Distinct novel quinazolinone exhibits selective inhibition in MGC-803 cancer cells by dictating mutant p53 function

Article abstract of DOI:10.1016/j.ejmech.2015.03.053

The mutant p53 proteins and their corresponding cellular response can be manipulated by novel quinazolinone derivatives 4-8 (a-i) in p53 mutant cancer cells. Of the two most potent compounds, 4a exhibited promising broad-spectrum anti-cancer effects, whereas 6c showed selective and exclusive inhibition activity in p53 mutant cancer cell lines but low toxicity to wild-type p53 cancer cell A375 and normal lung fibroblast WI-38 cells. Furthermore, 6c exhibited a more sophisticated mechanism for cell-destructive response by causing S/G2 phase arrest effect and cell size reduction. Compared with the cellular response of 6b and genetic background of cell lines studied, p53 mutation was found to be the key factor and main target for 6c evoked cell-destructive response. Molecular mechanism studies indicated that p53 phosphorylation and acetylation dual-targeting inhibitor 6c exerted anti-cancer activities with a special mechanism in evoking cell apoptosis by arresting mutant p53 function to trigger the deregulation of Cdk2 caused Bim-mediated apoptosis. To the best of our knowledge, 6c is the first quinazolinone derivative to dictate mutant p53 function for apoptotic cell death.

Full text of DOI:10.1016/j.ejmech.2015.03.053

European Journal of Medicinal Chemistry 95 (2015) 377e387
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Distinct novel quinazolinone exhibits selective inhibition in MGC-803
cancer cells by dictating mutant p53 function
Guo-Hai Zhang, Wen-Bin Xue, Yun-Feng An, Jing-Mei Yuan, Jiang-Ke Qin, Cheng-Xue Pan*,
Gui-Fa Su*
State Key Laboratory Cultivation Base for the Chemistry and Molecular Engineering of Medicinal Resources (Ministry of Science and Technology of China),
School of Chemistry & Pharmaceutical Sciences, Guangxi Normal University, Guilin 541004, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
The mutant p53 proteins and their corresponding cellular response can be manipulated by novel qui-
nazolinone derivatives 4e8 (aei) in p53 mutant cancer cells. Of the two most potent compounds, 4a
exhibited promising broad-spectrum anti-cancer effects, whereas 6c showed selective and exclusive
inhibition activity in p53 mutant cancer cell lines but low toxicity to wild-type p53 cancer cell A375 and
normal lung fibroblast WI-38 cells. Furthermore, 6c exhibited a more sophisticated mechanism for cell-
destructive response by causing S/G2 phase arrest effect and cell size reduction. Compared with the
cellular response of 6b and genetic background of cell lines studied, p53 mutation was found to be the
key factor and main target for 6c evoked cell-destructive response. Molecular mechanism studies indi-
cated that p53 phosphorylation and acetylation dual-targeting inhibitor 6c exerted anti-cancer activities
with a special mechanism in evoking cell apoptosis by arresting mutant p53 function to trigger the
deregulation of Cdk2 caused Bim-mediated apoptosis. To the best of our knowledge, 6c is the first
quinazolinone derivative to dictate mutant p53 function for apoptotic cell death.
Received 11 November 2014
Received in revised form
18 March 2015
Accepted 23 March 2015
Available online 24 March 2015
Keywords:
Mutant p53
Selective inhibition
Quinazolinone
Apoptosis
© 2015 Elsevier Masson SAS. All rights reserved.
1. Introduction
To develop novel tumor suppressors that target the oncogenic
functions of mutant p53 proteins, quinazolinone derivatives are
The remarkable progress made in understanding the oncogenic
effects of p53 mutants has afforded an important new avenue for
human cancer therapies [1e4]. Mutations in p53 are capable not only
to subvert the antiproliferative properties, but also to promote
various oncogenic responses through a gain-of-function activity so
as to modulate such critical factors as cell growth, metastasis, inva-
sion, apoptosis resistance, chemoresistance and genomic instability
[5e9]. Furthermore, the high frequency of p53 mutations in human
cancers [10] with genetic alterations was found widespread in
essentially every region of the proteins [11], indicating the important
role of p53 dysfunction in contributing to the malignant progression.
Consequently, pharmacological manipulation of the signaling
pathway of p53 mutants by non-toxic compounds is expected to be
an effective strategy in the treatment of various types of cancers.
considered as a subject of huge interest. As a result of their diverse
pharmacological activities such as anticancer [12,13], antimicrobial
[14e16], antimalarial [17,18], anti-inflammatory [19e22], antihy-
pertensive [23,24], anticonvulsant [25,26] and cholinesterase in-
hibition activities [27], these derivatives have received considerable
attention. However, a lack of studies is clearly noticed that are to
utilize the property that quinazolinone derivatives could arrest
mutant p53 while maintaining steady-state protein levels. The
mutant p53 launches a plethora of pro-oncogenic activities to
promote tumorigenesis when it is activated [28]. Resembling the
wild-type counterpart, mutant p53 is most likely regulated by a
barcode of post-translational modifications [29] including p53
phosphorylation [30] and acetylation [31e33] which are essential
in p53 response. Herein, we report the selective cytotoxicity and
the investigation of the anticancer mechanisms of action of a qui-
nazolinone derivative inhibiting p53 phosphorylation and acety-
lation in p53 mutant cell lines.
* Corresponding authors.
E-mail addresses: chengxuepan@163.com (C.-X. Pan), gfysglgx@163.com
(G.-F. Su).
http://dx.doi.org/10.1016/j.ejmech.2015.03.053
0223-5234/© 2015 Elsevier Masson SAS. All rights reserved.

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G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
2. Results and discussion
cytotoxicity. As far as the substituents at the styryl are concerned,
an electron-withdrawing group such as nitro, chloro and bromo
could enhance the cytotoxicity, while an electron-donating one
such as methoxyl, methylenedioxyl, or fluoro gave negative results.
In all of the compounds investigated, compound 4a and 6c
exhibited the most promising anti-cancer effects in MGC-803 cells,
which were chosen to further investigate the mechanism of its
cytotoxic effect.
2.1. Chemistry
The structure of the compounds investigated in this study is
depicted in Fig. 1.
The general synthetic route to 2-styryl-3H-quinazolin-4-one
derivatives is depicted in Scheme 1. Benzo[d][1,3]oxazin-4-one
derivatives 2 were prepared according to the reported methods
[34]. The synthesis of 2-methyl-3-alkylquinazolin- -4-one 3 and 2-
styryl-3-alkylquinazolin-4-one 4 was carried out as reported with
slight modifications [35].
2.2.2. 4a and 6c induce cell cycle arrest and cell size change in
MGC-803 cells
To study the mechanism of cell proliferation inhibition by
compounds 4a and 6c, we examined the change of cell cycle. As
shown in Fig. 2A and B, in MGC-803 cells, 4a or 6c treatment
Benzo[d][1,3]oxazin-4-one derivative 2 (4.0 mmol) and pri-
mary amine (10.0 mmol) in 15 mL of xylene were heated under
reflux for 10 h till the completion of the reaction (monitored by
TLC). The xylene was then removed under reduced pressure to
give the crude 2-methyl-3-alkylquinazolin-4-one derivative
which was purified by column chromatography on silica gel
(eluent: PE/EtOAc/MeOH) to afford 3 as yellow oil. Compound 3
and aromatic aldehyde (4.0 mmol) in 15 mL of acetic acid were
heated under reflux for 12 h (monitored by TLC), then the solvent
was removed under reduced pressure and the residues were
dissolved in 100 mL of DCM, washed successively with sodium
dicarbonate (3 ꢀ 20 mL) and brine (30 mL), dried (Na2SO4) and
filtered. The solvent was evaporated under vacuum to give the
crude product, which was purified by column chromatography on
silica gel (eluent: DCM/EtOAc/MeOH) to afford 4e8 as pale yellow
or yellow solids. All the synthesized compounds were character-
ized by 1H, 13C NMR spectroscopy, mass spectrometry and
elemental analysis.
(doses from 1 to 10 mM, 48 h) obviously increased the cell pro-
portion in sub-G1 phase (a specific phase of cell death), while the
cell proportions in S phase and G2 phase were markedly increased
in contrast to what was observed in G0/G1 phase after 6c treat-
ment. It indicated the S/G2 phase arrest effect. However, the S/G2
phase arrest effect was not so obvious in 4a-treated cells (Fig. 2C
and D).
Consistent with the obvious effects on cell cycle arrest, 6c
caused a more profound reduction in cell size (Fig. 2E, left). The
relative cell size (mean FSC-H) of 6cetreated MGC-803 cells was
59.4% of control cells (Fig. 2E, right). Collectively, these results
suggested that 6c exhibited a more profound mechanism in
response to its cell-destructive activities.
2.2.3. 6c induces AIF-mediated apoptosis by arresting the function
of p53 in MGC-803 cells
To gain further insight into the mechanism of action of 6c, we
are motivated to confirm firstly whether the sub-G1 phase cells
induced by 6c were due to apoptosis. The 6c-treated cells were
therefore investigated with Hoechst 33258 staining and AIF-
immunostaining for apoptosis identification. As depicted in
Fig. 3A, cells treated by 6c displayed obvious chromatin conden-
sation and fragmentation. Condensed bright apoptotic nuclei were
readily observed comparing to the control cells. Furthermore, after
the treatment of 6c, Apoptosis-inducing Factor (AIF) was released
from mitochondria and translocated to the nucleus to trigger
apoptosis. To further validate the authenticity that cell apoptosis
was induced by AIF-mediated apoptotic pathway, we also exam-
ined the caspase-3 activation by FACS analysis through FITC-DEVD-
FMK probe. As shown in Fig. 3B and C, nearly no markedly increased
actived-caspase-3 cells could be observed in 6c treated cells
compared to control group, as AIF is a caspase-independent death
effector [36].
