Synthesis and anticancer activities of 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.03.036

A series of 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline was prepared by conventional heating methods. Among these compounds, the crystal structure of compound 10o (CCDC: 916922) was determined by X-ray crystallography. Bioassay results showed that most target compounds had certain inhibition activities against proliferation of tumor cells, and some compounds even had good broad-spectrum inhibition activities. The ethoxyl series of compounds possessed higher inhibition activities against tumor cells than the methoxyl series of compounds. Bioactivity tests showed that the IC₅₀ values of compound 10s against PC3, MGC803, A375, and A549 cells were 1.8, 2.8, 1.3, and 2.9 μΜ, respectively, which were much higher than those of commercial gefitinib (7.2, 7.6, 7.2, and 9.8 μM, respectively). Conversely, the IC₅₀ values of compound 10s were very low against NH3T3, indicating only weak effect on normal cells as also proven by lactate dehydrogenase and acridine orange/ethidium bromide staining. Analyses of cell configuration and cell cycle revealed that compound 10s possibly caused cells to remain at G0/G1 phase by inhibiting cell proliferation for 24 h. Compound 10s also inhibited the phosphorylation of ERK1/2 and P38 with obvious concentration dependence. Thus, these compounds can inhibit the proliferation of A549 cells through the interruption of ERK1/2 and P38signaling pathways.

Full text of DOI:10.1016/j.ejmech.2014.03.036

European Journal of Medicinal Chemistry 78 (2014) 23e34
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and anticancer activities of 4-(4-substituted piperazin)-5,6,
7-trialkoxy quinazoline derivatives
,
,
Ying Zhang a 1, Yin-Jiu Huang b 1, Hong-Mei Xiang a, Pei-Yi Wang a, De-Yu Hu a, Wei Xue a,
Bao-An Song a , Song Yang
,
a,
*
*
a State Key Laboratory Breeding Base of Green Pesticide and Agricultural Bioengineering, Key Laboratory of Green Pesticide and Agricultural Bioengineering,
Ministry of Education, Center for R&D of Fine Chemicals of Guizhou Province, Guizhou University, Guiyang 550025, PR China
b Department of Biological Science, Bengbu Medical College, Bengbu 233030, 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 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline was prepared by conventional heating
methods. Among these compounds, the crystal structure of compound 10o (CCDC: 916922) was deter-
mined by X-ray crystallography. Bioassay results showed that most target compounds had certain in-
hibition activities against proliferation of tumor cells, and some compounds even had good broad-
spectrum inhibition activities. The ethoxyl series of compounds possessed higher inhibition activities
against tumor cells than the methoxyl series of compounds. Bioactivity tests showed that the IC50 values
Received 25 November 2013
Received in revised form
5 March 2014
Accepted 12 March 2014
Available online 13 March 2014
of compound 10s against PC3, MGC803, A375, and A549 cells were 1.8, 2.8, 1.3, and 2.9
which were much higher than those of commercial gefitinib (7.2, 7.6, 7.2, and 9.8
m
M, respectively,
Keywords:
Quinazoline
Anticancer
ERK1/2
m
M, respectively).
Conversely, the IC50 values of compound 10s were very low against NH3T3, indicating only weak effect
on normal cells as also proven by lactate dehydrogenase and acridine orange/ethidium bromide staining.
Analyses of cell configuration and cell cycle revealed that compound 10s possibly caused cells to remain
at G0/G1 phase by inhibiting cell proliferation for 24 h. Compound 10s also inhibited the phosphorylation
of ERK1/2 and P38 with obvious concentration dependence. Thus, these compounds can inhibit the
proliferation of A549 cells through the interruption of ERK1/2 and P38signaling pathways.
Ó 2014 Elsevier Masson SAS. All rights reserved.
P38
Antiphosphorylation
Cell cycle
1. Introduction
factor receptor PTKs. EGFR PTKs have been identified as explicit
targets for medicinal chemistry programs, particularly cancer
Protein tyrosine kinases (PTKs) are enzymes involved in many
cellular processes, such as cell proliferation, metabolism, survival,
and apoptosis. Several protein tyrosine kinases are known to be
activated in cancer cells and to drive tumor growth and progres-
sion. Thus, blocking tyrosine kinase activity can be a rational
approach to cancer therapy. PTKs catalyze the phosphorylation of
tyrosine and serine/threonine residues in various proteins involved
in the regulation of many functions [1]. Epidermal growth factor
receptor (EGFR) belongs to the family of transmembrane growth
therapy [2e6]. Since Fry et al. [7] discovered that the 4-
anilinoquinazoline derivative PD153035 possesses specific inhibi-
tion activity against EGFR PTKs, 4-anilinoquinazoline derivatives
have recently attracted increased attention from chemists and bi-
ologists. Indeed, numerous 4-anilinoquinazoline compounds
have been synthesized and found to show good anticancer activ-
ities [8e20]. Gefitinib (Iressa, ZD-1839) [21,22] and erlotinib (OSI-
774, Tarceva) [23,24] are first-generation EGFR-targeting 4-
anilinoquinazoline chemotherapeutics used to treat non-small-
cell lung cancer. Meanwhile, the second-generation EGFR-target-
ing chemotherapeutic BIBW2992 is a potent, irreversible EGFR in-
hibitor that has also been developed into phase-II clinical trials
against lung cancer [25,26]. Moreover, 2-chloro-6,7-dimethoxy-4-
(4-tosylpiperazin-1-yl)quinazoline reportedly shows good inhibi-
tion activity against MCF-7 [27]. In our previews work, a series of
5,6,7-trimethoxy-N-phenyl(ethyl)-4-aminoquinazoline has been
synthesized and found to show certain anticancer activities. Some
of the compounds possess strong inhibition activities against ERK1/
2 phosphorylation [28].
Abbreviationlist: AO/EB, acridine orange/ethidium bromide; 13C NMR, 13C Nu-
clear Magnetic Resonance; DMSO, dimethyl sulfoxide; LDH, lactate dehydrogenase;
EGF, epidermal growth factor; EGFR, epidermal growth factor receptor; ERK1/2,
extracellular regulated kinase1/2; P38, extracellular regulated kinase; ESI-MS,
electrospray ionization mass spectrometry; 1H NMR, Proton Nuclear Magnetic
Resonance; IR, Infra-red; MS, mass spectroscopy; MTT, 3-(4,5-Dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide.
* Corresponding authors.
E-mail addresses: jhzx.msm@gmail.com, yangsdqj2002@yahoo.com (S. Yang).
Both authors contributed equally to this work.
1
http://dx.doi.org/10.1016/j.ejmech.2014.03.036
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

24
Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
In the present study, we replaced the aniline moiety with
trimethoxy-quinazoline with substituted benzenesulfonyl chloride.
Meanwhile, target compounds 10fe10m were obtained by the
substitution reaction of 4-chloro-5,6,7-trimethoxyquinazoline with
substituted piperazine.
substituted piperazine fragments to determine if these compounds
would significantly affect the aforementioned inhibition activity.
On the other hand, trimethoxy and triethoxy substitution patterns
were synthesized to investigate whether the alkoxy substitution
would affect the activities of the target compounds. Thus, twenty-
six new 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazoline
derivatives were synthesized from 2,3,4-trihydroxybenzoic acid or
2,3,4-trimethoxybenzoic acid by the synthesis route shown in
Scheme 1. The structures of the title compounds were characterized
by 1H nuclear magnetic resonance (NMR), 13C NMR, and mass
spectroscopy (MS). The single-crystal structure of 10o was
confirmed by X-ray crystallography. The antiproliferation activities
of the title compounds against PC3, Bcap-37, MGC803, A375, and
BGC823 cells in vitro were evaluated by the 3-(4,5-Dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay.
Results showed that title compounds 10ae10z possessed weak to
strong anticancer activities.
For the ethoxyl series, the starting material 2,3,4-trihydro-
oxybenzoic acid was etherified with diethyl sulfate, esterified
with methanol in the presence of 98% sulfuric acid, nitrated
with 70% nitric acid, hydrogenated with Pd/C as catalyst in
ethanol, cyclized with formamide, and chlorinated with phos-
phorus oxychloride to give 4-choloro-5,6,7-triethoxy-quinazo-
line. The chlorinated product was reacted with N-Boc piperazine
to obtain 4-(N-Boc-piperazin)-5,6,7-triethoxy-quinazoline. Boc
was then removed to obtain 4-piperazin-5,6,7-triethoxy-quina-
zoline. Target compounds 10ne10r were obtained by the sub-
stitution reaction of 4-piperazin-5,6,7-triethoxy-quinazoline
with substituted benzenesulfonyl chloride. Meanwhile, target
compounds 10se10z were obtained by the substitution reaction
of 4-chloro-5,6,7-triethoxyquinazoline with substituted pipera-
zine (Scheme 1).
Probably due to the steric hindrance of the 5-ethoxyl group was
stronger than that of the methoxyl group, the yields of ethoxyl
series intermediates were lower than those of methoxyl series in-
termediates, and more byproducts were produced. Moreover, the
chlorinated product of the ethoxyl series cannot react with N-Boc
piperazine or substituted piperazine at refluxing temperature, but
the methoxyl series can. The ethoxyl series can only react with N-
Boc piperazine or substituted piperazine in the presence of DMF
and NaH.
The crystal structure of compound 10o (CCDC: 916922) was
determined by X-ray crystallography. The details of data collection
and structure refinement are listed in Table 1, and the X-ray
structures are shown in Fig. 1. Compound 10o crystallized in the
monoclinic space group P2(1)/c.
2. Results and discussion
2.1. Chemistry
For the methoxyl series, the starting material 2,3,4-trimethoxy-
benzoic acid was nitrated with 70% nitric acid, esterified with
methanol in the presence of 98% sulfuric acid, hydrogenated with
Pd/C as catalyst in ethanol, cyclized with formamide, and chlori-
nated with phosphorus oxychloride to give 4-choloro-5,6,7-
trimethoxy-quinazoline. The chlorinated product was reacted
with N-Boc piperazine to obtain 4-(N-Boc-piperazin)-5,6,7-
trimethoxy-quinazoline. Boc was then removed to obtain 4-
piperazin-5,6,7-trimethoxy-quinazoline. Target compounds 10ae
6e were obtained by the substitution reaction of 4-piperazin-5,6,7-
Scheme 1. Reagents and conditions of the synthesis of title compounds 10: (a) NaOH, H2O, (Et)2SO4, reflux, 5 h; (b) CH3OH, 98% H2SO4, reflux, 12 h; (c) nitric acid, 0 ꢀC, 1 h; (d) H2,
Pd/C, 95% C2H5OH, reflux, 12 h; (e) N2, toluene, formamide, POCl3, 102 ꢀC, 5 h; (f) N2, toluene, POCl3, 102 ꢀC, 3.5 h; (g) N-Boc piperazine, 95% C2H5OH, reflux, 4 h to obtain 8a; N-Boc
piperazine, DMF, NaH, 4 h to obtain 8b; (h) 18% HCl, 20 ꢀC, 4 h; (i) substituted benzene sulfochloride, 95% C2H5OH,Et3N, reflux, 3e5 h to obtain 10ae10e, 10ne10r; N-substituted
piperazine, DMF, NaH, 2e12 h to obtain 10fe10m, 10se10z.

Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
25
Table 1
inhibition rates of 78.0% ꢁ 2.1%, 73.4% ꢁ 2.5%, and 70.6% ꢁ 3.4%,
respectively, which were similar to that of gefitinib (68.9% ꢁ 3.2%).
The antiproliferation activities of 10a, 10h, 10i, 10s, 10u, and 10v
Details of data collection and structure refinement for compound 10o.
Compound
10o
against BGC823 cells at 10
m
M were 47.3% ꢁ 13.7%, 45.1% ꢁ 12.5%,
Empirical formula
Formula weight
Temperature (K)
C25 H29 F3 N4 O5
554.58
113 (2)
S
42.4% ꢁ 7.0%, 61.7% ꢁ 10.7%, 52.8% ꢁ 7.2%, and 42.7% ꢁ 10.7%,
respectively, which were similar to or even higher than that of
gefitinib (35.0% ꢁ 8.6%). Interestingly, among all title compounds,
10s showed the strongest and broad-spectrum inhibition activity,
as well as antiproliferation activity, against all cell lines (PC3,
Bcap37, MGC803, A375, and BGC823 cells) with inhibition rates of
64.0% ꢁ 2.0%, 58.0% ꢁ 7.0%, 85.6% ꢁ 1.4%, 78.0% ꢁ 2.1%, and
ꢀ
Wavelength (A)
0.71073 (MoK\a)
Crystal system, Space group
Crystal
Monoclinic, P2(1)/c
Colorless/prism
15.097 (6)
40.800 (16)
12.921 (5)
ꢀ
a (A)
ꢀ
b (A)
ꢀ
c (A)
a
b
g
(ꢀ)
(ꢀ)
(ꢀ)
90
61.7% ꢁ 10.7% at 10
mM, respectively. These values were relatively
92.977 (3)
90
7948 (5)
12/1.315
higher than those of gefitinib. Interestingly, it can be seen from
Table 2 that the antiproliferation activities of the triethoxy substi-
tution compounds were generally higher than those of the trime-
thoxy series (10aꢂ10m as compared to 10ne10z, only different at
trialkoxy), indicating that the alkoxy substitution pattern did affect
the activities.
3
ꢀ
Volume (A )
Z, Dcalc (mg mꢂ3
F (000)
)
3480
Crystal size (mm)
0.20 mm ꢄ 0.20 mm ꢄ 0.20 mm
q
Range for data
1.44e27.90
Collection (ꢀ)
ꢂ19 ꢅ h ꢅ 10
ꢂ50 ꢅ k ꢅ 53, ꢂ16 ꢅ l ꢅ 16
18,342/14,776 [Rint ¼ 0.0435]
14,776/78/1063
1.119
MTT method was applied to further detect the antiproliferation
activities of compounds 10o and 10s against A549 in vitro. The
antiproliferation activities of 10o and 10s against A549 cells at
Limiting indices
Reflections collected/unique
Data/restraints/parameters
Goodness-of-fit on F2
Final R indices[I > 2
Rindices (all data)
10
m
M were 60.6% ꢁ 5.7% and 90.4% ꢁ 7.7%, respectively. Compound
s(I)]
R1 ¼ 0.0679, wR2 ¼ 0.1423
10o was similar to that of gefitinib (63.3% ꢁ 7.6%), and compound
10s was higher than that of gefitinib. IC50 assays were further car-
ried out on 10o, 10s, and gefitinib. As shown in Table 3, the IC50
values of 10o against PC3 and A549 cells were 7.8 ꢁ 0.5 and
R1 ¼ 0.0886, wR2 ¼ 0.1532
2.2. Antiproliferation activities of title compounds against PC3,
Bcap-37, MGC803, A375, and BGC823 cells
7.8 ꢁ 0.9
mM, respectively, similar to those of gefitinib. Moreover,
the IC50 values of 10s against PC3, MGC803, A375, and A549 cells
were 1.8 ꢁ 0.1, 2.8 ꢁ 0.1, 1.3 ꢁ 0.1, and 2.9 ꢁ 0.1 M, respectively,
which were higher than those of gefitinib (7.2 ꢁ 0.8, 7.6 ꢁ 0.9,
7.2 ꢁ 1.1, and 9.8 ꢁ 1.1 M, respectively).
m
The antiproliferation activities of title compounds 10ae10z
were evaluated against PC3, MGC803, Bcap-37, A375, and BGC823
cells using gefitinib as a positive control. As shown in Table 2, the
antiproliferation activity of 10n, 10o, 10r, 10s, 10y, and 10z against
m
2.3. Preliminary investigation on the toxicity effect to A549 cells of
title compound 10s
PC3 cells at 10
m
M were 54.4% ꢁ 6.2%, 59.3% ꢁ 1.7%, 67.6% ꢁ 2.5%,
64.0% ꢁ 2.0%, 78.3% ꢁ 1.8%, and 56.8% ꢁ 3.7%, respectively, which
were similar to that of gefitinib (58.2% ꢁ 3.0%). Compounds 10n,
10p, 10r, 10s, 10v, 10y, and 10z displayed higher inhibition activities
2.3.1. LDH activities detecting the cytotoxicities of compound 10s
The cells toxicities of compound 10s were evaluated on A549
cells in terms of LDH activities using gefitinib and ZSC as a positive
control for 24 h Table 4 shows the difference analysis values (P1 and
P2) of compounds at different concentrations, as well as full (1/100)
and natural (DMSO) release groups, on the cytotoxicity of A549
cells. ZSC was found to possess higher cytotoxities on A549
against Bcap37 cells at 10
mM, with inhibition rates of 41.5% ꢁ 12.3%,
64.4% ꢁ 10.7%, 43.1% ꢁ 10.3%, 58.0% ꢁ 7.0%, 51.9% ꢁ 7.2%,
40.9% ꢁ 5.9%, and 47.4% ꢁ 5.5%, respectively, which were similar to
or even higher than that of gefitinib (31.6% ꢁ 6.9%). The anti-
proliferation activity of 10n, 10o, 10p, 10r, 10s, 10v, and 10y against
MGC803 cells at 10
mM were 72.6% ꢁ 5.4%, 93.6% ꢁ 0.8%,
(P2 < 0.05) at ꢃ1
mM. Cells were treated with 10s (0.01, 0.1, 1, 20,
M) for 24 h (all P1 ¼ 0, P2 > 0.05). Results showed that
65.3% ꢁ 4.8%, 67.3% ꢁ 5.6%, 85.6% ꢁ 1.4%, 84.6% ꢁ 1.4%, and
63.8% ꢁ 3.0%, respectively, similar to or even higher than that of
gefitinib (67.4% ꢁ 1.8%). Compounds 10s, 10v, and 10y displayed
and 40
m
cytotoxities were very low for compound 10s, similar to that of
gefitinib (P1 ¼ 0, P2 > 0.05).
higher inhibition activities against A375 cells at 10
mM, with
2.3.2. 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 frag-
ments, late apoptotic cells were densely stained orange or dis-
played orange fragments, and necrotic cells were stained orange
without condensed chromatin. The cytotoxicity of 10s at 1, 20, and
40
mM against A549 cells within 48 h was detected by AO/EB
staining, with DMSO used as a negative control. Results are shown
in Fig. 2.
Fig. 2 shows that the cells treated with 10s from 1 mM to 40 mM
for 48 h changed in A549 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.
Fig. 1. X-ray crystal structure and atom numbering schemes of compound 10o.

26
Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
Table 2
Growth inhibition ratios of title compounds against selected cell lines.
Compdsa
Inhibitory ratio (%)a
PC3
Bcap37
1
mM
10
m
M
1
m
M
10 mM
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
5.4 ꢁ 8.1
10.2 ꢁ 2.9
24.8 ꢁ 10.6
24.6 ꢁ 14.5
8.1 ꢁ 4.8
15.4 ꢁ 4.7
32.4 ꢁ 6.3
32.1 ꢁ 5.0
38.0 ꢁ 5.0
39.1 ꢁ 3.0
1.4 ꢁ 10.5
12.9 ꢁ 5.8
35.9 ꢁ 3.3
5.4 ꢁ 7.8
6.8 ꢁ 6.6
0.9 ꢁ 5.8
4.8 ꢁ 8.7
8.4 ꢁ 10.9
11.3 ꢁ 8.8
5.1 ꢁ 9.3
16.4 ꢁ 7.8
7.3 ꢁ 7.5
12.9 ꢁ 4.8
3.7 ꢁ 7.2
ꢂ3.5 ꢁ 9.7
7.7 ꢁ 6.6
1.1 ꢁ 5.9
11.7 ꢁ 11.9
10.2 ꢁ 7.4
28.0 ꢁ 8.8
16.7 ꢁ 11.3
14.7 ꢁ 12.0
4.7 ꢁ 14.0
4.3 ꢁ 10.9
25.7 ꢁ 8.8
41.5 ꢁ 12.3
34.3 ꢁ 11.8
64.4 ꢁ 10.7
5.4 ꢁ 10.4
43.1 ꢁ 10.3
58.0 ꢁ 7.0
16.9 ꢁ 14.5
23.7 ꢁ 12.9
51.9 ꢁ 7.2
18.0 ꢁ 2.8
32.0 ꢁ 10.7
40.9 ꢁ 5.9
47.4 ꢁ 5.5
31.6 ꢁ 6.9
5.0 ꢁ 8.7
15.8 ꢁ 7.8
7.6 ꢁ 8.8
20.3 ꢁ 9.6
26.9 ꢁ 12.1
3.6 ꢁ 5.5
ꢂ11.4 ꢁ 6.4
ꢂ12.1 ꢁ 6.3
ꢂ11.1 ꢁ 6.9
4.8 ꢁ 3.3
16.5 ꢁ 10.4
6.7 ꢁ 7.7
9.2 ꢁ 15.1
ꢂ6.7 ꢁ 9.2
19.9 ꢁ 6.3
0.3 ꢁ 11.0
5.0 ꢁ 6.4
22.2 ꢁ 5.5
17.4 ꢁ 7.0
54.4 ꢁ 6.2
59.3 ꢁ 1.7
26.9 ꢁ 2.8
17.1 ꢁ 3.1
67.6 ꢁ 2.5
64.0 ꢁ 2.0
40.4 ꢁ 4.2
21.9 ꢁ 8.0
42.3 ꢁ 2.4
23.7 ꢁ 8.8
9.0 ꢁ 4.4
14.9 ꢁ 2.0
18.9 ꢁ 4.5
6.6 ꢁ 3.1
18.5 ꢁ 14.5
19.6 ꢁ 14.2
26.7 ꢁ 9.6
1.7 ꢁ 3.4
6.4 ꢁ 7.7
15.3 ꢁ 11.7
13.6 ꢁ 6.6
11.6 ꢁ 7.1
ꢂ8.8 ꢁ 7.7
ꢂ2.3 ꢁ 9.6
ꢂ26.5 ꢁ 4.7
3.9 ꢁ 4.0
10s
10t
10.8 ꢁ 12.2
23.7 ꢁ 10.6
13.6 ꢁ 9.1
11.7 ꢁ 9.8
29.2 ꢁ 14.3
18.2 ꢁ 8.9
27.7 ꢁ 6.0
18.4 ꢁ 6.8
10u
10v
10w
10x
10y
10z
Gefitinib
2.6 ꢁ 5.5
78.3 ꢁ 1.8
56.8 ꢁ 3.7
58.2 ꢁ 3.0
ꢂ8.0 ꢁ 6.8
22.8 ꢁ 6.6
Compdsa
Inhibitory ratio (%)a
MGC803
A375
BGC823
1
mM
10
m
M
1
m
M
10
m
M
1
m
M
10 mM
10a
10b
10c
10d
10e
10f
10g
10h
10i
10j
10k
10l
10m
10n
10o
10p
10q
10r
6.6 ꢁ 9.1
20.8 ꢁ 10.8
15.8 ꢁ 2.0
13.0 ꢁ 3.4
4.1 ꢁ 4.2
8.7 ꢁ 6.1
15.0 ꢁ 6.9
16.1 ꢁ 6.8
