 ꢀ 5.4
NT
NT
13.8 ꢀ 6.5
NT
32.6 ꢀ 6.3
NT
7.4 ꢀ 8.1
2.4 ꢀ 6.4
1.0 ꢀ 9.3
5.9 ꢀ 4.9
2.1 ꢀ 4.4
15.5 ꢀ 3.0
3.9 ꢀ 5.0
17.4 ꢀ 6.5
24.3 ꢀ 8.8
23.9 ꢀ 3.2
23.2 ꢀ 3.0
43.0 ꢀ 5.9
41.9 ꢀ 4.9
68.4 ꢀ 10.7
51.9 ꢀ 7.2
88.2 ꢀ 6.1
34.3 ꢀ 9.2
58.9 ꢀ 6.1
32.6 ꢀ 6.5
14.9 ꢀ 11.4
16.9 ꢀ 5.6
ꢂ3.9 ꢀ 4.6
6.3 ꢀ 10.2
9.2 ꢀ 3.0
8.1 ꢀ 14.6
14.1 ꢀ 11.5
22.6 ꢀ 12.2
12.5 ꢀ 9.1
35.6 ꢀ 12.4
21.5 ꢀ 7.9
38.5 ꢀ 11.1
42.2 ꢀ 8.7
38.7 ꢀ 6.5
13.2 ꢀ 2.3
40.8 ꢀ 9.7
9.3 ꢀ 8.2
6y
6z
PD153035
a
Cells were treated with the title compounds or PD153035 at 1 and 10
m
M for 72 h and assayed by the MTT method. The data shown are the mean ꢀ SD of triplicate
experiments.
b
NT mean not tested.

Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
339
Table 4
10
increased in a dose-dependent and time-dependent manner. The
apoptosis ratios of 6x at 10 M for different treatment times (10.9%,
16.4%, and 31.8% at 24, 48, and 72 h, respectively) were markedly
higher than those of the positive control HCPT (5.7%, 8.3%, and 17.0%
mM. The apoptosis of PC3 cells treated with 6x gradually
IC50 values of 6x against three kinds of cells for 72 h in vitro.
Compound
IC50
PC3
(m
M)a
m
BGC823
Bcap37
6x
6.2 ꢀ 0.9
8.3 ꢀ 1.1
3.1 ꢀ 0.1
3.2 ꢀ 0.1
at 24, 48, and 72 h, respectively). Even at 1 mM, 6x also induced cell
apoptosis at a ratio of 11.6% after 72 h of treatment.
PD153035
14.4 ꢀ 1.5
15.7 ꢀ 1.2
a
IC50 means the concentration required to inhibit cell growth by 50% as deter-
mined from the doseeresponse curve. Each experiment was performed in triplicate.
3. Conclusion
A series of 5,6,7-trimethoxy-N-phenyl(ethyl)-4-aminoquinazo
line was prepared by MW and CH methods. The yields of the title
compounds increased from 30.1% to 71.6% under CH to 80.1%e90.0%
under MW with decreased reaction time from 2 to 12 h to 30 min.
Some of the title compounds were found to inhibit EGF-induced
ERK1/2 phosphorylation in PC3 cells effectively, which indicated
that the position of trimethoxy groups on the phenyl ring affected
the antiphosphorylation activities of 4-phenyl(ethyl)quinazoline
compounds. Preliminary structureeactivity relationship analysis
showed that the 4-phenyl ring containing a fluorine atom or tri-
fluoromethyl group generally had good antiphosphorylation ac-
tivities (6e, 6f, 6i, 6m, 6oe6q, and 6x). Further investigation
showed that 6p, 6q, and 6x showed strong inhibition activities
cells treated with the negative control dimethyl sulfoxide (DMSO)
were normally blue. On the other hand, the nuclei of cells treated
with HCPT appeared to be compact and condensed. After treatment
with 6x, the cells exhibited strong blue fluorescence and revealed
typical apoptotic morphology after 72 h at 10 mM. These findings
demonstrated that 6x induced apoptosis against PC3 cell lines,
consistent with the AO/EB double staining results.
2.4.2. DNA ladder assay
Endonucleases were activated when cells exhibited apoptosis.
DNA was selectively degraded to form 50e300 kb fragments and
was finally cleaved in the vicinity of the nucleosome in fragments
or multiples of 180e200 bp. These DNA fragments were then
extracted from cells. DNA fragmentation was detected by agarose
gel electrophoresis and Gold View staining. The results are shown
in Fig. 3.
Fig. 4 shows that the cells in the negative group did not show
any DNA ladder, but those treated with 6x had the typical DNA
ladder.
against ERK1/2 phosphorylation induced by EGF at 1.28 mM. Inter-
estingly, 4-substituted phenylethylamino-5,6,7-trimethoxyquinazo
line compounds also possessed strong antiphosphorylation activ-
ities, such as 6x. MTT assay showed that some of the compounds
had moderate to strong anticancer activities. The inhibition rate of
6x against PC3, BGC823, and PC3 cells at 10
88.2% ꢀ 6.1%, and 90.4% ꢀ 2.8%, respectively. Also, the IC50 values of
6x were 6.2 ꢀ 0.9, 3.2 ꢀ 0.1, and 3.1 ꢀ 0.1 M against PC3, BGC823,
and Bcap37 cells, respectively, which were much higher than those
M, respectively).
m
M were 90.6% ꢀ 9.1%,
m
2.4.3. Flow cytometry
The apoptosis ratios induced by 6x in tumor cells were quanti-
tatively assessed by flow cytometry. In the early stages of apoptosis,
phosphatidylserine was translocated from within the cell mem-
brane to the cell exterior. Annexin V, a calcium-dependent phos-
of PD153035 (8.3 ꢀ 1.1, 14.4 ꢀ 1.5, and 15.7 ꢀ 1.2
m
The apoptosis-inducing activity of 6x in PC3 cells was investigated
by AO/EB staining, Hoechst 33258 staining, DNA ladder, and flow
cytometry analyses. This compound clearly demonstrated cell
apoptosis-inducing effects, with the inducing ratio higher than
those ratios observed for the positive control HCPT. Moreover, the
pholipid-binding protein associated with
a high affinity for
phosphatidylserine, was used to detect early apoptotic cells. Pro-
pidine iodide (PI) is a red fluorescent dye that stains cells with lost
their membrane integrity. Cells stained with Annexin V-fluorescein
isothiocyanate and PI were classified as necrotic (Q1; Annexinꢂ/
PIþ), late apoptotic (Q2; Annexinþ/PIþ), intact (Q3; Annexinꢂ/PIꢂ),
orearly apoptotic (Q4; Annexinþ/PIꢂ) cells. The results are shown in
Fig. 4.
apoptosis ratio of 6x was 31.8% at 10
mM for 72 h, which was higher
than that of HCPT (17.0% at 10 M for 72 h). Further studies on the
m
specific mechanisms of 6x in PC3 cells are currently underway.