To determine how the compound 6c predominantly affected
cells to transfer proapoptotic stimuli, immunoblotting screening
followed by hit to lead discovery 6c as a novel p53 phosphorylation
and acetylation dual-targeting inhibitor. As shown in Fig. 3D, MGC-
803 cells incubated for 24 h with 6c showed a marked decrease in
expression of p21 but no alteration in the total p53 expression. This
implies that the function of p53 was arrested by 6c as to render it
incapable for transcription regulation. Further study revealed that
6c selectively inhibited P-p53 (s392) and Ac-p53 (k373) for p-53
function arrest. To investigate whether function-arrest of p53 is
essential for 6c induced cell-destructive activities in MGC-803 cells,
6b, an analog of 6c, was adopted to examine the relationship be-
tween functional p53-arrest and cell-destructive activities. As
shown in Fig. 3E and F, 6b displayed no obvious toxicity to MGC-803
cells (3E) combined with undetectable influence on p53 phos-
phorylation and acetylation (3F). These results clearly demon-
strated that deactivating p53 by 6c was responsible for the cell-
destructive activities in MGC-803 cancer cells.
2.2. Biological activity
2.2.1. Effects of 4e8 (aei) on cell proliferation in different cancer
cells
To study the potential use as therapeutic agents, the effect of
4e8 (aei) on cell proliferation after 48 h treatment was firstly
checked in various cancer cell lines. The IC50 values derived from
doseeresponse curves are summarized in Table 1.
The data indicated that the side chain attached to the N-3 po-
sition of the quinazolin-4-one had a critical effect on anticancer
activity. The presence of the dimethylamino substituent on this
alkyl side chain was a requirement for maintaining cytotoxicity.
When the side chain was replaced by n-butyl, as in the case of 8aei,
all the compounds would not exhibit valid cytotoxicity. The length
of the side chain also showed pronounced effect on activity. The
data indicated that a propylamino is more favorable than an
ethylamino.
The varied substituents at the benzene ring of quinazolin-4-one
or the styryl can also impact the activity. In generally, compounds
with methoxyl at the 6,7-position, or a chloro at 7-position of
quinazolin-4-one, as the case of 6 or 7, resulted in more potency on
O
R1 = H (4, 5, 8); 7-Cl (7);
6,7-OCH3 (6)
R1
N(CH2)nR
R = N(CH3)2 (4-7); CH3 (8)
n = 3 (4, 6-8); n = 2 (5)
N
Ar
4-8 (a-i)
Ar = 2,4-ClC6H3 (a); 4-FC6H4 (b); 4-ClC6H4 (c);
2-BrC6H4 (d); 4-NO2C6H4 (e);
3-NO2C6H4 (f); 3,4-(CH3O)2C6H3 (g);
3,4-OCH2OC6H3 (h); 3-HOC6H4 (i);
Fig. 1. Structures of quinazolin-4(3H)-one derivatives 4e8.

G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
379
O
N
O
N
R1
R1
R1
CO2H
NH2
Ac2O
reflux
R(CH2)nNH2
xylene, reflux
N(CH2)nR
O
1
2
3
R1= H (4,5,8); 6,7-(CH3O)2 (6); 7-Cl (7);
R= N(CH3)2 (4-7); CH3 (8); n =2 (5); 3 (4, 6-8);
Ar = 2,4-ClC6H3(a); 4-FC6H4(b); 4-ClC6H4(c);
2-BrC6H4(d); 4-NO2C6H4(e); 3-NO2C6H4(f);
3,4-CH3OC6H3(g); 3,4-OCH2OC6H3(h);
3-HOC6H4(i);
O
R1
ArCHO
AcOH, reflux
N(CH2)nR
Ar
N
4-8 (a-i)
Scheme 1. Synthesis of the quinazolin-4(3H)-one derivatives 4e8.
2.2.4. 6c arrests mutant p53 function for selective inhibition and
Cdk2-mediated apoptosis in MGC-803 cancer cells
NCIeH460, HepG2. However, invalid response in wild-type p53
cancer cell line (A375) after 6c treatment suggested the correla-
tion between p53 mutation and the selective activity against the
four tested cancer cell lines.
After confirming the underlying mechanism that governs p53
dysfunction, we sought to explore why 6c exhibited a selective
inhibitory effect on cell proliferation when tested in different
cancer cell lines. In contrast to the anti-cancer effect of 6c in the
four cancer cells studied above, p53 mutation maybe the key
factor of genetic background in 6c-mediated selective inhibition.
As our results found that 6c could significantly inhibited cell
proliferation in p53-mutant MGC-803 cancer cells [37,38] and
there was a mild inhibitory effect in the other two p53-mutant
cancer cell lines (NCIeH460 and HepG2) [39e41]. But no cyto-
toxicity was observed in the wild-type p53 A375 cancer cell lines
(Table 1). p53 mutation was a common phenomenon in human
cancers [10] with genetic alterations was found widespread in
essentially every region of the proteins [11], but only the most
frequent mutations have been investigated for their functional
effects. Nonsense mutations or frameshift with a result in the loss
of p53 expression were usually found in HepG2 cells, while
missense mutations in p53 leading to oncogenic responses
through a gain-of-function activity were usually occurred MGC-
803 cells. p53 mutation in cancer cells interacts with a wide va-
riety of other proteins, resulting in metastasis, drug resistance and
other oncogenic responses [9,37,39e41]. The diversity of anti-
cancer effect may be due to p53 mutation patterns and tumor
phenotype in p53-mutant cancer cell lines [42], such as MGC-803,
To further elaborate the contribution of p53 mutation to 6c-
mediated selective inhibition and therapeutic potential of 6c in
clinical study, we also conducted cytotoxicity study with normal
lung fibroblast WI-38 cells (wild-type p53). Generally speaking, if
p53 mutation was the key to selective inhibition in different cancer
cell lines, there should be low toxicity in 6c-treated normal lung
fibroblast cells without p53 mutation. In agreement with what was
expected, as shown in Fig. 4A, 6c displayed low toxicity in WI-38
cells, indicating selective toxicity for p53 mutant cancerous over
normal cells.
Multi mechanisms for mutant p53 oncogenic function have
been proposed so far [28,43]. However, there was still no clear
mechanism to explain how mutant p53 to promote cancer cell
survival and apoptosis resistance. Having confirmed the cell-
destructive activity by apoptosis after 6c-mediated pharmacolog-
ical manipulation of the mutant p53 pathway, it is important to
clarify the critical mechanism that triggers cell apoptosis.
Compared with the wild-type p53 function, there may be a highly
desirable and special mechanism to evoke cell apoptosis by
arresting mutant p53 function. p21 was the direct target of p53
regulatory circuit. The proapoptotic signals should cascade through
p21 into death effector. We therefore investigated the Cdk2 acti-
vation for death signal transduction. Since apoptosis appears linked
in some cases to aberrations in the activity of Cdk2 [44], Cdk2 is a
negative regulation target of p21. As shown in Fig. 4B, after the 6c
treatment, Cdk2 remained fairly unchanged coinciding with rapid
up regulation of Cdk2 activity. Thus, these results indicated that 6c
induced cell apoptosis by arresting mutant p53 function to dereg-
ulate Cdk2 activity through p21 for apoptotic signals cascading.
To verify which death effectors are dynamic participants in the
death signaling after deregulation Cdk2 activity. Bcl-2 family pro-
teins were firstly chosen to examine how abundance of likely
candidate proteins varied after 6c treatment. As shown in Fig. 4B,
the most increase was in the levels of Bim, while the most striking
decrease was in Bcl-2. BAX showed insignificant change. The pu-
tative mechanism of 6c-induced cell apoptosis is summarized in
Fig. 4C, where the role of mutant p53 functional arrest in MGC-803
cells was verified by examining the abundance and the activity of
some key p53 interacting proteins. This is an exciting discovery. To
the best of our knowledge, this is the first proof based on quina-
zolinone derivatives to manipulate mutant p53 pathway for Cdk2-
mediated apoptotic mechanism.
Table 1
IC50 of quinazolinone derivatives 4e8 (aei) in cancer cells HepG2, NCIeH460, MGC-
803 and A375 after 48 h of exposure to the derivatives.
Compound
IC50 (m
M)a
HepG2
NCIeH460
MGC-803
A375
4a
4d
4.20 0.41
5.06 0.59
>20
5.86 0.10
6.54 0.48
>20
3.89 0.47
14.57 0.07
>20
7.15 0.21
8.41 0.39
>20
4b, c
4eeh
4i
5aei
6a
6.10 0.44
>20
6.45 0.40
6.96 0.36
>20
>20
>20
6.37 0.23
7.15 0.13
>20
>20
>20
5.92 0.57
3.18 0.91
>20
>20
>20
12.74 0.28
>20
6c
6b
6dei
7a
7c
7d
7e
>20
8.58 0.62
6.43 1.18
8.10 0.28
7.41 0.62
6.13 0.86
>20
13.29 0.92
7.72 0.66
7.49 0.43
5.55 0.66
5.83 0.25
>20
5.75 0.68
6.88 0.31
12.59 0.63
8.28 0.37
4.14 0.42
>20
12.87 0.30
14.12 0.35
13.12 0.11
10.32 0.44
13.96 0.19
>20
7f
7b,7g,7h
7i
8aei
>20
>20
10.49 0.41
>20
>20
>20
>20
>20
3. Conclusions
a
Each IC50 value was calculated from 3 independent experiments performed in
triplicate.
A new series of quinazolinone derivatives have been

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G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
Fig. 2. Effects of 4a and 6c on cell cycle distribution and cell size in MGC-803 cells. (A, B) The regulation of cell cycle arrest by 4a (A) or 6c (B) was conducted by FACS analysis with PI
staining. (C) The effects of S phase arrest after 4a or 6c treatment. The ratio of S phase VS G1 phase in control group was assigned a value of 1. (D) The effects of G2 phase arrest after
4a or 6c treatment. The ratio of G2 phase VS G1 phase in control group was assigned a value of 1. (E) 6c reduced the cell size in MGC-803 cells. Cells were treated for 48 h with DMSO
or 1 mM compound (4a or 6c) and analyzed by FACS for relative cell size. Histograms of the forward scatter (FSCeH) for the treated cells and mean FSC-H values are shown. Con:
control.
synthesized and exhibited promising anti-cancer activities. Among
them compound 6c showed selective activity against p53 mutant
cell lines inhibiting mutant p53 phosphorylation and acetylation.
