2-Styryl-4-aminoquinazoline derivatives as potent DNA-cleavage, p53-activation and in vivo effective anticancer agents

Article abstract of DOI:10.1016/j.ejmech.2019.111851

Forty-eight analogues of CP-31398, an antitumor agent modulated the mutant p53 gene were synthesized and their cytotoxicities against four cancer cell lines with different p-53 status including bladder cell T24 (w-p53), gastric cell MGC-803 (m-p53), prostate cell DU145 (m-p53), prostate cell PC-3 (null-p53), lung cell A549 (w-p53) and normal liver cell line HL-7702 (w-p53) were examined. (E)-2-(4-Nitrostyryl)-4-(3-dimethylaminopropyl)-aminoquinazoline (10ah) was identified as the most potent compound in anti-proliferation against MGC-803 cells, with IC₅₀ lowed to 1.73 μM, far potency than that of CP-31398. Molecular mechanism study revealed that 10ah and CP-31398 differ greatly in mechanism to exert their antitumor properties. 10ah could intercalate into DNA and resulted in significant DNA double-strand break. 10ah-treatment in MGC-803 cells increased the expression of p53, phosphorylated p53 (p-p53), CDK4, p21 to cause cell cycle arrest at G2/M phase, significantly up-regulated the levels of pro-apoptosis proteins Bak, Bax, Bim while down-regulated the anti-apoptosis proteins Bcl-2, Bcl-xL and the levels of cyclin B1, fluctuated the intracellular reactive oxygen species (ROS), Ca²⁺ and mitochondrial membrane potential, activated Caspase-9 and Caspase-3 to induce apoptosis. 10ah also displayed potent anticancer efficiency against MGC-803 xenograft tumors models, with tumor growth inhibition (TGI) up to 61.8% at 20 mg/kg without obvious toxicity.

Full text of DOI:10.1016/j.ejmech.2019.111851

Journal Pre-proof
2-Styryl-4-aminoquinazoline derivatives as potent DNA-cleavage, p53-activation and
in vivo effective anticancer agents
Xin-Wei Wei, Jing-Mei Yuan, Wan-Yun Huang, Nan-Ying Chen, Xiao-Juan Li, Cheng-
Xue Pan, Dong-Liang Mo, Gui-Fa Su
PII:
S0223-5234(19)31003-7
DOI:
https://doi.org/10.1016/j.ejmech.2019.111851
EJMECH 111851
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 August 2019
Revised Date: 3 November 2019
Accepted Date: 3 November 2019
Please cite this article as: X.-W. Wei, J.-M. Yuan, W.-Y. Huang, N.-Y. Chen, X.-J. Li, C.-X. Pan, D.-
L. Mo, G.-F. Su, 2-Styryl-4-aminoquinazoline derivatives as potent DNA-cleavage, p53-activation
and in vivo effective anticancer agents, European Journal of Medicinal Chemistry (2019), doi: https://
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2-Styryl-4-aminoquinazoline derivatives as potent
DNA-cleavage, p53-activation and in vivo effective
anticancer agents
Xin-Wei Weia†, Jing-Mei Yuana†, Wan-Yun Huangb, Nan-Ying Chena, Xiao-Juan Lia,
Cheng-Xue Pana*, Dong-Liang Moa*, Gui-Fa Sua*
aState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,
School of Chemistry and Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004,
PR China
bDepartment of Pharmacology, Guilin Medical University, Guilin 541004, PR China
† These authors contributed equally to this work.
* Corresponding Authors: Professors Cheng-Xue Pan, Dong-Liang Mo, and Gui-Fa Su
Tel.
&
Fax:
+86-773-3696862,
Email:
chengxuepan@163.com
(C.-X.
Pan);
moeastlight@gxnu.edu.cn (D.-L. Mo); gfysglgx@163.com (G.-F. Su)

ABSTRACT
Forty-eight analogues of CP-31398, an antitumor agent modulated the mutant
p53 gene were synthesized and their cytotoxicities against four cancer cell lines with
different p-53 status including bladder cell T24 (w-p53), gastric cell MGC-803
(m-p53), prostate cell DU145 (m-p53), prostate cell PC-3 (null-p53), lung cell A549
(w-p53) and normal liver cell line HL-7702 (w-p53) were examined.
(E)-2-(4-Nitrostyryl)-4-(3-dimethylaminopropyl)-aminoquinazoline
(10ah)
was
identified as the most potent compound in anti-proliferation against MGC-803 cells,
with IC50 lowed to 1.73 µM, far potency than that of CP-31398.
Molecular mechanism study revealed that 10ah and CP-31398 differ greatly in
mechanism to exert their antitumor properties. 10ah could intercalate into DNA and
resulted in significant DNA double-strand break. 10ah-treatment in MGC-803 cells
increased the expression of p53, phosphorylated p53 (p-p53), CDK4, p21 to cause cell
cycle arrest at G2/M phase, significantly up-regulated the levels of pro-apoptosis
proteins Bak, Bax, Bim while down-regulated the anti-apoptosis proteins Bcl-2,
Bcl-xL and the levels of cyclin B1, fluctuated the intracellular reactive oxygen species
(ROS), Ca2+ and mitochondrial membrane potential, activated Caspase-9 and
Caspase-3 to induce apoptosis. 10ah also displayed potent anticancer efficiency
against MGC-803 xenograft tumors models, with tumor growth inhibition (TGI) up to
61.8% at 20 mg/kg without obvious toxicity.
Keywords: 2-Styryl-4-aminoquinazoline; CP-31398, DNA intercalator; Anticancer;
Synthesis

1. Introduction
2-Styryl-4-aminoquinazoline or 2-styryl-4-chloroquinazoline derivatives exhibit
diverse bioactivities such as antitumor [1-6], antibacterial [7-9], analgesic [10],
anti-inflammatory [11], antiprotozoal [12] and antiviral activity [13]. The most
famous compound was CP-31398, which had been reported to exert antitumor activity
via restoring the active conformation of mutated p53 to rescue its tumor suppressor
functions [2, 14-16], or targeting both the wild and mutant-type p53 and block the
growth of rhabdomyosarcoma [17]. Some more recent study also indicated that
CP-31398 is able to revers the epithelial mesenchymal transition via the
downregulation of PAX2s [18], or to down-regulate the p53 negative regulator
MDM2 in certain cancer [19]. Compound KIN-281 and KIN-236 were reported to
show effects on inhibiting a wide range of tumor-related kinase proteins [1-4] (Fig. 1).
Fig. 1. Representative bioactive 2-styryl-4aminoquinazoline derivatives reported in literature
Besides anticancer activity, other 2-styryl-4-aminoquinazoline derivatives such
as compound A was identified as promising anti-bacterial agent via inhibiting the cell
division protein FtsZ [8-9], compound B as a highly selective opioid receptor like-1
(ORL1) antagonists [10] (Fig. 1).
Based on the summarization above, it can be seen that slight change on the
substituents of 2-styryl-4-aminoquinazoline scaffold can results in great variety of

their bioactivity, which means that spent more efforts on the synthesis and bioactive
evaluation of more novel 2-styryl-4-aminoquinazolines derivatives, might help to
identify more novel candidates for drug development.
As our on going interest on the compounds that can regulate the p53 pathway
[20-21], we decided to synthesize more novel analogues of CP-31398 and then
explore their bioactivity in the aim of identifying new candidates for new antitumor
drug development. Herein, we report the synthesis, in vitro and in vivo anticancer
evaluation of a series of 2-styryl-4-aiminoquinazoline derivatives.
2. Results and discussion
2.1. Chemistry
Scheme 1. Synthesis of 2-styryl-4-aiminoquinazoline derivatives 9-11
The preparation of the target compounds were outlined in scheme 1. Stirring the
mixture of 2-aminobenzamide derivatives 1 and excess anhydride at room
temperature gave the compounds 2, which were further condensed with aromatic
aldehyde in acetic acid at 120 °C to provide the 3-5 in moderate yields.
Heating 3-5 with POCl3 in the presence of N,N-dimethylaniline in toluene at
115 °C offered the intermediate 6-8, respectively. In this step the presence of tertiary

amine as base was necessarily to smoothly finish the reaction, and we found that the
N,N-dimethylaniline was more preferable than triethylamine or diisopropyl
ethylamine. The intermediate 6-8 were very difficult to dissolve in any common
solvents, so their structures could not be characterized by NMR. Finally, refluxing of
6-8 with amine derivatives in toluene in the presence of catalytic amount of DMAP
furnished the targeted compounds 9-11 in moderate to high yields.
2.2. Biological activity
2.2.1. Antiproliferative activity of compounds 9-11
To obtain more information about the in vitro cytotoxic potency of the target
2-styryl-4-aminoquinazoline derivatives 9-11, five human cancer cell lines from four
different tissue, and in which also include the wild-type (w-p53), mutant-type (m-p53)
and null-p53 three status of p53 gene were selected to evaluate the cytotoxicity of
9-11 via MTT assay. Epirubicin and CP-31398 were used as the positive control. The
tested cell included the cancer cell lines T24 (from bladder, w-p53), MGC-803
(gastric cell, m-p53), DU145 (from prostate, m-p53), PC-3 (from prostate, null-p53),
A549 (from lung, w-p53), the normal cell line HL-7702 (from liver, w-p53) and the
IC50 values was summarized in table 1
Table 1 Structures and in vitro cytotoxicity of compounds 9–11
IC50 (µM)a
T24
w-p53
MGC-803
m-p53
DU145
m-p53
PC-3
null-p53 w-p53
A549 HL-7702
Comp.
R1, R2
R3
w-p53
H,
3,4-(OCH3)2
H,
3,4-(OCH3)2
H,
3,4-(OCH3)2
H,
3,4-(OCH3)2
H,
(CH3)2N(CH2)2
>100
>100
>100
>100
>100
>100
>100
>100
9aa
30.20
±2.98
6.50
±0.53
10.48
±0.61
31.4
50.32
±1.01
20.31
±0.64
78.54
±2.14
(C2H5)2N(CH2)2
>100
>100
9ab
9ac
9ad
±1.85
15.01
±1.22
71.63
±1.87
15.52
±0.72
33.23
±0.88
38.78
±2.01
82.67
±1.09
(i-Pr)2N(CH2)2
19.83
±0.99
>100
88.44
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9ae
9af
3,4-(OCH3)2
H,
(CH3)2N(CH2)3