0.6 ꢁ 8.0
7.5 ꢁ 6.7
ꢂ1.1 ꢁ 4.3
10.4 ꢁ 7.9
23.3 ꢁ 6.4
1.0 ꢁ 8.9
11.2 ꢁ 3.9
2.9 ꢁ 9.5
9.9 ꢁ 6.0
15.3 ꢁ 5.0
0.4 ꢁ 13.2
13.6 ꢁ 7.0
26.7 ꢁ 8.7
25.4 ꢁ 2.9
7.1 ꢁ 7.5
35.4 ꢁ 8.4
ꢂ3.0 ꢁ 8.7
ꢂ6.5 ꢁ 11.0
ꢂ2.0 ꢁ 8.9
21.5 ꢁ 8.0
28.9 ꢁ 7.6
16.0 ꢁ 7.1
15.8 ꢁ 7.3
30.0 ꢁ 5.7
16.6 ꢁ 4.2
5.1 ꢁ 12.5
1.6 ꢁ 11.5
6.8 ꢁ 4.2
NTb
47.3 ꢁ 13.7
ꢂ6.2 ꢁ 7.8
ꢂ7.5 ꢁ 10.6
19.1 ꢁ 7.1
22.8 ꢁ 3.3
25.8 ꢁ 6.7
30.4 ꢁ 6.0
45.1 ꢁ 12.5
42.4 ꢁ 7.0
39.6 ꢁ 7.4
28.2 ꢁ 14.0
38.4 ꢁ 7.6
13.8 ꢁ 3.1
NT
10.6 ꢁ 6.2
ꢂ1.9 ꢁ 3.6
ꢂ7.6 ꢁ 6.5
ꢂ1.0 ꢁ 4.1
13.4 ꢁ 5.8
28.9 ꢁ 13.5
39.6 ꢁ 8.9
27.7 ꢁ 10.3
37.0 ꢁ 14.3
18.3 ꢁ 8.7
2.3 ꢁ 5.5
17.5 ꢁ 6.8
35.6 ꢁ 5.4
29.3 ꢁ 11.9
55.2 ꢁ 6.6
36.7 ꢁ 12.9
39.1 ꢁ 13.8
23.6 ꢁ 6.2
39.6 ꢁ 8.4
19.3 ꢁ 7.1
72.6 ꢁ 5.4
93.6 ꢁ 0.8
65.3 ꢁ 4.8
33.0 ꢁ 8.2
67.3 ꢁ 5.6
85.6 ꢁ 1.4
56.1 ꢁ 5.5
28.4 ꢁ 9.7
84.6 ꢁ 1.4
30.3 ꢁ 9.7
29.7 ꢁ 7.5
63.8 ꢁ 3.0
26.3 ꢁ 7.1
67.4 ꢁ 1.8
12.8 ꢁ 5.8
3.4 ꢁ 3.8
19.1 ꢁ 8.1
29.7 ꢁ 5.5
0.3 ꢁ 4.4
7.7 ꢁ 3.9
0.6 ꢁ 5.2
14.1 ꢁ 8.6
37.7 ꢁ 2.3
23.0 ꢁ 3.9
41.3 ꢁ 8.1
40.3 ꢁ 5.6
30.3 ꢁ 5.7
23.5 ꢁ 3.1
78.0 ꢁ 2.1
23.3 ꢁ 5.8
29.4 ꢁ 9.2
73.4 ꢁ 2.5
16.9 ꢁ 7.3
20.3 ꢁ 9.4
70.6 ꢁ 3.4
35.8 ꢁ 4.8
68.9 ꢁ 3.2
5.6 ꢁ 15.0
15.6 ꢁ 7.1
18.5 ꢁ 8.2
9.8 ꢁ 9.4
ꢂ10.5 ꢁ 10.4
5.0 ꢁ 15.1
1.6 ꢁ 7.7
NT
NT
NT
NT
NT
NT
NT
NT
5.3 ꢁ 9.2
11.8 ꢁ 6.4
3.7 ꢁ 12.6
3.9 ꢁ 11.9
12.7 ꢁ 5.6
21.3 ꢁ 3.4
23.7 ꢁ 4.0
14.2 ꢁ 5.5
8.6 ꢁ 11.2
ꢂ2.7 ꢁ 8.6
12.3 ꢁ 7.8
1.9 ꢁ 1.3
10s
10t
19.4 ꢁ 2.2
14.1 ꢁ 11.8
21.4 ꢁ 6.7
4.0 ꢁ 3.5
27.1 ꢁ 6.0
6.5 ꢁ 14.6
24.5 ꢁ 9.3
17.6 ꢁ 14.3
ꢂ10.3 ꢁ 4.3
NT
ꢂ2.8 ꢁ 5.8
6.0 ꢁ 6.7
10.3 ꢁ 2.5
61.7 ꢁ 10.7
33.5 ꢁ 10.3
52.8 ꢁ 7.2
42.7 ꢁ 10.7
ꢂ1.6 ꢁ 4.6
NT
27.7 ꢁ 4.2
30.2 ꢁ 4.7
35.2 ꢁ 8.6
10u
10v
10w
10x
10y
10z
Gefitinib
3.0 ꢁ 3.3
23.0 ꢁ 8.2
8.7 ꢁ 7.6
4.3 ꢁ 6.5
13.2 ꢁ 6.2
a
Cells were treated with the title compounds or gefitinib 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.
Green live A549 cells with normal morphology were observed in
the negative control group. Cells treated with 10s displayed orange
dots and cell budding in A549 cells at 1 M and were stained light
yellow at 20 M after treatment. The emerging yellow dots at
40 M showed late apoptotic cells.
The results above indicated that the cells had apoptotic
morphology. The almost complete absence of red cells in 10s
indicated very low cytotoxicity. These findings showed that 10s was
likely to induce apoptosis with low cytotoxicity.
m
m
2.4. Flow cytometry
m
Flow cytometry was used to evaluate the effects of the active
compound 10s at a given concentration on the growth and division

Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
27
Table 3
steric hindrance of the ethoxyl group at the 5-position was stronger
than that of the methoxyl group. The structure of compound 10o
(CCDC: 916922) was determined by X-ray crystallography. Com-
pound 10o crystallized in the monoclinic space group P2(1)/c.
Bioassay results showed that most target compounds had certain
inhibition activities, and some compounds even displayed better
broad-spectrum inhibition activities (e.g., 10n, 10o, 10p, 10r, 10s,
10v, 10y, and 10z). Notably, the ethoxyl series of compounds
possessed higher inhibition activities than the methoxyl series of
compounds against tumor cells. Further bioactivity test showed
that the IC50 values of compound 10s against PC3, MGC803, A375,
IC50 values of 10o and 10s against a variety of cells for 72 h in vitro.
Compound
IC50
PC3
(m
M)a
MGC803
A375
A549
10s
10o
Gefitinib
1.8 ꢁ 0.1
7.8 ꢁ 0.5
7.2 ꢁ 0.8
2.8 ꢁ 0.1
NTb
7.6 ꢁ 0.9
1.3 ꢁ 0.1
NT
7.2 ꢁ 1.1
2.9 ꢁ 0.2
7.8 ꢁ 0.9
9.8 ꢁ 1.1
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.
b
NT mean not tested.
and A549 cells were 1.8, 2.8, 1.3, and 2.9
were much higher than those of gefitinib (7.2, 7.6, 7.2, and 9.8
respectively). Moreover, the IC50 values of compound 10o against
PC3 nd A549 cells were 7.8 and 7.8 M, respectively, which were
m
M, respectively, which
of A549 cells using gefitinib as a positive control for 24 h, by
measuring the DNA content of eukaryotic cells. Results showed the
proportion of cells emitting a given fluorescent level proportional
to the DNA content, as shown in Fig. 3.
Fig. 4 shows that changes occurred in A549 cell cycle distribu-
tion after treatment with 10s at 2, 5, and 10 mM for 24 h. Unlike
antimitotic agents that accumulate their DNA content at the G2/M
phase of the cell cycle, the results in Table 5 showed that compound
10s caused significant cell cycle arrest at G0/G1 phase with
increased compound concentration. As shown in Table 5, com-
pound 10s caused significant cell cycle arrest at G0/G1 phase (the
proportion of cells increased from 33.9% to 90.7%), with a corre-
sponding decrease in the proportion of cells at G2/M phase
compared with control cultures (the proportion of cells decreased
from 63.2% to 3.1%). This result was consistent with the behavior of
gefitinib (the proportion of cells increased from 84.2% to 87.0% at
G0/G1 phase, and proportion of cells decreased from 12.0% to 3.7%
at G2/M).
mM,
m
similar to those of gefitinib. The inhibition activities of compound
10s were lower against NH3T3, indicating a weaker effect on
normal cells. Thus, compound 10s was used to study the antitumor
(A549) mechanism of action. LDH and AO/EB staining proved that
the toxicity to normal cells was weak. Analyses of cell configuration
and cell cycle revealed that the compound possibly caused cells to
remain at G0/G1 phase by inhibiting cell proliferation for 24 h. The
inhibition activities of Erk1/2 and p38 protein phosphorylation
were also tested by Western blot analysis. Results showed that the
compound inhibited the phosphorylation of ERK1/2 and P38 with
obvious concentration dependence. Thus, these compounds can
inhibit the proliferation of A549 cells through the mediated
signaling pathway of ERK1/2 and P38.
4. Experimental
2.5. Inhibition activity of title compounds against ERK1/2 and P38
phosphorylation
4.1. Materials and instruments
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 an 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. 1HNMRand 13CNMR spectra
(solvent CD3OD, or CDCl3) were recorded on a JEOL-ECX 500NMR
spectrometer at room temperature using tetramethylsilane as an
internal standard. Elemental analysis was performed with an Ele-
mentar Vario-III CHN analyzer.
Western blot analysis demonstrated that compound 10s had
significant inhibition activities against EGF-induced ERK1/2 and
P38 phosphorylation in A549 cells. Fig. 4 shows that 10s inhibited
ERK1/2 and P38 phosphorylation in A549 cells induced by EGF at 5,
10, 20, and 40
mM. Compound 10s possessed obvious inhibition
activities against ERK1/2 and p38 phosphorylation at 20 and 40
m
M,
respectively, with obvious concentration dependency. Thus, these
compounds can inhibit the proliferation of A549 cells through the
mediated signaling pathway of ERK1/2 and P38.
4.2. Chemistry
3. Conclusion
4.2.1. Preparation of methoxyl series intermediate
4.2.1.1. Preparation of 2,3,4-trimethoxy-6-nitrobenzoic acid (3a) [29].
2,3,4-Trimethoxybenzoic acid was added in portions to 45 mL of
A series of 4-(4-substituted piperazin)-5,6,7-trialkoxy quinazo-
line was prepared by conventional methods. The ethoxyl series of
compounds were more difficult to prepare because the effect of
Table 4
Cell toxicity of compounds tested on A549 cells by LDH at different concentrations within 24 h.