Molecular mechanism studies indicated that arresting mutant p53
function to trigger the deregulation of Cdk2, was essential for 6c-
induced apoptosis.

G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
381
Fig. 3. 6c induced apoptosis by dictating p53 function in MGC-803 cells. (A) The induction of apoptosis by 6c was examined with Hoechst 33258 and AIF-immunostaining in MGC-
803 cells. (B, C) 6c induced apoptosis without triggering Caspase-3 activities in MGC-803 cells. MGC-803 cells were incubated with 5 M 6c for 24 h. Arrow showed the Caspase-3-
m
activited cells. (D) The effects of 6c on p53 phosphorylation and acetylation in MGC-803 cells. (E) Cell proliferative inhibition effect of 6b in MGC-803 cancer cells. (F) The effects of
6b on p53 phosphorylation and acetylation in MGC-803 cells. Whole-cell extracts were prepared and analyzed by Western blot analysis using antibodies against proteins indicated.
The same blots were stripped and reprobed with b-actin antibody to show equal protein loading.
In summary, we presented a small-molecule platform able to
induce cell apoptosis by dictating mutant p53 functional arrest and
Cdk2-mediated apoptotic signaling pathway. The information
gained by our study provides a reference toward discovering novel
mutant p53 function-targeted anti-cancer agents.
IA apparatus and uncorrected. Thin layer chromatography (TLC)
was performed on silica gel plates 60 F-254 and column chroma-
tography on silica gel (200e300 mesh) from Branch of Qindao
Marine Chemical Co., Ltd. NMR spectra were recorded in DMSO-d6
or CDCl3 on Bruker Advance (500 MHz) with TMS as internal
standard. Chemical shifts were recorded in
d values. ESI-MS (elec-
trospray ionization mass spectrum) were recorded on a Bruker HCT
Electrospray Ionization Mass Spectrometer. Microanalyses were
performed by PerkineElmer 240 series II analyzer.
4. Experimental
4.1. General
All chemicals employed in this work are commercially available
and were used as received. All reactions were carried out under an
atmosphere of dry nitrogen. Melting points were recorded on WRS-
4.2. Compound characterization
4a: pale yellow solid, yield 27%, m.p. 132e136 ꢁC, 1H NMR

382
G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
Fig. 4. 6c arrested mutant p53 function for selective inhibition and Cdk2-mediated apoptosis in MGC-803 cancer cells. (A) 6c exhibited low toxicity to the normal lung fibroblast
WI-38 cells. (B) Immunoblotting analysis of proteins related to the p53-arrest-caused Cdk2-mediated apoptotic pathway. Whole-cell extracts were prepared and analyzed by
Western blot analysis using antibodies against proteins indicated. The same blots were stripped and reprobed with b-actin antibody to show equal protein loading. (C) Proposed
molecular mechanism explaining the induction of apoptasis by 6c. *p < 0.05 and **p < 0.01.
(500 MHz, CDCl3)
d
: 8.25e8.19 (m, 2H, ArH, ¼CH), 7.72e7.69 (m, 2H,
J ¼ 7.4 Hz, 2H, NCH2), 2.42 (t, J ¼ 6.5 Hz, 2H, CH2N), 2.29 (s, 6H,
N(CH3)2), 2.05e1.96 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d:
ArH), 7.66 (d, J ¼ 8.5 Hz, 1H, ArH), 7.45e7.35 (m, 3H, ArH, ¼CH),
7.25e7.23 (m, 1H, ArH), 4.27 (t, J ¼ 7.4 Hz, 2H, NCH2), 2.38 (t,
J ¼ 6.5 Hz, 2H, CH2N), 2.23 (s, 6H, N(CH3)2), 2.00e1.92 (m, 2H, CH2);
164.5, 162.2, 152.3, 147.6, 139.9, 134.2, 131.9, 129.7, 129.6, 127.2,
126.8, 126.4, 120.6, 119.2, 116.1, 115.9, 56.4, 45.6, 45.6, 42.1, 27.0.
APCI-MS m/z: 352.10 (Mþ1)þ. Anal. Calcd for C21H22FN3O: C, 71.77;
H, 6.31; N, 11.96. Found: C, 71.85; H, 6.22; N, 12.05.
13C NMR (125 MHz, CDCl3)
d: 162.1, 151.8, 147.4, 135.8, 135.6, 135.2,
134.2, 132.5, 130.0, 128.3, 127.5, 127.5, 126.7, 126.7, 122.7, 120.7, 56.3,
45.4, 45.4, 42.2, 26.9. APCI-MS m/z: 402.10 (Mþ1)þ. Anal.Calcd for
4c: pale yellow solid, yield 46%, m.p. 115e118 ꢁC, 1H NMR
C
21H21Cl2N3O: C, 62.69; H, 5.26; N, 10.44. Found: C, 62.72; H, 5.32;
(500 MHz, CDCl3)
d
: 8.26 (dd, J ¼ 8.0, 1.0 Hz, 1H, ArH), 7.95 (d,
N, 10.35.
4b: pale yellow solid, yield 23%, m.p. 90e93 ꢁC, 1H NMR
(500 MHz, CDCl3)
J ¼ 15.3 Hz, 1H, ¼CH), 7.77e7.37 (m, 7H, ArH), 7.34 (d, J ¼ 15.3 Hz,
1H, ¼CH), 4.31 (t, J ¼ 7.7 Hz, 2H, NCH2), 2.61 (t, J ¼ 7.1 Hz, 2H, CH2N),
2.38 (s, 6H, N(CH3)2), 2.13e2.02 (m, 2H, CH2); 13C NMR (125 MHz,
d
: 8.29 (d, J ¼ 7.4 Hz, 1H, ArH), 7.97 (d, J ¼ 15.3 Hz,
1H, ¼CH), 7.79e7.59 (m, 4H, ArH), 7.46 (ddd, J ¼ 8.1, 6.7, 1.6 Hz, 1H,
CDCl3)
d: 176.1, 162.2, 152.0, 147.5, 140.3, 135.6, 134.4, 134.0, 129.3,
ArH), 7.37 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.16e7.10 (m, 2H, ArH), 4.35 (t,
129.1, 127.3, 126.8, 126.6, 120.4, 120.0, 119.4, 55.6, 44.2, 44.2, 41.8,

G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
383
26.1. APCI-MS m/z: 368.15 (Mþ1)þ. Anal. Calcd for C21H22ClN3O: C,
CDCl3)
d
: 8.24 (d, J ¼ 7.8 Hz, 1H, ArH), 8.19 (d, J ¼ 15.4 Hz, 1H, ¼CH),
68.56; H, 6.03; N, 11.42. Found: C, 68.67; H, 5.92; N, 11.33.
7.74e7.70 (m, 2H, ArH), 7.59 (d, J ¼ 8.5 Hz, 1H, ArH), 7.46e7.40 (m,
2H, ArH), 7.28e7.22 (m, 2H, ArH, ¼CH), 4.31 (t, J ¼ 7.1 Hz, 2H, NCH2),
2.65 (t, J ¼ 7.1 Hz, 2H, CH2N), 2.32 (s, 6H, N(CH3)2); 13C NMR
4d: pale yellow solid, yield 52%, 1H NMR (500 MHz, CDCl3)
d:
8.33e8.23 (m, 2H, ArH, ¼CH), 7.79e7.70 (m, 3H, ArH), 7.65 (dd,
J ¼ 8.0, 1.2 Hz, 1H, ArH), 7.46 (ddd, J ¼ 8.1, 6.2, 2.0 Hz, 1H, ArH),
7.41e7.33 (m, 2H, ArH, ¼CH), 7.26e7.22 (m, 1H, ArH), 4.32 (t,
J ¼ 7.4 Hz, 2H, NCH2), 2.40 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.26 (s, 6H,
(125 MHz, CDCl3) d: 162.1, 151.8, 147.4, 135.9, 135.7, 135.2, 134.4,
132.4, 130.1, 128.3, 127.6, 127.6, 126.8, 122.7, 120.6, 120.0, 57.9, 46.0,
46.0, 42.3. APCI-MS m/z: 388.05 (Mþ1)þ. Anal. Calcd for
N(CH3)2), 2.06e1.94 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d:
C20H19Cl2N3O: C, 61.86; H, 4.93; N,10.82. Found: C, 61.72; H, 4.76; N,
162.2,151.9, 147.5, 139.6,135.7,134.2,133.5, 130.6,127.8, 127.6, 127.6,
126.7, 126.6, 125.2, 122.5, 120.7, 56.4, 45.5, 45.5, 42.3, 27.0. APCI-MS
m/z: 412.00, 414.00 (Mþ1)þ. Anal. Calcd for C21H22BrN3O: C, 61.17;
H, 5.38; N, 10.19. Found: C, 61.25; H, 5.32; N, 10.35.
11.90.
5b: yellow solid, yield 57%, m.p. 136e139 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.28e8.24 (m, 1H, ArH), 7.93 (d, J ¼ 15.4 Hz, 1H, ¼CH),
7.76e7.68 (m, 2H, ArH), 7.62e7.56 (m, 2H, ArH), 7.44 (ddd, J ¼ 8.1,
6.7, 1.5 Hz, 1H, ArH), 7.18 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.13e7.08 (m, 2H,
ArH), 4.36 (t, J ¼ 7.2 Hz, 2H, NCH2), 2.69 (t, J ¼ 7.2 Hz, 2H, CH2N),
4e: pale yellow solid, yield 77%, m.p. 165e166 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.30e8.24 (m, 3H, ArH), 8.00 (d, J ¼ 15.3 Hz,
1H, ¼CH), 7.83e7.69 (m, 4H, ArH), 7.64 (d, J ¼ 15.3 Hz, 1H, ¼CH),
7.47 (ddd, J ¼ 8.1, 6.8, 1.4 Hz, 1H, ArH), 4.32 (t, J ¼ 7.4 Hz, 2H, NCH2),
2.46 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.30 (s, 6H, N(CH3)2), 2.06e1.97 (m,
2.37 (s, 6H, N(CH3)2); 13C NMR (125 MHz, CDCl3)
d: 164.6, 162.2,
152.2, 147.5, 139.9, 134.3, 131.7, 129.6, 129.6, 127.2, 126.9, 126.5,
120.5, 119.0, 116.2, 116.0, 57.8, 45.9, 45.9, 42.1. APCI-MS m/z: 338.10
(Mþ1)þ. Anal. Calcd for C20H20FN3O: C, 71.20; H, 5.97; N, 12.45.