3,4-(OCH3)2
H,
3,4-(OCH3)2
H,
±1.96
>100
(C2H5)2N(CH2)3
(CH2)4NH2
>100
>100
>100
>100
>100
>100
>100
>100
>100
9ag
9ah
30.89
±0.49
8.95
70.46
±0.53
14.37
±1.07
3,4-(OCH3)2
15.74
±0.94
51.09
±1.38
33.94
±1.44
17.96
±1.39
44.46
±1.25
8.96
H, p-NO2
H, p-NO2
H, p-NO2
H, p-NO2
H, p-NO2
H, p-NO2
H, p-NO2
H, p-NO2
(CH3)2N(CH2)2
(C2H5)2N(CH2)2
(i-Pr)2N(CH2)2
10aa
10ab
10ac
10ad
10ae
10af
10ag
10ah
11aa
11ab
11ac
11ad
11ae
11af
11ag
11ah
9ba
±0.84
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
14.23
±0.47
36.16
±1.33
29.05
±1.88
27.57
±1.18
±0.49
25.61
±0.97
28.23
±1.41
>100
>100
>100
>100
>100
>100
9.16
±0.83
5.42
±0.23
1.79
±0.16
66.44
±1.78
8.68
±0.47
17.50
±1.46
6.38
15.73
±1.47
8.25
±0.36
1.73
24.40
±1.04
12.30
±1.30
4.15
59.54
±1.90
21.40
±0.56
5.30
±1.48
7.46
±0.31
57.84
±1.01
20.71
±1.43
21.24
±1.56
9.91
(CH3)2N(CH2)3
(C2H5)2N(CH2)3
(CH2)4NH2
>100
15.43
±0.76
4.53
>100
5.48
±0.98
±0.19
±0.32
±0.28
H,
3,4-OCH2O-
H,
3,4-OCH2O-
H,
3,4-OCH2O-
H,
3,4-OCH2O-
H,
3,4-OCH2O-
H,
3,4-OCH2O-
H,
3,4-OCH2O-
H,
3,4-OCH2O-
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
Cl,
3,4-(OCH3)2
(CH3)2N(CH2)2
(C2H5)2N(CH2)2
(i-Pr)2N(CH2)2
>100
>100
>100
>100
9.88
±0.19
11.32
±0.84
10.70
±0.56
16.34
±0.99
33.86
±1.96
46.90
±2.17
>100
>100
>100
8.53
±0.23
12.16
±0.46
39.64
±1.01
±0.76
±0.21
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
8.81
±0.99
13.95
±1.42
25.40
±1.89
14.49
±2.21
(CH3)2N(CH2)3
(C2H5)2N(CH2)3
(CH2)4NH2
>100
>100
>100
>100
>100
>100
41.18
±0.93
12.10
±0.88
12.17
±0.77
14.85
±0.43
7.64
24.58
±0.49
11.84
±0.79
16.75
±0.38
15.56
±0.75
13.51
±0.99
17.86
±1.35
23.55
±1.49
21.34
±1.12
80.47
±2.34
91.20
±0.21
(CH3)2N(CH2)2
(C2H5)2N(CH2)2
(i-Pr)2N(CH2)2
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
9bb
>100
9bc
17.24
±1.07
21.17
±2.11
13.86
±0.39
9bd
±0.22
91.80
±1.65
>100
>100
>100
>100
>100
>100
>100
>100
9be
(CH3)2N(CH2)3
(C2H5)2N(CH2)3
(CH2)4NH2
>100
9bf
3.51
±0.26
7.82
±0.32
6.97
±0.24
11.04
±0.77
56.64
±1.57
34.34
±0.79
8.42
±0.76
14.14
±0.72
4.68
5.35
±0.39
19.99
±1.45
6.76
±1.20
12.41
±1.64
6.25
±0.84
5.17
±0.73
9bg
50.50
±4.45
>100
>100
9bh
8.02
±0.78
71.34
±1.49
Cl, p-NO2
Cl, p-NO2
Cl, p-NO2
Cl, p-NO2
Cl, p-NO2
Cl, p-NO2
Cl, p-NO2
(CH3)2N(CH2)2
(C2H5)2N(CH2)2
(i-Pr)2N(CH2)2
10ba
10bb
10bc
10bd
10be
10bf
10bg
>100
>100
>100
±0.17
7.12
>100
>100
>100
>100
>100
>100
>100
>100
>100
±0.34
17.27
±1.43
19.97
±0.47
>100
>100
>100
41.08
±1.47
>100
>100
>100
7.42
±0.49
7.12
6.67
±0.39
7.37
8.49
±0.54
8.12
5.32
±0.56
6.89
15.83
±0.76
76.43
±3.33
(CH3)2N(CH2)3
(C2H5)2N(CH2)3
14.97
±0.56
±0.22
±0.24
±0.37
±0.33

5.62
±0.55
9.61
±0.94
8.17
±0.88
7.46
±0.82
9.22
±0.89
38.32
±3.89
8.12
±0.67
9.74
±0.37
5.65
±0.24
20.34
±2.07
1.12
9.16
±0.67
9.46
±0.78
11.12
±0.27
8.57
±0.48
14.66
±1.01
10.03
±1.67
Cl, p-NO2
(CH2)4NH2
>100
>100
>100
>100
>100
>100
10bh
11ba
11bb
11bc
11bd
11be
11bf
11bg
11bh
Cb
Cl,
3,4-OCH2O-
Cl,
3,4-OCH2O-
Cl,
3,4-OCH2O-
Cl,
3,4-OCH2O-
(CH3)2N(CH2)2
(C2H5)2N(CH2)2
(C2H5)2N(CH2)2
>100
>100
>100
>100
>100
>100
>100
7.56
±0.47
8.78
14.21
±1.56
10.57
±0.68
15.91
±2.37
>100
>100
>100
>100
>100
>100
±0.57
>100
>100
>100
>100
>100
>100
>100
>100
>100
Cl,
>100
3,4-OCH2O-
Cl,
3,4-OCH2O-
Cl,
3,4-OCH2O-
Cl,
3,4-OCH2O-
17.23
±2.42
12.94
±1.20
10.31
±0.39
18.27
±1.02
1.20
(CH3)2N(CH2)3
(C2H5)2N(CH2)3
(CH2)4NH2
50.29
±1.20
15.34
±2.03
2.01
9.82
±1.08
24.78
±2.07
0.82
16.34
±1.43
2.31
25.34
±0.49
2.10
Dc
±0.33
±0.31
±0.47
±0.51
±0.17
±0.51
a Results were expressed as means ± SD (standard deviation) of four independent experiments.
b C: CP-31398; c D: Epirubicin
As shown in table 1, most target compounds displayed moderate to potent
inhibitory activities against the tested cancer cell lines. Compounds 10ah, 9bg, 10ba,
10bf, 10bg and 11bc exhibited especially potent cytotoxicity and 10ah display
exceptional efficacy against MGC-803 and T24 cancer cell lines, with IC50 values low
to 1.73 and 1.79 µM, respectively, almost 8 and 11 folds potency than that of its
analogue CP-31398 and close to that of the clinical antitumor drug Epirubicin.
The data in table 1 revealed that the series compounds display no much
selectivity to the different p53 status of the cancer cell, indicating they can exert their
anti-proliferation via a p53 independent manner. In 2015 and most recently, some p53
independent antitumor styrylquinoline derivatives were also reported by Musiol and
Mrozek-Wilcziewicz [22-23], which suggested that these styrylquinazoline
derivatives, though different in structure to the styrylquinoline derivatives reported by
Musiol and Mrozek-Wilcziewicz, might share some common mechanism of action.
Since mutations in the p53 gene are very common in cancers and are closely related to
drug resistance and poor prognosis, compounds displaying anti-proliferation in a p53
independent manner are often considered to be a promising class of compounds that
deserve further exploration[23]. In addition, the series compounds demonstrate
somewhat selectivity to certain tissue of cancer cell. In general, the anti-proliferation
against the T24 and MGC-803 cell lines are more potent than that against the other

three tested cell lines DU145, PC-3 and A549.
The results in table 1 indicated that the side chains at the 2-styryl-4-
aminoquinazoline scaffold, the substituent on the phenyl ring of quinazoline motifs or
the styryl all played very important roles in the anti-proliferation, but the substituents
on the three position mentioned above seem significantly influence each other, which
means that a seemingly favorable substituent at certain position, might probably
mismatch with another favorable substituent at the other position. These complicated
results of structure-activity relationship (SAR), might attribute to the fact that this
2-styryl-4-aminoquinazoline scaffold, might usually act as many kinase inhibitors,
and a very subtle change in structure of the compounds would results in the mismatch
with the structural domain of the certain kinase [2].
The data in table 1 also indicated that R1 as Cl (b series) seemed more favorable
than H (a series), and R2 as 4-NO2 (10 series) more desirable than 3,4-(OCH3)2 (9
series) or 3,4-(OCH2CH2O)- (11 series) for the cytotoxic activities of the targeted
compounds in general. As far as the effects of R3 on the cytotoxicity was concerned,
when R3 is a primary amine (series xxh), a dialkylamine (series xxa-c and xxf-g ) or a
piperidinyl (series 9~11xe), the cytotoxicity of the compounds and the kinds of R3
seems no much obvious regularities and the only obvious SAR of R3 able to
summarize in table 1 was that compounds with a morpholinyl (series 9-11xe) as R3
almost display no any anti-proliferation activity. The complicated SAR results of this
series 2-styryl-4-aminoquinazoline, suggested that to identify more potent antitumor
drug candidates based on the 2-styryl-4-aminoquinazoline scaffold, more attention
should be paid to the optimal matching of 2-styryl-4-aminoquinazoline matrix
substituents at different positions.
Since compound 10ah displayed the highest cytotoxicity against the tested tumor
cells, especially to the MGC-803 and T24 cell lines, it was used as a representative
compound to carry out the mechanism studies in MGC-803 cell.
2.2.2. 10ah intercalate into DNA and significantly induce DNA double-strand break
DNA is believed to be one of the primary targets of the antitumor drug [24-27].
As many studies reported in literature mainly focused on how the 2-styryl-4-amino-
quinazoline derivatives affecting and regulating the certain protein kinases [1-4], what

