Synthesis and bioactivities of 6,7,8-trimethoxy-N-aryl-4-aminoquinazoline derivatives

Article abstract of DOI:10.1016/j.bmc.2007.07.006

A series of 4-aminoquinazoline derivatives is prepared by the nucleophilic substitution reaction of 6,7,8-trimethoxy-4-chloroquinazoline and aryl amine. The structures of the compounds are confirmed by elemental analysis, IR, and ¹H NMR spectral data. The compounds are also evaluated for their ability to inhibit tumor cells PC3, A431, Bcap-37, and BGC823 by MTT assays. Among them, 6b and 6e are found as potent inhibitors, with IC₅₀ values ranging from 5.8 to 9.8 μM, in vitro assay.

Full text of DOI:10.1016/j.bmc.2007.07.006

Bioorganic & Medicinal Chemistry 15 (2007) 6608–6617
Synthesis and bioactivities of 6,7,8-trimethoxy-N-
aryl-4-aminoquinazoline derivatives
Gang Liu, De-Yu Hu, Lin-Hong Jin, Bao-An Song,* Song Yang, Ping-Shen Liu,
Pinaki S. Bhadury, Yao Ma, Hui Luo and Xian Zhou
Center for Research and Development of Fine Chemicals, Key Laboratory of Green Pesticide and Bioengineering,
Ministry of Education, Key Laboratory of Fine Chemicals of Guizhou Province, Guizhou University, Guiyang 550025, PR China
Received 29 April 2007; revised 30 June 2007; accepted 6 July 2007
Available online 25 July 2007
Abstract—A series of 4-aminoquinazoline derivatives is prepared by the nucleophilic substitution reaction of 6,7,8-trimethoxy-4-
chloroquinazoline and aryl amine. The structures of the compounds are confirmed by elemental analysis, IR, and 1H NMR spectral
data. The compounds are also evaluated for their ability to inhibit tumor cells PC3, A431, Bcap-37, and BGC823 by MTT assays.
Among them, 6b and 6e are found as potent inhibitors, with IC50 values ranging from 5.8 to 9.8 lM, in vitro assay.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
ies have assumed enormous importance. Certain
compounds containing 3,4,5,-trimethoxyphenyl moiety
have been reported to be biologically active, for exam-
ple, Combretastatin A-4 (CA-4) is an excellent substance
for antitumor activity, which was first isolated from the
South African bush willow tree Combretum caffrum.
Due to its structural simplicity and potent cytotoxicity,
many synthetic analogues of CA-4 have been devel-
oped.9–11 The existing study has revealed that the group
of 3,4,5-trimethoxyphenyl is a crucial pharmacophore
for antitumor activities of CA-4 analogues.12 4-alkyl(ar-
yl)thio-quinazoline derivatives reported by us earlier
were shown to have antitumor effects.13 and that 4-ani-
linoquinazoline pharmacophore has been shown to be
effective inhibitors of EGF and PDGF.14,15 We reasoned
that the compounds containing both active groups
might generate a new group of biologically active
compounds.
Many components of mitogenic signaling pathways in
normal and neoplastic cells have been identified. These
include the large family of protein kinases, which func-
tion as components of signal transduction pathways,
playing a central role in diverse biological processes,
such as control of cell growth, metabolism, differentia-
tion, and apoptosis.1–3 The development of selective
protein kinase inhibitors that can block or modulate dis-
eases caused by abnormalities in these signaling path-
ways is widely considered as a promising approach for
drug development. Due to their deregulation in human
cancers, protein kinases, such as EGF, PDGF, belong-
ing to the epidermal growth factor-receptor family are
considered as prime targets for the development of selec-
tive inhibitors. Therefore, tyrosine kinases are expected
to be promising targets for cancer chemotherapy.4,5
The development of specific inhibitors of the epidermal
growth factor receptor (EGFR) tyrosine kinase has re-
ceived wide attention, and several elaborations of the
fundamental 4-anilinoquinazoline pharmacophore have
now been reported as potent and selective inhibitor of
this class of enzymes.6–8 With growing interest in qui-
nazoline compounds, their synthesis and biological stud-
Therefore, in continuation to our research for finding
new anticancer agents, we have synthesized new quinaz-
oline compounds (6a–6s) with the methoxy (OCH3)
groups present in the phenyl ring. Starting from gallic
acid, the synthetic route to 6,7,8-tri-methoxy-N-aryl-4-
aminoquinazoline derivatives is shown in Schemes 1
and 2. The structure of title compounds is characterized
1
by IR, H NMR, 13C NMR, and elemental analysis.
Keywords: Quinazoline; Amino moiety; Gallic acid; Synthesis; Anti-
cancer activities.
Following the reported method,16,17 the synthetic meth-
od under microwave irradiation is rapid and high yield-
ing. While in classical method, an yield of 19.9–66.2% is
*
Corresponding author. Tel.: +86 851 3620521; fax: +86 851
3622211; e-mail: songbaoan22@yahoo.com
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.07.006

G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
6609
OMe
OMe
OMe
OMe
MeO
OMe
MeO
a
OMe
NO2
MeO
OMe
NH2
MeO
OMe
NO2
c
b
COOCH3
COOCH3
COOH
COOH
2
3
1
Scheme 1. Synthesis of 2-amino-3,4,5,-trimethoxybenzoic acid. Reagents and conditions: (a) nitric acid, 40–50 ꢁC, 1–2 h; (b) 0.8 mol/L sodium
hydroxide, 95% ethanol, 45–50 ꢁC, 1–2 h; 35% HCl, pH 2–3; (c) Sn, HCl, 20 ꢁC, 4–5 h, then 80 ꢁC, 20 min.
R1
N
Cl
N
O
N
HN
MeO
MeO
MeO
MeO
COOH
NH2
MeO
MeO
MeO
MeO
c
a
b
N
NH
N
OMe
OMe
OMe
OMe
3
4
5
6
Scheme 2. Synthesis of N-arylsubstituted-4-aminoquinazoline derivatives. Reagents and conditions: (a) formamide, 120–140 ꢁC, 5–6 h; (b) POCl3,
DMF, 100–105 ꢁC, 2–3 h; (c) aryl amine, 2-propanol, MW, 50 W, 80 ꢁC, 10 min.
achieved in 4–24 h, a considerable improvement in yield
(55.6–92.5%) is noticed in just 10 min under microwave
condition.
Table 1. Reaction conditions used for the microwave assisted synthesis
of 6a–6s
Product
Microwave methodb
Classical methodc
a
Yield (%)
Reaction
time (min)
Yielda (%)
Reaction
time (h)
The compounds were evaluated for their antiprolifera-
tive activities against PC3, A431, Bcap-37, and
BGC823 cells hyperplasia in vitro by MTT method.
Among them, 6a and 6d were found highly effective
against PC3 cells and 6e showed moderate activities
against Bcap37 and BGC823 cells. The IC50 values of
potent inhibitors 6b and 6e against PC3 cells were 9.8,
8.9 lM, respectively.