4. Experimental
Fig. 4 shows that 6x induced apoptosis in PC3 cells at 1 mM. As
shown in Table 5, the apoptosis ratios (including the early and late
4.1. Materials and instruments
apoptosis ratios) after 72 h of treatment with 6x were 11.6%, 19.8%,
Unless specifically stated, all reagents and solvents used were
obtained from commercial suppliers without further purification.
All melting points of the products were determined on a XT-4
binocular microscope (Beijing Tech Instrument Co., China) and
were not corrected. The IR spectra were recorded on a Bruker
VECTOR22 spectrometer in KBr disks. 1H NMR and 13C NMR spectra
(solvent DMSO-d6, CD3OD, or CDCl3) were recorded on a JEOL-ECX
500NMR spectrometer at room temperature using tetramethylsi-
lane as an internal standard. Elemental analysis was performed
with an Elementar Vario-III CHN analyzer. Microwave reactions
were performed on a variable power Focused Microwave Synthesis,
DiscoverÔ LabMate equipped with a high-sensitivity IR sensor for
temperature control and measurement.
and 31.8% at 1, 5, and 10
mM, respectively. For the positive control
HCPT, the apoptosis ratio was only 17.0% after 72 h of treatment at
4.2. Chemistry
4.2.1. Preparation of 2,3,4-trimethoxy-6-nitrobenzoic acid (1) [31]
2,3,4-Trimethoxybenzoic acid was added in portions to 45 mL of
concentrated 70% nitric acid at 0 ꢁC (5.0 g, 0.02 mol). After stirring
Fig. 3. Effect of 6x on DNA fragmentation in PC3 cells at10 mM.

340
Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
Fig. 4. Annexin V/PI apoptosis ratio detection assay. (1) Cells were treated with 0.1% DMSO for 72 h (2) Cells were treated with HCPT (10
6x at 1, 5, and 10 M, respectively for 72 h.
mM) for 72 h (3e5) Cells were treated with
m
at 0 ꢁC for 10 min, the mixture was diluted with 500 mL of cold
water. Stirring was continued at 0 ꢁC for 1 h, and the resulting light
orange precipitate was filtered and washed well with water to
produce 3.60 g of 1. An additional 0.58 g of product was obtained by
extracting the aqueous filtrate with methylene chloride and then
evaporating the organic solvent. Total yield: 69.0%; mp: 150e
ethanol, and the mixture was stirred in the presence of H2 at room
temperature for 12 h. Then, the mixture was filtered and the ob-
tained solids were purified by silica gel column chromatography to
produce 4.0 g of white solid 3. Yield: 83.2%; mp 93e95 ꢁC. 1H NMR
(CDCl3, 500 MHz):
2H), 5.95 (s, 1H). IR (KBr)
1616.4, 1685.8, 2980.0, 3005.7, 3373.5, 3477.7 cmꢂ1. Anal. calcd. for
11H15NO5 (%): C, 54.77; H, 6.27; N, 5.81. Found: C, 54.84; H, 6.02;
d
3.76 (s, 3H), 3.98e4.02 (3s, 9H, 3OCH3), 5.34 (s,
n
: 771.5, 1130.3, 1273.0, 1452.4, 1500.6,
152 ꢁC. 1H NMR (CDCl3, 500 MHz):
d 3.98e4.15 (3s, 9H, 3OCH3), 7.55
(s, 1H), 9.52 (s, 1H). IR (KBr)
n
: 735.6, 1112.9, 1305.8, 1334.7, 1496.8,
C
1523.8 1710.9, 2953.0, 3010.8 cmꢂ1. Anal. calcd. for C10H11NO7 (%):
C, 46.70; H, 4.31; N, 5.45. Found: C, 47.02; H, 4.64; N, 5.13.
N, 5.96.
4.2.4. Preparation of 5,6,7-trimethoxyquinazoline-4-one (4)
4.2.2. Preparation of methyl 2,3,4-trimethoxy-6-nitrobenzoate (2)
2,3,4-Trimethoxy-6-nitrobenzoic acid (5.0 g, 0.02 mol) was
added to 30 mL of methanol and 2.5 mL of 98% sulfuric acid with
stirring at refluxing temperature for 8 h. Another 2 mL of 98%
sulfuric acid was added, the temperature was rapidly increased to
110 ꢁC, and the water generated in the reaction was separated by
azeotropic distillation. When the mixture became a thick brown
liquid, the mixture was cooled down and placed in a refrigerator at
0 ꢁC for 12 h. The resulting solid was then filtered and washed well
with water to give 3.25 g of 2. Yield: 60.4%; mp: 66e69 ꢁC. 1H NMR
Methanol sodium (0.45 g, 0.01 mol) and 4 mL of methanol were
added to a solution of methyl 6-amino-2,3,4-trimethoxybenzoate
(2.0 g, 0.008 mol) in 12 mL of N,N-dimethylformamide (DMF) and
8 mL of formamide with stirring at 150ꢁCfor 24 h. Then, the mixture
was cooled to room temperature and purified by silica gel column
chromatography to produce1.05 g of white solid 4. Yield: 53.6%; mp
182e183 ꢁC. 1H NMR (CDCl3, 500 MHz):
3OCH3), 7.02 (s, 1H), 8.02 (s, 1H), 11.84 (s, 1H, NH). 13C NMR (CDCl3,
500 MHz): 56.3, 61.6, 62.3, 105.2, 110.9, 142.3, 143.9, 147.9, 152.9,
159.0, 161.2. IR (KBr) : 800.5, 1141.8, 1263.3, 1427.5, 1620.2, 1684.6,
d 3.95e4.01 (3s, 9H,
d
n
(CDCl3, 500 MHz):
1H). IR (KBr)
d
3.63 (s, 3H), 3.83e3.89 (3s, 9H, 3OCH3), 7.53 (s,
2881.6, 3200.0, 3302.1. Anal. calcd. for C11H12N2O4 (%): C, 55.93; H,
5.12; N, 11.86. Found: C, 55.76; H, 5.48; N, 11.67.
n
: 852.6, 1116.8, 1312.8, 1352.1, 1683.4, 2943.4, 3003.3
cmꢂ1. Anal. calcd. for C11H13NO7 (%): C, 48.71; H, 4.83; N, 5.16.
Found: C, 48.43; H, 4.57; N, 5.32.