most interest us was whether 10ah probably exerted its antitumor activity via target
the DNA and to trigger the cascade reaction of antitumor-related pathway in tumor
cells to induce apoptosis [28].
UV–Vis and fluorescence spectrophotometry were firstly used to investigate the
interaction of 10ah with ct-DNA. The UV–Vis spectra of 10ah in the absence (dashed
line) and presence of ct-DNA were shown in Fig. 2A. As the Fig. 2A indicated, 10ah
exhibit characteristic absorption peak at ca. 320~335 nm. With the addition of DNA, a
corresponding decrease in the absorption intensity was observed, suggesting the 10ah
could bind to the DNA and result in a hypochromicity.
To confirm the interaction mode of 10ah with DNA, competitive binding assay
was further performed using the classic DNA intercalator ethidium bromide (EB) as a
probe. As shown in Fig. 2B, the addition of 10ah (the ratio of [10ah]/[DNA]/[EB]
increasing from 0.5: 10: 1 to 5: 10: 1) could obviously result in the quenching of
ct-DNA–EB fluorescence intensity. The result indicated that 10ah could compete with
EB and replace it to intercalate into ct-DNA [29] and the quenching constant Kq,
according to the Stern-Volmer quenching equation, was calculated to be 1.08×104.
To determine whether 10ah can intercalate into intact DNA, agarose gel
electrophoresis assay was further used to investigate the interaction of 10ah with
pBR332 plasmid DNA, EB, a well-known of DNA intercalator able to directly
intercalate into intact DNA, was used as the positive control. As indicated in Fig. 2C,
a noticeable retardation of DNA movement was observed when the concentration of
10ah was up to 100 µM, suggesting that 10ah can directly intercalate into the intact
DNA [29].
The level of γH2AX, a biomarker of DNA cleavage, was also measured by
Western blotting assay in 10ah-treated MGC-803 cells. As depicted in Fig. 2D,
significantly increase in the level of γH2AX in 10ah-treated MGC-803 cells was
observed at 2.5 µM of 10ah, indicating that 10ah not only able to intercalate into
DNA but induce substantial DNA double-strand break in MGC-803 cells. By
comparing the performance of CP-31398 with that of compound 10ah in Fig. 2C and
D, suggesting that even very similarly in structure, 10ah and CP-31398 might exert
their antitumor properties via a completely different manner.

Fig. 2. DNA-binding properties of 10ah. (A) UV/Vis spectra of compound 10ah (20 µM) in
Tris-HCl buffer with increasing amounts of ct-DNA ([ct-DNA]/[10ah] = 0~1.0) at 25 °C. (B)
Fluorescence emission spectra of EB bound with ct-DNA in the absence (dashed line) and
presence of compound 10ah as competitive agent with increasing [10ah]/[EB] ratios of 0.5: 1 to 5:
1. Inset: Plot of I0/I versus [10ah] the quenching constant (Kq) of 10ah towards ct-DNA-EB
solution. (C) Gel electrophoresis of pBR322 DNA after being incubated with 10ah at the
concentrations of 50−200 µM for 3 h in Tris-HCl bu er. 1% DMSO + pBR322 DNA in lane 1 as
negative control, EB + pBR322 DNA in lane 2 as positive control, Lane 3 is the mixture of
pBR322 DNA and 100 µM CP-31398. Other lanes are the mixture of pBR322 DNA and 10ah (50,
100, 200 µM), respectively. (D) Immunoblotting analysis of γH2AX in CP-31398 and
10ah-treated MGC-803 cells. Whole cell extracts were prepared and analyzed by Western blot.
The data are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001
vs the negative control.
2.2.3. 10ah activate the p53 and induces cell cycle arrest in MGC-803 cells
Since damage in DNA might usually result in the activation of p53 [30] to result

in cell cycle arrest, 10ah and CP-31398 appear differently in interaction with DNA,
the different effects of 10ah and CP-31398 in the regulation of some key proteins in
p53 signal pathway and cell cycle were firstly investigated. As shown in Fig. 3A-B,
10ah substantially up-regulated the p53 and p-p53 (s15) levels in MGC-803 cells at
lowed concentration while slightly enhanced the expression of its negative regulator
MDM2 in p53 signal pathway. The level of some key cyclin, cyclin dependent kinases
(CDK) or CDK inhibitor such as cyclin B1, CDK4 and p21 that are closely-related to
the development in tumor in cell were also regulated by 10ah. The cyclin B1 was
significantly down-regulated and the expression of CDK4 and p21 were increased.
Fig. 3. (A) The expression levels of p53, p-p53 after treating of 10ah. (B) The expression levels of
p21, MDM2, CDK4 and Cyclin B1 after treating of 10ah. CP-31398 was used as a positive
control. β-actin was used as the loading control. (C) Compound 10ah induced cell-cycle arrest in
MGC-803 cells. (D) Cell cycle analysis of MGC-803 cells treated with various concentrations of
CP-31398 for 48 h by flow cytometry. The data are representative of three independent
experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the negative control.

To further determine whether 10ah inhibits the proliferation of MGC-803 cells
through cell cycle arrest, flow cytometry was used to examine the cell cycle
distribution of 10ah-treated MGC-803 cells. As shown in Fig. 3C, with the increase of
concentrations of 10ah, the percentage of MGC-803 cells at G2/M phase was
dramatically increased from 7.94% (Control group ) to 18.16% (0.125 µM), 55.43%
(0.25 µM) and 72.67% (0.5 µM) after 10ah-treatment, indicating that 10ah-treatment
would result in remarked G2/M phase arrest in a dose–dependent manner.
In comparation with the results of CP-31398 treatment in MGC-803 cells, the
expression of p53, p-p53, MDM2, cyclin B1 and p21 seems not to be obviously
affected by CP-31398 at a concentration of 5.0 µM, only a slight increasing in CDK4
and the cell cycle is arrest in S and G2/M phase (Fig. 3D), suggested that the
mechanism of 10ah to exert it anticancer quite differ from that of CP-31398. As the
report [12], PLC/PRL/5 cells (from liver, m-p53) treated by CP-31398 in a dose of 10
µg/mL (27.6 µM) would result in significantly up-regulating in the MDM2 and p21,
while down-regulating in the cyclin B1 and inducing a G1 arrest, indicating that
different cell treated by CP-31398 might have different performance in their p53
pathway and distribution in cell cycle.
2.2.4. 10ah affect the intracellular ROS, Ca2+ and MMP
The balance of intracellular reactive oxygen species (ROS) [31] and Ca2+ [32]
are very meaningful for the live and the normal physical activity of cells, an abnormal
fluctuation of them in cell usually the indicator of apoptosis. To determine whether
10ah-treatment MGC-803 cells would induce the production of ROS and fluctuation
of Ca2+, MGC-803 cells were treated with 10ah and stained with
2′,7′-dichloro-dihydrofluorescein diacetate (DCFH-DA, fluorescent indicators for
ROS) or Fluo-3 AM (fluorescent indicators for Ca2+) at different doses for 24 h and
the fluorescence intensities were measured.
As depicted in Fig. 4A, the fluorescence intensities indicating the amount of
ROS were significantly increased, especially when the dose of 10ah changed from 0
to 0.5 µM. While in Fig. 4B, the fluorescence intensities which indicated the level of

intracellular Ca2+ also increased steadily, suggesting that 10ah-treament in MGC-803
cells would result in the increase of intracellular ROS and Ca2+ levels which may be
responsible for apoptosis induction by 10ah.
Fig. 4. (A) The generation of ROS after treatment with 10
ah for 24 h. (B) Effects of compound
10
a
h on the intracellular Ca2+ level in MGC-803 cells. (C) The collapses of mitochondrial
membrane potential treatment with compound 10ah for 24 h. The data are representative of three
independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the negative control.
Mitochondrial membrane potential (MMP) is a critical parameter for
understanding mitochondrial function [33] and the loss of MMP usually indicate the
mitochondrial dysfunction associated with intrinsic mitochondrial apoptosis pathway
[34]. To further get the information related to the pathway that mediated the cell
apoptosis induced by 10ah, the change of MMP of 10ah-treated MGC-803 cells was
measured by the fluorescent probe JC-1 (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethyl-
benzimidazolylcarbocyanine). As shown in Fig. 4C, in control group, weak green
fluorescence (p2 region) which indicated a high ∆Ψm was observed. While in
10ah-treated cells the green fluorescence obviously increased in a dose-dependent
manner, indicating the loss in MMP after 10ah treatment in MGC-803 cells.