Found: C, 71.32; H, 5.82; N, 12.50.
2H, CH2); 13C NMR (125 MHz, CDCl3)
d: 162.0, 151.4, 147.9, 147.2,
141.7,138.4,134.4,128.4,128.4,127.3,126.9,126.8,124.1, 124.1,123.7,
120.6, 55.8, 45.1, 45.1, 42.1, 26.6. APCI-MS m/z: 379.17 (Mþ1)þ. Anal.
Calcd for C21H22N4O3: C, 66.65; H, 5.86; N, 14.81. Found: C, 66.72; H,
5.72; N, 14.90.
5c: yellow solid, yield 60%, m.p. 140e143 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.26 (dd, J ¼ 8.0, 1.0 Hz, 1H, ArH), 7.91 (d, J ¼ 15.4 Hz,
1H, ¼CH), 7.76e7.66 (m, 2H, ArH), 7.56e7.50 (m, 2H, ArH), 7.44
(ddd, J ¼ 8.1, 6.8, 1.5 Hz, 1H, ArH), 7.41e7.35 (m, 2H, ArH), 7.23 (d,
J ¼ 15.3 Hz, 1H, ¼CH), 4.35 (t, J ¼ 7.3 Hz, 2H, NCH2), 2.68 (t,
J ¼ 7.3 Hz, 2H, CH2N), 2.37 (s, 6H, N(CH3)2);13C NMR (125 MHz,
4f: pale yellow solid, yield 46%, m.p. 140e143 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.54 (s, 1H, ArH), 8.27 (d, J ¼ 8.0 Hz, 1H, ArH),
8.21 (d, J ¼ 8.2 Hz,1H, ArH), 8.04e7.98 (m, 2H, ArH, ¼CH), 7.78e7.70
(m, 3H, ArH), 7.66 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.63e7.44 (m, 2H, ArH),
4.33 (t, J ¼ 7.3 Hz, 2H, NCH2), 2.58 (t, J ¼ 6.9 Hz, 2H, CH2N), 2.39 (s,
CDCl3) d: 162.1, 152.1, 147.5, 139.8, 135.6, 134.3, 134.0, 129.2, 129.0,
127.3, 126.9, 126.6, 120.5, 120.0, 119.9, 57.8, 45.9, 45.9, 42.1. APCI-MS
m/z: 354.10 (Mþ1)þ. Anal. Calcd for C20H20ClN3O: C, 67.89; H, 5.70;
N, 11.88. Found: C, 67.72; H, 5.72; N, 11.97.
6H, N(CH3)2), 2.14e2.01 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d:
176.2, 162.2, 151.7, 148.7, 147.4, 138.7, 137.3, 134.5, 134.0, 129.9, 127.4,
126.8, 124.0,122.4, 122.3, 120.6, 55.5, 44.5, 44.5, 42.0, 26.2. APCI-MS
m/z: 379.15 (Mþ1)þ. Anal. Calcd for C21H22N4O3: C, 66.65; H, 5.86;
N, 14.81. Found: C, 66.73; H, 5.81; N, 14.75.
5d: yellow solid, yield 54%, m.p. 94e96 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.30e8.21 (m, 2H, ArH, ¼CH), 7.79e7.71 (m, 2H, ArH), 7.66
(ddd, J ¼ 11.7, 7.9, 1.2 Hz, 1H, ArH), 7.46 (ddd, J ¼ 8.1, 5.4, 2.9 Hz, 2H,
ArH), 7.36 (t, J ¼ 7.5 Hz, 1H, ArH), 7.25e7.16 (m, 2H, ArH, ¼CH), 4.35
(t, J ¼ 7.2 Hz, 2H, NCH2), 2.69 (t, J ¼ 7.2 Hz, 2H, CH2N), 2.35 (s, 6H,
4g: yellow solid, yield 44%, m.p. 187e188 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.29e8.24 (m, 1H, ArH), 7.96 (d, J ¼ 15.2 Hz, 1H, ¼CH),
7.76e7.68 (m, 2H, ArH), 7.43 (ddd, J ¼ 8.1, 6.6, 1.7 Hz, 1H, ArH),
7.26e7.20 (m, 2H, ArH, ¼CH), 7.15 (d, J ¼ 2.0 Hz, 1H, ArH), 6.91 (d,
J ¼ 8.3 Hz, 1H, ArH), 4.34 (t, J ¼ 7.5 Hz, 2H, NCH2), 3.96 (s, 3H, OCH3),
3.94 (s, 3H, OCH3), 2.41 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.28 (s, 6H,
N(CH3)2); 13C NMR (125 MHz, CDCl3)
d: 162.2, 151.9, 147.5, 139.6,
135.7, 134.3, 133.5, 130.7, 127.8, 127.7, 127.6, 126.8, 126.7, 125.1, 122.5,
120.6, 57.8, 45.9, 45.9, 42.3. APCI-MS m/z: 398.00, 400.00 (Mþ1)þ.
Anal. Calcd for C20H20BrN3O: C, 60.31; H, 5.06; N, 10.55. Found: C,
60.42; H, 4.92; N, 10.43.
N(CH3)2), 2.03e1.96 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d:
162.3, 152.6, 150.7, 149.2, 147.7, 141.3, 134.1, 128.7, 127.1, 126.8, 126.1,
121.8, 120.4, 117.0, 111.2, 110.3, 56.5, 56.0, 56.0, 45.5, 45.5, 42.1, 26.9.
APCI-MS m/z: 394.22 (Mþ1)þ. Anal. Calcd for C23H27N3O3: C, 70.21;
H, 6.92; N, 10.68. Found: C, 70.12; H, 6.79; N, 10.87.
5e: pale yellow solid, yield 75%, m.p. 178e180 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.26 (d, J ¼ 8.7 Hz, 3H, ArH), 8.03 (d, J ¼ 15.3 Hz,
1H, ¼CH)), 7.88 (d, J ¼ 8.7 Hz, 2H, ArH), 7.80e7.68 (m, 2H, ArH), 7.53
(d, J ¼ 15.3 Hz, 1H, ¼CH), 7.47 (ddd, J ¼ 8.1, 6.9, 1.4 Hz, 1H, ArH), 4.46
(t, J ¼ 7.5 Hz, 2H, NCH2), 2.82 (t, J ¼ 7.5 Hz, 2H, CH2N), 2.47 (s, 6H,
4h: yellow solid, yield 42%, m.p. 177e179 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.26 (dd, J ¼ 8.0, 0.9 Hz, 1H, ArH), 7.91 (d, J ¼ 15.2 Hz,
N(CH3)2); 13C NMR (125 MHz, CDCl3)
d: 162.0, 151.3, 148.1, 147.3,
1H, ¼CH), 7.77e7.65 (m, 2H, ArH), 7.42 (ddd, J ¼ 8.1, 6.6, 1.6 Hz, 1H,
ArH), 7.25 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.17 (d, J ¼ 1.6 Hz, 1H, ArH), 7.10
(dd, J ¼ 8.0, 1.5 Hz, 1H, ArH), 6.84 (d, J ¼ 8.0 Hz, 1H, ArH), 6.02 (s, 2H,
OCH2O), 4.31 (t, J ¼ 7.4 Hz, 2H, NCH2), 2.39 (t, J ¼ 6.5 Hz, 2H, CH2N),
2.28 (s, 6H, N(CH3)2), 2.02e1.93 (m, 2H, CH2); 13C NMR (125 MHz,
141.6,138.9,134.6,128.7,128.7,127.5,127.1,126.9,124.2,124.2,123.3,
120.6, 56.9, 45.1, 45.1, 41.3. APCI-MS m/z: 365.11 (Mþ1)þ. Anal.
Calcd for C20H20N4O3: C, 65.92; H, 5.53; N,15.38. Found: C, 65.81; H,
5.66; N, 15.40.
5f: pale yellow solid, yield 69%, m.p. 185e188 ꢁC, 1H NMR
CDCl3)
d
: 162.3, 152.6, 149.1, 148.4, 147.7, 140.9, 134.1, 130.2, 127.1,
(500 MHz, CDCl3)
d
: 8.50 (s, 1H, ArH), 8.28 (dd, J ¼ 7.8, 0.8 Hz, 1H,
126.8, 126.1, 124.1, 120.5, 117.4, 108.6, 106.3, 101.5, 56.5, 45.6, 45.6,
42.1, 27.0. APCI-MS m/z: 378.19 (Mþ1)þ. Anal. Calcd for C22H23N3O3:
C, 70.01; H, 6.14; N, 11.13. Found: C, 70.12; H, 6.07; N, 11.24.