4.2.5. Preparation of 4-chloro-5,6,7-trimethoxyquinazoline (5)
A suspension of trimethoxyquinazolin-4-one (0.5 g, 0.002 mol)
and POCl3 (2 mL) in toluene (12 mL) were added dropwise to
triethylamine (6.0 mL). The resulting solution was heated at 102 ꢁC
under N2 atmosphere for 3.5 h, cooled, and concentrated. The
4.2.3. Preparation of methyl 6-amino-2,3,4-trimethoxybenzoate (3)
Pd/C (10%, 0.54 g) was added to a solution of methyl 2,3,4-
trimethoxy-6-nitrobenzoate (5.4 g, 0.02 mol) in 50 mL of 95%
Table 5
Apoptosis rate of compound 6x and HCPT tested on PC3 cells.
Compd
24 h
48 h
72 h
6x
1
m
M
5
m
M
10
10.9%
30
11.9%
m
M
1
m
M
5
m
M
10
16.4%
30
12.8%
m
M
1
m
M
5
m
M
10
31.8%
30
38.2%
m
M
5.1%
10
5.7%
8.2%
20
7.4%
9.5%
10
8.3%
11.1%
20
11.9%
11.6
10
17.0%
19.8%
20
19.8%
HCPT
m
M
m
M
mM
m
M
mM
m
M
m
M
m
M
m
M

Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
341
resulting solid was then diluted with CH2Cl2. The solution was
173 ꢁC. 1H NMR (CDCl3, 500 MHz):
d
2.41 (s, 1H, CH3), 3.97e4.28 (3s,
neutralized with ammonia water and extracted with methylene
chloride. The obtained organic layer was then passed through a
short column of silica gel and then sequentially washed with
CH2Cl2 and CH2Cl2/EtOAc (3:1) to produce the chlorinated product
0.395 g. Yield: 73.3%; mp 113e115 ꢁC. 1H NMR (CDCl3, 500 MHz):
9H, 3OCH3), 7.28 (d, 2H, J ¼ 8.0 Hz), 7.53 (d, 2H, J ¼ 8.0 Hz), 7.79 (s,
1H), 8.54 (s, 1H), 10.54 (s, 1H). 13C NMR (CDCl3, 500 MHz):
21.2,
57.4, 61.6, 62.9, 98.0, 102.0, 123.4, 130.1, 133.3, 133.4, 137.3, 137.5,
141.8, 149.3, 158.7, 160.9. IR (KBr) : 810.0, 1153.4, 1290.4, 1494.8,
1631.7, 2889.4, 2945.3, 3007.6, 3281.1, 3443.3 cmꢂ1. Anal. calcd. for
18H20ClN3O3 (%): C, 59.75; H, 5.57; N, 11.61. Found: C, 59.50; H,
d
n
d
4.00e4.05 (3s, 9H, 3OCH3), 7.23 (s, 1H), 8.84 (s, 1H). 13C NMR
C
(CDCl3, 500 MHz):
d
56.5, 61.3, 62.0, 114.9, 104.2, 144.2, 148.3, 150.4,
5.76; N, 11.48.
153.1, 157.8, 160.0. IR (KBr)
n
: 783.1, 1140.0, 1249.9, 1427.3, 1604.8,
2845.0, 2945.3, 3100.2 cmꢂ1. Anal. calcd. for C11H11ClN2O3 (%): C,
51.88; H, 4.35; N, 11.00. Found: C, 52.05; H, 4.63; N, 11.28.
4.2.6.5. N-(4-Fluorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
(6e). Yield: 89.0%; light yellow solid; mp: 172e175 ꢁC. 1H NMR
(CDCl3, 500 MHz):
J ¼ 8.6 Hz), 7.63e7.66 (m, 2H), 7.80 (s, 1H), 8.54 (s, 1H), 10.56 (s, 1H).
13C NMR (CDCl3, 500 MHz):
57.4, 61.6, 62.9, 97.7,101.8,116.2, 116.4,
125.4, 125.5, 131.8, 137.1, 141.8, 149.2, 158.8, 160.0, 161.1, 162.0. IR
d 3.97e4.31 (3s, 9H, 3OCH3), 7.18 (t, 2H,
4.2.6. General procedure for the synthesis of 5,6,7-trimethoxy-N-
aryl-4-aminoquinazolines (6ae6z)
d
In the MW method, a mixture of 4-chloro-5,6,7-trimethoxy
quinazoline (3.0 mmol) and aryl amine (3.0 mmol) in 2-propanol
(10 mL) was stirred for 3 min and then reacted under MW at
100 W and 80 ꢁC for 20 min. Upon reaction completion as moni-
tored by thin-layer chromatography (TLC), the solvent was
removed under reduced pressure. The residue was dissolved with
methanol or chloroform and purified by TLC (petroleum ether/ethyl
acetate, 1:2, V:V). The target compound was eluted from the silica
gel with ethyl acetate to give the title compounds.
In the CH method, a solution of 4-chloro-5,6,7-trimethoxy
quinazoline (4.0 mmol) and aryl amine (4.0 mmol) in 2-propanol
(6 mL) was stirred under reflux for 2e12 h. Upon reaction
completion as monitored by TLC, the solvent was removed under
reduced pressure. The resulting solid was purified by silica gel or
recrystallized with ethanol or methanol to give the title
compounds.
(KBr) n: 802.8, 1155.4, 1290.4, 1504.5, 1631.8, 2851.2, 2980.0, 3021.5,
3285.6, 3437.3 cmꢂ1. Anal. calcd. for C17H16FN3O3 (%): C, 62.00; H,
4.90; N, 12.76. Found: C, 61.89; H, 4.96; N, 12.52.