2.2.5. 10ah regulate the apoptosis related proteins to induce intrinsic apoptosis
Fig. 5. 10ah induces cell apoptosis. (A) Apoptosis was detected by Annexin V-FITC/PI staining
after cells treated with the indicated concentration of 10 h for 24 h. (B) Morphology detection
with Hoechst 33258 staining. (C) The activation of Caspase-3/9 by compound 10 h in MGC-803
cells after treatment with 2.5 µM for 24 h. (D) Immunoblotting analysis of proteins related to the
mitochondria-mediated intrinsic apoptotic pathway mediated by 10 h and CP-31398. The data are
a
a
a
representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001 vs the
negative control.
Apoptosis-inducing is still the basic way of the majority of clinical
chemotherapeutic agent to exert their antitumor. To determine whether cell cycle
arrest in MGC-803 cells result in apoptosis, Annexin V–FITC/PI staining and cell
morphology analysis assay were used to evaluate the apoptosis-inducing effect. As

shown in Fig. 5A, with the increasing of 10ah from 0 µM, 0. 5 µM, 1.0 µM to 2.5 µM,
the percentages of the apoptotic cells increase dramatically from 1.7%, 20.9%, 42.5%
to 83.9%, suggested that 10ah could strongly induce apoptosis in MGC-803 cells. The
result of cell morphology analysis via Hoechst 33258 staining depicted in Fig. 5B,
also clearly indicated that 10ah-treament result in cell shrinkage, nuclear
fragmentation, chromatin condensation while non-treated cells displayed dispersal
uniform fluorescence.
To gain more information about the pathway by which 10ah mediated the
apoptosis, the proportion of Caspase-3/9 activated cells after treatment with 10ah was
examined. As shown in Fig. 5C, the activation of Caspase-3 and Caspase-9 induced
by 10ah was clearly observed, as the proportion of Caspase-3 activated cells
increased from 1.47% to 22.98% and the Caspase-9 activated cells from 2.82% to
57.35% when treating by 2.5 µM of 10ah. These results indicated that 10ah induced
apoptosis via a caspase-dependent pathway in MGC-803 cells.
To gain further insight into the different mechanisms of tumor suppression
activity of 10ah and CP-31398 and which death effectors were the dynamic
participants in the apoptosis signaling after 10ah or CP-31398 treatment, first the
levels of Bcl-2 family proteins in MGC-803 cells were measured after treated by 10ah
or CP-31398. As shown in Fig. 5D, the expression levels of pro-apoptotic proteins
Bim and Bax were significantly up–regulated while the levels of anti-apoptotic
proteins Bcl-2 were down–regulated both in the 10ah or CP-31398 treatment cells,
merely in CP-31398 treatment cells it needed more high concentration of drug, quite
similar to the results in PLC/PRF/5 cell treated with CP-31398 report in literature [12].
The most different performance between the 10ah and CP-31398 treatment cells
could be observed in their levels of Bak and Bcl-xL, which were changed obviously
in 10ah treatment cells while almost unchanged in CP-31398 treatment.
2.2.5. 10ah shows potential anticancer potency in vivo
The anticancer efficiency of 10ah in vivo was examined in MGC-803 xenograft
tumors. As shown in Fig. 6A-C, The MGC-803 xenograft tumor-bearing nude mice
treated by 10ah (10 or 20 mg/kg/2 days) displayed significant tumor growth inhibition.
The in vivo tumor growth inhibition (TGI) of 10ah and clinical drug Epirubicin were

35.9% (10ah, 10 mg/kg/2 days), 61.8% (10ah, 20 mg/kg/2 days), and 67.2%
(Epirubicin, 10 mg/kg/2 days), respectively. By compared with the dose of CP-31398
(0.5 mg/mouse, about 30 mg/kg) treated in PLC/PRL/5 xenograft tumors to reach
about 60% TGI [12], 10ah need less dose than CP-31398 to obtain satisfaction
treatment result, confirming the promising anticancer efficiency of 10ah in vivo. The
potential toxicity of 10ah was also investigated by monitoring the weight loss of all
tested nude mice. As depicted in Fig. 6D, no obvious changes in body weight were
found in control group and treatment group during the treatment, indicating the low
toxicity of 10ah at the therapeutic dosage.
Fig. 6. The antitumor efficacy of 10ah in human gastric carcinoma xenograft models in vivo. (A)
Changes in tumor volume of mice from different groups over the observation period. (B) Tumor
weight and tumor growth inhibition after treating with 10ah at the end of treatment. (C)
Photographs of excised tumor from vehicle group and treatment groups. (D) Body weight of mice
at the end of treatment. Results are expressed as the mean ± SD, error bars represented SD, n = 6,
(∗∗) p < 0.01 vs vehicle.
3. Conclusions
In this report, forty eight 2-styryl-4-aminoquinazolines derivatives as the
analogue of CP-31398 were synthesized. Cytotoxicity evaluation identified the 10ah
as the most promising compound with IC50 values lowed to 1.73 µM against

MGC-803 cell lines. The molecular mechanism studies revealed that 10ah and
CP-31398 differ in their mode to interact with DNA and their mechanism on
regulation p53 signal pathway, cell cycle and cell apoptosis to exert their tumor
suppression activity. The study also revealed that 10ah might exert its antitumor
activity via intercalating into DNA, breaking the DNA double-strand to activate the
p53 to cause cell cycle arrest, inducing fluctuation in the level of intracellular ROS,
Ca2+and MMP, up-regulating the pro-apoptosis proteins Bak, Bax and Bim while
down-regulation the anti-apoptosis proteins Bcl-2 and Bcl-xL to mediate the intrinsic
apoptosis. Furthermore, 10ah also displayed promising anticancer efficiency in vivo
with tumor growth inhibition (TGI) up to 61.8% without obvious toxicity. These
results suggest that 10ah may serve as a promising candidate in development of novel
therapeutic agent to treat gastric cancer.
4. Experimental
4.1. General information
All chemicals employed in this work are commercially available and were used
as received. Melting points were recorded on WRSIA apparatus and uncorrected. Thin
layer chromatography (TLC) was performed on silica gel plates 60 F-254 and column
chromatography on silica gel (200~300 mesh) from Branch of Qindao Marine
Chemical Co., Ltd. NMR spectra were recorded in CDCl3, CD3OD and DMSO-d6 on
Bruker Advance (400 MHz, 500 MHz, 600 MHz) with TMS as internal standard.
Chemical shifts were recorded in δ values. HRMS were measured in ESI mode and
the mass analyzer of the HRMS was TOF.
4.2. Compounds synthesis and characterization
4.2.1. General procedure for synthesis of 2
Compound 1 (36.7 mmol) added to acetic anhydride (30 mL) and then stirred at

room temperature for 0.5 h (monitored by TLC, VEA/VPE = 1/2). After completion, 30
mL of water was added to the reaction mixture and precipitations occured. The
precipitate was filtered off and washing successively with saturated NaHCO3 and
ethyl acetate to give white solid 2.
2-Methyl-4-hydroxyquinazoline (2a). White solid, yield 67%, m.p. 174~176
1
(lit [35]. m.p. 176.5 °C); H NMR (400 MHz, CD3OD) δ 8.35 (d, J = 8.3 Hz, 1H),
7.72 (dd, J = 7.9, 1.4 Hz, 1H), 7.49~7.43 (m, 1H), 7.15~7.11 (m, 1H), 2.15 (s, 3H).
6-Chloro-2-methyl-4-hydroxyquinazoline (2b). White solid, yield 79%, m.p.
281~283 °C (lit [36]. m.p. 287 °C). 1H NMR (400 MHz, CDCl3) δ 10.96 (s, 1H), 8.61
(d, J = 8.9 Hz, 1H), 7.73~7.34 (m, 1H), 2.20 (s, 3H).
4.2.2. General procedure for synthesis of 3~5
The mixture of 2 (18.7 mmol) and aromatic aldehyde (22.5 mmol) in AcOH was
stirred and refluxed for about 12 h (monitored by TLC, VEA/VPE = 1/2). After
completion, 20 mL of water was added, the resultant precipitate was filtered off and
washing successively with saturated NaHCO3 and water to afford 3~5.
(E)-2-(3,4-Dimethoxystyryl)-4-hydroxyquinazoline (3a). Pale yellow solid, yield
43%, m.p. 251~253 °C (lit [37]. m.p. 265~266 °C), 1H NMR (500 MHz, DMSO-d6) δ
12.21 (s, 1H), 8.10~8.09 (m, 1H), 7.89 (d, J = 16.1 Hz, 1H), 7.82~7.77 (m, 1H), 7.64
(d, J = 8.2 Hz, 1H), 7.47~7.44 (m, 1H), 7.28 (s, 1H), 7.23~7.17 (m, 1H), 7.03 (d, J =
8.2 Hz, 1H), 6.90 (d, J = 16.1 Hz, 1H), 3.84 (s, 3H), 3.81 (s, 3H).
(E)-2-(4-Nitrostyryl)-4-hydroxyquinazoline (4a). Yellow solid, yield 60%, m.p.
315~317 °C (lit [38]. m.p. >350 °C). 1H NMR (400 MHz, DMSO-d6) δ 12.44 (s, 1H),
8.30 (d, J = 8.6 Hz, 2H), 8.13 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 16.2 Hz, 1H), 7.93 (d, J
= 8.6 Hz, 2H), 7.86~7.82 (m, 1H), 7.71 (d, J = 8.0 Hz, 1H), 7.54~7.50 (m, 1H), 7.20
(d, J = 16.2 Hz, 1H).
(E)-2-(3,4-Methylenedioxystyryl)-4-hydroxyquinazoline (5a). Gray solid, yield
50%, m.p. 215~217 °C (lit [39]. m.p. 216~217 °C). 1H NMR (400 MHz, DMSO-d6) δ
8.09 (dd, J = 8.0, 1.5 Hz, 1H), 7.86 (d, J = 16.1 Hz, 1H), 7.81~7.77 (m, 1H), 7.64 (d, J
= 8.1 Hz, 1H), 7.49~7.43 (m, 1H), 7.27 (d, J = 1.5 Hz, 1H), 7.15 (dd, J = 8.0, 1.5 Hz,
1H), 6.99 (d, J = 8.0 Hz, 1H), 6.84 (d, J = 16.1 Hz, 1H), 6.09 (s, 2H).