4i: pale yellow solid, yield 13%, m.p. 163e165 ꢁC, 1H NMR
ArH), 8.25e8.18 (m, 1H, ArH), 7.99 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.89 (d,
J ¼ 7.8 Hz, 1H, ArH), 7.82e7.68 (m, 2H, ArH), 7.64e7.56 (m, 1H, ArH),
7.53 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.49e7.41 (m, 1H, ArH), 4.36 (t,
J ¼ 6.8 Hz, 2H, NCH2), 2.72 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.39 (s, 6H,
(500 MHz, DMSO-d6)
d: 9.67 (s, 1H, OH), 8.13 (d, J ¼ 7.9 Hz, 1H, ArH),
N(CH3)2); 13C NMR (125 MHz, CDCl3)
d: 162.1, 151.6, 148.8, 147.4,
7.87e7.48 (m, 4H, ArH, ¼CH), 7.39 (d, J ¼ 15.3 Hz, 1H, ¼CH),
7.27e7.20 (m, 3H, ArH), 6.85 (d, J ¼ 6.8 Hz, 1H, ArH), 4.33 (t,
J ¼ 7.1 Hz, 2H, NCH2), 2.71 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.42 (s, 6H,
138.1, 137.3, 134.4, 133.6, 130.0, 127.4, 126.9, 123.9, 122.9, 121.9,
120.7, 58.2, 46.0, 46.0, 42.4. APCI-MS m/z: 365.12 (Mþ1)þ. Anal.
Calcd for C20H20N4O3: C, 65.92; H, 5.53; N,15.38. Found: C, 66.04; H,
5.40; N, 15.23.
N(CH3)2), 2.00e1.90 (m, 2H, CH2); 13C NMR (125 MHz, DMSO-d6)
d:
161.8,158.3,152.6,147.6,141.2,137.0,134.9,130.3,127.5,126.8,120.4,
119.9, 119.7, 117.5, 115.0, 55.6, 44.2, 44.2, 41.2, 25.9. APCI-MS m/z:
350.19 (Mþ1)þ. Anal. Calcd for C21H23N3O2: C, 72.18; H, 6.63; N,
12.03. Found: C, 72.32; H, 6.72; N, 11.90.
5g: yellow solid, yield 42%, m.p. 113e115 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.24 (d, J ¼ 8.0 Hz, 1H, ArH), 7.98 (d, J ¼ 15.2 Hz, 1H, ¼CH),
7.76e7.61 (m, 2H, ArH), 7.47e7.37 (m, 1H, ArH), 7.36e7.22 (m, 2H,
ArH), 7.17 (d, J ¼ 15.2 Hz, 1H, ¼CH), 6.90 (d, J ¼ 8.3 Hz, 1H, ArH), 4.48
(t, J ¼ 7.7 Hz, 2H, NCH2), 3.97 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 2.82
5a: yellow solid, yield 69%, m.p. 149e151 ꢁC, 1H NMR (500 MHz,

384
G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
(t, J ¼ 7.7 Hz, 2H, CH2N), 2.48 (s, 6H, N(CH3)2); 13C NMR (125 MHz,
3H, OCH3), 4.00 (s, 3H, OCH3), 2.39 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.24 (s,
6H, N(CH3)2), 2.01e1.94 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d:
CDCl3)
d
: 162.3, 152.5, 150.8, 149.3, 147.7, 142.0, 134.4, 128.5, 127.2,
126.8, 126.3, 122.3, 120.2, 116.3, 111.1, 110.4, 56.6, 56.1, 56.0, 44.9,
161.5,154.9,150.8,149.1, 143.9,138.8,135.8, 133.5, 130.5,127.7, 127.7,
125.1,122.6,114.2,107.9,105.7, 56.4, 56.3, 56.3, 45.5, 42.2, 27.1. APCI-
MS m/z: 472.10, 474.10 (Mþ1)þ. Anal. Calcd for C23H26BrN3O3: C,
58.48; H, 5.55; N, 8.90. Found: C, 58.39; H, 5.67; N, 8.92.
44.9, 40.8. APCI-MS m/z: 380.10 (Mþ1)þ. Anal. Calcd for
C
22H25N3O3: C, 69.64; H, 6.64; N, 11.07. Found: C, 69.72; H, 6.70; N,
10.90.
5h: yellow solid, yield 46%, m.p. 199e203 ꢁC, 1H NMR (500 MHz,
CDCl3)
6e: pale yellow solid, yield 48%, m.p. 199e202 ꢁC, 1H NMR
d
: 8.30e8.19 (m, 1H, ArH), 7.89 (d, J ¼ 15.2 Hz, 1H, ¼CH),
(500 MHz, CDCl3)
d
: 8.29 (d, J ¼ 8.8 Hz, 2H, ArH), 7.96 (d, J ¼ 15.4 Hz,
7.77e7.62 (m, 2H, ArH), 7.42 (ddd, J ¼ 8.1, 6.8, 1.5 Hz, 1H, ArH),
7.13e7.08 (m, 2H, ArH), 7.06 (d, J ¼ 15.2 Hz, 1H, ¼CH), 6.84 (d,
J ¼ 8.0 Hz, 1H, ArH), 6.02 (s, 2H, OCH2O), 4.36 (t, J ¼ 7.4 Hz, 2H,
NCH2), 2.68 (t, J ¼ 7.4 Hz, 2H, CH2N), 2.38 (s, 6H, N(CH3)2); 13C NMR
1H, ¼CH), 7.79 (d, J ¼ 8.8 Hz, 2H, ArH), 7.70 (d, J ¼ 15.4 Hz,1H, ¼CH),
7.62 (s, 1H, ArH), 7.15 (s, 1H, ArH), 4.36 (t, J ¼ 7.2 Hz, 2H, NCH2), 4.04
(s, 3H, OCH3), 4.03 (s, 3H, OCH3), 2.41 (t, J ¼ 6.3 Hz, 2H, CH2N), 2.30
(s, 6H, N(CH3)2), 2.07e1.98 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
(125 MHz, CDCl3)
d: 162.3, 152.4, 149.2, 148.4, 147.6, 141.0, 134.3,
d: 161.3, 155.1, 150.4, 149.4, 147.9, 143.6, 142.0, 137.3, 128.3, 128.3,
130.0, 127.2, 126.8, 126.3, 124.1, 120.4, 117.1, 108.7, 106.3, 101.6, 57.7,
124.3, 124.3, 124.2, 114.4, 107.6, 105.7, 56.4, 56.4, 56.1, 45.6, 45.6,
45.9, 45.9, 42.0. APCI-MS m/z: 364.10 (Mþ1)þ. Anal. Calcd for
42.1, 27.0. APCI-MS m/z: 439.20 (Mþ1)þ. Anal. Calcd for
C
21H21N3O3: C, 69.41; H, 5.82; N, 11.56. Found: C, 69.52; H, 5.70; N,
C23H26N4O5: C, 63.00; H, 5.98; N, 12.78. Found: C, 62.89; H, 6.12; N,
11.43.
5i: pale yellow solid, yield 73%, m.p. 217e220 ꢁC, 1H NMR
(500 MHz, DMSO-d6)
12.90.
6f: pale yellow solid, yield 43%, m.p. 188e191 ꢁC, 1H NMR
(500 MHz, DMSO-d6)
d
: 9.63 (s, 1H, OH), 8.12 (dd, J ¼ 8.0, 1.1 Hz, 1H,
d
: 8.58 (t, J ¼ 2.0 Hz, 1H, ArH), 8.20 (dd, J ¼ 8.1,
ArH), 7.85e7.74 (m, 2H, ArH, ¼CH), 7.68 (d, J ¼ 8.1 Hz, 1H, ArH), 7.48
(ddd, J ¼ 8.1, 7.2, 1.1 Hz, 1H, ArH), 7.37 (d, J ¼ 15.3 Hz, 1H, ¼CH),
7.29e7.11 (m, 3H, ArH), 6.83 (ddd, J ¼ 8.0, 2.3, 0.9 Hz, 1H, ArH), 4.38
(t, J ¼ 6.8 Hz, 2H, NCH2), 2.53 (t, J ¼ 6.8 Hz, 2H, CH2N), 2.22 (s, 6H,
2.0 Hz, 2H, ArH), 7.94 (d, J ¼ 15.3 Hz, 1H), 7.73 (t, J ¼ 8.0 Hz,
1H, ¼CH), 7.69 (d, J ¼ 15.4 Hz, 1H, ¼CH), 7.40 (s, 1H, ArH), 7.12 (s, 1H,
ArH), 4.32 (t, J ¼ 7.2 Hz, 2H, NCH2), 3.92 (s, 3H, OCH3), 3.87 (s, 3H,
OCH3), 2.49 (2H, CH2N, overlap by solvent peak), 2.31 (s, 6H,
N(CH3)2); 13C NMR (125 MHz, DMSO-d6)
d: 161.7, 158.2, 152.7, 147.5,
N(CH3)2), 1.93e1.83 (m, 2H, CH2); 13C NMR (125 MHz, DMSO-d6)
d:
140.6, 137.0, 134.9, 130.5, 127.5, 126.9, 126.8, 120.3, 120.2, 119.6,
117.4, 114.6, 58.1, 46.0, 46.0, 41.2. APCI-MS m/z: 336.10 (Mþ1)þ.
Anal. Calcd for C20H21N3O2: C, 71.62; H, 6.31; N, 12.53. Found: C,
71.69; H, 6.22; N, 12.50.
160.9, 155.2, 150.6, 149.2, 148.8, 143.6, 137.7, 137.3, 134.8, 130.9,
124.1, 123.3, 122.3, 113.8, 108.1, 105.7, 56.4, 56.2, 55.9, 44.8, 44.8,
41.3, 26.4. APCI-MS m/z: 439.18 (Mþ1)þ. Anal. Calcd for
C
23H26N4O5: C, 63.00; H, 5.98; N, 12.78. Found: C, 63.11; H, 5.86; N,
6a: pale yellow solid, yield 51%, m.p. 153e155 ꢁC, 1H NMR
12.87.