4.2.6.6. N-(3-Fluorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
hydrochloride (6f). Yield: 37.2% (only obtained under CH); pale
yellow solid; mp: 176e179 ꢁC. 1H NMR (DMSO-d6, 500 MHz):
d
3.76e4.18 (3s, 9H, 3OCH3), 6.72 (s, 1H), 7.08 (dt, 1H, J ¼ 8.6 Hz,
1.7 Hz), 7.39 (d, 1H, J ¼ 8.6 Hz), 7.47 (dd, 1H, J ¼ 14.9 Hz, 8.6 Hz), 7.81
(d, 1H, J ¼ 10.9 Hz), 8.64 (s, 1H), 12.75 (b, 1H). 13C NMR (DMSO-d6,
500 MHz):
d 56.8, 60.6, 60.7, 90.5, 100.8, 110.5, 110.7, 112.2, 112.4,
119.5, 131.0, 131.1, 135.8, 150.9, 159.7, 160.5, 163.6. IR (KBr) n: 808.7,
1153.4, 1294.5, 1492.9, 1614.4, 2829.6, 2935.7, 3032.6, 3312.5,
3400.2 cmꢂ1. Anal. calcd. for C17H17FClN3O3 (%): C, 55.82; H, 4.68; N,
11.49. Found: C, 55.67; H, 5.01; N, 11.60.
4.2.6.1. 5,6,7-Trimethoxy-N-(4-nitrophenyl)quinazoline-4-amine
4.2.6.7. N-(3-Chlorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
(6a). Yield: 85.8%; pale yellow needles; mp: 170e173 ꢁC. 1H NMR
(6g). Yield: 81.9% (only obtained under MW); white solid; mp:
(CDCl3, 500 MHz):
d
4.00e4.36 (3s, 9H, 3OCH3), 7.90 (s, 1H), 7.99 (d,
122e124 ꢁC. 1H NMR (CDCl3, 500 MHz):
d 3.96e4.17 (3s, 9H,
2H, J ¼ 9.2 Hz), 8.35 (d, 2H, J ¼ 9.2 Hz), 8.70 (s, 1H), 10.94 (s, 1H). 13
C
3OCH3), 7.08 (s, 1H), 7.09 (d, 1H, J ¼ 8.0 Hz), 7.31 (t, 1H, J ¼ 8.0 Hz),
NMR (CDCl3, 500 MHz):
d 57.6, 61.8, 63.3, 98.1, 102.5, 122.8, 125.2,
7.62 (d, 1H, J ¼ 8.0 Hz), 7.99 (s,1H), 8.61 (s,1H), 9.93 (s, 1H). 13C NMR
137.7, 141.9, 142.4, 145.2, 148.8, 148.9, 158.7, 161.7. IR (KBr)
n
: 812.4,
(CDCl3, 500 MHz): d 57.1, 158.0, 61.6, 63.0, 104.3, 104.6, 119.3, 121.3,
1141.9, 1268.5,1456.3,1619.3, 2876.8, 2998.2, 3010.0, 3312.2, 3460.5
cmꢂ1. Anal. calcd. for C17H16N4O5 (%): C, 57.30; H, 4.53; N, 15.72.
Found: C, 56.95; H, 4.71; N, 15.65.
123.7, 129.9, 134.6, 140.2, 140.8, 148.4, 148.6, 154.7, 157.0. IR (KBr) n:
805.7, 1149.2, 1280.8, 1491.5, 1633.6, 2926.4, 2982.3, 3005.7, 3286.7,
3425.3 cmꢂ1. Anal. calcd. for C17H16ClN3O3 (%): C, 59.05; H, 4.66; N,
12.15. Found: C, 59.26; H, 4.42; N, 11.85.
4.2.6.2. 4-(5,6,7-Trimethoxyquinazoline-4-yl-amino)phenol hydro-
chloride (6b). Yield: 89.7%; pale brown needles; mp: 207e209 ꢁC.
4.2.6.8. 5,6,7-Trimethoxy-N-(3-nitrophenyl)quinazoline-4-amine
1H NMR (CD3OD, 500 MHz):
d
3.93e4.18 (3s, 9H, 3OCH3), 6.68 (dd,
(6h). Yield: 85.0%; yellow solid; mp: 192e194 ꢁC. 1H NMR (CDCl3,
2H, J ¼ 6.9 Hz, 2.3 Hz), 6.79 (d, 1H, J ¼ 9.2 Hz), 6.84 (dd, 2H,
500 MHz): d 3.97e4.21 (3s, 9H, 3OCH3), 7.12 (s, 1H), 7.55 (t, 1H,
J ¼ 6.9 Hz, 2.9 Hz), 6.99 (s, 1H), 7.47 (d, 1H, J ¼ 8.6 Hz), 8.30 (s, 1H).
J ¼ 8.1 Hz), 7.96 (dd, 1H, J ¼ 8.1 Hz, 1.8 Hz), 8.15 (dd, 1H, J ¼ 8.1 Hz,
13C NMR (CD3OD, 500 MHz):
d
56.1, 60.6, 62.9, 95.5, 101.8, 115.2,
1.8 Hz), 8.66 (s, 1H), 8.80 (s, 1H), 10.14 (s, 1H). 13C NMR (CDCl3,
125.6, 127.9, 136.4, 141.5, 149.4, 150.4, 156.5, 158.6, 161.2. IR (KBr)
n
:
500 MHz):
d
76.8, 77.1, 77.4, 104.2, 104.7, 115.9, 118.1, 126.8, 129.7,
: 800.5,
805.2, 1151.5, 1290.4, 1494.8, 1629.8, 2867.4, 2990.3, 3009.7, 3288.6,
3423.3 cmꢂ1. Anal. calcd. for C17H18ClN3O4 (%): C, 56.13; H, 4.99; N,
11.55. Found: C, 56.37; H, 4.88; N, 11.51.
140.3, 141.0, 148.3, 148.7, 148.8, 154.5, 156.9, 158.3. IR (KBr)
n
1141.9, 1280.6, 1494.3, 1617.6, 2851.1, 2979.4, 3020.6, 3323.3, 3435.4
cmꢂ1. Anal. calcd. for C17H16N4O5 (%): C, 57.30; H, 4.53; N, 15.72.
Found: C, 57.03; H, 4.57; N, 15.42.
4.2.6.3. 5,6,7-Trimethoxy-N-(2-nitrophenyl)quinazoline-4-amine
(6c). Yield: 86.1%; yellow solid; mp: 124e127 ꢁC. 1H NMR (CDCl3,
4.2.6.9. N-(2-Fluorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
500 MHz):
d
3.97e4.28 (3s, 9H, 3OCH3), 7.13 (s, 1H), 7.18 (dt, 1H,
(6i). Yield: 88.9%; white solid; mp: 139e142 ꢁC. 1H NMR (CDCl3,
J ¼ 8.0 Hz,1.7 Hz), 7.68 (dt,1H, 8.0 Hz,1.7 Hz), 8.19 (dd,1H, J ¼ 8.0 Hz,
500 MHz):
1H), 8.52e8.55 (m, 1H), 8.67 (s, 1H), 11.09 (s, 1H). 13C NMR (CDCl3,
500 MHz): 57.5, 61.7, 62.9, 97.8, 102.5, 115.3, 115.6, 124.3, 125.0,
127.2, 137.3, 142.0, 149.2, 153.2, 155.1, 158.5, 161.3. IR (KBr) : 792.7,
d 3.98e4.33 (3s, 9H, 3OCH3), 7.25e7.29 (m, 3H), 7.81 (s,
1.7 Hz), 8.65 (s,1H), 8.97 (dd,1H, J ¼ 8.0 Hz,1.7 Hz),11.97 (s,1H, NH).
13C NMR (CDCl3, 500 MHz):
d
56.3, 61.6, 62.3, 104.8, 105.4, 122.8,
d
124.7,125.8,134.6,135.1,138.9,140.9,148.6,149.2,153.9,156.7,158.7.
n
IR (KBr)
n
: 807.5, 1149.6, 1290.4, 1498.7, 1606.7, 2833.4, 2941.4,
1145.6, 1296.2, 1498.7, 1635.6, 2883.6, 2951.1, 3020.5, 3281.4, 3487.2
cmꢂ1. Anal. calcd. for C17H16FN3O3 (%): C, 62.00; H, 4.90; N, 12.76.