6a
6b
6c
6d
6e
6f
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
80.9
78.0
80.1
59.0
89.0
86.0
92.5
82.3
79.0
89.6
89.9
75.4
58.6
55.6
77.7
80.3
79.5
78.8
80.2
4
5
51.7
46.8
61.2
19.9
65.6
63.4
36.1
55.4
63.8
66.2
64.4
54.3
34.5
32.7
64.0
41.9
58.0
48.9
61.4
5
5
5
12
12
12
24
12
8
6g
6h
6i
6j
2. Chemistry
6k
6l
12
12
24
24
5
Compound 3 was synthesized from gallic acid as de-
scribed in Scheme 1. O-methylation of the phenol with
dimethyl sulfate followed by esterification using metha-
nol produced methyl 3,4,5-trimethoxybenzoate. Reac-
tion of the ester with concentrated 70% nitric acid in
acetic acid at 40–50 ꢁC gave the methyl 2-nitro-3,4,5-
trimethoxybenzoate 1. Hydrolysis of this nitro ester fol-
lowed by reduction of the nitro group by tin dichloride
at 80 ꢁC generated 2-amino-3,4,5-trimethoxybenzoic
acid. Cyclization of the latter by formamide and subse-
quent chlorination by phosphorus oxychloride provided
4-chloro-6,7,8-trimethoxyquinazoline (Scheme 2). The
desired products (6a–6s) were then obtained under
microwave irradiation by a nucleophilic substitution
reaction involving an aryl amine and the chloro-quinaz-
oline. The results of microwave reaction are compared
with those obtained under classical reactions (Table 1).
6m
6n
6o
6p
6q
6r
6s
8
24
24
a Yields of isolated products.
b Reaction conditions: i-PrOH, reflux under MW (50 W power).
c Reaction conditions: i-PrOH, reflux temperature.
ferent conditions. The results are shown in Table 2.
Without microwave irradiation (Table 1) compound 6f
could be obtained in 63.4% after 12 h. When the reac-
tion was carried out under microwave irradiation at
80 ꢁC, the yield of 6f was increased to 80.0% in 5 min
and to 86.0% in 10 min. With longer reaction time (Ta-
ble 2, entry 3), slight lowering in the yield was observed
and might be attributed to the formation of some
byproduct. On increasing the microwave power from
40 to 50, 80, and 100 W, 70.9%, 86.0%, 88.0%, and
87.2% yields were obtained, respectively (Table 2, entries
2 and 4–6). No significant-improvement was thus no-
ticed at higher power settings when compared to that
at 50 W. When the reaction temperature was increased
It can be seen that with one step microwave assisted
approach, not only is the reaction time significantly
reduced from 4–24 h to 10 min, but the yields are also
considerably improved (55.6–92.5% with microwave
against 19.9–66.2% with classical method).
In order to optimize the reaction parameters, we selected
compound 6f and subjected it to further study under dif-

6610
G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
Table 2. Different conditions used for the microwave assisted synthesis
of 6f
from 30 ꢁC to 50 ꢁC, 70 ꢁC, and 80 ꢁC, 6f was obtained
in 56.9%, 77.0%, 80.9%, and 86.0% yields, respectively
(Table 2, entry 2 and entries 7–9). So, under optimal
condition, when reacted with the appropriate amine
and 4-chloro-6,7,8-trimethoxyquinazoline, 6f can be ob-
tained in 86.0% yield with a microwave irradiation at
50 W for 10 min at 80 ꢁC.
Entry Reaction
time (min)
Power (W) Reaction
temperature (ꢁC)
Yielda (%)
1
2
3
4
5
6
7
8
9
5
10
20
10
10
10
10
10
10
50
50
80
80.0
86.0
84.5
70.9
88.0
87.2
56.9
77.0
80.9
80
80
80
80
80
30
50
70
50
40
80
100
50
3. Anticancer activity
50
50
Some potential compounds for treatment of cancer have
been identified by studying their anti-cancer activities on
3 human tumor cells. Series of the test compounds
including 6a–6s and their commercial analogue
a Yields of isolated products. Each reaction was repeated three times
and the average yield was recorded.
Table 3. Growth inhibition of selected cell lines
HN R1
H3CO
H3CO
N
N
OCH
3
6a∼s
Compounda
R1
IC50 (lM)
b
Bcap-37c
31.2
PC3d
23.4
A431e
42.1
BGC823f
23.8
6a
6b
Br
·2HCl
O
8.1
9.8
9.0
9.9
H3CO
·2HCl
·HCl
6c
6d
6e
6f
21.0
21.0
12.3
22.1
21.0
28.7
19.0
12.6
8.9
32.1
11.0
7.6
19.0
12.1
5.8
Cl
Br
·HCl
Br
·HCl
25.3
35.6
24.5
29.7
29.7
29.0
28.9
29.9
39.6
F
6g
6h
F
F
6i
63.2
18.8
72.1
36.7
·HCl
Br
F
6j
89.0
76.5
21.0
30.0
51.5
65.4
31.0
23.1
·HCl
Cl
OCH3
OCH3
OCH3
6k
·HCl

G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
6611
Table 3 (continued)
Compounda
R1
IC50 (lM)
b
Bcap-37c
28.9
PC3d
25.5
A431e
45.2
BGC823f
6l
32.2
39.0
21.0
37.7
34.4
35.4
12.0
11.9
16.8
NO
2
·HCl
·HCl
6m
31.1
29.9
65.4
52.2
45.5
23.2
12.9
13.7h
24.4
31.2
36.7
31.0
20.1
18.8
10.0
8.9
37.7
31.0
21.2
19.0
22.2
21.0
12.0
6.9i
NO
2
·HCl
6n
O N
2
6o
6p
Cl ·2HCl
6q
·2HCl
OH
H
6r
R-(-)
S-(+)
CH3
H
6s
CH3
PD 153035g
a These compounds were tested as the free base.
b IC50concentrations needed to inhibit cell growth by 50% as determined from the dose–response curve. Determined by three separate experiments
and each was performed in triplicate.
c Breast cancer.
d Prostate cancer.
e Uterus cancer.
f Stomach cancer.
g The standard compound used for comparison of activity.
h The value was determined by using our assay protocol.
i The IC50 value of 3.0 lM for A431 cells reported by Ras et al.19
PD153035 [6,7-dimethoxy-N-(3-bromophenyl)-4-amino-
quinazoline]18 were evaluated for their anti-proliferation
activities against four types of human cancer cell lines,
breast cancer, prostate cancer, uterus cancer, and stom-
ach cancer cells (Table 3).