Concentration (
m
M)
0.01
0.1
1
20
40
Natural release
Full release
Gefitinib (u/L)
P1
1277.2 ꢁ 268.2
1054.1 ꢁ 78.4
689.0 ꢁ 231.0
1182.7 ꢁ 217.2
941.7 ꢁ 124.9
821.1 ꢁ 112.4
3611.8 ꢁ 231.8
0
0
0
0
0
0
P2
0.3
0.5
0.7
0.2
0.9
0
ZSC (u/L)
P1
1014.7 ꢁ 163.8
1159.1 ꢁ 142.8
1163.9 ꢁ 113.4
1589.0 ꢁ 112.8
1989.8 ꢁ 121.4
821.1 ꢁ 112.4
3611.8 ꢁ 231.8
0
0
0
0
0
0
P2
0.4
0.2
0.03
0.01
0
0
10s (u/L)
P1
P2
868.2 ꢁ 108.3
644.2 ꢁ 126.8
1211.2 ꢁ 139.1
896.2 ꢁ 134.8
1225.2 ꢁ 121.8
804.4 ꢁ 102.1
3608.8 ꢁ 145.7
0
0
0
0
0
0
0.9
0.8
0.2
0.8
0.3
0
“u/L” enzyme activity unit.
“P1” Difference analysis of compounds at different concentration and full release (1/100) group on the cytotoxicity of A549 cells.
“P2” Difference analysis of compounds at different concentration and natural release (DMSO) group on the cytotoxicity of A549 cells.
“P < 0.05” significant difference.

28
Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
Fig. 2. AO/EB staining of 10s in A549 cells. (1) Cells were treated with 0.1% DMSO for 48. (2e4) Cells were treated with 10s (1, 20, and 40 mM) for 48 h.
concentrated 70% nitric acid at 0 ꢀC (5.0 g, 0.02 mol). After stirring
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
152 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 3.98e4.15 (3s, 9H, 3OCH3), 7.55
Fig. 3. Cell cycle analysis histogram. (1) Cells were treated with 10s at 2, 5, and 10 mM for 24 h (2) Cells were treated with gefitinib (10, 20, and 40 mM) for 24 h.

Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
29
4.2.1.4. Preparation of 5,6,7-trimethoxyquinazoline-4-one (6a).
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 150 ꢀC for 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):
9H, 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,
d 3.95e4.01 (3s,
d
n
1684.6, 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.
4.2.1.5. Preparation of 4-chloro-5,6,7-trimethoxyquinazoline (7a).
A suspension of trimethoxyquinazolin-4-one (0.5 g, 0.002 mol) and
POCl3 (2 mL) in toluene (12 mL) were added dropwise to triethyl-
amine (6.0 mL). The resulting solution was heated at 102 ꢀC under
N2 atmosphere for 3.5 h, cooled, and concentrated. The resulting
solid was then diluted with CH2Cl2. The solution was 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:
Fig. 4. Dose effect of compound 10s (different concentrations, 60 min treatment) on
their inhibition activities against ERK 1/2 and p38 phosphorylation in A549 cells
treated by EGF (10 ng/mL, 10 min).
(s, 1H), 9.52 (s, 1H). IR (KBr) n: 735.6, 1112.9, 1305.8, 1334.7, 1496.8,
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.
73.3%; mp 113e115 ꢀC. 1H NMR (CDCl3, 500 MHz):
9H, 3OCH3), 7.23 (s, 1H), 8.84 (s, 1H). 13C NMR (CDCl3, 500 MHz):
56.5, 61.3, 62.0, 114.9, 104.2, 144.2, 148.3, 150.4, 153.1, 157.8, 160.0.
d 4.00e4.05 (3s,
4.2.1.2. Preparation of methyl 2,3,4-trimethoxy-6-nitrobenzoate (4a).
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
d
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.
4.2.1.6. Preparation of tert-butyl 4-(5,6,7-trimethoxyquinazolin-4-yl)
piperazine-1-carboxylate (8a). A solution of 4-chloro-5,6,7-
trimethoxyquinazoline (1.0 mmol) and tert-butyl piperazine-1-
carboxylate (1.2 mmol) in ethanol (8 mL) and triethylamine
(1.2 mmol) was stirred under reflux for 4 h. Upon reaction
completion as monitored by TLC, the solvent was removed under
reduced pressure. The resulting oil matter was purified by silica gel
to give 0.3 g the intermediate 8a. Yield: 75.0%; mp: 121e132 ꢀC. 1H
(CDCl3, 500 MHz):
1H). IR (KBr)
d 3.63 (s, 3H), 3.83e3.89 (3s, 9H, 3OCH3), 7.53 (s,
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.
NMR (CDCl3, MHz):
d 1.47 (s, 9H, COOC(CH3)3), 3.80, 3.96, 3.98 (3s,
9H, 3OCH3), 3.49 (s, 4H, H-3,5 of piperazin), 3.58 (s, 4H, H-2,6 of
4.2.1.3. Preparation of methyl 6-amino-2,3,4-trimethoxybenzoate
(5a). 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%
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
piperazin), 7.08 (s, 1H, H-8 of quinazoline), 8.53 (s, 1H, H-2 of qui-
nazoline);13C NMR(CDCl3, MHz):
d 28.5, 28.5, 28.5, 50.3, 50.3, 56.3,
56.3, 56.3, 61.8, 62.7, 80.1, 103.9, 106.3, 142.2, 148.2, 150.4, 153.0,
154.9, 158.3, 162.7. MS(ESI): m/z (M þ H)þ 405.2.
4.2.1.7. Preparation of 4 piperazin-5,6,7-trimethoxyquinazoline (9a).
A solution of tert-butyl 4-(5,6,7-trimethoxyquinazolin-4-yl)pipera-
zine-1-carboxylate (1.0 mmol) and 18% HCl (8 mL) was stirred at
25 ꢀC for 2 h. Upon reaction completion as monitored by TLC, the
solution was neutralized with ammonia water and extracted with
methylene chloride. The obtained organic layer was purified by
silica gel to give 0.23 g white solid 9a. Yield: 75.9%; mp: 158e
(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; N,
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,
C
5.96.
159 ꢀC. 1H NMR (CDCl3, MHz):
d 3.79, 3.95, 3.97 (3s, 9H, 3OCH3),
3.04 (s, 4H, H-2,6 of piperazine), 3.53 (s, 4H, H-3,5 of piperazine),
7.05 (s, 1H, H-8 of quinazoline), 8.51 (s, 1H, H-2 of quinazoline), 2.92
Table 5
Cell cycle distribution by flow cytometry in A549 cells treated for 24 h.
(s, 1H, NH of piperazine). 13C NMR (CDCl3, MHz):
d 46.0, 46.0, 51.4,
Compound
Concentration (
m
M)
G2/M (%)
G1/G0 (%)
S (%)
51.4, 56.2, 61.8, 62.8, 103.9, 106.1, 142.0, 148.2, 150.6, 153.1, 158.1,
162.6; MS(ESI): m/z (M þ H)þ 305.2.
Control
10s
63.2
43.5
10.1
3.1
12.0
13.9
6.2
33.9
52.8
81.3
90.7
84.2
83.0
90.0
87.0
2.9
3.7
8.6
6.2
3.8
3.1
3.8
9.3
2
5
10
4.2.2. Preparation of ethoxyl series intermediate
4.2.2.1. Preparation
of
2,3,4-triethoxybenzoic
acid
(2b).
Control
Gefitinib
10
20
40
2,3,4-Trihydroxybenzoic acid (5.0 g, 29.4 mmol) was added in
portions to 20 mL water with stirring at normal temperatures,
25 mL NaOH solution (4 mol/L) and diethyl sulfate were added
3.7

30
Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
dropwise to above solution. After refluxing temperature for 6 h,
cooled and neutralized by diluted HCl until 7 of PH. The resulting
solid was then separated out and filtered. The obtained solid was
then passed through a column of silica gel and then sequentially
washed with CHCl3 to produce the etherified product 4.0 g. Yield:
(m, 4H, 3OCH2CH3); 1.39, 1.48, 1.54 (3t, 9H, 3OCH2CH3, J ¼ 6.9 Hz,
6.8 Hz, 6.8 Hz); 13C NMR (CDCl3, 500 MHz):
14.6, 15.8, 15.9, 64.6,
d
69.8, 71.0, 105.6, 111.0, 141.7, 143.5, 147.7, 152.4, 158.8, 161.2;
MS(ESI): m/z (M þ H)þ 279.2.
53.5%; mp 128e130 ꢀC. 1H NMR (CDCl3, 500 MHz)
d: 11.40 (s, 1H,
4.2.2.6. Preparation of 4-chloro-5,6,7-triethoxyquinazoline (7b).
A suspension of triethoxyquinazolin-4-one (0.5 g, 1.8 mmol) and
POCl3 (1 mL) in toluene (8 mL) were added dropwise to triethyl-
amine (3.0 mL). The resulting solution was heated at 102 ꢀC under
N2 atmosphere for 3.5 h, cooled, and concentrated. The resulting
solid was then diluted with CH2Cl2. The solution was 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 (5:1) to produce the chlorinated product 0.4 g. Yield: 75.1%;
Ph-COOH), 6.77 (d, 1H, Ph-H-5, J ¼ 8.0 Hz), 7.86 (d, 1H, Ph-H-6,
J ¼ 8.0 Hz), 4.04, 4.15, 4.46 (3q, 6H, 3OCH2CH3 J ¼ 7.4 Hz, 6.9 Hz,
7.4 Hz); 1.36, 1.46 (2t, 9H, 3OCH2CH3, J ¼ 6.8 Hz, 7.4 Hz); 13C NMR
(CDCl3, 500 MHz):
d 14.8, 15.6, 15.7, 64.7, 69.5, 71.8, 108.8, 114.6,
128.2, 140.3, 152.0, 157.9, 165.4. MS(ESI): m/z (M þ Na)þ 277.2.