Found: C, 61.78; H, 5.17; N, 12.92.
3007.6, 3288.6, 3400 cmꢂ1. Anal. calcd. for C17H16N4O5 (%): C, 57.30;
H, 4.53; N, 15.72. Found: C, 57.06; H, 4.21; N, 15.98.
4.2.6.4. 5,6,7-Trimethoxy-N-(4-methylphenyl)quinazoline-4-amine
4.2.6.10. N-(2-Bromo-4,6-dimethylphenyl)-5,6,7-trimethoxyquinazo
hydrochloride (6d). Yield: 84.8%; light yellow needles; mp: 171e
line-4-amine hydrochloride (6j). Yield: 83.2%; white solid; mp: 245e

342
Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
247 ꢁC.1H NMR (CD3OD, 500 MHz):
d
2.24, 2.34 (2s, 6H, 2CH3), 3.94e
1H), 7.84 (d, 2H, J ¼ 8.6 Hz), 7.95 (d, 2H, J ¼ 8.6 Hz), 8.83 (s, 1H),
10.83 (s, 1H). 13C NMR (DMSO-d6, 500 MHz):
56.7, 56.8, 60.7, 91.6,
101.1, 123.6, 125.8, 126.6, 126.7, 135.8, 137.6, 142.5, 143.2, 151.1, 153.3,
4.20 (3s, 9H, 3OCH3), 7.02 (s, 1H), 7.15 (s, 1H), 7.39 (s, 1H), 8.16 (s, 1H).
13C NMR (CD3OD, 500 MHz):
17.7, 19.5, 56.2, 60.6, 62.2, 91.6, 96.4,
101.7, 121.7, 122.1, 130.4, 130.6, 138.1, 139.9, 141.7, 150.1, 150.7, 159.9,
d
d
159.6. IR (KBr) n: 810.6, 1149.6, 1280.3, 1475.2, 1625.3, 2935.6,
161.4. IR (KBr)
n
: 812.0, 1149.6, 1298.1, 1490.9, 1633.7, 2916.4, 2982.0,
2981.9, 3162.9, 3270.3, 3418.6 cmꢂ1. Anal. calcd. for C18H16F3N3O3
(%): C, 56.99; H, 4.25; N, 11.08. Found: C, 56.66; H, 4.09; N, 11.40.
3170.7, 3269.6, 3408.3 cmꢂ1. Anal. calcd. for C19H21BrClN3O3 (%): C,
50.18; H, 4.65; N, 9.24. Found: C, 50.30; H, 4.45; N, 9.11.
4.2.6.17. N-(3-Trifluoromethylphenyl)-5,6,7-trimethoxyquinazoline-
4-amine (6q). Yield: 85.1%; yellow solid; mp: 163e166 ꢁC. 1H NMR
4.2.6.11. N-(2-Chloro-4-bromophenyl)-5,6,7-trimethoxyquinazoline-
4-amine (6k). Yield: 81.0%; white needles; mp: 195e196 ꢁC. 1H
(CDCl3, 500 MHz):
1H, J ¼ 8.0 Hz), 7.47 (t, 1H, J ¼ 8.1 Hz), 7.58 (d, 1H, J ¼ 8.1 Hz), 7.83 (s,
1H), 8.78 (s, 1H), 10.69 (s, 1H). 13C NMR (CDCl3, 500 MHz):
57.4,
61.7, 63.2, 97.1, 102.6, 124.2, 125.2, 126.7, 130.9, 131.2, 133.4, 138.2,
138.8, 141.2, 150.1, 150.6, 159.2, 160.7. IR (KBr) : 809.5, 1157.1,
1295.4, 1496.2, 1635.7, 2889.3, 2956.8, 3009.6, 3254.4, 3420.2 cmꢂ1
d 3.85e4.18 (3s, 9H, 3OCH3), 7.31 (s, 1H), 7.35 (d,
NMR (DMSO-d6, 500 MHz): d 3.88e4.16 (3s, 9H, 3OCH3), 7.13 (s,1H),
7.62 (dd, 1H, J ¼ 9.2 Hz, 2.3 Hz), 7.85 (d, 1H J ¼ 2.3 Hz), 8.55 (s, 1H),
d
8.80 (d, 1H, J ¼ 9.2 Hz), 10.45 (s, 1H). 13C NMR (DMSO-d6, 500 MHz):
d
56.8, 61.5, 63.1, 104.2, 104.6, 115.2, 124.9, 125.0, 131.1, 131.8, 136.0,
n
140.9, 148.3, 148.7, 154.1, 156.7, 158.6. IR (KBr)
n
: 808.7, 1153.4,
.
1294.2, 1469.8, 1629.5, 2939.5, 2977.8, 3010.9, 3309.3, 3420.5 cmꢂ1
.
Anal. calcd. for C18H16F3N3O3 (%): C, 56.99; H, 4.25; N, 11.08. Found:
C, 56.78; H, 4.45; N, 11.26.
Anal. calcd. for C17H15BrClN3O3 (%): C, 48.08; H, 3.56; N, 9.89.
Found: C, 47.78; H, 3.83; N, 10.04.