PD153035 is totally similar except that PD153035 con-
tains 6,7-dimethoxy substitution while 6b is 6,7,8-tri-
methoxy derivative, and the cell growth inhibition
activity of 6b is better than that of PD153035. An inter-
esting observation noted was that in case of 6,7,8-tri-
methoxy-N-(3-bromophenyl)-4-aminoquinazoline, the
hydrochloride salt of the amine (6e) was more effective
than the free amine (6d). Compound 6d has the average
IC50values ranging from 12.1 to 21.0 lM which are con-
siderably lower than those of compound 6e. This finding
is thought to be beneficial for further SAR studies of
quinazoline analogues.
As shown in Table 3, the nature of group attached at the
N-atom present at the side chain of 4 position of quinaz-
oline ring greatly influences the anti-tumor properties of
these compounds. The compounds 6a, 6c, 6f–6q, and 6r
showed weak to moderate anti-tumor activities against
all the tested tumor cells. Compounds 6b, 6e, and 6s
bearing 2-methoxydibenzofuran-3-yl, 3-bromophenyl,
(S)-a-methylbenzyl at the 4-position in quinazoline
show best anti-tumor activities with average IC50 rang-
ing from 5.8 to 12.9 lM over all the cancer cell lines.
Remarkably, these values are comparable to the average
IC50 of PD 153035 (6.9–16.8 lM). It is clear that the cell
growth inhibition bioactivity of some title compounds,
such as 6b, and 6e, is better than their commercial ana-
logue PD153035. For example, the structure of 6b and
In order to evaluate biological activity of these identified
compounds, we carried out several ERK phosphoryla-
tion experiments. First, we sought to determine effects
of these compounds on (epidermal growth factor)
EGF-induced ERK1/2 phosphorylation. PC3 cells were
pretreated with 20 lM of these compounds, respectively,
for 60 min at 37 ꢁC in media with serum, followed by
treatment with 40 ng/mL EGF for 10 min. The result

6612
G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
5. Experimental
5.1. Analysis and instruments
Unless otherwise stated, all common reagents and sol-
vents were used as obtained from commercial suppliers
without further purification. All melting points of the
products were determined on a XT-4 binocular micro-
scope (Beijing Tech Instrument Co., China) and are
not corrected. The infrared spectra were recorded on a
Bruker VECTOR22 spectrometer in KBr disks. 1H
NMR (solvent DMSO-d6) and 13C NMR spectra (sol-
vent DMSO-d6) were recorded on a JEOL-ECX 500
NMR spectrometer at room temperature using TMS
as an internal standard. D2O exchange was used to con-
firm the assignment of NH proton signals. Elemental
analysis was performed by an Elementar Vario-III
CHN analyzer. Microwave reactions were performed
on a variable power Focused Microwave Synthesis, Dis-
coverTM LabMate equipped with a high sensitivity IR
sensor for temperature control and measurement.
Figure 1. Effect of quinazoline derivatives on phosphorylation of
EGFR and ERK 1/2 triggered by EGF.
5.2. Preparation of methyl 2-nitro-3,4,5-trimethoxybenzoate
(1)
is shown in Figure 1. The blot was in sequence, negative
control (control), positive control (EGF), and com-
pound plus EGF (6d+E, 6b+E, PD153035+E, 6f+E,
6a+E, 6e+E, and 6s+E). It can be seen that the com-
pounds 6a, 6b, 6d, 6e, 6f, and 6s at 20 lM had no signif-
icant inhibitory effect on EGF-induced ERK1/2
phosphorylation in PC3 cells. Results in Figure 1 indi-
cate that PD153035at 20 lM blocked ERK1/2 phos-
phorylation in PC3 cells induced by EGF.
To a three-necked 100 mL round-bottom flask equipped
with a magnetic stirrer bar were added methyl 3,4,5-
trimethoxybenzoate20 and 35% acetic acid (10 mL).
The solution was stirred at 10–30 ꢁC on an ice bath
and 70% concentrated nitric acid (2.5 mL) was added
dropwise over a period of 30 min. The mixture was di-
luted with cold water (60 mL), and the resulting precip-
itate was filtered and washed well with water, dried to
give compound as pale yellow solid (0.61 g), yield,
The preliminary bioassay demonstrated that these com-
pounds possess no significant inhibitory activities
against EGFR, while the 4-anilinoquinazolines usually
do, suggesting that cytotoxicity may not result from
inhibiting EGFR and there is probably a new mecha-
nism which accounts for their activity.
1
25.4%; mp 62–63 ꢁC; H NMR (DMSO-d6, 500 MHz):
d 3.83 (s, 3H, COOCH3), 3.94 (t, 9H, 3OCH3,
J = 25 Hz), 7.34 (s, 1H, Ph-H); 13C NMR (DMSO-d6,
500 MHz): d 53.7, 57.1, 61.6, 63.1, 109.2, 117.7, 139.6,
145.5, 146.2, 154.7, 163.3; IR (KBr): 866.0, 1112.9,
1232.5, 1344.4, 1496.8, 1544.9, 1579.7, 2846.9, 2962.7,
3012.8 cmꢀ1; Anal. Calcd for C11H13NO7: C, 48.71; H,
4.83; N, 5.16. Found: C, 48.47; H, 5.02; N, 4.81.
PC3 cells cultured in 12-well were pretreated with qui-
nazoline derivatives (20lM) for 60 min followed by
treatment with EGF (60 ng/mL for 10 min). Cells were
harvested, resuspended in 2· sample buffer (Tris–HCl,
pH 7.4, 4% SDS, glycerine, glycerol 20%, bromophenol
blue 1%, and b-mercaptoethanol 5%), sonicated, and
denatured at 90 ꢁC water for 5 min. Proteins were sepa-
rated on 10% SDS–PAGE and subjected to Western blot
analyses. Antibodies used for Western blot analyses were
anti-phosphotyrosine, anti-pErk1/2, and anti-caveolin.
5.3. Preparation of 2-nitro-3,4,5-trimethoxybenzoic acid
(2)
To a 250 mL three-necked-round-bottom flask equipped
with a magnetic stirrer were added methyl 2-nitro-3,4,5-
trimethoxybenzoate (14.0 g, 50 mmol) and 0.8 mol/L
sodium hydroxide solution (120 mL, 100 mmol) in 95%
ethanol (60 mL). The mixture was stirred at 45–50 ꢁC
for 1–2 h and the course of the reaction was followed
by TLC (eluent: petroleum/ethyl acetate, 1:1, v:v). After
the completion of the reaction, the mixture was cooled
to room temperature and acidified with 35% HCl solu-
tion until a pH of 2–3 was obtained. The resulting pre-
cipitate was filtered and washed with water, dried to
give compound as pale yellow solid (1.2 g), yield,
92.3%; mp 160–161 ꢁC; 1H NMR (DMSO-d6,
500 MHz): d 3.88 (t, 9H, 3OCH3,J = 25 Hz), 7.32 (s,
1H, Ph-H); 13C NMR (DMSO-d6, 500 MHz): d 57.0,
61.5, 63.1, 109.3, 118.8, 139.8, 145.3, 145.8, 154.5,
164.1; IR (KBr): 725.2, 1116.8, 1246.0, 1340.5, 1496.8,
4. Conclusion
In summary, the current method offers a novel approach
for the preparation of new N-aryl-4-aminoquinazoline
derivatives 6a–6s under microwave irradiation. The syn-
thesis from gallic acid is rapid and provides higher yield
as compared to the classical method. All compounds
are fully characterized by spectroscopic methods. The
compounds 6b and 6e were found to be potent inhibitors
of tumor cells, with IC50 values ranging from 5.8 to 9.8 lg/
mL in vitro assay.