(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-hydroxyquinazoline (3b). Yellow solid,
yield 37%, m.p. 294~296 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.36 (s, 1H), 8.02 (s,
1H), 7.90 (d, J = 16.1 Hz, 1H), 7.81 (d, J = 8.6 Hz, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.26
(s, 1H), 7.20 (d, J = 8.3 Hz, 1H), 7.03 (d, J = 8.3 Hz, 1H), 6.88 (d, J = 16.1 Hz, 1H),
3.83 (s, 3H), 3.81 (s, 3H).
(E)-6-Chloro-2-(4-nitrostyryl)-4-hydroxyquinazoline (4b). Yellow solid, yield
49%, m.p. 352~355 °C. 1H NMR (400 MHz, DMSO-d6) δ 12.57 (s, 1H), 8.27 (d, J =
8.8 Hz, 2H), 8.06~7.97 (m, 2H), 7.89 (d, J = 8.8 Hz, 2H), 7.83(dd, J = 8.7, 2.5 Hz 1H),
7.70 (d, J = 8.7 Hz, 1H), 7.16 (d, J = 16.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6) δ
161.1, 151.7, 148.1, 148.0, 141.8, 136.7, 135.2, 131.4, 130.0, 129.1, 125.6, 125.4,
124.7, 123.0.
(E)-6-Chloro-2-(3,4-methylenedioxystyryl)-4-hydroxyquinazoline (5b). Yellow
solid, yield 39%, m.p. 357~359 °C. 1H NMR (400 MHz, DMSO-d6) δ 7.95 (d, J = 2.5
Hz, 1H), 7.75 (d, J = 16.0 Hz, 1H), 7.61 (dd, J = 8.7, 2.5 Hz, 1H), 7.52 (d, J = 8.7 Hz,
1H), 7.27 (d, J = 1.5 Hz, 1H), 7.11 (dd, J = 8.0, 1.5 Hz, 1H), 6.97 (d, J = 8.0 Hz, 1H),
6.82 (d, J = 16.0 Hz, 1H), 6.06 (s, 2H) (the resonance of O−H was not observed in
DMSO-d6).
4.2.3. General procedure for synthesis of 6~8
The mixture of 3~5 (6.5 mmol), POCl3 (32.4 mmol), N,N-dimethylaniline in
toluene was stirred and refluxed for 10 h (monitored by TLC, VEA/VPE = 1/2). After
completion, the solvent was removed under reduced pressure and the residue was
purified on silica gel to provide 6~8.
(E)-4-Chloro-2-(3,4-dimethoxystyryl)quinazoline (6a). Yellow solid, yield 76%,
m.p. 167~169 .
(E)-4-Chloro-2-(4-nitrostyryl)quinazoline (7a). Yellow solid, yield 94%, m.p.
154~156 .
(E)-4-Chloro-2-(3,4-Methylenedioxystyryl)quinazoline (8a). reddish-brown solid,
yield 90%, m.p. 105–107 .
(E)-4,6-Dichloro-2-(3,4-dimethoxystyryl)quinazoline (6b). Yellow solid, 44%
yield, m.p. 165~167 .

(E)-4,6-Dichloro-2-(4-nitrostyryl)quinazoline (7b). Gray solid, 51% yield, m.p.
173~175 .
(E)-2-(3,4-Methylenedioxystyryl)-4,6-dichloroquinazoline (8b). Yellow solid, 94%
yield, m.p. 236~238 .
These products hardly dissolved in the common solvents and can’t be further
structurally characterized by NMR.
4.2.4. General procedure for synthesis of 9~11
The mixture of 6~8 (1.4 mmol), DMAP (0.3 mmol), TEA, amine (0.5 mmol) in
toluene was stirred and refluxed for 12 h (monitored by TLC, VMeOH/VDCM = 1/10).
The solvent was evaporated under reduced pressure when the reaction was completed,
and the residue was purified by column chromatography on silica gel (VMeOH/VDCM
1/20) to afford 9~11. The structures were confirmed by NMR and HRMS.
=
(E)-2-(3,4-Dimethoxystyryl)-4-(2-dimethylaminoethyl)aminoquinazoline (9aa).
Yellow solid, yield 31%, m.p. 154~156 °C. 1H NMR (400 MHz, CDCl3) δ 7.96 (d, J =
15.7 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H), 7.72 (d, J = 8.2 Hz, 1H), 7.70~7.66 (m, 1H),
7.40~7.36 (m, 1H), 7.23 (d, J=1.4 Hz, 1H), 7.16 (dd, J=8.2, 1.4 Hz, 1H), 7.11 (d, J =
15.7 Hz, 1H), 6.87 (d, J = 8.2 Hz, 1H), 6.52 (s,1H), 3.93 (s, 3H), 3.90 (s, 3H),
3.82~3.76 (m, 2H), 2.66 (t, J = 5.8 Hz, 2H), 2.33 (s, 6H). 13C NMR (100 MHz, CDCl3)
δ 161.1, 159.1, 150.3, 149.7, 149.1, 136.8, 132.5, 129.8, 128.1, 127.3, 125.1, 121.6,
121.0, 114.0, 111.1, 109.2, 57.6, 55.9, 55.8, 45.3, 38.2. HRMS (ESI) m/z calcd for
C22H27N4O2 [M+H]+ 379.2129, found 379.2119.
(E)-2-(3,4-Dimethoxystyryl)-4-(2-diethylaminoethyl)aminoquinazoline
(9ab).
Yellow solid, yield 35%, m.p. 141~143 °C. 1H NMR (500 MHz, CDCl3) δ 7.97 (d, J =
15.7 Hz, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.70~7.67 (m, 2H), 7.41~7.37 (m, 1H), 7.24 (d,
J = 1.7 Hz, 1H), 7.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.11 (d, J = 15.7 Hz, 1H), 6.88 (d, J =
8.3 Hz, 1H), 6.72 (s, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.80~3.76 (m, 2H), 2.81 (t, J =
13
6.0 Hz, 2H), 2.65 (q, J = 7.1 Hz, 4H), 1.10 (t, J = 7.1 Hz, 6H). C NMR (100 MHz,
CDCl3) δ 161.1, 159.0, 150.2, 149.7, 149.1, 136.7, 132.4, 129.8, 128.1, 127.3, 125.2,
121.5, 120.8, 114.1, 111.0, 109.1, 55.9, 55.8, 51.1, 46.8, 38.1, 12.0. HRMS (ESI) m/z

calcd for C24H31N4O2 [M+H]+ 407.2442, found 407.2433.
(E)-2-(3,4-Dimethoxystyryl)-4-(2-diisopropylaminoethyl)aminoquinazoline (9ac).
1
Gray solid, yield 25%, m.p. 124~126 °C. H NMR (500 MHz, CDCl3) δ 7.96 (d, J =
15.7 Hz, 1H), 7.79 (d, J = 8.3 Hz, 1H), 7.69~7.65 (m, 2H), 7.39~7.36 (m, 1H), 7.23 (s,
1H), 7.16 (d, J = 8.3 Hz, 1H), 7.10 (d, J = 15.7 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H), 3.93
(s, 3H), 3.90 (s, 3H), 3.75~3.69 (m, 2H), 3.18~3.12 (m, 2H), 2.86 (t, J = 5.4 Hz, 2H),
1.10 (d, J = 6.6 Hz, 12H). 13C NMR (125 MHz, CDCl3) δ 161.1, 158.9, 150.2, 149.7,
149.1, 136.7, 132.3, 129.8, 128.1, 127.3, 125.1, 121.5, 120.6, 114.1, 111.1, 109.2,
55.9, 55.8, 38.8, 20.8. HRMS (ESI) m/z calcd for C26H35N4O2 [M+H]+ 435.2755,
found 435.2747 (the resonance of O-H was not observed in CDCl3).
(E)-2-(3,4-dimethoxystyryl)-4-(1-piperidinylethyl)aminoquinazoline
(9ad).
1
Yellow solid, yield 43%, m.p. 127~129 °C. H NMR (400 MHz, CDCl3) δ 7.96 (d, J
= 15.7 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.75~7.65 (m, 2H), 7.43~7.35 (m, 1H), 7.23
(d, J = 1.8 Hz, 1H), 7.16 (dd, J = 8.3, 1.8 Hz, 1H), 7.10 (d, J = 15.7 Hz, 1H), 6.87 (d,
J = 8.3 Hz, 1H), 6.78 (s, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.82~3.79 (m, 2H), 2.71 (t, J
= 6.0 Hz, 2H), 2.57~2.46 (m, 4H), 1.68~1.61 (m, 4H), 1.54~1.46 (m, 2H). 13C NMR
(100 MHz, CDCl3) δ 161.1, 159.0, 150.2, 149.7, 149.1, 136.8, 132.4, 129.8, 128.1,
127.3, 125.1, 121.6, 120.8, 114.1, 111.0, 109.1, 56.7, 55.9, 55.8, 54.2, 37.3, 26.2, 24.4.
HRMS (ESI) m/z calcd for C25H31N4O2 [M+H]+ 419.2442, found 419.2434.
(E)-2-(3,4-Dimethoxystyryl)-4-[2-(morpholinyl)ethyl]-aminoquinazoline (9ae).
1
Yellow solid, yield 38%, m.p. 145~147 °C. H NMR (400 MHz, CDCl3) δ 7.96 (d, J
= 15.7 Hz, 1H), 7.81 (d, J = 8.2 Hz, 1H), 7.72~7.67 (m, 2H), 7.43~7.39 (m, 1H), 7.23
(d, J = 1.7 Hz, 1H), 7.15 (dd, J = 8.2, 1.7 Hz, 1H), 7.11 (d, J = 15.7 Hz, 1H), 6.87 (d,
J = 8.2 Hz, 1H), 6.52 (s, 1H), 3.93 (s, 6H), 3.86~3.81 (m, 2H), 3.79~3.75 (m, 4H),
13
2.76 (t, J = 6.0 Hz, 2H), 2.60~2.55 (m, 4H). C NMR (100 MHz, CDCl3) δ 161.1,
159.0, 150.2, 149.8, 149.2, 137.0, 132.6, 129.7, 128.2, 127.2, 125.3, 121.6, 120.7,
113.9, 111.1, 109.1, 67.1, 56.7, 55.9, 55.8, 53.4, 37.0. HRMS (ESI) m/z calcd for
C24H29N4O3 [M+H]+ 421.2234, found 421.2224.
(E)-2-(3,4-Dimethoxystyryl)-4-(3-dimethylaminopropyl)aminoquinazoline (9af).
Yellow solid, yield 33%, m.p. 172~174 °C. 1H NMR (400 MHz, CD3OD) δ 8.20~8.18