4.2.6.18. N-(3-Chlorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
4.2.6.12. N-(2-Bromo-5-fluorophenyl)-5,6,7-trimethoxyquinazoline-
4-amine (6l). Yield: 80.1%; pale yellow solid; mp: 158e161 ꢁC. 1H
hydrochloride (6r). Yield: 45.8% (only obtained under CH); yellow
solid; mp: 192e195 ꢁC. 1H NMR (DMSO-d6, 500 MHz):
d 3.88e4.14
NMR (DMSO-d6, 500 MHz):
d
3.91e4.26 (3s, 9H, 3OCH3), 7.22 (dt,
(3s, 9H, 3OCH3), 7.10 (s, 1H), 7.16 (dd,1H, J ¼ 8 Hz,1.2 Hz), 7.40 (t,1H,
1H, J ¼ 8.6 Hz, 2.9 Hz), 7.42 (s, 1H), 7.84 (dt, 1H, J ¼ 8.6 Hz, 2.9 Hz),
J ¼ 8.0 Hz), 7.72 (dd, 1H, J ¼ 8.0 Hz, 1.2 Hz), 8.21 (d, 1H, J ¼ 1.2 Hz),
8.06 (dd, 1H, J ¼ 10.3 Hz, 2.9 Hz), 8.87 (s, 1H), 10.90 (s, 1H). 13C NMR
8.53 (s, 1H), 10.01 (s, 1H). 13C NMR (DMSO-d6, 500 MHz):
d 56.3,
(DMSO-d6, 500 MHz):
d
57.4, 61.8, 63.5, 97.4, 102.6, 114.9, 115.2,
60.1, 62.3, 95.4, 101.7, 118.6, 121.0, 122.0, 124.1, 131.0, 133.7, 135.5,
115.7, 134.4, 137.6, 141.4, 149.9, 150.7, 159.2, 160.6, 161.0, 162.6. IR
141.5, 150.1, 152.9, 156.0, 158.7. IR (KBr) n: 803.6, 1147.6, 1278.5,
(KBr)
n
: 821.7, 1147.7, 1294.2, 1496.7, 1626.0, 2941.2, 2947.2, 3008.9,
1485.2, 1643.4, 2931.8, 2968.5, 3015.6, 3290.3, 3417.7 cmꢂ1. Anal.
calcd. for C17H17Cl2N3O3 (%): C, 53.42; H, 4.48; N, 10.99. Found: C,
53.30; H, 4.26; N, 10.70.
3240.3, 3410.5 cmꢂ1. Anal. calcd. for C17H15BrFN3O3 (%): C, 50.02; H,
3.70; N, 10.29. Found: C, 50.34; H, 3.43; N, 10.51.
4.2.6.13. N-(3-Fluorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
4.2.6.19. N-(2-Bromo-5-trifluoromethylphenyl)-5,6,7-trimethoxyqui
nazoline-4-amine (6s). Yield: 83.6%; pale yellow solid; mp: 185e
(6m). Yield: 81.9% (only obtained under MW); white solid; mp:
158e161 ꢁC. 1H NMR (CDCl3, 500 MHz):
d
3.98e4.31 (3s, 9H,
188 ꢁC. 1H NMR (DMSO-d6, 500 MHz):
d 3.88e4.12 (3s, 9H, 3OCH3),
3OCH3), 7.04 (t, 1H, J ¼ 8.0 Hz), 7.36 (d, 1H, J ¼ 8.0 Hz), 7.45 (dd, 1H,
7.13 (s, 1H), 7.40 (dd, 1H, J ¼ 8.6 Hz, 2.3 Hz), 7.96 (d, 1H, J ¼ 8.1 Hz),
J ¼ 14.9 Hz, 8.6 Hz), 7.68 (d, 1H, J ¼ 10.0 Hz), 7.85 (s, 1H), 8.62 (s, 1H),
8.57 (s, 1H), 9.21 (s, 1H), 10.50 (s, 1H). 13C NMR (DMSO-d6,
10.68 (s, 1H). 13C NMR (CDCl3, 500 MHz):
d
57.4, 61.6, 62.9, 97.9,
500 MHz):
d 57.2, 61.3, 63.4, 92.5, 100.4, 123.5, 124.1, 124.6, 125.4,
102.0,110.8,113.9,118.5,130.5,137.4,142.0,148.9,149.0,158.6,161.2,
134.6, 135.9, 137.7, 141.4, 149.8, 150.6, 160.1, 160.5, 161.1. IR (KBr) n:
161.9, 163.9. IR (KBr)
n
: 801.7, 1159.2, 1292.3, 1487.1, 1631.8, 2856.8,
792.7, 1145.8, 1287.5, 1464.0, 1627.9, 2887.4, 2954.9, 3037.9, 3238.5,
3405.1 cmꢂ1. Anal. calcd. for C18H15BrF3N3O3 (%): C, 47.18; H, 3.30;
N, 9.17. Found: C, 47.36; H, 3.38; N, 9.07.
2983.9, 3012.5, 3271.3, 3451.2 cmꢂ1. Anal. calcd. for C17H16FN3O3
(%): C, 62.00; H, 4.90; N, 12.76. Found: C, 61.76; H, 5.01; N, 12.50.
4.2.6.14. N-(4-Bromophenyl)-5,6,7-trimethoxyquinazoline-4-amine
4.2.6.20. N-(4-Chlorophenyl)-5,6,7-trimethoxyquinazoline-4-amine
(6n). Yield: 82.4%; light yellow needles; mp: 143e145 ꢁC. 1H NMR
dihydrochloride (6t). Yield: 90.0%; light yellow needles; mp: 161e
(DMSO-d6, 500 MHz):
(d, 2H, J ¼ 9.2 Hz), 7.87 (d, 2H, J ¼ 8.6 Hz), 8.49 (s, 1H), 9.98 (s, 1H).
13C NMR (DMSO-d6, 500 MHz):
57.3, 61.7, 62.7, 103.9, 104.6, 115.6,
124.1, 131.9, 138.8, 140.8, 148.4, 148.6, 154.3, 156.9, 158.4. IR (KBr)
d
3.87e4.13 (3s, 9H, 3OCH3), 7.09 (s, 1H), 7.56
163 ꢁC. 1H NMR (CDCl3, 500 MHz):
d
3.97e4.30 (3s, 9H, 3OCH3);
7.44 (d, 2H, J ¼ 8.6 Hz), 7.65 (d, 2H, J ¼ 8.6 Hz), 7.80 (s, 1H), 8.59 (s,
1H), 10.59 (s, 1H). 13C NMR (CDCl3, 500 MHz):
54.3, 62.9, 63.5,
98.8, 102.3, 124.3, 129.5, 132.0, 134.9, 138.8, 141.9, 149.1, 149.8, 158.5,
d
d
n
:
817.8, 1139.9, 1298.1, 1467.8, 1608.6, 2947.2, 2990.6, 3003.2, 3294.6,
3411.7 cmꢂ1. Anal. calcd. for C17H16BrN3O3 (%): C, 52.32; H, 4.13; N,
10.77. Found: C, 52.05; H, 4.15; N, 10.87.
160.8. IR (KBr) n: 809.8, 1155.4, 1294.2, 1490.9, 1616.6, 2893.2,
2987.7, 3012.8, 3271.1, 3416.4 cmꢂ1. Anal. calcd. for C17H18Cl3N3O3
(%): C, 48.77; H, 4.33; N, 10.04. Found: C, 48.81; H, 4.60; N, 9.75.