4.2.2.2. Preparation of methyl 2,3,4-triethoxybenzoate (3b).
2,3,4-Triethoxybenzoic acid (1.2 g, 4.7 mmol) was added to 5 mL of
methanol and 0.5 mL of 98% sulfuric acid with stirring at refluxing
temperature for 10 h. The mixture was cooled down and extracted
with methylene chloride. The obtained organic layer was purified
by silica gel column chromatography to produce the oil product
mp 83e84 ꢀC. 1H NMR (CDCl3, 500 MHz)
d: 8.79 (s, 1H, quinazoline
H-2), 7.17 (s, 1H, quinazoline H-8), 4.15 (q, 2H, OCH2CH3, J ¼ 6.9 Hz),
4.21e4.26 (m, 4H, 3OCH2CH3); 1.41, 1.47, 1.55 (3t, 9H, 3OCH2CH3,
0.9 g of 2. Yield: 71.8%. 1H NMR (CDCl3, 500 MHz)
d: 3.86 (s, 3H,
J ¼ 7.4 Hz, 6.8 Hz, 6.9 Hz); 13C NMR (CDCl3, 500 MHz):
d 14.5, 15.8,
COOCH3), 6.66 (d, 1H, Ph-H-5, J ¼ 6.8 Hz), 7.57 (d, 1H, Ph-H-6,
J ¼ 7.0 Hz), 4.04, 4.39, 4.45 (3q, 6H, 3OCH2CH3, J ¼ 6.8 Hz, 6.8 Hz,
6.9 Hz); 1.36,1.45,1.48 (3t, 9H, 3OCH2CH3, J ¼ 7.4 Hz, 6.2 Hz, 6.9 Hz);
15.9, 65.0, 69.8, 70.8, 104.5, 115.1, 143.5, 147.7, 150.5, 152.9, 157.9,
159.7. MS(ESI): m/z (M þ H)þ 297.1.
13C NMR (CDCl3, 500 MHz):
d
14.8, 15.7, 15.8, 51.5, 65.0, 69.5, 69.9,
4.2.2.7. Preparation of tert-butyl 4-(5,6,7-triethoxyquinazolin-4-yl)
piperazine-1-carboxylate (8b). A solution of 4-chloro-5,6,7-
triethoxyquinazoline (1.2 mmol) and tert-butyl piperazine-1-
carboxylate (1.8 mmol) in DMF (8 mL) and NaH (1.8 mmol) was
stirred at 25 ꢀC for 4 h. Upon reaction completion as monitored by
TLC, washed by saturated ammonia chloride solution, and extracted
with acetic ether, the solvent was removed under reduced pressure.
The resulting oil matter was purified by silica gel to give 0.38 g the
98.5, 104.6, 127.2, 140.8, 153.1, 157.8, 166.8. MS(ESI): m/z (M þ Na)þ
291.2.
4.2.2.3. Preparation of 2,3,4-triethoxy-6-nitrobenzoic acid (4b) [28].
Methyl 2,3,4-triethoxybenzoate was added in portions to 10 mL of
concentrated 70% nitric acid at 0 ꢀC (1.2 g, 4.5 mmol). After stirring
at 0 ꢀC for 10 min, the mixture was diluted with 50 mL of cold water.
Stirring was continued at 0 ꢀC for 1 h, and the resulting light orange
oil matter was extracted with methylene chloride. The obtained
organic layer was purified by silica gel column chromatography to
produce the oil product 0.9 g of 3. Yield: 64.2%. 1H NMR (CDCl3,
intermediate 8b. Yield: 71.4%. 1H NMR (CD3OD, MHz):
d 1.46 (s, 9H,
COOC(CH3)3),1.28e1.50 (3t, 9H, 3OCH2CH3), 3.56 (s, 8H, H-2,3,5,6 of
piperazin), 3.95e4.20 (m, 6H, 3OCH2CH3), 6.98 (s, 1H, H-8 of qui-
nazoline), 8.37 (s, 1H, H-2 of quinazoline);13C NMR(CDCl3, MHz):
500 MHz)
d
: 3.87 (s, 3H, COOCH3), 7.41 (s, 1H, Ph-H-5), 3.98 (q, 2H,
d 13.5,14.7,14.7, 27.3, 27.3, 27.3, 47.5, 47.5, 49.8, 49.8, 64.5, 69.5, 71.0,
OCH2CH3, J ¼ 6.8 Hz), 4.06e4.11 (m, 4H, 2OCH2CH3); 1.23, 1.37, 1.44
80.2, 102.6, 105.9, 141.6, 147.6, 149.3, 152.0, 155.1, 158.5, 162.6.
(3t, 9H, 3OCH2CH3, J ¼ 6.8 Hz, 6.8 Hz, 7.4 Hz); 13C NMR (CDCl3,
MS(ESI): m/z (M þ H)þ 447.3.
500 MHz): d 14.5, 15.5, 15.6, 53.0, 65.1, 69.7, 70.8, 104.3, 119.3, 140.0,
147.2, 150.2, 153.3. MS(ESI): m/z (M þ H)þ 314.2.
4.2.2.8. Preparation of 4-piperazin-5,6,7-triethoxyquinazoline (9b).
A solution of tert-butyl 4-(5,6,7-triethoxyquinazolin-4-yl)piperazine-
1-carboxylate (1.0 mmol) and 18% HCl (8 mL) was stirred at 25 ꢀC for
2 h. Upon reaction completion as monitored by TLC, the solution
was neutralized with ammonia water and extracted with methy-
lene chloride. The obtained organic layer was purified by silica gel
to give 0.25 g oil matter 9b. Yield: 72.7%. 1H NMR(CDCl3, MHz):
4.2.2.4. Preparation of methyl 6-amino-2,3,4-trimethoxybenzoate
(5b). Pd/C (10%, 0.12 g) was added to a solution of methyl 2,3,4-
triethoxy-6-nitrobenzoate (1.3 g, 4.2 mmol) in 5 mL of 95%
ethanol, and the mixture was stirred in the presence of H2 at room
temperature for 24 h. Then, the mixture was filtered and the ob-
tained solids were purified by silica gel column chromatography to
produce 0.8 g of oil matter 4. Yield: 68.1%. 1H NMR (CDCl3,
d
1.30e1.48 (3t, 9H, 3OCH2CH3), 3.90e4.16 (m, 6H, 3OCH2CH3), 2.99
(s, 4H, H-2,6 of piperazine), 3.52 (s, 4H, H-3,5 of piperazine), 6.98 (s,
1H, H-8 of quinazoline), 8.44 (s, 1H, H-2 of quinazoline), 2.54 (s, 1H,
500 MHz) d: 5.90 (s, 1H, Ph-H), 5.30 (s, 2H, NH2), 3.91, 3.96, 4.05 (3q,
6H, 3OCH2CH3, J ¼ 7.4 Hz, 7.4 Hz, 7.4 Hz); 1.30, 1.34, 1.40 (3t, 9H,
NH of piperazine). 13C NMR(CDCl3, MHz):
d 14.6, 15.8, 15.8, 45.9,
3OCH2CH3, J ¼ 6.9 Hz, 6.8 Hz, 6.9 Hz), 3.84 (s, 3H, COOCH3). 13C NMR
45.9, 51.2, 51.2, 64.5, 69.8, 71.2, 104.4, 106.3, 141.1, 147.6, 150.4, 152.8,
(CDCl3, 500 MHz):
d
14.7, 15.7, 15.8, 51.6, 64.0, 69.2, 69.9, 95.6, 101.2,
157.8, 162.6. MS(ESI): m/z (M þ H)þ 347.3.
133.6, 147.4, 154.3, 157.2, 168.6. MS(ESI): m/z (M þ H)þ 284.2.
4.2.3. General procedure for the synthesis of 5,6,7-trimethoxy-N-
aryl-4-aminoquinazolines (10ae10z)
4.2.2.5. Preparation of 5,6,7-triethoxyquinazoline-4-one (6b).
Formamide (10 mL) and 3 mL of POCl3 were added dropwise to a
solution of methyl 6-amino-2,3,4-triethoxybenzoate (5.0 g,
0.008 mol) in 25 mL of toluene with stirring at 100 ꢀC under N2
atmosphere for 5 h. Then, the mixture was cooled to room tem-
perature and concentrated. The resulting solid was then diluted
with water. The solution was neutralized with ammonia water and
extracted with methylene chloride. The obtained organic layer was
then purified by silica gel column chromatography to produce 3.3 g
of white solid 5. Yield: 67.2%; mp 192e195 ꢀC. 1H NMR (CDCl3,
A solution of 4-piperazin-5,6,7-trialkoxyquinazoline (1.0 mmol)
and substituted benzene sulfochloride (1.2 mmol) in ethanol (8 mL)
and triethylamine (1.2 mmol) was stirred under reflux for 3e5 h.
Upon reaction completion as monitored by TLC, the solvent was
removed under reduced pressure. The resulting oil matter was
purified by silica gel to give the title compounds (10ae10e, 10ne
10r).
A solution of 4-chloro-5,6,7-trialkoxyquinazoline (1.0 mmol)
and substituted piperazine (1.2 mmol) in DMF (8 mL) and NaH
(1.5 mmol) was stirred at 25 ꢀC for 4e8 h. Upon reaction completion
as monitored by TLC, washed by saturated ammonia chloride
500 MHz)
d: 11.69 (s, 1H, NH), 7.98 (s, 1H, quinazoline H-2), 6.98 (s,
1H, quinazoline H-8), 4.13 (q, 2H, OCH2CH3, J ¼ 6.8 Hz), 4.19e4.23

Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
31
solution, and extracted with acetic ether, the solvent was removed
under reduced pressure. The resulting oil matter was purified by
silica gel to give the title compounds (10fe10m, 10se10z).
4. 2. 3 . 7 . 4 - ( 4 - ( 4-F l uoro phe nyl)piperaz in-1-yl)-5, 6, 7-
trimethoxyquinazoline (10g). Yield: 73.8%; brown solid; mp: 122e
124 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 3.80, 3.98, 3.99 (3s, 9H, 3OCH3),
3.27 (t, 4H, J ¼ 5.6 Hz, piperazine-H), 3.70 (s, 4H, piperazine-H), 7.09
(s, 1H, quinazoline-H), 6.92e6.94 (m, 2H, AreH), 8.56 (s, 1H,
quinazoline-H), 6.97e7.00 (m, 2H, AreH). 13C NMR (CDCl3, MHz):
4.2.3.1. 5,6,7-Trimethoxy-4-(4-(2-trifluoromethylphenylsulfonyl)
piperazin-1-yl)quinazoline (10a). Yield: 67.2%; yellow oil matter. 1H
d
50.3, 50.3, 50.5, 50.5, 56.3, 61.8, 62.8, 104.0, 106.2, 115.6, 115.8,
NMR (CDCl3, 500 MHz):
d 3.73, 3.89, 3.97 (3s, 9H, 3OCH3), 3.39 (t,
118.0, 118.1, 142.2, 147.9, 148.2, 150.5, 153.1, 156.5, 158.2, 162.6.
4H, J ¼ 4.9 Hz, piperazine-H), 3.59 (s, 4H, piperazine-H), 7.07 (s, 1H,
quinazoline-H), 8.15 (t, 1H, J ¼ 6.9 Hz, AreH), 8.51 (s, 1H,
quinazoline-H), 7.97 (t, 1H, J ¼ 6.9 Hz, AreH), 7.72e7.74 (m, 2H, Are
MS(ESI): m/z (M þ H)þ 399.3.
H). 13C NMR (CDCl3, MHz):
d 45.7, 45.7, 50.0, 50.0, 56.3, 61.8, 62.7,
4 . 2 . 3 . 8 . 4 - ( 4 - ( 4 - N i t r o p h e n yl ) p i p e ra z i n - 1 - yl ) - 5 , 6 , 7 -
trimethoxyquinazoline (10h). Yield: 77.5%; brown solid; mp: 210e
104.1,106.2,121.5,123.6,128.1,128.8,132.3,133.0, 137.4,142.3,147.9,
213 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 3.81, 3.98, 3.99 (3s, 9H, 3OCH3),
150.5, 153.0, 158.4, 162.3. MS(ESI): m/z (M þ H)þ 513.2.