(m, 1H), 8.09 (d, J = 15.6 Hz, 1H), 7.86~7.81 (m, 1H), 7.69 (d, J = 8.0 Hz, 1H),
7.57~7.53 (m, 1H), 7.30~7.25 (m, 2H), 7.00~6.95 (m, 2H), 3.96 (t, J = 6.7 Hz, 2H),
13
3.89 (s, 3H), 3.86 (s, 3H), 3.65~3.55 (m, 2H), 2.89 (s, 6H), 2.28~2.24 (m, 2H). C
NMR (100 MHz, CD3OD) δ 160.1, 158.8, 151.4, 149.4, 143.6, 142.0, 134.3, 128.2,
126.6, 122.9, 122.5, 122.2, 120.0, 113.0, 111.4, 110.3, 55.4, 55.2, 55.1, 42.2, 38.1,
24.3. HRMS (ESI) m/z calcd for C23H29N4O2 [M+H]+ 393.2285, found 393.2274.
(E)-2-(3,4-Dimethoxystyryl)-4-(3-diethylaminopropyl)aminoquinazoline (9ag).
Yellow sticky solid. This compound deteriorates easily and can’t be further
structurally characterized by NMR. HRMS (ESI) m/z calcd for C25H33N4O2 [M+H]+
421.2598, found 421.2592.
(E)-2-(3,4-Dimethoxystyryl)-4-(4-aminobutyl)aminoquinazoline (9ah). Yellow
solid, yield 41%, m.p. 177~179 °C. 1H NMR (400 MHz, CD3OD) δ 8.03 (dd, J = 8.1,
3.9 Hz, 1H), 7.97~7.86 (m, 1H), 7.71~7.63 (m, 2H), 7.45~7.35 (m, 1H), 7.30~7.09 (m,
2H), 7.00~6.91 (m, 2H), 3.93 (s, 3H), 3.87 (s, 3H), 3.80~3.75 (m, 2H), 2.88~2.84 (m,
13
2H), 1.89~1.82 (m, 2H), 1.77~1.62 (m, 2H). C NMR (100 MHz, CD3OD) δ 161.0,
159.7, 150.3, 149.3, 137.6, 135.4, 132.5, 129.5, 126.0, 125.4, 125.1, 121.9, 121.4,
113.9, 111.4, 109.8, 55.1, 55.0, 40.2, 40.0, 27.4, 26.1. HRMS (ESI) m/z calcd for
C22H27N4O2 [M+H]+ 379.2129, found 379.2125.
(E)-2-(4-Nitrostyryl)-4-(2-dimethylaminoethyl)aminoquinazoline (10aa). Yellow
solid, yield 45%, m.p. 156~158 °C. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.6 Hz,
2H), 8.02 (d, J = 15.8 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.79 (d, J = 8.2 Hz, 1H),
7.76~7.69 (m, 3H), 7.48~7.43 (m, 1H), 7.34 (d, J = 15.8 Hz, 1H), 6.68 (s, 1H),
13
3.82~3.77 (m, 2H), 2.71~2.67 (m, 2H), 2.35 (s, 6H). C NMR (125MHz, CDCl3) δ
159.9, 150.0, 147.3, 143.2, 134.1, 133.8, 132.8, 129.2, 128.4, 127.9, 125.9, 124.1,
121.1, 114.2, 57.5, 45.2, 38.2. HRMS (ESI) m/z calcd for C20H22N5O2 [M+H]+
364.1768, found 364.1751.
(E)-2-(4-Nitrostyryl)-4-(2-diethylaminoethyl)aminoquinazoline (10ab). Brown
solid, yield 59%, m.p. 151~153 °C. 1H NMR (400 MHz, CDCl3) δ 8.23 (d, J = 8.8 Hz,
2H), 8.03 (d, J = 15.8 Hz, 1H), 7.83 (d, J = 8.3 Hz, 1H), 7.76~7.69 (m, 4H),
7.48~7.43 (m, 1H), 7.34 (d, J = 15.8 Hz, 1H), 6.83 (s, 1H), 3.79~3.74 (m, 2H),

2.84~2.80 (m, 2H), 2.66 (q, J = 7.1 Hz, 4H), 1.11 (t, J = 7.1 Hz, 6H). 13C NMR (100
MHz, CDCl3) δ 159.9, 159.1, 149.9, 147.2, 143.1, 134.0, 133.7, 132.6, 128.3, 127.8,
125.9, 124.0, 120.8, 114.2, 51.0, 46.7, 38.1, 12.0. HRMS (ESI) m/z calcd for
C22H26N5O2 [M+H]+ 392.2081, found 392.2072.
(E)-2-(4-Nitrostyryl)-4-(2-diisopropylaminoethyl)aminoquinazoline
(10ac).
1
Yellow solid, yield 45%, m.p. 214~216 °C. H NMR (400 MHz, CDCl3) δ 8.68 (s,
1H), 8.28 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 8.8 Hz, 2H), 7.91 (d, J = 15.9 Hz, 1H), 7.79
(d, J = 7.8 Hz, 1H), 7.73~7.70 (m, 3H), 7.53~7.45 (m, 1H), 7.32 (d, J = 15.9 Hz, 1H),
4.29~4.21 (m, 2H), 3.64~3.55 (m, 2H), 3.36~3.29 (m, 2H), 1.41 (d, J = 5.6 Hz, 12H).
13C NMR (100 MHz, CDCl3) δ 159.4, 159.2, 150.0, 147.4, 143.1, 133.8, 133.7, 133.2,
127.9, 127.8, 126.9, 124.1, 122.8, 114.4, 55.7, 47.3, 38.3, 18.8. HRMS (ESI) m/z
calcd for C24H30N5O2 [M+H]+ 420.2394, found 420.2386.
(E)-2-(4-Nitrostyryl)-4-(1-piperidinylethyl)aminoquinazoline (10ad). Yellow
solid, yield 38%, m.p. 166~168 °C. 1H NMR (500 MHz, CDCl3) δ 8.22 (d, J = 8.8 Hz,
2H), 8.01 (d, J = 15.9 Hz, 1H), 7.82 (d, J = 8.0 Hz, 1H), 7.75~7.70 (m, 4H),
7.49~7.44 (m, 1H), 7.33 (d, J = 15.9 Hz, 1H), 6.82 (s, 1H), 3.80~3.76 (m, 2H), 2.71 (t,
J = 6.0 Hz, 2H), 2.55~2.47 (m, 4H), 1.68~1.62 (m, 4H), 1.54~1.48 (s, 2H). 13C NMR
(125 MHz, CDCl3) δ 160.0, 159.1, 150.1, 147.4, 143.2, 134.1, 133.9, 132.6, 128.5,
127.9, 126.0, 124.1, 120.9, 114.3, 56.3, 54.3, 37.4, 26.2, 24.4. HRMS (ESI) m/z calcd
for C23H26N5O2 [M+H]+ 404.2081, found 404.2075.
(E)-2-(4-Nitrostyryl)-4-[2-(morpholinyl)ethyl]-aminoquinazoline (10ae). Yellow
solid, yield 41%, m.p. 189~191 °C. 1H NMR (400 MHz, CDCl3) δ 8.21 (d, J = 8.8 Hz,
2H), 8.00 (d, J = 15.9 Hz, 1H), 7.84 (d, J = 7.9 Hz, 1H), 7.79~7.66 (m, 4H),
7.51~7.43 (m, 1H), 7.33 (d, J = 15.9 Hz, 1H), 6.60 (s, 1H), 3.85~3.76 (m, 6H), 2.78 (t,
J = 6.0 Hz, 2H), 2.61~2.56 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 159.8, 159.1,
150.0, 147.3, 143.1, 134.1, 133.6, 132.7, 128.5, 127.8, 126.0, 124.0, 120.7, 114.1,
67.0, 56.5, 53.3, 36.9. HRMS (ESI) m/z calcd for C22H24N5O3 [M+H]+ 406.1874,
found 406.1869.
(E)-2-(4-Nitrostyryl)-4-(3-dimethylaminopropyl)aminoquinazoline
(10af).
1
Yellow solid, yield 58%, m.p. 161~163 °C. H NMR (500 MHz, CDCl3) δ 8.62 (s,