4.2.6.15. N-((2-Fluoro-5-trifluoromethyl)phenyl)-5,6,7-trimethoxy
quinazoline-4-amine (6o). Yield: 80.7%; white solid; mp: 173e
4.2.6.21. N-(2-Fluoro-3-(trifluoromethyl)phenyl)-5,6,7-trimethoxyqu
inazolin-4-amine hydro-chloride (6u). Yield: 81.2%, yellow solid;
176 ꢁC. 1H NMR (DMSO-d6, 500 MHz):
d
3.90e4.22 (3s, 9H, 3OCH3),
7.39 (s, 1H), 7.67 (t, 1H, J ¼ 9.7 Hz), 7.79e7.81 (m, 1H), 8.36e8.37 (m,
1H), 8.85 (s, 1H), 10.80 (s, 1H). 13C NMR (DMSO-d6, 500 MHz):
57.3,
61.7, 63.1, 97.6, 102.6, 117.4, 117.6, 123.8, 125.1, 125.8, 126.9, 138.9,
141.3, 149.9, 151.0, 157.2, 159.4, 160.9. IR (KBr) : 810.1, 1161.0,
1292.3, 1489.0, 1627.9, 2941.4, 2970.4, 3030.2, 3277.2, 3430.9 cmꢂ1
mp: 152e154 ꢁC. 1H NMR (CDCl3, 500 MHz):
d 3.99e4.36 (3s, 9H,
3OCH3), 7.27 (s, 1H), 7.41 (t, 1H, J ¼ 8.0 Hz), 7.52 (t, 1H, J ¼ 7.5 Hz),
8.71 (s, 1H), 8.86 (t, 1H, J ¼ 7.5 Hz), 11.19 (s, 1H); 13C NMR (CDCl3,
500 MHz): 57.5, 61.7, 63.1, 98.2, 102.7, 121.1, 123.4, 124.8, 124.9,
126.5, 126.6, 127.8, 138.1, 142.2,149.0, 149.3, 158.6,161.5. Anal. calcd.
for C18H16ClF4N3O3: C, 49.84; H, 3.72; N, 9.69. Found C, 49.54; H,
4.01; N, 9.48.
d
n
.
Anal. calcd. for C18H15F4N3O3 (%): C, 54.41; H, 3.81; N, 10.58. Found:
C, 54.48; H, 4.10; N, 10.35.
4.2.6.22. N-(2-Bromo-4,6-difluorophenyl)-5,6,7-trimethoxyquinazolin-
4-amine (6v). Yield, 84.8%; yellow oil; 1H NMR (CDCl3, 500 MHz):
4.2.6.16. 5,6,7-Trimethoxy-N-(4-(trifluoromethyl)phenyl)quinazo-
line-4-amine (6p). Yield: 81.1%; yellow solid; mp: 143e145 ꢁC. 1H
d
3.97e4.20 (3s, 9H, 3OCH3), 6.97 (dt,1H, J ¼ 9.2 Hz, 2.9 Hz), 7.09 (s,1H),
NMR (DMSO-d6, 500 MHz):
d 3.90e4.23 (3s, 9H, 3OCH3), 7.38 (s,
7.26e7.29 (m,1H), 8.47 (s,1H), 9.07 (s,1H); 13C NMR (CDCl3, 500 MHz):

Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
343
56.4, 61.4, 62.4,104.1,104.3,104.6,115.8,116.0,122.4,123.5,140.6,148.7,
148.8, 155.0, 157.9, 158.3, 159.9. Anal. calcd. for C17H14BrF2N3O3: C,
47.91; H, 3.31; N, 9.86. Found C, 47.95; H, 3.59; N, 9.58.
used to treat cultured cells. Tested cells were plated in 96-well
plates at a density of 2 ꢃ 103 cells per well per 100
mL of the proper
culture medium and treated with the compounds at 1e100
mM for
72 h. In parallel, cells treated with 0.1% DMSO served as a control.
An MTT assay (Roche Molecular Biochemicals, 1465-007) was per-
formed 30 h later according to the manufacturer’s instructions. This
assay was based on the cellular cleavage of MTT into formazan,
which is soluble in cell culture medium. Any absorbance caused by
formazan was measured at 595 nm with a microplate reader (BIO-
RAD, model 680), and this absorbance was directly proportional to
the number of living cells in culture. The experiment was per-
formed in triplicate. The percentage cytotoxicity was calculated
using the formula.
4.2.6.23. 4-Iodo-2-(5,6,7-trimethoxyquinazolin-4-ylamino)benzoic
acid hydrochloride (6w). Yield, 80.5%; white solid; mp: 195e198 ꢁC.
1H NMR (CDCl3, 500 MHz):
d 3.91e4.24 (3s, 9H, 3OCH3), 7.39 (s, 1H),
8.01 (dd, 1H, J ¼ 8.6 Hz, 1.7 Hz), 8.17 (d, 1H, J ¼ 8.6 Hz), 8.28 (d, 1H,
J ¼ 1.7 Hz), 8.86 (s, 1H), 12.26 (s, 1H); 13C NMR (CDCl3/CD3OD,
500 MHz): 57.2, 61.5, 62.5, 89.4, 96.3,103.0,123.0,126.6,137.2,140.1,
141.3, 141.6, 148.6, 150.0, 158.2, 161.5, 167.4. Anal. calcd. for
C
18H17ClIN3O5: C, 41.76; H, 3.31; N, 8.12. Found C, 41.86; H, 3.19;
N, 8.09.
ðControl abs ꢂ Blank absÞ ꢂ ðTest abs ꢂ Blank absÞ
ðControl abs ꢂ Blank absÞ
%Cytotoxicity ¼
ꢃ 100
4.2.6.24. N-(2-Fluorophenylethyl)-5,6,7-trimethoxyquinazolin-4-
4.3.3. AO/EB staining
amine (6x). Yield: 83.9%; yellow oil; 1H NMR (CDCl3, 500 MHz):
Cells were seeded at a concentration of 5 ꢃ 104 cell/ml in a
volume of 0.6 mL on a sterile cover slip in six-well tissue culture
plates. After incubation, the culture medium was removed and
replaced with fresh medium plus 10% FBS and then supplemented
with tested compound. After the treatment period, the cover slip
d
3.08 (t, 2H, J ¼ 6.9 Hz), 3.89 (q, 2H, J ¼ 6.3 Hz), 3.80e3.96 (3s, 9H,
3OCH3), 6.98 (s, 1H), 7.04e7.10 (m, 2H), 7.20e7.23 (m, 1H), 7.26e
7.28 (m, 1H), 7.80 (t, 1H, J ¼ 5.2 Hz), 8.46 (s, 1H); 13C NMR (CDCl3,
500 MHz): 40.9, 56.1, 61.2, 61.8,103.1,104.1,115.4,124.2,126.1,128.3,
131.2, 131.3, 139.9, 147.9, 149.2, 152.8, 157.5, 159.3, 162.4. Anal. calcd.
for C19H20FN3O3: C, 63.85; H, 5.64; N, 11.76. Found C, 63.61; H, 5.52;
N 11.99.
with monolayer cells was inverted on the glass slide with 20
mL of
AD/EB stain (100 g/mL). Fluorescence was read on an IX71SIF-3
m
fluorescence microscope (OLYMPUS Co., Japan).