3.58 (t, 4H, J ¼ 5.8 Hz, piperazine-H), 3.70 (s, 4H, piperazine-H), 7.10
(s, 1H, quinazoline-H), 6.87 (d, 2H, J ¼ 9.7 Hz, AreH), 8.56 (s, 1H,
quinazoline-H), 8.13 (d, 2H, J ¼ 9.2 Hz, AreH). 13C NMR (CDCl3,
4.2.3.2. 5,6,7-Trimethoxy-4-(4-(4-trifluoromethylphenylsulfonyl)
piperazin-1-yl)quinazoline (10b). Yield: 74.1%; white solid; mp:
211e213 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 3.60, 3.94, 3.97 (3s, 9H,
MHz):
d 47.2, 47.2, 49.7, 49.7, 56.3, 61.8, 62.8, 104.2, 106.4, 112.9,
112.9, 126.0, 126.0, 138.9, 142.3, 148.1, 150.6, 153.1, 154.9, 158.4,
3OCH3), 3.20 (t, 4H, J ¼ 5.4 Hz, piperazine-H), 3.64 (s, 4H,
162.5. MS(ESI): m/z (M þ H)þ 426.3.
piperazine-H), 7.06 (s, 1H, quinazoline-H), 7.83 (d, 2H, J ¼ 8.0 Hz,
AreH), 8.50 (s,1H, quinazoline-H), 7.92 (d, 2H, J ¼ 8.6 Hz, AreH). 13
C
4.2.3.9. 5,6,7-Trimethoxy-4-(4-(pyridin-2-yl)piperazin-1-yl)quinazo-
NMR (CDCl3, MHz):
d 46.1, 46.1, 49.6, 49.6, 56.3, 61.8, 62.6, 104.2,
line (10i). Yield 66.7%; yellow solid; mp: 134e136 ꢀC. 1H NMR
106.1, 124.3, 126.4, 126.4, 128.3, 128.3, 134.9, 139.5, 142.3, 147.7,
150.6, 153.0, 158.4, 162.1. MS(ESI): m/z (M þ H)þ 513.2.
(CDCl3, 500 MHz):
d 3.81, 3.98, 3.99 (3s, 9H, 3OCH3), 3.67 (t, 4H,
J ¼ 4.0 Hz, piperazine-H), 3.71 (s, 4H, piperazine-H), 7.08 (s, 1H,
quinazoline-H), 8.22, 8.21 (dd, 1H, J ¼ 2.9 Hz, 2.0 Hz, pyridine-H),
8.56 (s, 1H, quinazoline-H), 7.50e7.53 (m, 1H, pyridine-H), 6.72
4.2.3.3. 5,6,7-Trimethoxy-4-(4-(4-methylphenylsulfonyl)piperazin-1-
yl)quinazoline (10c). Yield: 74.1%; white solid; mp: 175e177 ꢀC. 1H
(d, 1H, J ¼ 8.6 Hz, pyridine-H), 6.66e6.68 (m, 1H, pyridine-H). 13
C
NMR (CDCl3, 500 MHz):
d 3.59, 3.94, 3.96 (3s, 9H, 3OCH3), 3.14 (t,
NMR (CDCl3, MHz):
d 45.6, 45.6, 50.1, 50.1, 56.3, 61.8, 62.7, 104.0,
4H, J ¼ 5.4 Hz, piperazine-H), 3.63 (s, 4H, piperazine-H), 2.43 (s, 3H,
CH3-Ph), 7.34 (d, 2H, J ¼ 8.0 Hz, AreH), 8.49 (s, 1H, quinazoline-H),
7.67 (d, 2H, J ¼ 8.0 Hz, AreH), 7.05 (s, 1H, quinazoline-H). 13C NMR
106.3, 107.4, 113.8, 137.7, 142.1, 148.1, 148.3, 150.5, 153.1, 158.2, 159.6,
162.7. MS(ESI): m/z (M þ H)þ 382.3.
(CDCl3, MHz):
d 21.6, 46.1, 46.1, 49.5, 49.5, 56.2, 61.8, 62.6, 104.1,
4.2.3.10. 4-(4-Benzylpiperazin-1-yl)-5,6,7-trimethoxyquinazoline
106.0, 127.9, 127.9, 129.8, 129.8, 132.5, 142.2, 143.9, 147.8, 150.6,
(10j). Yield 79.6%; brown solid; mp: 86e89 ꢀC; 1H NMR (CDCl3,
153.0, 162.0. MS(ESI): m/z (M þ H)þ 459.2.
500 MHz):
d
3.77, 3.93, 3.96 (3s, 9H, 3OCH3), 2.60 (t, 4H, J ¼ 4.8 Hz,
piperazine-H), 3.59 (s, 4H, piperazine-H), 3.57 (s, 2H, CH2eAr), 7.04
(s, 1H, quinazoline-H), 8.49 (s, 1H, quinazoline-H), 7.30e7.33 (m,
4.2.3.4. 5,6,7-Trimethoxy-4-(4-(4-methoxyphenylsulfonyl)piperazin-
1-yl)quinazoline (10d). Yield: 75.0%; white solid; mp: 176e178 ꢀC.
5H, AreH). 13C NMR (CDCl3, MHz):
d 50.1, 50.1, 53.3, 53.3, 56.2, 61.8,
1H NMR (CDCl3, 500 MHz):
d 3.59, 3.94, 3.96 (3s, 9H, 3OCH3), 3.14 (t,
62.7, 63.2, 103.7, 105.9, 127.3, 128.4, 128.4, 129.4, 129.4, 137.8, 141.9,
148.3, 150.5, 153.1, 158.1, 162.3. MS(ESI): m/z (M þ H)þ 395.2.
4H, J ¼ 5.4 Hz, piperazine-H), 3.63 (s, 4H, piperazine-H), 3.60 (s, 3H,
OCH3-Ph), 7.01 (d, 2H, J ¼ 8.6 Hz, AreH), 8.49 (s, 1H, quinazoline-H),
7.72 (d, 2H, J ¼ 9.2 Hz, AreH), 7.05 (s, 1H, quinazoline-H). 13C NMR
4.2.3.11. 5,6,7-Trimethoxy-4-(4-(3-morpholinopropyl)piperazin-1-yl)
quinazoline (10k). Yield: 51.6%; brown solid; mp: 102e105 ꢀC. 1H
(CDCl3, MHz):
d 46.2, 46.2, 49.5, 49.5, 55.7, 56.3, 61.8, 62.6, 104.1,
106.1, 114.4, 114.4, 127.1, 130.0, 130.0, 142.2, 147.8, 150.6153.0, 158.3,
NMR (CDCl3, 500 MHz):
d 1.71e1.77 (m, 2H, CH2CH2CH2), 2.39 (t,
162.1, 163.2. MS(ESI): m/z (M þ H)þ 475.2.
4H, J ¼ 7.6 Hz, CH2CH2CH2), 2.44 (t, 4H, J ¼ 4.2 Hz, morpholino-H),
3.71 (t, 4H, J ¼ 8.0 Hz, morpholino-H), 3.78, 3.95, 3.97 (3s, 9H,
3OCH3), 2.60 (s, 4H, J ¼ 5.0 Hz, piperazine-H), 3.60 (t, 4H,
piperazine-H), 7.05 (s, 1H, quinazoline-H), 8.50 (s, 1H, quinazoline-
4.2.3.5. 5,6,7-Trimethoxy-4-(4-(phenylsulfonyl)piperazin-1-yl)quina-
zoline (10e). Yield: 68.4%; yellow solid; mp: 112e114 ꢀC. 1H NMR
H). 13C NMR (CDCl3, MHz):
d 23.8, 50.0, 53.4, 53.4, 53.8, 53.8, 56.2,
(CDCl3, 500 MHz):
d 3.57, 3.94, 3.96 (3s, 9H, 3OCH3), 3.17 (t, 4H,
J ¼ 5.9 Hz, piperazine-H), 3.62 (s, 4H, piperazine-H), 7.06 (s, 1H,
quinazoline-H), 7.79 (d, 2H, J ¼ 8.0 Hz, AreH), 8.49 (s, 1H,
quinazoline-H), 7.62 (t, 1H, J ¼ 7.4 Hz, AreH), 7.56 (t, 2H, J ¼ 8.0 Hz,
56.7, 57.1, 57.1, 61.8, 62.7, 67.0, 67.0, 103.8, 106.0, 141.9, 148.3, 150.6,
153.1, 158.1, 162.2. MS(ESI): m/z (M þ H)þ 432.4.
AreH). 13C NMR (CDCl3, MHz):
d 46.2, 46.2, 49.6, 49.6, 56.3, 61.8,
4. 2. 3.12. 4-(4-(4-Chlorobenzyl)piperazin-1-yl)-5, 6, 7-
62.6, 104.1, 106.1, 127.9, 127.9, 129.2, 129.2, 133.1, 135.5, 142.4, 147.8,
trimethoxyquinazoline (10l). Yield: 51.8%; yellow solid; mp: 124e
150.6, 153.0, 158.4, 162.1. MS(ESI): m/z (M þ H)þ 445.2.
127 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 3.78, 3.94, 3.98 (3s, 9H, 3OCH3),
2.58 (t, 4H, J ¼ 6.4 Hz, piperazine-H), 3.59 (s, 4H, piperazine-H), 3.53
4.2.3.6. 5,6,7-Trimethoxy-4-(4-(3-methoxyphenyl)piperazin-1-yl)
(s, 2H, CH2-Ar), 7.06 (s, 1H, quinazoline-H), 7.30 (s, 4H, AreH), 8.50
quinazoline (10f). Yield: 75.0%; yellow oil matter. 1H NMR (CDCl3,
(s, 1H, quinazoline-H); 13C NMR (CDCl3, MHz):
d 50.1, 53.3, 53.3,
500 MHz):
d
3.80, 3.98, 3.99 (3s, 9H, 3OCH3), 3.36 (t, 4H, J ¼ 5.0 Hz,
56.2, 56.2, 61.8, 62.4, 62.7, 103.8, 106.0, 128.5, 128.5, 130.5, 130.5,
133.0, 136.5, 141.9, 148.3, 150.6, 153.1, 158.1, 162.3. MS(ESI): m/z
(M þ H)þ 429.3.
piperazine-H), 3.70 (s, 4H, piperazine-H), 3.80 (s, 3H, OCH3eAr),
7.09 (s, 1H, quinazoline-H), 7.20 (t, 1H, J ¼ 8.0 Hz, H-3 of AreH), 8.55
(s, 1H, quinazoline-H), 6.45, 6.44 (dd, 1H, J ¼ 8.0 Hz, 2.0 Hz, AreH),
6.60, 6.59 (dd, 1H, J ¼ 8.0 Hz, 2.0 Hz, AreH), 6.52 (t, 1H, J ¼ 2.5 Hz,
4.2.3.13. 5,6,7-Trimethoxy-4-(4-(4-fluorobenzyl)piperazin-1-yl)qui-
nazoline (10m). Yield: 61.5%; yellow solid; mp: 142e145 ꢀC. 1H
AreH). 13C NMR (CDCl3, MHz):
d 49.4, 49.4, 50.2, 50.2, 55.3, 56.3,
61.8, 62.8, 102.7, 104.0, 104.9, 106.3, 109.0, 130.0, 142.1, 148.2, 150.6,
NMR (CDCl3, 500 MHz):
2H, CH2-Ar), 2.57 (t, 4H, J ¼ 5.6 Hz, piperazine-H), 3.58 (s, 4H,
d 3.78, 3.94, 3.97 (3s, 9H, 3OCH3), 3.53 (s,
152.6, 153.1, 158.2, 160.7, 162.6. MS(ESI): m/z (M þ H)þ 411.3.