G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
6613
1546.9, 1577.8, 1695.4, 2850.8, 3107.3 cmꢀ1; Anal. Calcd
for C10H11NO7: C, 46.70; H, 4.31; N, 5.45. Found: C,
46.77; H, 4.14; N, 5.32.
poured into ice water (30 g), neutralized with saturated
NaHCO3 solution to a pH of 4–5, and filtered. The
mother liquor was extracted with chloroform (2·
20 mL), and the combined organic layers were dried
over anhydrous magnesium sulfate and filtered again.
After removal of the chloroform under reduced pres-
sure, the gray solid was recrystallized from petroleum
ether to afford a brown solid (0.4 g), yield, 62.5%;
mp100–103 ꢁC;1H NMR (DMSO-d6, 500 MHz): d 8.93
(s, 1H, H-2 of quinazoline), 7.29 (s, 1H, H-5 of quinaz-
oline), 4.01 (t, 9H, 3OCH3, J = 25 Hz); 13C NMR
(DMSO-d6, 500 MHz): d 56.3, 61.1, 62.2, 98.6, 120.1,
142.7, 146.3, 148.1, 150.9, 154.3, 158.9; IR (KBr):
786.9, 1136.1, 1246.0, 1467.8, 1602.9, 2845.0, 2945.3,
3099.6 cmꢀ1; Anal. Calcd for C11H12N2O4: C, 51.88;
H, 4.35; N, 11.00. Found: C, 52.02; H, 4.21; N, 10.94.
5.4. Preparation of 2-amino-3,4,5-trimethoxybenzoic acid
(3)
To a 100 mL three-necked-round-bottom flask equipped
with a magnetic stirrer was added Sn powder (5.3 g,
45 mmol) in concentrated HCl (18 mL) and stirred at
20 ꢁC for 4–5 h until a homogeneous solution was ob-
tained. 2-Nitro-3,4,5-trimethoxybenzoic acid (2.6 g,
10 mmol) was then added and the resulting mixture
was heated at 80 ꢁC for 20 min. Concentrated HCl
(5 mL) solution was added and again stirred for
10 min. The mixture was cooled to 0–10 ꢁC and white so-
lid was filtered and washed with concentrated HCl. The
solid was then neutralized with 10% K2CO3 solution un-
til a pH of 9–10 was obtained. The resulting mixture was
stirred for 1–2 h and filtered to give a gray solid. The so-
lid was again treated with 10% K2CO3solution, acidified
with 30% HOAc solution to adjust the pH in the range 2–
3. The resulting precipitate was filtered and washed with
water, dried to give compound as white solid (1.4 g),
yield, 61.7%; mp126–128 ꢁC;1H NMR (DMSO-d6,
500 MHz): d 3.82 (t, 9H, 3OCH3J = 25 Hz), 3.45 (s,
2H, NH2), 7.07 (s, 1H, Ph-H); 13C NMR (DMSO-d6,
500 MHz): d 56.6, 60.6, 60.9, 104.5, 110.9, 140.4, 141.6,
142.9, 147.6, 169.4; IR (KBr): 744.5, 1149.6, 1273.0,
1458.2, 1575.8, 1670.4, 3200.0, 3379.3, 3487.3,
3379.3 cmꢀ1; Anal. Calcd for C10H13NO5: C, 52.86; H,
5.77; N, 6.16. Found: C, 52.74: H, 5.43; N, 6.02.
5.7. General procedure for 6,7,8-trimethoxy-N-aryl-4-
aminoquinazolines (6a–6s) (microwave method)
A
mixture of 4-chloro-6,7,8-trimethoxyquinazoline
(4.0 mmol) and aryl amine (4.0 mmol) in 2-propanol
(10 mL) was stirred for three minutes, then the mixture
was irradiated in the microwave oven at 50 W for 10 min
at 80 ꢁC. Upon completion of the reaction, as monitored
by TLC, the solvent was removed under reduced pres-
sure and the residue was washed with water, filtered
off, and purified by silica gel column chromatography
(petroleum ether/ethyl acetate, 5:1, v:v) to give the title
compounds.
5.7.1. General procedure for 6,7,8-trimethoxy-N-aryl-4-
amino quinazolines (6a–6s) (classical method). A solution
of 4-chloroquinazoline (4.0 mmol) and aryl amine
(4.0 mmol) in 2-propanol (100 mL) was stirred under
reflux for 4–24 h. The work-up was carried out as
described for the microwave method.
5.5. Preparation of 6,7,8-trimethoxyquinazoline-4-one (4)
To a 100 mL three-necked-round-bottom flask equipped
with a magnetic stirrer were added 2-amino-3,4,5-tri-
methoxybenzoic acid (1.1 g, 5 mmol) and formamide
(2 mL). The resulting solution was refluxed at 120–
140 ꢁC for 5–6 h, then cooled to 100 ꢁC. Water (10 mL)
was added dropwise, cooled to 60 ꢁC, and again another
10 mL of water was added in one shot. The reaction mix-
ture was brought to room temperature, filtered to give a
gray solid (0.9 g), which was recrystallized from anhy-
drous ethanol to finally afford a white solid (0.3 g), yield,
25.4%; mp 220–222 ꢁC; 1H NMR (DMSO-d6, 500 MHz):
d 3.88 (t, 9H, 3OCH3, J = 25 Hz), 7.35 (s, 1H, H-5 of qui-
nazolone), 8.01 (s, 1H, H-2 of quinazolone),12.21 (s, 1H,
NH); 13C NMR (DMSO-d6, 500 MHz): d 56.4, 61.4,
62.4, 101.7, 119.2, 139.0, 143.3, 147.7, 148.3, 152.7,
160.6; IR (KBr): 798.5, 1126.4, 1286.5, 1473.6, 1612.5,
1664.6, 3163.3, 3200.0, 3304.1 cmꢀ1; Anal. Calcd for
C11H12N2O4: C, 55.93; H, 5.12; N, 11.86. Found: C,
56.48; H, 5.18; N,12.04.