6g: pale yellow solid, yield 44%, m.p. 173e175 ꢁC, 1H NMR
(500 MHz, CDCl3)
(500 MHz, CDCl3)
d
: 8.18 (d, J ¼ 15.3 Hz,1H, ¼CH), 7.68 (d, J ¼ 8.5 Hz,
1H, ArH), 7.58 (s, 1H, ArH), 7.45 (d, J ¼ 1.9 Hz, 1H, ArH), 7.39 (d,
J ¼ 15.3 Hz, 1H, ¼CH), 7.28 (dd, J ¼ 8.5, 1.8 Hz, 1H, ArH), 7.14 (s, 1H,
ArH), 4.29 (t, J ¼ 7.2 Hz, 2H, NCH2), 4.01 (s, 3H, OCH3), 3.99 (s, 3H,
OCH3), 2.40 (t, J ¼ 6.5 Hz, 2H, CH2N), 2.26 (s, 6H, N(CH3)2),
d
: 7.86 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.57 (s, 1H, ArH),
7.22e7.20 (m, 1H, ArH), 7.19 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.12 (d,
J ¼ 1.8 Hz, 1H, ArH), 7.10 (s, 1H, ArH), 6.89 (d, J ¼ 8.4 Hz, 1H, ArH),
4.33 (t, J ¼ 7.3 Hz, 2H, NCH2), 4.00 (s, 3H, OCH3), 3.98 (s, 3H, OCH3),
3.94 (s, 3H, OCH3), 3.92 (s, 3H, OCH3), 2.42 (t, J ¼ 6.5 Hz, 2H, CH2N),
2.28 (s, 6H, N(CH3)2), 2.02e1.96 (m, 2H, CH2); 13C NMR (125 MHz,
2.02e1.94 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d: 161.4, 155.0,
150.6, 149.2, 143.8, 135.5, 135.2, 135.0, 132.6, 130.0, 128.2, 127.5,
122.8, 114.2, 107.8, 105.6, 56.4, 56.3, 56.3, 45.4, 45.4, 42.1, 26.9. APCI-
MS m/z: 462.16 (Mþ1)þ. Anal. Calcd for C23H25Cl2N3O3: C, 59.75; H,
5.45; N, 9.09. Found: C, 59.72; H, 5.36; N, 8.94.
CDCl3) d: 161.6, 154.9, 151.5, 150.5, 149.1, 148.8, 144.0, 140.4, 128.7,
121.7, 117.1, 113.9, 111.1, 110.1, 107.4, 105.7, 56.5, 56.3, 56.3, 56.0, 56.0,
45.5, 45.5, 42.0, 26.9. APCI-MS m/z: 454.24 (Mþ1)þ. Anal. Calcd for
6b: pale yellow solid, yield 40%, m.p. 86e89 ꢁC, 1H NMR
C25H31N3O5: C, 66.21; H, 6.89; N, 9.27. Found: C, 66.09; H, 6.96; N,
(500 MHz, CDCl3)
d
: 7.85 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.61e7.56 (m, 3H,
9.38.
6h: yellow solid, yield 64%, m.p. 167e170 ꢁC, 1H NMR (500 MHz,
CDCl3)
ArH), 7.28 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.10e7.06 (m, 3H, ArH), 4.30 (t,
J ¼ 7.3 Hz, 2H, NCH2), 3.99 (s, 3H, OCH3), 3.98 (s, 3H, OCH3), 2.41 (t,
J ¼ 6.6 Hz, 2H, CH2N), 2.27 (s, 6H, N(CH3)2), 2.01e1.93 (m, 2H, CH2);
d
: 7.82 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.57 (s, 1H, ArH), 7.20 (d,
J ¼ 15.2 Hz, 1H), 7.16 (d, J ¼ 1.5 Hz, 1H, ¼CH), 7.10 (s, 1H, ArH), 7.07
(dd, J ¼ 8.0, 1.5 Hz, 1H, ArH), 6.82 (d, J ¼ 8.0 Hz, 1H, ArH), 6.01 (s, 2H,
OCH2O), 4.31 (t, J ¼ 7.3 Hz, 2H, NCH2), 4.00 (s, 3H, OCH3), 3.99 (s, 3H,
OCH3), 2.44 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.31 (s, 6H, N(CH3)2),
13C NMR (125 MHz, CDCl3)
d: 164.4, 162.4, 161.5, 155.0, 151.1, 149.0,
143.8, 139.1, 132.0, 129.6, 129.5, 119.2, 116.1, 115.9, 114.0, 107.5, 105.7,
56.3, 56.3, 45.3, 45.3, 42.0, 26.9. APCI-MS m/z: 412.10 (Mþ1)þ. Anal.
Calcd for C23H26FN3O3: C, 67.14; H, 6.37; N,10.21. Found: C, 67.02; H,
6.45; N, 10.09.
2.03e1.95 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d: 161.6, 154.9,
151.4,149.0,148.8,148.3,144.0,140.1,130.2,124.0,117.4,113.9,108.6,
107.4,106.3,105.7, 101.5, 56.4, 56.3, 56.3, 45.4, 45.4, 42.0, 26.9. APCI-
MS m/z: 438.20 (Mþ1)þ. Anal. Calcd for C24H27N3O5: C, 65.89; H,
6.22; N, 9.60. Found: C, 65.80; H, 6.12; N, 9.54.
6c: pale yellow solid, yield 43%, m.p. 118e120 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 7.85 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.58 (s, 1H, ArH),
7.55 (d, J ¼ 8.5 Hz, 2H, ArH), 7.37 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.36 (d,
J ¼ 8.5 Hz, 2H, ArH), 7.11 (s, 1H, ArH), 4.31 (t, J ¼ 7.3 Hz, 2H, NCH2),
4.00 (s, 3H, OCH3), 3.99 (s, 3H, OCH3), 2.43 (t, J ¼ 6.5 Hz, 2H, CH2N),
2.29 (s, 6H, N(CH3)2), 2.03e1.95 (m, 2H, CH2); 13C NMR (125 MHz,
6i: pale yellow solid, yield 30%, m.p. 168e171 ꢁC, 1H NMR
(500 MHz, DMSO-d6)
d
: 9.71 (s, 1H, OH), 7.80 (d, J ¼ 15.3 Hz,
1H, ¼CH), 7.43 (s, 1H, ArH), 7.34 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.26e7.24
(m, 2H, ArH), 7.19 (s, 1H, ArH), 7.16 (s, 1H, ArH), 6.85 (d, J ¼ 6.7 Hz,
1H, ArH), 4.33 (t, J ¼ 7.0 Hz, 2H, NCH2), 3.92 (s, 3H, OCH3), 3.87 (s,
3H, OCH3), 2.86 (t, J ¼ 6.6 Hz, 2H, CH2N), 2.53 (s, 6H, N(CH3)2),
CDCl3) d: 161.5, 155.1, 151.0, 149.1, 143.8, 138.9, 135.3, 134.3, 129.1,
129.1, 128.9, 128.9, 120.2, 114.1, 107.6, 105.8, 56.4, 56.3, 56.3, 45.4,
45.4, 42.0, 26.9. APCI-MS m/z: 428.16 (Mþ1)þ. Anal. Calcd for
C
23H26ClN3O3: C, 64.55; H, 6.12; N, 9.82. Found: C, 64.62; H, 6.05; N,
2.00e1.97 (m, 2H, CH2); 13C NMR (125 MHz, DMSO-d6)
d: 161.1,
9.90.
6d: pale yellow solid, yield 32%, m.p. 81e83 ꢁC, 1H NMR
(500 MHz, CDCl3)
1.5 Hz, 1H, ArH), 7.63 (dd, J ¼ 8.0, 1.0 Hz, 1H, ArH), 7.60 (s, 1H, ArH),
7.36 (d, J ¼ 7.4 Hz, 1H, ArH), 7.32 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.26e7.20
(m, 1H, ArH), 7.16 (s, 1H, ArH), 4.30 (t, J ¼ 7.4 Hz, 2H, NCH2), 4.02 (s,
155.3, 155.3, 151.0, 149.1, 143.8, 140.3, 137.1, 130.3, 119.8, 119.5, 117.3,
114.9, 113.5, 108.2, 105.9, 56.5, 56.5, 56.2, 55.3, 43.7, 43.7, 25.6. APCI-
MS m/z: 410.19 (Mþ1)þ. Anal. Calcd for C23H27N3O4: C, 67.46; H,
6.65; N, 10.26. Found: C, 67.39; H, 6.72; N, 10.34.
d
: 8.21 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.71 (dd, J ¼ 7.8,
7a: yellow solid, yield 43%, m.p. 156e158 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.28 (d, J ¼ 15.3 Hz, 1H, ¼CH), 8.19 (d, J ¼ 8.5 Hz, 1H, ArH),

G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
385
7.75 (d, J ¼ 2.1 Hz, 1H, ArH), 7.68 (d, J ¼ 8.5 Hz, 1H, ArH), 7.48 (d,
J ¼ 2.1 Hz, 1H, ArH), 7.42 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.39 (dd, J ¼ 8.5,
2.1 Hz, 1H), 7.30 (dd, J ¼ 8.5, 2.1 Hz, 1H), 4.28 (t, J ¼ 7.3 Hz, 2H,
NCH2), 2.37 (t, J ¼ 6.4 Hz, 2H, CH2N), 2.24 (s, 6H, N(CH3)2), 1.99e1.93
7.70 (d, J ¼ 2.0 Hz, 1H, ArH), 7.35 (dd, J ¼ 8.5, 2.0 Hz, 1H, ArH), 7.24
(dd, J ¼ 8.4, 2.0 Hz, 1H, ArH), 7.21 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.14 (d,
J ¼ 2.0 Hz, 1H, ArH), 6.91 (d, J ¼ 8.4 Hz, 1H, ArH), 4.32 (t, J ¼ 7.4 Hz,
2H, NCH2), 3.96 (s, 3H, OCH3), 3.94 (s, 3H, OCH3), 2.40 (t, J ¼ 6.5 Hz,
2H, CH2N), 2.27 (s, 6H, CH3), 2.01e1.94 (m, 2H, CH2); 13C NMR
(m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d: 161.6,153.0,148.4,140.4,
136.6, 135.9, 135.4, 132.3, 130.1, 128.3, 128.3, 127.5, 127.2, 127.0,
122.3, 119.1, 56.3, 45.5, 45.5, 42.3, 26.9. APCI-MS m/z: 436.00
(Mþ1)þ. Anal. Calcd for C21H20Cl3N3O: C, 57.75; H, 4.62; N, 9.62.
Found: C, 57.68; H, 4.71; N, 9.79.