32
Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
piperazine-H), 7.05 (s, 1H, quinazoline-H), 8.50 (s, 1H, quinazoline-
4.2.3.19. 5,6,7-Triethoxy-4-(4-(3-methoxyphenyl)piperazin-1-yl)qui-
H), 7.00 (t, 2H, J ¼ 8.6 Hz, AreH), 7.30 (q, 2H, J ¼ 5.2 Hz, AreH). 13
C
nazoline (10s). Yield: 57.4%; yellow oil matter. 1H NMR (CDCl3,
NMR (CDCl3, MHz):
d
50.1, 50.1, 53.2, 56.2, 56.2, 61.8, 62.4, 62.7,
500 MHz):
d
1.32, 1.44, 1.52 (3t, 9H, J ¼ 7.4 Hz, 6.9 Hz, 6.8 Hz,
103.9, 106.0, 115.1, 115.3, 130.7, 130.8, 133.6, 141.9, 148.3, 150.6, 153.1,
3OCH2CH3), 4.18 (q, 2H, J ¼ 7.4 Hz, 2OCH2CH3), 3.97 (q, 2H,
J ¼ 7.4 Hz, OCH2CH3), 3.33 (t, 4H, J ¼ 4.9 Hz, piperazine-H), 3.71 (s,
4H, piperazine-H), 3.79 (s, 3H, OCH3-Ar), 7.04 (s,1H, quinazoline-H),
7.19 (t, 1H, J ¼ 8.0 Hz, AreH), 8.51 (s, 1H, quinazoline-H), 6.44, 6.43
(dd, H, J ¼ 8.0 Hz, 2.6 Hz, AreH), 6.58, 6.57 (dd, H, J ¼ 8.0 Hz, 2.6 Hz,
153.1, 158.1, 162.3. MS(ESI): m/z (M þ H)þ 413.2.
4.2.3.14. 5,6,7-Triethoxy-4-(4-(2-(trifluoromethyl)phenylsulfonyl)
piperazin-1-yl)quinazoline (10n). Yield: 70.3%; white solid; mp:
154e157 ꢀC. 1H NMR (CDCl3, 500 MHz):
d
1.39, 1.45, 1.51 (3t, 9H,
AreH), 6.50 (t, H, J ¼ 2.2 Hz, AreH). 13C NMR (CDCl3, MHz):
d 14.6,
J ¼ 7.4 Hz, 6.9 Hz, 6.8 Hz, 3OCH2CH3), 3.89 (q, 2H, J ¼ 7.4 Hz,
OCH2CH3), 4.12e4.18 (m, 4H, 2OCH2CH3), 3.38 (s, 4H, piperazine-H),
3.62 (s, 4H, piperazine-H), 7.02 (s, 1H, quinazoline-H), 8.13 (d, 1H,
J ¼ 5.0 Hz, AreH), 8.46(s, 1H, quinazoline-H), 7.90 (d, 1H, J ¼ 6.8 Hz,
15.9, 15.9, 49.2, 49.2, 50.0, 50.0, 55.3, 64.6, 69.8, 71.4, 102.8, 104.1,
104.8, 106.4, 109.1, 130.0, 141.4, 147.6, 150.2, 152.7, 152.8, 157.9, 160.7,
162.5. MS(ESI): m/z (M þ H)þ 453.3.
AreH), 7.69e7.72 (m, 2H, AreH). 13C NMR (CDCl3, MHz):
d
14.6,15.7,
4.2.3.20. 4-(4-(4-Fluorophenyl)piperazin-1-yl)-5,6,7-
15.8, 45.5, 45.5, 49.8, 49.8, 64.6, 69.9, 71.3, 104.3, 106.4, 128.7, 132.2,
132.2, 132.3, 133.0, 133.0,137.3, 141.6, 147.3, 150.4,152.7, 158.1, 162.2.
MS(ESI): m/z (M þ H)þ 555.2.
triethoxyquinazoline (10t). Yield: 84.0%; yellow solid; mp: 118e
121 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 1.34, 1.44, 1.52 (3t, 9H,
J ¼ 7.4 Hz, 6.8 Hz, 6.8 Hz, 3OCH2CH3), 3.98 (q, 2H, J ¼ 6.8 Hz,
OCH2CH3), 4.15e4.22 (m, 4H, 2OCH2CH3), 3.25 (t, 4H, J ¼ 4.6 Hz,
piperazine-H), 3.74 (t, 4H, J ¼ 5.6 Hz, piperazine-H), 7.05 (s, 1H,
quinazoline-H), 8.52 (s, 1H, quinazoline-H), 6.91e6.93 (m, 2H, Are
4.2.3.15. 5,6,7-Triethoxy-4-(4-(4-(trifluoromethyl)phenylsulfonyl)
piperazin-1-yl)quinazoline (10o). Yield: 62.5%; white solid; mp:
172e174 ꢀC. 1H NMR (CDCl3, 500 MHz):
d
1.02, 1.41, 1.50 (3t, 9H,
H), 6.96e7.00 (m, 2H, AreH). 13C NMR (CDCl3, MHz):
d 14.6, 15.8,
J ¼ 6.8 Hz, 6.8 Hz, 7.4 Hz, 3OCH2CH3), 3.76 (q, 2H, J ¼ 6.8 Hz,
OCH2CH3), 4.10e4.18 (m, 4H, 2OCH2CH3), 3.17 (s, 4H, piperazine-H),
3.63 (s, 4H, piperazine-H), 7.00 (s, 1H, quinazoline-H), 7.82 (d, 2H,
J ¼ 8.2 Hz, AreH), 8.46 (s, 1H, quinazoline-H), 7.92 (d, 2H, J ¼ 8.6 Hz,
15.8, 50.1, 50.1, 50.4, 50.4, 64.5, 69.8, 71.3, 104.2, 106.5, 115.6, 115.8,
118.1, 118.1, 141.5, 147.6, 148.0, 150.4, 152.9, 156.5, 157.9, 162.5.
MS(ESI): m/z (M þ H)þ 441.3.
AreH). 13C NMR (CDCl3, MHz):
d
14.6, 15.5, 15.8, 46.0, 46.0, 49.3,
4. 2 . 3 . 21. 4 - ( 4 - ( 4 - N i t ro p h e nyl ) p i p e raz i n - 1 - yl ) - 5 , 6 , 7-
49.3, 64.6, 69.8, 71.2, 104.5, 106.4, 124.3, 126.4, 126.4, 128.4, 128.4,
134.7, 139.0, 141.6, 147.3, 150.4, 152.8, 158.1, 162.1. ESI-MS m/z:
(M þ H)þ 555.2.
triethoxyquinazoline (10u). Yield: 71.4%; yellow solid; mp: 203e
206 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 1.33, 1.42, 1.52 (3t, 9H,
J ¼ 6.8 Hz, 6.8 Hz, 6.9 Hz, 3OCH2CH3), 3.98 (q, 2H, J ¼ 7.4 Hz,
OCH2CH3), 4.13e4.22 (m, 4H, 2OCH2CH3), 3.58 (t, 4H, J ¼ 5.0 Hz,
piperazine-H), 3.74 (s, 4H, piperazine-H), 7.06 (s, 1H, quinazoline-
H), 8.54 (s, 1H, quinazoline-H), 6.87 (d, 2H, J ¼ 8.0 Hz, AreH), 8.14
4.2.3.16. 5,6,7-Triethoxy-4-(4-(4-(methyl)phenylsulfonyl)piperazin-
1-yl)quinazoline (10p). Yield: 69.2%; white solid; mp: 164e166 ꢀC.
1H NMR (CDCl3, 500 MHz):
d
1.04, 1.40, 1.50 (3t, 9H, J ¼ 7.4 Hz,
(d, 2H, J ¼ 7.2 Hz, AreH). 13C NMR (CDCl3, MHz):
d 14.6, 15.8, 15.8,
6.9 Hz, 6.8 Hz, 3OCH2CH3), 3.79 (q, 2H, J ¼ 6.9 Hz, OCH2CH3), 4.11e
4.17 (m, 4H, 2OCH2CH3), 3.12 (s, 4H, piperazine-H), 3.63 (s, 4H,
piperazine-H), 2.42 (s, 3H, CH3-Ph), 7.00 (s, 1H, quinazoline-H), 7.33
(d, 2H, J ¼ 8.0 Hz, AreH), 8.44 (s, 1H, quinazoline-H), 7.65 (d, 2H,
47.1, 47.1, 49.5, 49.5, 64.6, 69.8, 71.3, 104.4, 106.6, 112.9, 112.9, 126.0,
126.0,138.9,141.6,147.4,150.4,152.8,154.9,158.1,162.4. MS(ESI): m/
z (M þ H)þ 468.3.
J ¼ 7.4 Hz, AreH). 13C NMR (CDCl3, MHz):
d
14.6, 15.5, 15.8, 21.6,
4.2.3.22. 5,6,7-Triethoxy-4-(4-(pyridin-2-yl)piperazin-1-yl)quinazo-
46.0, 46.0, 49.4, 49.4, 64.6, 69.8, 71.2, 104.3, 106.3, 128.0, 128.0,
129.8, 129.8, 132.2, 141.5, 144.0, 147.4, 150.4, 152.8, 158.0, 162.1.
MS(ESI): m/z (M þ H)þ 501.2.
line (10v). Yield 61.2%; yellow oil matter. 1H NMR (CDCl3,
500 MHz):
d
1.32, 1.43, 1.52 (3t, 9H, J ¼ 7.4 Hz, 7.4 Hz, 6.8 Hz,
3OCH2CH3), 4.18 (q, 2H, J ¼ 7.4 Hz, 2OCH2CH3), 3.98 (q, 2H,
J ¼ 7.4 Hz, OCH2CH3), 3.69 (s, 8H, piperazine-H), 7.04 (s, 1H,
quinazoline-H), 8.21, 8.20 (dd, 1H, J ¼ 3.4 Hz, 2.0 Hz, pyridine-H),
8.52 (s, 1H, quinazoline-H), 7.49e7.52 (m, 1H, pyridine-H), 6.70
(d, 1H, J ¼ 8.6 Hz, pyridine-H), 6.65 (q, 1H, J ¼ 2.2 Hz, pyridine-H).
4.2.3.17. 5,6,7-Triethoxy-4-(4-(4-(methoxyl)phenylsulfonyl)piper-
azin-1-yl)quinazoline (10q). Yield: 66.7%; yellow solid; mp: 148e
151 ꢀC. 1H NMR (CDCl3, 500 MHz):
d 1.07, 1.41, 1.50 (3t, 9H,
J ¼ 6.8 Hz, 6.9 Hz, 6.9 Hz, 3OCH2CH3), 3.78 (q, 2H, J ¼ 7.4 Hz,
OCH2CH3), 4.10e4.17 (m, 4H, 2OCH2CH3), 3.11 (s, 4H, piperazine-H),
3.63 (s, 4H, piperazine-H), 3.86 (s, 3H, OCH3-Ph), 7.01 (s, 1H,
quinazoline-H), 7.71 (d, 2H, J ¼ 8.6 Hz, AreH), 8.45 (s, 1H,
quinazoline-H), 6.99 (d, 2H, J ¼ 4.6 Hz, AreH). 13C NMR (CDCl3,
13C NMR (CDCl3, MHz):
d 14.6, 15.8, 15.8, 45.3, 45.3, 49.9, 49.9, 64.5,
69.8, 71.3, 104.2, 106.6, 107.3, 113.7, 137.6, 141.4, 147.7, 148.1, 150.4,
152.9, 157.9, 159.6, 162.7. MS(ESI): m/z (M þ H)þ 424.3.