5.7.2. 6,7,8-Trimethoxy-N-(4-bromophenyl)-4-aminoqui-
nazoline hydrochloride (6a). Pale yellow needles, yield,
80.9%; mp170–173 ꢁC; 1H NMR (DMSO-d6,
500 MHz): d 4.02 (t, 9H, 3OCH3, J = 15 Hz), 7.71 (s,
4H, Ph-H), 8.14 (s, 1H, H-5 of quinazoline) 8.76 (s,
1H, H-2 of quinazoline), 11.53 (s, 1H, NH); 13C NMR
(DMSO-d6, 500 MHz): d 57.8, 61.9, 62.7, 100.6, 109.9,
127.3, 132.2, 136.5, 142.1, 148.2, 149.6, 154.5, 158.9;
IR (KBr): 804.3, 1128.4, 1288.5, 1489.1, 1627.9,
2847.0, 2947.2, 3010.9, 3200.0, 3400 cmꢀ1; Anal. Calcd
for C17H18BrCl2N3O3: C, 44.09; H, 3.92; N 9.07. Found:
C, 43.82; H, 4.08; N, 9.04.
5.7.3. 6,7,8-Trimethoxy-N-(2-methoxydibenzofurane-3-
yl)-4-aminoquinazoline hydrochloride (6b). Pale yellow
needles; yield, 78.0%; mp160–162 ꢁC; 1H NMR
(DMSO-d6, 500 MHz):
d 4.04 (t, 12H, 4OCH3,
5.6. Preparation of 4-chloro-6,7,8-trimethoxyquinazoline
(5)
J = 55 Hz), 7.47 (t, 1H, H-6 of dibenzofurane,
J = 10 Hz), 7.55 (t, 1H, H-7 of dibenzofurane,
J = 15 Hz), 7.72 (d, 1H, H-8 of dibenzofurane,
J = 7.45 Hz), 7.88 (s, 1H, H-4 of dibenzofurane), 8.03
(s, 1H, H-1 of dibenzofurane), 8.09 (s, 1H, H-5 of qui-
nazoline), 8.22 (d, 1H, H-5 of dibenzofurane,
J = 7.45 Hz), 8.68 (s, 1H, H-2 of quinazoline), 11.45 (s,
1H, NH); 13C NMR (DMSO-d6, 500 MHz): d 56.3,
A mixture of 6,7,8-trimethoxyquinazoline-4-one (0.6 g,
2.5 mmol) in POCl3(25 mL) and DMF (0.12 mL) was
stirred under reflux at 100–105 ꢁC for 2–3 h. The solvent
was then removed under reduced pressure. The residue
was added into chloroform (20 mL). The mixture was

6614
G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
56.9, 61.3, 62.0, 99.8, 103.6, 108.6, 111.4, 111.7, 121.3,
123.1, 123.3, 123.5, 127.7, 141.4, 145.3, 147.7, 148.9,
150.9, 154.0, 156.3; IR (KBr): 763.8, 1124.5, 1286.5,
1473.6, 1626.0, 2850.8, 2949.2, 3010.9, 3200.0,
3600 cmꢀ1; Anal. Calcd for C25H27Cl2N3O5: C, 57.70;
H, 5.23; N, 8.07. Found: C, 57.50; H, 5.03; N, 8.16.
130.9, 131.0, 148.3, 149.7, 154.6, 159.0, 161.4; IR
(KBr): 1134.1, 1288.5, 1489.1, 1627.9, 2852.6, 2951.1,
3012.8, 3419.8 cmꢀ1; Anal. Calcd for C17H17ClFN3O3:
C, 55.82; H, 4.68; N, 11.49. Found: C, 55.77; H, 4.60;
N, 11.33.
5.7.8. 6,7,8-Trimethoxy-N-(3-fluorophenyl)-4-aminoqui-
nazoline (6g). Pale yellow needles, yield, 92.5%; mp
252–254 ꢁC, 1H NMR (DMSO-d6, 500 MHz): d 3.93
(t, 9H, 3OCH3, J = 40 Hz), 6.96 (t, 1H, H-2 of Ph-H,
J = 8.0 Hz), 7.43 (q, 1H, H-5 of Ph-H, J = 5 Hz), 7.63
(d, 1H, H-6 of Ph-H, J = 8.0 Hz), 7.71 (s, 1H, H-5 of
quinazoline), 7.88 (d, 1H, H-4 of Ph-H, J = 12.1 Hz),
8.56 (s, 1H, H-2 of quinazoline), 9.69 (s, 1H, NH); 13C
NMR (DMSO-d6, 500 MHz): d 56.2, 60.7, 61.6, 97.6,
108.4, 109.6, 117.4, 129.6, 129.7, 146.0, 151.4, 156.1,
160.7, 162.7; IR (KBr): 1118.7, 1271.1, 1454.3, 1618.3,
5.7.4. 6,7,8-Trimethoxy-N-(3-chlorophenyl)-4-aminoqui-
nazoline hydrochloride (6c). Pale yellow needles, yield,
80.1%, mp 172 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz) d: 4.02 (t, 9H, 3OCH3, J = 15 Hz), 7.41 (d,
1H, H-6 of Ph-H, J = 9.15), 7.53 (t, 1H, H-5 of Ph-H,
J = 5 Hz), 7.71 (d, 1H, H-4 of Ph-H, J = 9.2 Hz), 7.90
(s, 1H, H-2 of Ph-H), 8.81 (s, 1H, H-2 of quinazoline),
11.43 (s, 1H, NH);13C NMR (DMSO-d6, 500 MHz): d
57.1, 61.2, 62.0, 99.8, 109.3, 123.0, 124.2, 126.2, 130.4,
132.8, 138.1, 147.6, 149.2, 153.9, 158.4; IR (KBr):
785.0, 1126.4, 1288.5, 1487.1, 2846.9, 2947.2, 3012.8,
3012.8, 3600.0 cmꢀ1. Anal. Calcd for C17H17Cl2N3O3:
C, 53.42; H, 4.48; N, 10.99. Found: C, 53.49; H, 4.60;
N, 11.00.
2833.4, 2933.7, 3074.5 cmꢀ1
;
Anal. Calcd for
C17H16FN3O3: C, 62.00; H, 4.90; N, 12.76. Found: C,
61.97; H, 4.85; N, 12.59.