4.2.6.25. 4,5-Dimethoxy-2-(5,6,7-trimethoxyquinazolin-4-ylamino)
4.3.4. Western blot analysis
benzoic acid hydrochloride (6y). Yield, 85.4%; white solid; mp:
PC3 cells were seeded on a six-well plate and incubated in RPMI
1640 medium plus 10% FBS at 37 ꢁC. After incubation for 36e48 h,
the medium was removed and the cells were incubated with
serum-free medium for 24 h. Then, the cells were treated with the
title compounds at different concentrations for 60 min followed by
10 ng/mL EGF for 10 min. The plate was then immediately placed on
ice to quench the phosphorylation process. The medium was
removed and the cells were rinsed with ice-cold phosphate-buff-
ered saline (PBS) buffer twice. Then, the cells were treated with
lysis buffer [1%NP-40, 0.1% sodium dodecyl sulfate (SDS), 150 mM
NaCl, 10 mM TriseHCl, 1 mM EDTA, 0.6 mM Na3VO4, 10 mM NaF,
207e210 ꢁC. 1H NMR (CDCl3, 500 MHz):
d 3.94e4.24 (3s, 9H,
3OCH3), 4.47 (s, 6H, 2OCH3), 7.48 (s, 1H), 7.62 (s, 1H), 8.33 (s, 1H),
8.56 (s, 1H); 13C NMR (CDCl3/CD3OD, 500 MHz): 60.0, 60.0, 60.3,
65.1, 66.0, 105.2, 109.3, 111.0, 112.8, 117.0, 123.8, 125,3, 139.2, 144.7,
148.7, 153.5, 156.4, 161.2, 163.1, 172.8. Anal. calcd. for C20H22ClN3O7:
C, 53.16; H, 4.91; N, 9.30. Found C, 53.45; H, 4.70; N, 9.49.
4.2.6.26. N-(3-Fluorophenylethyl)-5,6,7-trimethoxyquinazolin-4-
amine (6z). Yield: 83.7%; yellow oil; 1H NMR (CDCl3, 500 MHz):
d
3.03 (t, 2H, J ¼ 6.9 Hz), 3.89 (q, 2H, J ¼ 6.3 Hz), 3.77e3.96 (3s, 9H,
3OCH3), 6.92e7.01 (m, 2H), 6.98 (s, 1H), 7.07 (d, 1H, J ¼ 7.5 Hz),
7.27e7.32 (m, 1H), 7.77 (t, 1H, J ¼ 5.2 Hz), 8.47 (s, 1H); 13C NMR
(CDCl3, 500 MHz): 41.6, 56.2, 61.2, 61.5, 104.0, 104.1, 113.4, 115.7,
124.6, 130.2, 139.9, 141.9, 148.0, 149.2, 155.3, 157.6, 159.3, 162.1,
164.0. Anal. calcd. for C19H20FN3O3: C, 63.85; H, 5.64; N, 11.76.
Found C, 64.09; H, 5.93; N, 11.44.
10 mM
b
-glycerophosphate, 1 mM dithiothreitol, 10
mg/mL leu-
peptin, 10
m
g/mL pepstatin, and 40 g/mL phenylmethanesulfonyl
m
fluoride] and then with the sample buffer, followed by denaturation
at 90 ꢁC water for 5 min.
The previously prepared cell lysates were subjected to 10% SDSe
polyacrylamide gel electrophoresis, and proteins were transferred
onto poly(vinylidenedifluoride) membranes (Bio-Rad). The mem-
brane was blocked with 5% nonfat dried milk freshly made in PBS
plus 0.2% Tween 20 and then incubated with monoclonal antibody
(anti-pErk1/23, Stanta Cruz Biotechnology; or anti-actin) overnight
at 4 ꢁC. The membrane was then washed for 3e5 min with PBS plus
0.2% Tween 20, incubated again with second antibody for 2e3 h at
25 ꢁC, and washed three times with PBS plus 0.2% Tween 20. The
signal was detected by enhanced chemical luminescence detection
system (PIERCE) [32].
4.3. Anticancer activity bioassay
4.3.1. Cell culture
The human prostate cancer cell line PC3, breast cancer cell line
Bcap-37, and gastric cancer cell line BGC823 were purchased from
the Institute of Biochemistry and Cell Biology, China Academy of
Sciences and cultured in RPMI 1640 medium supplemented with
10% heat-inactivated fetal bovine serum (FBS). All cell lines were
maintained at 37 ꢁC in a humidified 5% carbon dioxide and 95% air
incubator.
4.3.5. Flow cytometry
Prepared PC3 cells (1 ꢃ 106/mL) were washed twice with cold
4.3.2. MTT assay
PBS and then re-suspended gently in 200 mL of binding buffer.
All tested compounds were dissolved in DMSO (1e100
mM so-
Thereafter, cells were stained in 5
mL of Annexin V-FITC and shaken
lution) and subsequently diluted in culture medium before being
well. Finally, the cells were mixed with 5
mL of PI, incubated for

344
Y. Zhang et al. / European Journal of Medicinal Chemistry 66 (2013) 335e344
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Conflicts of interest
None.
Author’s contributions
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Yang Zhang synthesized the compounds and carried out part of
the bioassay experiments. Linhong Jin did part of the bioassay ex-
periments. Hongmei Xiang also did part of the bioassay experi-
ments. Jian Wu and Deyu Hu took part in the compound structural
elucidation and bioassay experiments. Wei Xue carried out some
structure elucidation experiments. Prof. Song Yang is the corre-
sponding author for this work.
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This work was financially supported by the National Key Pro-
gram for Basic Research (Grant No. 2010CB126105) and the Na-
tional Nature Science Foundation of China (Grant Nos. 21172048
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Appendix A. Supplementary data
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