4.2.3.23. 4-(4-Benzylpiperazin-1-yl)-5,6,7-triethoxyquinazoline
MHz):
d
14.6, 15.5, 15.8, 46.0, 46.0, 49.4, 49.4, 55.7, 64.6, 69.8, 71.2,
(10w). Yield 84.9%; brown oil matter. 1H NMR (CDCl3, 500 MHz):
104.3, 106.3, 114.4, 114.4, 126.8, 130.1, 130.1, 141.5, 147.4, 150.4, 152.8,
d
1.31, 1.40, 1.50 (3t, 9H, J ¼ 6.8 Hz, 6.8 Hz, 6.8 Hz, 3OCH2CH3), 3.92,
158.0, 162.1, 163.3. MS(ESI): m/z (M þ H)þ 517.3.
4.12, 4.16 (3q, 6H, J ¼ 6.8 Hz, 6.8 Hz, 6.8 Hz, 3OCH2CH3), 2.57 (t, 4H,
J ¼ 5.3 Hz, piperazine-H), 3.61 (s, 4H, piperazine-H), 3.57 (s, 2H,
CH2-Ar), 7.00 (s, 1H, quinazoline-H), 8.46 (s, 1H, Quinazoline-H),
7.26e7.34 (m, 5H, H-2,3,4,5,6 of AreH). 13C NMR (CDCl3, MHz):
4.2.3.18. 5,6,7-Triethoxy-4-(4-(phenylsulfonyl)piperazin-1-yl)quina-
zoline (10r). Yield: 62.3%; white solid, mp: 170e173 ꢀC. 1H NMR
(CDCl3, 500 MHz):
d
1.01, 1.39, 1.48 (3t, 9H, J ¼ 6.8 Hz, 6.8 Hz, 7.4 Hz,
d 14.6, 15.8, 15.8, 50.1, 50.1, 53.1, 53.1, 63.2, 64.5, 69.8, 71.2, 104.0,
3OCH2CH3), 3.75, 4.10, 4.14 (3q, 6H, J ¼ 7.3 Hz, 6.9 Hz, 6.9 Hz,
3OCH2CH3), 3.14 (s, 4H, piperazine-H), 3.61 (s, 4H, piperazine-H),
6.99 (s, 1H, quinazoline-H), 7.77 (d, 2H, J ¼ 7.4 Hz, AreH), 8.44 (s,
1H, quinazoline-H), 7.60 (t, 1H, J ¼ 7.4 Hz, AreH), 7.55 (d, 2H,
106.2, 127.2, 128.4, 128.4, 129.3, 129.3, 137.8, 141.2, 147.7, 150.4,
152.9, 157.7, 162.2. MS(ESI): m/z (M þ H)þ 437.3.
4.2.3.24. 5,6,7-Triethoxy-4-(4-(3-morpholinopropyl)piperazin-1-yl)
J ¼ 8.0 Hz, AreH). 13C NMR (CDCl3, MHz):
d
14.6, 15.5, 15.8，46.0,
quinazoline (10x). Yield 46.9%; yellow oil matter. 1H NMR
46.0, 49.4, 49.4, 64.6, 69.8, 71.2, 104.3, 106.3, 127.9, 127.9, 129.2,
129.2,133.1, 135.3,141.5,147.4,150.4,152.8,158.0,162.2. MS(ESI): m/
z (M þ H)þ 487.2.
(500 MHz, CDCl3): d 1.70e1.76 (m, 2H, CH2CH2CH2), 2.34e2.41 (m,
4H, CH2CH2CH2), 2.44 (t, 4H, J ¼ 5.0 Hz, morpholino-H), 3.70 (t, 4H,
J ¼ 7.6 Hz, morpholino-H), 1.30, 1.40, 1.49 (3t, 9H, J ¼ 6.9 Hz, 6.8 Hz,

Y. Zhang et al. / European Journal of Medicinal Chemistry 78 (2014) 23e34
33
7.4 Hz, 3OCH2CH3), 3.89 (q, 2H, J ¼ 7.4 Hz, OCH2CH3), 4.10e4.17 (m,
4H, 2OCH2CH3), 2.57 (s, 4H, piperazine-H), 3.62 (s, 4H, piperazine-
H), 6.99 (s, 1H, quinazoline-H), 8.44 (s, 1H, quinazoline-H). 13C NMR
the number of living cells in culture. The experiment was per-
formed in triplicate. The percentage cytotoxicity was calculated
using the formula.
ðControl abs ꢂ Blank absÞ ꢂ ðTest abs ꢂ Blank absÞ
% Cytotoxicity ¼
ꢄ 100
ðControl abs ꢂ Blank absÞ
(CDCl3, MHz):
d
14.6, 15.8, 23.7, 29.8, 49.8, 53.2, 53.2, 53.8, 53.8,
4.3.3. LDH release assay
56.6, 57.0, 57.0, 64.5, 66.9, 66.9, 69.8, 71.2, 103.9, 106.1, 141.2, 147.6,
1.0 ꢄ 105 cells per well (total volume 1.6 mL) were plated into a 6
well plate and incubated for 24 h at 37 ꢀC in 5% CO2. Cytotoxicity was
assessed based on measuring the release of LDH after 24 h compound
treatment. Prior to each assay, the cells were lysed with 2% (V/V)
Triton X-100 in culture media for 10 min at 37 ꢀC to obtain a repre-
sentative maximal LDH release as the positive control with 100%
toxicity and cells in culture media alone was the low control. The
amounts of LDH in the supernatant were determined and calculated
as kit instructions. All tests were performed in triplicate and assay
was repeated three times independently with similar results.
150.2, 152.7, 157.8, 162.1. MS(ESI): m/z (M þ H)þ 474.4.
4.2.3.25. 4-(4-(4-Chlorobenzyl)piperazin-1-yl)-5,6,7-
triethoxyquinazoline (10y). Yield 63.0%; yellow solid; mp: 99e
101 ꢀC. 1HNMR (CD3OD, 500 MHz):
d 1.29, 1.37, 1.47 (3t, 9H,
J ¼ 6.8 Hz, 6.8 Hz, 6.9 Hz, 3OCH2CH3), 3.91, 4.11, 4.17 (3q, 6H,
J ¼ 6.9 Hz, 6.9 Hz, 6.8 Hz, 3OCH2CH3), 2.58 (s, 4H, piperazine-H),
3.60 (s, 4H, piperazine-H), 3.55 (s, 2H, CH2-Ph), 6.95 (s, 1H,
quinazoline-H), 7.29e7.33 (m, 4H, AreH), 8.32 (s, 1H, quinazoline-
H). 13C NMR (CD3OD, MHz):
d 13.5, 14.7, 14.7, 49.6, 49.6, 52.5, 61.6,
61.6, 64.4, 69.5, 71.0, 102.5, 105.7, 128.1, 128.1, 130.8, 130.8, 132.9,
135.9, 141.3, 147.7, 149.3, 152.0, 158.4, 162.3. MS(ESI): m/z (M þ H)þ
471.2.
4.3.4. AO/EB staining
Cells were seeded at a concentration of 5 ꢄ 104 cells/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
4.2.3.26. 5,6,7-Triethoxy-4-(4-(4-fluorobenzyl)piperazin-1-yl)quina-
zoline (10z). Yield 65.0%; yellow solid; mp: 85e88 ꢀC. 1H NMR
with monolayer cells was inverted on the glass slide with 20
mL of
(CDCl3, 500 MHz):
d
1.32, 1.41, 1.50 (3t, 9H, J ¼ 7.4 Hz, 6.8 Hz, 6.8 Hz,
AD/EB stain (100 g/mL). Fluorescence was read on an IX71SIF-3
m
3OCH2CH3), 3.92, 4.13, 4.17 (3q, 6H, J ¼ 7.4 Hz, 6.8 Hz, 6.9 Hz,
3OCH2CH3), 2.55 (s, 4H, piperazine-H), 3.60 (s, 4H, piperazine-H),
3.52 (s, 2H, CH2-Ph), 7.00 (s, 1H, quinazoline-H), 6.99 (t, 2H,
J ¼ 9.2 Hz, AreH), 7.28e7.31 (q, 2H, J ¼ 2.8 Hz, AreH), 8.47 (s, 1H,
fluorescence microscope (OLYMPUS Co., Japan).
4.3.5. Cell cycle analysis
quinazoline-H). 13C NMR (CDCl3, MHz):
d 14.6, 15.8, 15.8, 50.0, 50.0,
Test substances were dissolved in DMSO and diluted to given
concentration. A549 cells (1.6 ꢄ 105 cells/mL) were cultured six-well
plate for 24 h with test compounds. The cells were harvested, washed
with PBS and centrifuged. The fixation of cells was realized through
the addition of 0.6 mL ethanol (70% ice-cold) and then keeping cells
at ꢂ20 ꢀC overnight until DNA staining. The fixed cells were treated
with 10 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
eukaryotic cells was then measured with flow cytometery.
53.0, 62.4, 62.4, 64.5, 69.8, 71.2, 104.0, 106.2, 115.1, 115.2, 130.7,
130.8, 133.6, 141.2, 147.7, 150.4, 152.8, 157.8, 161.2, 162.3. MS(ESI): m/
z (M þ H)þ 455.3.
4.3. Anticancer activity bioassay
4.3.1. Cell culture
The human prostate cancer cell line PC3, breast cancer cell line
Bcap-37, gastric cancer cell line MGC803 and BGC823, malignant
melanoma cell line A375 and nonsmall-cell lung cancer A549 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.
4.3.6. Western blot analysis
A549 cells were seeded on a six-well plate and incubated in
RPMI 1640 medium plus 10% FBS at 37 ꢀC. After incubation for 36e
48 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,
4.3.2. MTT assay
All tested compounds were dissolved in DMSO (1e100 mM so-
lution) and subsequently diluted in culture medium before being
used to treat cultured cells. Tested cells were plated in 96-well
plates at a density of 2 ꢄ 103 cells per well per 100
mL of the proper
culture medium and treated with the compounds at 1e100
m
M for
10 mM
b
-glycerophosphate, 1 mM dithiothreitol, 10
mg/mL leu-
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 formazane,
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
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% SDS-
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

34
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Conflicts of interest
None.
Author’s contributions
Yang ZHANG synthesized the compounds and carried out part of
the bioassay experiments. Yin-Jiu HUANG did part of the bioassay
experiments. Hong-Mei XIANG also did part of the bioassay ex-
periments. De-Yu HU took part in the compound structural eluci-
dation and bioassay experiments. Wei XUE carried out some
structure elucidation experiments. Prof. Bao-An Sopng and Prof.
Song Yang are the corresponding authors for this work.
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Acknowledgments
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This work was financially supported by the National Key Pro-
gram for Basic Research (Grant No. 2010CB126105), National Nature
Science Foundation of China (Grant Nos. 21172048), Key Technol-
ogies R&D Program (Nos. 2011BAE06B02-23), the Research Project
of Chinese Ministry of Education (No. 213033A), Guizhou Key S&T
Project (Nos. [2012]6012, [2008]70011, [2011]3016), and the Nat-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.03.036.
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