1H), 8.33 (d, J = 8.2 Hz,1H), 8.23 (d, J = 8.0 Hz, 2H), 8.06~8.01 (m, 3H), 7.79~7.74
(d, J = 7.0 Hz, 1H), 7.71 (d, J = 8.3 Hz, 1H), 7.52~7.48 (m, 1H), 7.37 (d, J = 16.0 Hz,
1H), 3.79~3.74 (m, 2H), 3.21~3.17 (m, 2H), 2.75 (s, 6H), 2.17~2.11 (m, 2H). 13C
NMR (125 MHz, CDCl3) δ 164.5, 164.5, 164.4, 154.8, 152.1, 148.1, 139.1, 138.9,
138.0, 133.7, 132.7, 129.2, 128.2, 119.3, 59.9, 47.3, 42.9, 29.0. HRMS (ESI) m/z
calcd for C21H24N5O2 [M+H]+ 378.1925, found 378.1921.
(E)-2-(4-Nitrostyryl)-4-(3-diethylaminopropyl)aminoquinazoline (10ag). Yellow
1
solid, yield 31%, m.p. 130~132 °C. H NMR (600 MHz, CDCl3) δ 8.90 (s, 1H), 8.21
(d, J = 8.7 Hz, 2H), 7.99 (d, J = 15.8 Hz, 1H), 7.82~7.77 (m, 2H), 7.73 (d, J = 8.7 Hz,
2H), 7.70~7.67 (m, 1H), 7.43~7.39 (m, 1H), 7.32 (d, J = 15.8 Hz, 1H), 3.88~8.85 (m,
2H), 2.83~2.79 (m, 2H), 2.76 (q, J = 7.1 Hz, 4H), 2.01~1.96 (m, 2H), 1.17 (t, J = 7.1
Hz, 6H). 13C NMR (150 MHz, CDCl3) δ 160.0, 159.4, 149.9, 147.2, 143.3, 134.0,
133.8, 132.5, 128.1, 127.8, 125.7, 124.0, 121.7, 114.5, 52.9, 46.8, 41.9, 24.0, 10.9.
HRMS (ESI) m/z calcd for C23H28N5O2 [M+H]+ 406.2238, found 406.2231.
(E)-2-(4-Nitrostyryl)-4-(4-aminobutyl)aminoquinazoline (10ah). Yellow solid,
yield 57%, m.p. 172~174 °C, 1H NMR (400 MHz, CD3OD) δ 8.08 (d, J = 8.8 Hz, 2H),
7.98 (d, J = 7.7 Hz, 1H), 7.89 (d, J = 15.8 Hz, 1H), 7.71~7.61 (m, 4H), 7.42~7.38 (m,
1H), 7.13 (d, J = 15.8 Hz, 1H), 3.76~3.71 (m, 2H), 2. 87~2.82 (m, 2H), 1.87~1.78 (m,
13
2H), 1.74~1.66 (m, 2H). C NMR (100 MHz, CD3OD) δ 159.8, 159.6, 149.1, 147.4,
142.6, 134.7, 132.6, 131.9, 127.7, 126.3, 125.8, 123.5, 122.0, 114.1, 40.3, 40.2, 27.8,
26.0. HRMS (ESI) m/z calcd for C20H22N5O2 [M+H]+ 364.1768, found 364.1761 (the
resonance of O−H and H−N−H were not observed in CD3OD).
(E)-2-(3,4-Methylenedioxystyryl)-4-(2-dimethylaminoethyl)aminoquinazoline
(11aa). Yellow solid, yield 45%, m.p. 156~158 °C. 1H NMR (500 MHz, DMSO-d6) δ
8.33 (s, 1H), 8.24 (d, J = 8.2 Hz, 1H), 7.87 (d, J = 15.8 Hz, 1H), 7.75~7.71 (m, 1H),
7.67 (d, J = 8.2 Hz, 1H), 7.47~7.42 (m, 1H), 7.40~7.38 (m, 1H), 7.14 (d, J = 8.0 Hz,
1H), 7.02 (d, J = 15.8 Hz, 1H), 6.96 (d, J = 8.0 Hz, 1H), 6.08 (s, 2H), 3.87~3.83 (m,
2H), 2.92~2.86 (m, 2H), 2.50 (s, 6H). 13C NMR (125 MHz, DMSO-d6) δ 160.6, 159.5,
150.4, 148.5, 148.4, 136.5, 133.0, 131.1, 127.9, 127.8, 125.4, 123.5, 123.3, 114.4,

108.9, 106.6, 101.7, 57.4, 44.8, 26.8. HRMS (ESI) m/z calcd for C21H23N4O2 [M+H]+
363.1816, found 363.1816.
(E)-2-(3,4-Methylenedioxystyryl)-4-(2-diethylaminoethyl)aminoquinazoline
1
(11ab). Yellow solid, yield 30%, m.p. 141~143 °C. H NMR (400 MHz, CDCl3) δ
7.90 (d, J = 15.8 Hz, 1H), 7.82~7.77 (m, 2H), 7.68~7.64 (m, 1H), 7.40~7.36 (m, 1H),
7.16 (d, J = 1.5 Hz, 1H), 7.07~7.02 (m, 2H), 6.80 (d, J = 8.0 Hz, 1H), 5.97 (s, 2H),
3.83 (t, J = 5.6 Hz, 2H), 3.38 (s, 1H), 2.92~2.86 (m, 2H), 2.72 (q, J = 7.2 Hz, 4H),
13
1.13 (t, J = 7.2 Hz, 6H). C NMR (100 MHz, CDCl3) δ 161.1, 159.0, 150.3, 148.2,
148.1, 136.6, 132.4, 131.3, 128.2, 127.6, 125.2, 123.0, 120.7, 114.1, 108.4, 106.4,
101.2, 77.3, 77.0, 76.7, 51.1, 46.8, 38.1, 12.1. HRMS (ESI) m/z calcd for C23H27N4O2
[M+H]+ 391.2129, found 391.2129.
(E)-2-(3,4-Methylenedioxystyryl)-4-(2-diisopropylaminoethyl)aminoquinazoline
1
(11ac). Yellow solid, yield 60%, m.p. 169~171 °C. H NMR (400 MHz, CDCl3) δ
7.93 (d, J = 15.8 Hz, 1H), 7.79 (d, J = 7.9 Hz, 1H), 7.70~7.65 (m, 2H), 7.41~7.36 (m,
1H), 7.18 (d, J = 1.6 Hz, 1H), 7.10~7.03 (m, 2H), 6.82 (d, J = 7.9 Hz, 1H), 5.99 (m,
2H), 3.80~3.65 (m, 2H), 3.25~3.10 (m, 2H), 2.95~2.75 (m, 2H), 1.12 (d, J = 5.4 Hz,
12H). 13C NMR (100 MHz, CDCl3) δ 161.1, 159.0, 150.3, 148.2, 148.1, 136.6, 132.4,
131.3, 128.2, 127.6, 125.3, 123.0, 120.7, 114.2, 108.4, 106.4, 101.2, 47.7, 42.7, 38.7,
29.7, 20.8. HRMS (ESI) m/z calcd for C25H31N4O2 [M+H]+ 419.2442, found 419.2434
(the resonance of O-H was not observed in CDCl3).
(E)-2-(3,4-Methylenedioxystyryl)-4-(1-piperidinylethyl)aminoquinazoline (11ad).
1
Yellow solid, yield 29%. m.p, 169~171 °C. H NMR (400 MHz, CDCl3) δ 7.92 (d, J
= 15.7 Hz, 1H), 7.78 (dd, J = 8.3, 0.7 Hz, 1H), 7.74 (dd, J = 8.3, 0.7 Hz, 1H),
7.68~7.64 (m, 1H), 7.39~7.35 (m, 1H), 7.17 (d, J = 1.6 Hz, 1H), 7.08~7.02 (m, 2H),
6.85 (s, 1H), 6.80 (d, J = 8.0 Hz, 1H), 5.97 (s, 2H), 3.82~3.77 (m, 2H), 2.71~2.67 (m,
2H), 2.53~2.45 (m, 4H), 1.67~1.60 (m, 4H), 1.52~1.45 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 161.0, 159.0, 150.1, 148.1, 148.1, 136.7, 132.4, 131.2, 128.0, 127.4, 125.1,
122.9, 121.1, 114.1, 108.4, 106.3, 101.2, 56.9, 54.2, 37.3, 26.0, 24.3. HRMS (ESI)
m/z calcd for C24H27N4O2 [M+H]+ 403.2129, found 403.2129.
(E)-2-(3,4-Methylenedioxystyryl)-4-[2-(morpholinyl)ethyl]-aminoquinazoline

1
(11ae). Yellow solid, yield 38%, m.p. 245~247 °C. H NMR (400 MHz, CDCl3) δ
7.93 (d, J = 15.7 Hz, 1H), 7.81 (d, J = 8.3 Hz, 1H), 7.72~7.66 (m, 2H), 7.43~7.37 (m,
1H), 7.17 (d, J = 1.6 Hz, 1H), 7.08~7.03 (m, 2H), 6.81 (d, J = 8.0 Hz, 1H), 6.54 (s,
1H), 5.98 (s, 2H), 3.85~3.79 (m, 2H), 3.77 (t, J = 4.4 Hz, 4H), 2.75 (t, J = 5.9 Hz, 2H),
2.60~2.54 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 160.9, 159.0, 150.1, 148.2, 136.9,
132.5, 131.1, 128.1, 127.2, 125.2, 123.0, 120.7, 113.9, 108.4, 106.3, 101.2, 67.0, 56.6,
53.3, 36.9. HRMS (ESI) m/z calcd for C23H25N4O2 [M+H]+ 405.1921, found
405.1918.
(E)-2-(3,4-Methylenedioxystyryl)-4-(3-dimethylaminopropyl)aminoquinazoline
(11af). White solid, yield 43%, m.p. 133~135 °C. 1H NMR (500 MHz, CDCl3) δ 8.55
(s, 1H) , 7.95 (d, J = 15.7 Hz, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H),
7.59 (d, J= 8.0 Hz, 1H), 7.37 (t, J = 7.5 Hz, 1H), 7.20 (s, 1H), 7.11~7.06 (m, 2H), 6.83
(d, J = 7.5 Hz, 1H), 6.00 (s, 2H), 3.86 (d, J = 4.4 Hz, 2H), 2.60 (d, J = 4.8 Hz, 2H),
2.38 (s, 6H), 1.91 (s, 2H). 13C NMR (125 MHz, CDCl3) δ 161.2, 159.3, 150.2, 148.3,
148.2, 136.5, 132.2, 131.3, 128.0, 127.7, 125.0, 123.0, 121.1, 114.4, 108.4, 106.4,
101.2, 59.9, 45.6, 42.3, 24.9. HRMS (ESI) m/z calcd for C22H25N4O2 [M+H]+
377.1972, found 377.1967.
(E)-2-(3,4-Methylenedioxystyryl)-4-(3-diethylaminopropyl)aminoquinazoline
1
(11ag). Yellow solid, yield 49%, m.p. 161~163 °C. H NMR (400 MHz, CDCl3) δ
8.75 (s, 1H), 7.93 (d, J = 15.7 Hz, 1H), 7.77 (d, J = 8.2 Hz, 1H), 7.67~7.63 (m, 2H),
7.38~7.31 (m, 1H), 7.18 (d, J = 1.5 Hz, 1H), 7.08 (dd, J = 8.1, 1.5 Hz, 1H), 7.05 (d, J
= 15.7 Hz, 1H), 6.81 (d, J = 8.1 Hz, 1H), 5.98 (s, 2H), 3.87~3.83 (m, 2H), 2.74~2.71
13
(m, 2H), 2.67 (q, J = 7.1 Hz, 4H), 1.93~1.87 (m, 2H), 1.12 (t, J = 7.1 Hz, 6H). C
NMR (100 MHz, CDCl3) δ 161.1, 159.2, 150.2, 148.2, 148.1, 136.5, 132.2, 131.3,
128.0, 127.7, 124.8, 122.9, 121.4, 114.4, 108.4, 106.4, 101.2, 53.6, 47.1, 42.7, 24.5,
11.5. HRMS (ESI) m/z calcd for C24H29N4O2 [M+H]+ 405.2285, found 405.2278.
(E)-2-(3,4-Methylenedioxystyryl)-4-(4-aminobutyl)aminoquinazoline
(11ah).
Yellow solid, yield 31%, m.p. 182~184 °C. 1H NMR (400 MHz, CD3OD) δ: 8.18 (d, J
= 7.8 Hz, 1H), 8.02 (d, J = 15.5 Hz, 1H), 7.84~7.80 (m, 1H), 7.67 (d, J = 8.2 Hz, 1H),
7.56~7.52 (m, 1H), 7.20 (d, J = 1.4 Hz, 1H), 7.14 (dd, J = 8.0, 1.4 Hz, 1H), 6.90 (d, J