5.7.9. 6,7,8-Trimethoxy-N-(4-fluorophenyl)-4-aminoqui-
nazoline (6h). Pale yellow needles, yield, 82.3%; mp
184–186 ꢁC; 1H NMR (DMSO-d6, 500 MHz): d 4.02
(t, 9H, 3OCH3, J = 15 Hz), 7.34 (t, 2H, H-2, 6 of Ph-
H, J = 20 Hz), 7.71–7.74 (m, 2H, H-3, 5 of Ph-H), 8.17
(s, 1H, H-5 of quinazoline), 8.73 (s, 1H, H-2 of quinaz-
oline), 11.61 (s, 1H, NH); 13C NMR (DMSO-d6,
500 MHz): d 57.8, 61.8, 62.7, 100.7, 109.7, 116.1,
116.2, 127.6, 127.7, 133.3, 142.1, 148.1, 149.7, 154.5,
159.1, 159.8, 161.7; IR (KBr): 788.9, 1126.4, 1282.7,
5.7.5. 6,7,8-Trimethoxy-N-(3-bromophenyl)-4-aminoqui-
nazoline (6d). Pale yellow needles, yield, 59.0%,
mp126–127 ꢁC; 1H NMR (DMSO-d6, 500 MHz): d
4.02 (t, 9H, 3OCH3, J = 15 Hz), 7.46 (t, 1H, H-5 of
Ph-H, J = 20.0 Hz),11.51 (s, 1H, NH), 7.54 (d, 1H, H-
6 of Ph-H, J = 8.05 Hz), 7.76 (d, 1H, H-4 of Ph-H,
J = 8.05 Hz), 8.02 (s, 1H, H-5 of quinazoline), 8.13 (d,
J = 4.0 Hz, 1H, H-2 of Ph-H), 8.81 (s, 1H, H-2 of qui-
nazoline); 13C NMR (DMSO-d6, 500 MHz): d 57.7,
61.9, 62.7, 100.6, 109.9, 121.8, 124.1, 127.7, 129.7,
131.3, 138.8, 148.2, 149.8, 154.5,159.0; IR (KBr):
788.9, 1126.4, 1288.5, 1487.1, 1627.9, 2852.7, 2941.4,
3010.9, 3307.9 cmꢀ1; Anal. calcd for C17H16BrN3O3:
C, 52.32; H, 4.13; N, 10.77. Found: C, 52.49; H, 4.00;
N, 10.90.
1487.1, 1626.0, 2856.6, 2947.2, 3016.7, 3356.1 cmꢀ1
;
Anal. Calcd for C17H16FN3O3: C, 62.00; H, 4.90; N,
12.76. Found: C, 61.99; H, 4.75; N, 12.69.
5.7.10. 6,7,8-Trimethoxy-N-(2-fluorophenyl)-4-aminoqui-
nazoline hydrochloride (6i). Pale yellow needles, yield,
79.0%, m.p.194 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz) d: 4.03 (t, 9H, 3OCH3, J = 15 Hz), 7.36-7.56
(m, 4H, H-3,4,5, 6 of Ph-H), 8.23 (s, 1H, H-5 of quinaz-
oline), 8.72 (s, 1H, H-2 of quinazoline), 11.82 (s, 1H,
NH); 13C NMR (DMSO-d6, 500 MHz): d 57.1, 61.2,
62.0, 100.2, 108.9, 116.2, 116.3, 128.6, 129.3, 141.7,
147.7, 149.2, 153.9, 155.9, 157.8, 159.3; IR (KBr):
759.9, 1010.7, 1126.4, 1257.6, 1487.1, 1629.9, 2854.7,
5.7.6. 6,7,8-Trimethoxy-N-(3-bromophenyl)-4-aminoqui-
nazoline hydrochloride (6e). Pale yellow needles, yield,
89.0%; mp 180 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz): d 4.02 (t, 9H, 3OCH3, J = 5 Hz), 7.46 (t, 1H,
H-5 of Ph-H, J = 15 Hz), 7.54 (d, 1H, H-6 of Ph-H,
J = 8.0 Hz), 7.77 (d, 1H, H-4 of Ph-H, J = 8.0 Hz), 8.03
(s, 1H, H-2, of Ph-H), 8.20 (s, 1H, H-5 of quinazoline),
8.81 (s, 1H, H-2 of quinazoline),11.62 (s, 1H, NH); 13C
NMR (DMSO-d6, 500 MHz) d 57.2, 61.3, 62.1, 100.2,
109.3, 121.2, 123.6, 127.1, 129.1, 130.7, 138.8, 141.5,
147.7, 149.1, 154.0, 158.5; IR (KBr): 786.9, 1126.4,
1286.5, 1487.1, 1626.0, 2848.9, 2945.3, 3010.9,
3600.0 cmꢀ1; Anal. Calcd for C17H17BrClN3O3: C,
47.85; H, 4.02; N, 9.85. Found: C, 47.80; H, 4.09; N, 9.75.
2945.3,
3363.9,
3600.1 cmꢀ1
;
Anal.Calcd
for
C17H17ClFN3O3: C, 55.82; H, 4.68; N, 11.49. Found:
C, 55.66; H, 4.86; N, 11.31.
5.7.11. 6,7,8-Trimethoxy-N-(2-chloro-4-bromophenyl)-4-
aminoquinazoline hydrochloride (6j). White needles,
1
yield, 89.6%; mp 178 ꢁC (dec); H NMR (DMSO-d6,
500 MHz): d 4.02 (t, 9H, 3OCH3, J = 5 Hz), 7.54 (d,
1H, H-6 of Ph-H, J = 8.6 Hz), 7.74 (d, 1H, H-5 of Ph-
H, J = 8.6 Hz), 8.00 (d, 1H, H-3 of Ph-H, J = 2.3 Hz),
8.04 (s, 1H, H-5 of quinazoline), 8.71 (s, 1H, quinazoline
H-2); 13C NMR (DMSO-d6, 500 MHz): d 62.3, 66.7,
67.4, 104.9, 105.1, 114.1, 126.4, 136.6, 137.7, 138.8,
147.2, 153.3, 154.4, 159.8, 164.8; IR (KBr): 1126.4,
1286.5, 1487.1, 1626.0, 2848.9, 2945.3, 3014.7,
3365.8 cmꢀ1; Anal. Calcd for C17H16BrCl2N3O3: C,
44.28; H, 3.50; N, 9.11. Found: C, 43.96; H, 3.96; N,
9.01.
5.7.7. 6,7,8-Trimethoxy-N-(3-fluorophenyl)-4-aminoqui-
nazoline hydrochloride (6f). Pale yellow needles, yield,
86.0%; mp 232–234 ꢁC; 1H NMR (DMSO-d6,
500 MHz): d 4.03 (t, 9H, 3OCH3, J = 15 Hz), 7.20 (t,
1H, H-2 of Ph-H, J = 8.0 Hz), 7.54 (q, 1H, H-5 of Ph-
H, J = 15 Hz), 7.73 (d, 1H, H-6 of Ph-H, J = 8.0 Hz),
7.73 (d, 1H, H-4 of Ph-H, J = 10.9 Hz), 8.21 (s, 1H,
H-5 of quinazoline), 8.81 (s, 1H, H-2 of quinazoline),
11.63 (s, 1 H, NH); 13C NMR (DMSO-d6, 500 MHz) d
57.8, 61.9, 62.7, 100.7, 109.9, 112.3, 112.5, 121.1,

G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
6615
5.7.12. 6,7,8-Trimethoxy-N-(3,4,5-trimethoxyphenyl)-4-
aminoquinazoline hydrochloride (6k). Yellow needles,
yield, 89.9%; mp 195 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz): d 3.71 (s, 3H, 4-CH3 of Ph-H), 3.81 (s, 6H,
3, 5–2CH3 of Ph-H), 4.02 (t, 9H, 3OCH3, J = 20 Hz),
11.52 (s, 1H, NH), 7.09 (s, 2H, H-1,6 of Ph-H), 8.17
(s, 1H, H-5 of quinazoline), 8.75 (s, 1H, H-2 of quinaz-
oline); 13C NMR (DMSO-d6, 500 MHz): d 56.6, 57.8,
60.7, 61.2, 62.7, 100.8, 103.5, 109.7, 132.8, 136.6,
148.1, 149.6, 153.3, 154.5, 159.0; IR (KBr): 1130.3,
J = 5.7 Hz,), 7.23 (t, 1H, 4-H of Ph-H, J = 7.8 Hz),
7.31–7.37 (m, 4H, 2, 3, 5, 6-H of Ph-H), 7.54 (s, 1H,
H-5 of quinazoline), 8.36 (s, 1H, H-2 of quinazoline),
8.60 (t, 1H, NH, J = 5.7 Hz); 13C NMR (DMSO-d6,
500 MHz) d 42.1, 56.7, 61.4, 62.2, 98.5, 111.7, 127.7,
128.8, 140.2, 140.9, 146.3, 147.6, 152.2, 153.1,
159.1 cmꢀ1; Anal. Calcd for C18H19N3O3: C, 66.45; H,
5.89; N, 12.91. Found: C, 66.56; H, 5.95; N, 12.93.