(125 MHz, CDCl3) d: 161.8, 153.8, 150.9, 149.2, 148.7, 142.1, 140.3,
128.5, 128.3, 126.6, 126.5, 122.0, 118.8, 116.6, 111.2, 110.4, 56.5, 56.0,
56.0, 45.6, 45.6, 42.1, 26.8. APCI-MS m/z: 428.10 (Mþ1)þ. Anal. Calcd
for C23H26ClN3O3: C, 64.55; H, 6.12; N, 9.82. Found: C, 64.48; H,
6.06; N, 9.73.
7b: yellow solid, yield 40%, m.p. 113e116 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.18 (d, J ¼ 8.5 Hz, 1H, ArH), 7.97 (d, J ¼ 15.3 Hz, 1H, ¼CH),
7h: yellow solid, yield 33%, m.p. 113e115 ꢁC, 1H NMR (500 MHz,
7.70 (d, J ¼ 2.0 Hz, 1H, ArH), 7.63 (dd, J ¼ 8.6, 5.4 Hz, 2H, ArH), 7.37
(dd, J ¼ 8.5, 2.0 Hz, 1H, ArH), 7.31 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.11 (t,
J ¼ 8.6 Hz, 2H, ArH), 4.30 (t, J ¼ 7.4 Hz, 2H, NCH2), 2.42 (t, J ¼ 6.5 Hz,
2H, CH2N), 2.28 (s, 6H, N(CH3)2), 2.01e1.94 (m, 2H, CH2); 13C NMR
CDCl3)
d
: 8.17 (d, J ¼ 8.5 Hz, 1H, ArH), 7.92 (d, J ¼ 15.2 Hz, 1H, ¼CH),
7.69 (d, J ¼ 2.0 Hz, 1H, ArH), 7.35 (dd, J ¼ 8.5, 2.0 Hz, 1H, ArH), 7.23
(d, J ¼ 15.2 Hz,1H, ¼CH), 7.17 (d, J ¼ 1.5 Hz,1H, ArH), 7.10 (dd, J ¼ 8.0,
1.5 Hz, 1H, ArH), 6.84 (d, J ¼ 8.0 Hz, 1H, ArH), 6.03 (s, 2H, OCH2O),
4.30 (t, J ¼ 7.4 Hz, 2H, NCH2), 2.39 (t, J ¼ 6.4 Hz, 2H, CH2N), 2.28 (s,
(125 MHz, CDCl3) d: 161.7, 153.4, 148.5, 140.8, 140.4, 131.7, 129.9,
129.8, 128.3, 126.9, 126.6, 119.0, 118.7, 116.2, 116.0, 56.2, 45.4, 45.4,
6H, CH3), 1.99e1.93 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d:
42.1, 26.8. APCI-MS m/z: 386.10 (Mþ1)þ. Anal. Calcd for
161.8, 153.7, 149.3, 148.7, 148.4, 141.8, 140.3, 130.0, 128.3, 126.7,
126.5, 124.4, 118.9, 116.9, 108.6, 106.3, 101.6, 56.4, 45.6, 45.6, 42.1,
26.9. APCI-MS m/z: 412.10 (Mþ1)þ. Anal. Calcd for C22H22ClN3O3: C,
64.15; H, 5.38; N, 10.20. Found: C, 64.28; H, 5.26; N, 10.13.
C
21H21ClFN3O: C, 65.37; H, 5.49; N, 10.89. Found: C, 65.49; H, 5.54;
N, 10.98.
7c: yellow solid, yield 40%, m.p. 115e117 ꢁC, 1H NMR (500 MHz,
CDCl3)
d
: 8.18 (d, J ¼ 8.5 Hz, 1H, ArH), 7.95 (d, J ¼ 15.3 Hz, 1H, ¼CH),
7i: pale yellow solid, yield 38%, m.p. 112e115 ꢁC, 1H NMR
7.70 (d, J ¼ 2.0 Hz, 1H, ArH), 7.56 (d, J ¼ 8.5 Hz, 2H, ArH), 7.41e7.36
(m, 4H, ArH, ¼CH), 4.30 (t, J ¼ 7.3 Hz, 2H, NCH2), 2.39 (t, J ¼ 6.5 Hz,
2H, CH2N), 2.27 (s, 6H, N(CH3)2), 2.00e1.93 (m, 2H, CH2); 13C NMR
(500 MHz, CDCl3)
d
: 8.17 (d, J ¼ 8.6 Hz, 1H, ArH), 7.85 (d, J ¼ 15.2 Hz,
1H, ¼CH), 7.71 (d, J ¼ 1.9 Hz, 1H, ArH), 7.37 (dd, J ¼ 8.5, 1.9 Hz, 1H,
ArH), 7.16 (d, J ¼ 7.8 Hz, 1H, ArH), 7.12 (d, J ¼ 15.2 Hz, 1H, ¼CH), 7.08
(t, J ¼ 1.9 Hz, 1H, ArH), 7.04 (d, J ¼ 7.7 Hz, 1H, ArH), 6.74 (dd, J ¼ 7.9,
1.9 Hz, 1H, ArH), 4.28 (t, J ¼ 7.5 Hz, 2H, NCH2), 2.48 (t, J ¼ 7.1 Hz, 2H,
CH2N), 2.32 (s, 6H, CH3), 2.04e1.96 (m, 2H, CH2); 13C NMR
(125 MHz, CDCl3) d: 161.7, 153.3, 148.5, 140.6, 140.4, 135.7, 134.0,
129.2, 129.2, 129.1, 129.1, 128.3, 127.0, 126.7, 119.6, 119.0, 56.3, 45.5,
45.5, 42.2, 26.8. APCI-MS m/z: 402.10 (Mþ1)þ. Anal. Calcd for
C
21H21Cl2N3O: C, 62.69; H, 5.26; N, 10.44. Found: C, 62.76; H, 5.12;
(125 MHz, CDCl3) d: 161.8, 157.2, 153.4, 148.5, 142.4, 140.5, 136.8,
N, 10.51.
7d: yellow solid, yield 51%, m.p. 141e143 ꢁC, 1H NMR (500 MHz,
CDCl3)
129.9, 128.3, 127.0, 126.6, 120.4, 118.8, 118.4, 117.3, 114.6, 56.4, 45.2,
45.2, 41.9, 26.9. APCI-MS m/z: 384.10 (Mþ1)þ. Anal. Calcd for
d: 8.31 (d, J ¼ 15.2 Hz, 1H, ¼CH), 8.19 (d, J ¼ 8.5 Hz, 1H, ArH),
C21H22ClN3O2: C, 65.71; H, 5.78; N, 10.95. Found: C, 65.59; H, 5.76;
7.75 (d, J ¼ 2.0 Hz, 1H, ArH), 7.71 (dd, J ¼ 7.8, 1.6 Hz, 1H, ArH), 7.65
(dd, J ¼ 8.0, 1.2 Hz, 1H, ArH), 7.40e7.36 (m, 2H, ArH), 7.34 (d,
J ¼ 15.1 Hz, 1H, ¼CH), 7.25e7.21 (m, 1H, ArH), 4.29 (t, J ¼ 7.3 Hz 2H,
NCH2), 2.38 (t, J ¼ 6.5 Hz, 2H, CH2N), 2.24 (s, 6H, N(CH3)2),
N, 10.89.
8a: pale yellow solid, yield 30%, m.p. 99e102 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.27 (d, J ¼ 8.1 Hz, 1H, ArH), 8.24 (d, J ¼ 15.3 Hz,
1H, ¼CH), 7.77e7.71 (m, 2H, ArH), 7.59 (d, J ¼ 8.4 Hz, 1H, ArH),
7.49e7.43 (m, 2H, ArH), 7.31 (d, J ¼ 8.5 Hz, 1H, ArH), 7.10 (d,
J ¼ 15.3 Hz, 1H, ¼CH), 4.23 (t, J ¼ 7.7 Hz, 2H, NCH2), 1.82e1.74 (m,
2H, CH2), 1.53e1.43 (m, 2H, CH2), 1.00 (t, J ¼ 7.3 Hz, 3H, CH3); 13C
2.00e1.93 (m, 2H, CH2); 13C NMR (125 MHz, CDCl3)
d: 161.7, 153.1,
148.5,140.4,140.3,135.5,133.6,130.8,128.3,127.8,127.7,127.1,127.0,
125.3, 122.0, 119.1, 56.4, 45.5, 45.5, 42.3, 26.9. APCI-MS m/z: 446.00,
448.00 (Mþ1)þ. Anal. Calcd for C21H21BrClN3O: C, 56.46; H, 4.74; N,
9.41. Found: C, 56.51; H, 4.65; N, 9.52.
NMR (125 MHz, CDCl3) d: 162.0, 151.4, 147.4, 136.0, 135.7, 135.3,
134.3,132.4,130.2,128.4,127.6,127.5,126.9,126.8,122.3,120.7, 43.5,
7e: yellow solid, yield 69%, m.p. 159e163 ꢁC, 1H NMR (500 MHz,
31.3, 20.2, 13.8. APCI-MS m/z: 373.07 (Mþ1)þ. Anal. Calcd for
CDCl3)
d
: 8.27 (d, J ¼ 8.6 Hz, 2H, ArH), 8.19 (d, J ¼ 8.5 Hz, 1H, ArH),
C20H18Cl2N2O: C, 64.35; H, 4.86; N, 7.50. Found: C, 64.48; H, 4.98; N,
8.01 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.78 (d, J ¼ 8.6 Hz, 2H, ArH), 7.71 (s,
1H, ArH), 7.66 (d, J ¼ 15.3 Hz, 1H, CH]CH), 7.40 (d, J ¼ 8.5 Hz, 1H,
ArH), 4.31 (t, J ¼ 7.2 Hz, 2H, NCH2), 2.39 (t, J ¼ 6.3 Hz, 2H, CH2N),
2.28 (s, 6H, N(CH3)2), 2.02e1.95 (m, 2H, CH2); 13C NMR (125 MHz,
7.43.