= 15.5 Hz, 2H), 6.84(d, J= 8.0 Hz, 2H), 6.00 (s, 2H), 3.88 (t, J = 6.6 Hz, 2H),
3.04~3.00 (m, 2H), 1.93~1.86 (m, 2H), 1.84~1.79 (m, 2H). HRMS (ESI) m/z calcd for
C21H23N4O2 [M+H]+ 363.1816, found 363.1809. Because of its poor solubility,
compound 11ah could not be characterized by13C NMR.
(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-(2-dimethylaminoethyl)aminoquinazoline
(9ba). Yellow solid, yield 48%, m.p. 170~171 °C. 1H NMR (400 MHz, CDCl3) δ 7.95
(d, J = 15.7 Hz, 1H), 7.74~7.70 (m, 2H), 7.63~7.60 (m, 1H), 7.22 (d, J = 1.8 Hz, 1H),
7.16 (dd, J = 8.3, 1.8 Hz, 1H), 7.07 (d, J = 15.7 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H),
6.48 (s, 1H), 3.94 (s, 3H), 3.91 (s, 3H), 3.82~3.77 (m, 2H), 2.70~2.66 (m, 2H), 2.35 (s,
13
6H). C NMR (100 MHz, CDCl3) δ 161.3, 158.2, 149.9, 149.2, 148.9, 137.3, 133.2,
130.3, 129.8, 129.6, 126.9, 121.7, 120.6, 114.7, 111.1, 109.2, 57.5, 55.9, 55.9, 45.2,
38.3. HRMS (ESI) m/z calcd for C22H26ClN4O2 [M+H]+ 413.1739, found 413.1735.
(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-(2-diethylaminoethyl)aminoquinazoline
(9bb). Yellow solid, yield 35%, m.p. 133~135 °C. 1H NMR (400 MHz, CDCl3) δ 7.95
(d, J = 15.7 Hz, 1H), 7.72 (d, J = 8.7 Hz, 1H), 7.64~7.60 (m, 2H), 7.22 (d, J = 1.7 Hz,
1H), 7.16 (dd, J = 8.3, 1.7 Hz, 1H), 7.07 (d, J = 15.7 Hz, 1H), 6.88 (d, J = 8.3 Hz, 1H),
6.64 (s, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.79~3.74 (m, 2H), 2.81 (t, J = 5.9 Hz, 2H),
2.66 (q, J = 7.1 Hz, 4H), 1.11 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ
161.4, 158.2, 149.9, 149.2, 148.9, 137.3, 133.1, 130.3, 129.8, 129.6, 126.9, 121.7,
120.4, 114.8, 111.1, 109.2, 55.9, 55.9, 51.0, 46.8, 38.3, 12.0. HRMS (ESI) m/z calcd
for C24H30ClN4O2 [M+H]+ 441.2052, found 441.2052.
(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-(2-diisopropylaminoethyl)aminoquinazo-
-line (9bc). Yellow solid, yield 28%, m.p. 182~185 °C. 1H NMR (400 MHz, CDCl3) δ
7.96 (d, J = 15.7 Hz, 1H), 7.72 (d, J = 9.2 Hz, 1H), 7.63~7.58 (m, 2H), 7.22 (d, J =
1.8 Hz, 1H), 7.16 (dd, J = 8.3, 1.8 Hz, 1H), 7.07 (d, J = 15.7 Hz, 1H), 6.88 (d, J = 8.3
Hz, 1H), 6.70 (s, 1H), 3.94 (s, 3H), 3.91 (s, 3H), 3.74~3.66 (m, 2H), 3.19~3.12 (m,
13
2H), 2.89~2.83 (m, 2H), 1.12 (d, J = 6.5 Hz, 12H). C NMR (100 MHz, CDCl3) δ
161.5, 158.1, 149.9, 149.2, 148.8, 137.3, 133.1, 130.4, 129.8, 129.6, 126.9, 121.7,
120.3, 114.8, 111.1, 109.2, 56.0, 55.9, 47.8, 39.0, 30.9, 20.9. HRMS (ESI) m/z calcd
for C26H34ClN4O2 [M+H]+ 469.2365, found 469.2364.

(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-(1-piperidinylethyl)aminoquinazoline
(9bd). Yellow solid, yield 64%, m.p. 85~87 °C. 1H NMR (400 MHz, CDCl3) δ 7.95 (d,
J = 15.7 Hz, 1H), 7.74~7.70 (m, 2H), 7.63~7.59 (m, 1H), 7.22 (d, J = 1.8 Hz, 1H),
7.16 (dd, J = 8.3, 1.8 Hz, 1H), 7.06 (d, J = 15.7 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H),
6.75 (s, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.83~3.78 (m, 2H), 2.72 (t, J = 5.9 Hz, 2H),
2.57~2.50 (m, 4H), 1.70~1.63 (m, 4H), 1.55~1.48 (m, 2H). 13C NMR (100 MHz,
CDCl3) δ 161.3, 158.2, 149.9, 149.2, 148.8, 137.3, 133.1, 130.3, 129.7, 129.6, 126.9,
121.7, 120.8, 114.8, 111.1, 109.2, 56.8, 55.9, 55.9, 54.3, 37.4, 26.0, 24.3. HRMS (ESI)
m/z calcd for C25H30ClN4O2 [M+H]+ 453.2052, found 453.2050.
(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-[2-(morpholinyl)ethyl]-aminoquinazoline
(9be). Yellow solid, yield 52%, m.p. 159~161 °C. 1H NMR (400 MHz, CDCl3) δ 7.94
(d, J = 15.7 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.67~7.60 (m, 2H), 7.21 (d, J = 1.8 Hz,
1H), 7.15 (dd, J = 8.3, 1.8 Hz, 1H), 7.06 (d, J = 15.7 Hz, 1H), 6.87 (d, J = 8.3 Hz, 1H),
6.43 (s, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.85~3.77 (m, 6H), 2.76 (t, J = 6.0 Hz, 2H),
2.61~2.56 (m, 4H). 13C NMR (100 MHz, CDCl3) δ 161.3, 158.1, 150.0, 149.2, 148.8,
137.5, 133.3, 130.4, 129.8, 129.5, 126.7, 121.7, 120.4, 114.6, 111.1, 109.1, 67.0, 56.7,
55.9, 55.8, 53.4, 37.1. HRMS (ESI) m/z calcd for C24H28ClN4O3 [M+H]+ 455.1844,
found 455.1849 (the resonance of O–H was not observed in CDCl3).
(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-(3-dimethylaminopropyl)aminoquinazoline
(9bf). Yellow solid, yield 53%, m.p. 76~78 °C. 1H NMR (400 MHz, CDCl3) δ 8.88 (s,
1H), 7.93 (d, J = 15.7 Hz, 1H), 7.69 (d, J = 8.8 Hz, 1H), 7.62~7.57 (m, 2H), 7.22 (d, J
= 1.8 Hz, 1H), 7.15 (dd, J = 8.3, 1.8 Hz, 1H), 7.06 (d, J = 15.7 Hz, 1H), 6.87 (d, J =
8.3 Hz, 1H), 3.93 (s, 3H), 3.91 (s, 3H), 3.87~3.82 (m, 2H), 2.70~2.66 (m, 2H), 2.44 (s,
13
6H), 1.97~1.91 (m, 2H). C NMR (100 MHz, CDCl3) δ 161.5, 158.6, 149.8, 149.1,
148.7, 137.1, 132.8, 130.2, 129.7, 129.5, 127.1, 121.7, 121.2, 115.2, 111.1, 109.2,
59.4, 55.9, 55.9, 45.2, 42.1, 24.3. HRMS (ESI) m/z calcd for C23H28ClN4O2 [M+H]+
427.1895, found 427.1896.
(E)-6-Chloro-2-(3,4-dimethoxystyryl)-4-(3-diethylaminopropyl)aminoquinazoline
(9bg). Yellow solid, yield 24%, m.p. 78~80 °C. 1H NMR (500 MHz, CDCl3) δ 9.03 (s,
1H), 7.85 (d, J = 15.7 Hz, 1H), 7.61~7.56 (m, 2H), 7.48~7.43 (m, 1H), 7.13~7.10 (m,