5.7.17. 6,7,8-Trimethoxy-N-(4-chlorophenyl)-4-aminoqui-
nazoline hydrochloride (6p). Pale yellow needles, yield,
80.3%; mp175 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz): d 4.02 (t, 9H, 3OCH3, J = 20 Hz), 7.57 (d,
2H, 2, 6-H of Ph-H, J = 8.6 Hz), 7.77 (d, 2H, 3,5-H of
Ph-H, J = 8.6 Hz), 8.23 (s, 1H, H-5 of quinazoline),
8.75 (s, 1H, H-2 of quinazoline), 11.57 (s, 1H, NH);
13C NMR (DMSO-d6, 500 MHz): d 57.2, 61.2, 62.0,
100.2, 109.9, 126.4, 128.6, 135.8, 147.5, 149.3, 153.7,
158.4; IR (KBr): 806.3, 1130.3, 1286.5, 1471.7, 1627.9,
2852.7, 2947.2, 3010.9, 3419.8 cmꢀ1; Anal. Calcd for
C17H18Cl3N3O3: C,48.77; H, 4.33; N, 10.04. Found: C,
48.53; H, 4.93; N, 9.97.
1284.6, 1456.3, 1633.7, 2839.2, 2947.2, 3419.8 cmꢀ1
;
Anal. Calcd for C20H24ClN3O6: C, 54.86; H, 5.52; N,
9.60. Found: C, 54.80; H, 5.51; N, 9.50.
5.7.13. 6,7,8-Trimethoxy-N-(4-nitrophenyl)-4-aminoqui-
nazoline hydrochloride (6l). Pale yellow needles, yield,
75.4%; mp 212 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz) d: 4.02 (t, 9H, 3OCH3, J = 20 Hz), 8.06 (s,
1H, H-5 of quinazoline), 8.15 (d, 2H, 2, 6-H of Ph-H,
J = 8.6 Hz), 8.36 (d, 2H, 3, 5-H of Ph-H, J = 8.6 Hz),
8.82 (s, 1H, H-2 of quinazoline), 11.21 (s, 1H, NH);
13C NMR (DMSO-d6, 500 MHz): d 56.9, 61.2, 62.0,
99.3, 110.5, 123.2, 124.4, 143.9, 147.4, 149.7, 153.6,
157.8; IR (KBr): 1130.3, 1284.6, 1340.5, 1487.1,
5.7.18. 6,7,8-Trimethoxy-N-(4-hydroxyphenyl)-4-amino-
quinazoline hydrochloride (6q). Pale yellow needles, yield,
79.5%; mp 153 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz): d 4.00 (t, 9H, 3OCH3, J = 15 Hz), 6.87 (d,
2H, 2, 6-H of Ph-H, J = 8.6 Hz), 7.43 (d, 2H, 3, 5-H
of Ph-H, J = 8.6 Hz), 8.15 (s, 1H, H-5 of quinazoline),
8.66 (s, 1H, H-2 of quinazoline), 9.75 (s, 1H, OH),
11.42 (s, 1H, NH); 13C NMR (DMSO-d6, 500 MHz): d
57.1, 61.2, 61.9, 100.2, 109.1, 115.2, 126.3, 127.6,
141.6, 147.3, 149.1, 153.7, 156.1, 158.3; IR (KBr):
1128.4, 1273.0, 1473.6, 1626.0, 2854.7, 2949.2, 3030.2,
1514.1, 1627.9, 2839.0, 2951.1, 3010.9, 3365.8 cmꢀ1
;
Anal. Calcd for C17H17ClN4O5: C, 51.98; H, 4.36; N,
14.26. Found: C, 51.63; H, 4.72; N, 14.12.
5.7.14. 6,7,8-Trimethoxy-N-(3-nitrophenyl)-4-aminoqui-
nazoline hydrochloride (6m). Pale yellow needles, yield,
58.6%; mp 204–206 ꢁC; 1H NMR (DMSO-d6,
500 MHz): d 4.03 (t, 9H, 3OCH3, J = 25 Hz), 7.79 (t,
1H, 5-H of Ph-H, J = 15 Hz), 8.18 (d, 1H, 6-H of Ph-
H, J = 9.7 Hz), 11.84 (s, 1H, NH), 8.26 (s, 1H, H-5 of
quinazoline), 8.29 (d, 1H, 4-H of Ph-H, J = 9.2 Hz),
8.72 (s, 1H, 2-H of Ph-H), 8.86 (s, 1H, H-2 of quinazo-
line); 13C NMR (DMSO-d6, 500 MHz): d 57.9, 61.9,
62.7, 100.8, 110.1, 121.3, 130.7, 131.2, 138.6, 142.3,
148.3, 149.8, 154.6, 159.1; IR (KBr): 1120.6, 1284.6,
1346.3, 1485.2, 1469.8, 1631.8, 2843.1, 2947.2, 3014.7,
3419.8 cmꢀ1; Anal. Calcd for C17H17ClN4O5: C, 51.98;
H, 4.36; N, 14.26. Found: C, 51.71; H, 4.55; N, 14.32.
3226.9, 3419.8, 3600.0 cmꢀ1
;
Anal. Calcd for
C17H19Cl2N3O4: C, 51.01; H, 4.78; N, 10.50. Found:
C, 51.24; H, 5.14; N, 10.52.
5.7.19. 6,7,8-Trimethoxy-(R)-N-(4-a-methylbenzyl)-4-amino-
quinazoline hydrochloride (6r). White needles, yield,
D
78.8%; mp 207–209 ꢁC; ½aꢁ ꢀ213.0ꢁ(c = 0.01 mol/L,
22
EtOH), 1H NMR (DMSO-d6, 500 MHz): d 1.60 (d,
3H, CH3, J = 7.5 Hz), 3.87 (t, 9H, 3OCH3, J = 45 Hz
), 5.62 (d, 1H, CH, J = 7.45 Hz), 7.21 (t, 1H, 4-H of
Ph-H, J = 7.5 Hz), 7.32 (t, 2H, 3, 5-H of Ph-H,
J = 7.5 Hz), 7.42 (d, 2H, 2, 6-H of Ph-H, J = 7.5 Hz),
7.64 (s, 1H, H-5 of quinazoline), 8.22 (d, 1H, NH,
J = 7.45 Hz), 8.31 (s, 1H, H-2 of quinazoline);
13C NMR (DMSO-d6, 500 MHz): d 22.9, 49.6, 56.9,
61.4, 62.2, 98.7, 111.6, 126.6, 127.1, 128.8, 140.9,
145.4, 146.3, 147.6, 152.2, 153.0, 158.3; IR (KBr):
702.1, 1120.6, 1280.7, 1477.5, 1612.5, 2831.5, 2831.5,
5.7.15. 6,7,8-Trimethoxy-N-(2-nitrophenyl)-4-aminoqui-
nazoline hydrochloride (6n). White needles, yield,
55.6%; mp 210 ꢁC (dec); 1H NMR (DMSO-d6,
500 MHz): d 4.02 (t, 9H, 3OCH3, J = 20 Hz), 7.65 (t,
1H, 5-H of Ph-H, J = 7.8 Hz), 7.77 (d, 1H, 6-H of Ph-
H, J = 7.5 Hz), 7.90 (t, 1H, 4-H of Ph-H, J = 9.2 Hz,),
8.17 (d, 1H, 3-H of Ph-H, J = 7.5 Hz), 8.18 (s, 1H, H-
5 of quinazoline), 8.66 (s, 1H, H-2 of quinazoline),
12.01 (s, 1H, NH); 13C NMR (DMSO-d6, 500 MHz): d
57.1, 61.2, 62.0, 99.9, 109.3, 125.2, 128.1, 128.9, 134.3,
144.8, 146.3, 147.8, 149.3, 153.9, 158.7; IR (KBr):
792.7, 1130.3, 1286.5, 1355.9, 1458.2, 1489.0, 1642.0,
2845.0, 2945.3, 3018.6, 3200.2 cmꢀ1; Anal. Calcd for
C17H17ClN4O5: C, 51.98; H, 4.36; N, 14.26. Found; C,
52.28:, H, 4.57; N, 14.29.
2933.7, 3026.3, 3261.6 cmꢀ1
;
Anal. Calcd for
C19H21N3O3: C, 67.24; H, 6.24; N, 12.38. Found: C,
67.31; H, 5.97; N, 12.23.
5.7.20. 6,7,8-Trimethoxy-(S)-N-(4-a-methylbenzyl)-4-amino-
quinazoline hydrochloride (6s). White needles, yield,
D
80.2%; mp 205–206 ꢁC; ½aꢁ +208.0 (c = 0.01 mol/L,
22
5.7.16. 6,7,8-Trimethoxy-N-benzyl-4-aminoquinazoline
hydrochloride (6o). White needles, yield, 77.7%;
mp173–175 ꢁC; 1H NMR (DMSO-d6, 500 MHz): d
3.87 (t, 9H, 3OCH3, J = 50 Hz), 4.79 (d, 2H, CH2,
EtOH), 1H NMR (DMSO-d6, 500 MHz): d 1.60 (d,
3H, CH3, J = 6.9 Hz), 3.96 (t, 9H, 3OCH3, J = 5 Hz),
5.62 (d, 1H, CH, J = 7.45 Hz), 7.21 (t, 1H, 4-H of Ph-
H, J = 7.45 Hz), 7.32 (t, 2H, 3, 5-H of Ph-H,

6616
G. Liu et al. / Bioorg. Med. Chem. 15 (2007) 6608–6617
J = 7.45 Hz), 7.42 (d, 2H, 2, 6-H of Ph-H, J = 7.45 Hz),
7.64 (s, 1H, H-5 of quinazoline), 8.22 (d, 1H, NH,
J = 7.45 Hz), 8.31 (s, 1H, H-2 of quinazoline); 13C
NMR (DMSO-d6, 500 MHz): d 22.9, 49.6, 56.9, 61.4,
62.2, 98.7, 111.6, 126.6, 127.1, 128.8, 140.9, 145.4,
146.3, 147.6, 152.2, 153.0, 158.3; IR (KBr): 700.2,
1130.3, 1311.3, 1311.6, 1477.5, 1612.5, 2831.5, 2935.7,
freshly made in PBS plus 0.2% Tween 20, then incubated
with monoclonal antibody (anti-phosphotyrosin, anti-
pErk1/2 or anti-caveolin) overnight at 4 ꢁC. The mem-
brane was then washed for 3· 5 min with PBS plus
0.2% Tween 20. It was incubated again with second anti-
body for 2–3 h at 25 ꢁC, washed three times with PBS
plus 0.2% Tween 20, and the signal was detected by en-
hanced chemical luminescence (ECL) detection system
(PIERCE).22
2972.3, 3028.2, 3242.3 cmꢀ1
.
Anal. Calcd for
C19H21N3O3: C, 67.24; H, 6.24; N, 12.38; Found: C,
67.43; H, 6.23; N, 12.37.
5.8. MTT assay against cancer cell proliferation
Acknowledgments
All tested compounds were dissolved in DMSO
(1–100 lM solution) and subsequently diluted in the
culture medium before treatment of the cultured cells.
Tested cells were plated in 96-well plates at a density
2 · 103 cells/well/100 lL of the proper culture medium
and treated with the compounds at 1–100 lM for 72 h.
In parallel, the cells treated with 0.1% DMSO served as
control. An MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-di-
phenyl tetrazoliumbromide] assay (Roche Molecular
Biochemicals, 1465-007) was performed 30 h later
according to the instructions provided by Roche. This
assay is based on the cellular cleavage of MTT into
formazan which is soluble in cell culture medium.
And the absorbance caused by formazan was measured
at 595 nm with a microplate reader (Bio-Rad, model
680), which is directly proportional to the number of
living cells in culture. Three types of cells were used
in these assays, A431(uterus cancer), PC3 (prostate
cancer), BGC 823 (human gastric cancer), and Bcap37
(breast cancer) cell lines, provided by ATCC and culti-
vated in RPMI 1640 (for PC3, A431, BGC823, and
Bcap37) supplemented with 10% fetal bovine serum.
Tissue culture reagents were obtained from Gibco
BRL.21
We thank the National Key Project for International
Cooperation of Science and Technology (Grant No.
2005DFA30650) and the National Nature Science
Foundation of China (Grant No. 20562002), the Foun-
dation for New Century Talent in Universities of China,
and the Foundation for Science and Technology Excel-
lent Talent in Guizhou Province, China, for their finan-
cial support to carry out the research.
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