8b: pale yellow solid, yield 48%, m.p. 107e110 ꢁC, 1H NMR
(500 MHz, DMSO-d6)
d: 8.27 (dd, J ¼ 8.2, 1.1 Hz, 1H, ArH), 7.97 (d,
J ¼ 15.3 Hz, 1H, ¼CH), 7.76e7.68 (m, 2H, ArH), 7.61e7.56 (m, 2H,
ArH), 7.46e7.41 (m, 1H, ArH), 7.15e7.08 (m, 2H, ArH), 7.02 (d,
J ¼ 15.3 Hz, 1H, ¼CH), 4.25 (t, J ¼ 7.8 Hz, 2H, NCH2), 1.83e1.75 (m,
2H, CH2), 1.55e1.45 (m, 2H, CH2), 1.02 (t, J ¼ 7.4 Hz, 3H, CH3); 13C
CDCl3) d: 161.5, 152.7, 148.3, 148.1, 141.7, 140.5, 138.9, 128.5, 128.5,
128.4, 127.4, 126.8, 124.2, 124.2, 123.7, 119.2, 56.1, 45.6, 45.6, 42.3,
26.8. APCI-MS m/z: 413.10 (Mþ1)þ. Anal. Calcd for C21H21ClN4O3: C,
61.09; H, 5.13; N, 13.57. Found: C, 61.21; H, 5.04; N, 13.73.
NMR (125 MHz, CDCl3) d: 164.6, 162.6, 162.1, 151.9, 147.5, 139.9,
7f: pale yellow solid, yield 74%, m.p. 118e122 ꢁC, 1H NMR
134.2, 129.6, 129.5, 127.2, 126.9, 126.4, 120.6, 118.7, 116.2, 116.0, 43.4,
(500 MHz, CDCl3)
d
: 8.56 (t, J ¼ 1.9 Hz, 1H, ArH), 8.24e8.21 (m, 1H,
31.3, 20.2, 13.8. APCI-MS m/z: 323.10 (Mþ1)þ. Anal. Calcd for
ArH), 8.20 (d, J ¼ 8.5 Hz,1H, ArH), 8.01 (d, J ¼ 15.3 Hz,1H, ¼CH), 7.88
(d, J ¼ 7.7 Hz, 1H, ArH), 7.79 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.71 (d,
J ¼ 1.9 Hz, 1H, ArH), 7.61 (t, J ¼ 7.9 Hz, 1H, ArH), 7.40 (dd, J ¼ 8.5,
2.0 Hz, 1H, ArH), 4.33 (t, J ¼ 7.0 Hz, 2H, NCH2), 2.36 (t, J ¼ 6.0 Hz, 2H,
CH2N), 2.30 (s, 6H, N(CH3)2), 2.04e1.97 (m, 2H, CH2); 13C NMR
C20H19FN2O: C, 74.51; H, 5.94; N, 8.69. Found: C, 74.46; H, 6.05; N,
8.73.
8c: pale yellow solid, yield 45%, m.p. 100e102 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.28e8.26 (m, 1H, ArH), 7.95 (d, J ¼ 15.3 Hz,
1H, ¼CH), 7.74e7.69 (m, 2H, ArH), 7.54e7.52 (m, 2H, ArH), 7.46e7.42
(m, 1H, ArH), 7.41e7.38 (m, 2H, ArH), 7.07 (d, J ¼ 15.3 Hz, 1H, ¼CH),
4.25 (t, J ¼ 7.8 Hz, 2H, NCH2), 1.82e1.75 (m, 2H, CH2), 1.54e1.45 (m,
(125 MHz, CDCl3) d: 161.6, 153.0, 148.8, 148.4, 140.5, 139.0, 137.3,
134.3, 130.0, 128.3, 127.3, 126.8, 124.0, 122.6, 121.6, 119.2, 55.9, 45.4,
45.4, 42.3, 26.6. APCI-MS m/z: 413.10 (Mþ1)þ. Anal. Calcd for
2H, CH2), 1.02 (t, J ¼ 7.4 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3)
d:
C
21H21ClN4O3: C, 61.09; H, 5.13; N, 13.57. Found: C, 61.02; H, 5.14; N,
162.1, 151.7, 147.5, 139.8, 135.6, 134.2, 134.0, 129.2, 129.2, 128.9, 128.9,
127.2, 126.9, 126.5, 120.6, 119.5, 43.4, 31.3, 20.2, 13.8. APCI-MS m/z:
339.09 (Mþ1)þ. Anal. Calcd for C20H19ClN2O: C, 70.90; H, 5.65; N,
8.27. Found: C, 70.78; H, 5.76; N, 8.13.
13.45.
7g: yellow solid, yield 26%, m.p. 102e105 ꢁC, 1H NMR (500 MHz,
CDCl3)
: 8.18 (d, J ¼ 8.5 Hz, 1H, ArH), 7.98 (d, J ¼ 15.2 Hz, 1H, ¼CH),
d

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G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
8d: pale yellow solid, yield 55%, m.p. 78e84 ꢁC, 1H NMR
(500 MHz, CDCl3)
20.0, 14.1. APCI-MS m/z: 321.11 (Mþ1)þ. Anal. Calcd for C20H20N2O2:
d: 8.30e8.26 (m, 2H, ArH, ¼CH), 7.76e7.72 (m,
C, 74.98; H, 6.29; N, 8.74. Found: C, 75.09; H, 6.26; N, 8.73.
2H, ArH), 7.66e7.64 (m, 2H, ArH), 7.47e7.44 (m, 1H, ArH), 7.37 (t,
J ¼ 7.5 Hz, 1H, ArH), 7.26e7.22 (m, 1H, ArH), 7.06 (d, J ¼ 15.3 Hz,
1H, ¼CH), 4.24 (t, J ¼ 7.8 Hz, 2H, NCH2), 1.83e1.75 (m, 2H, CH2),
1.53e1.44 (m, 2H, CH2), 1.00 (t, J ¼ 7.4 Hz, 3H, CH3); 13C NMR
4.3. Materials and cell lines
Caspase-3 assay kit was purchased from Biovision (Milpitas, CA,
USA). Anti-phospho p53 (s392) (P-p53), anti-phospho Cdk2 (T14)
(p-Cdk2) anti-actin, anti-p53, anti-p21, anti-Bim, anti-Bax and anti-
AIF antibodies were purchased from Abcam (Cambridge, MA). Anti-
Bcl-2 antibody, and anti-rabbit and -mouse secondary antibodies
were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX,
USA). RNase A, Propidium iodide (PI), 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide (MTT) and Hoechst 33258 were
purchased from Sigma (St. Louis, MO).
MGC-803, HepG2, NCIeH460, A375 and WI-38 cell lines were
purchased from ATCC (Manassas, VA). All cancer cell lines were
cultured in RPMI 1640 medium (GIBCO-BRL, Grand Island, NY)
supplemented with 10% fetal calf serum (FCS), while the normal
lung fibroblast WI-38 cells were cultured in DMEM medium
(GIBCO-BRL, Grand Island, NY) supplemented with 10% fetal calf
serum (FCS).
(125 MHz, CDCl3) d: 162.1, 151.6, 147.5, 139.7, 135.7, 134.2, 133.6,
130.7, 127.8, 127.8, 127.6, 126.9, 126.6, 125.1, 122.1, 120.7, 43.6, 31.3,
20.2, 13.8. APCI-MS m/z: 383.00, 385.00 (Mþ1)þ. Anal. Calcd for
C
20H19BrN2O: C, 62.67; H, 5.00; N, 7.31. Found: C, 62.58; H, 5.09; N,
7.22.
8e: pale yellow solid, yield 52%, m.p. 141e143 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.29 (d, J ¼ 8.7 Hz, 3H, ArH), 8.04 (d, J ¼ 15.4 Hz,
1H, ¼CH), 7.79e7.70 (m, 4H, ArH), 7.50e7.46 (m, 1H, ArH), 7.24 (d,
J ¼ 15.4 Hz, 1H, ¼CH), 4.27 (t, J ¼ 7.8 Hz, 2H, NCH2), 1.84e1.76 (m,
2H, CH2), 1.56e1.46 (m, 2H, CH2), 1.02 (t, J ¼ 7.4 Hz, 3H, CH3); 13C
NMR (125 MHz, CDCl3) d: 161.9, 151.0, 148.1, 147.3, 141.6, 138.3,
134.4,128.3,128.3,127.3,127.0,127.0,124.3, 124.3,123.2,120.8, 43.6,
31.4, 20.2, 13.8. APCI-MS m/z: 350.10 (Mþ1)þ. Anal. Calcd for
C
20H19N3O3: C, 68.75; H, 5.48; N, 12.03. Found: C, 68.64; H, 5.36; N,
11.95.
8f: pale yellow solid, yield 25%, m.p. 121e123 ꢁC, 1H NMR
(500 MHz, CDCl3)
d: 8.46 (t, J ¼ 1.9 Hz, 1H, ArH), 8.29 (dd, J ¼ 8.0,
4.4. Cell viability assay
0.9 Hz, 1H, ArH), 8.24 (dd, J ¼ 8.2, 1.3 Hz, 1H, ArH), 8.05 (d,
J ¼ 15.4 Hz, 1H, ¼CH), 7.90 (d, J ¼ 7.7 Hz, 1H, ArH), 7.79e7.70 (m, 2H,
ArH), 7.62 (t, J ¼ 7.9 Hz, 1H, ArH), 7.50e7.45 (m, 1H, ArH), 7.23 (d,
J ¼ 15.3 Hz, 1H, ¼CH), 4.28 (t, J ¼ 7.8 Hz, 2H, NCH2), 1.85e1.77 (m,
2H, CH2), 1.56e1.47 (m, 2H, CH2), 1.03 (t, J ¼ 7.4 Hz, 3H, CH3); 13C
MGC-803, HepG2, NCIeH460, A375 and WI-38 cells were har-
vested from culture dishes by trypsinization and seeded into 96-
well microculture plates with supplemented culture medium
(200
m
L/well) at a density of 2 ꢀ 103 cells for 24 h. Then, the me-
NMR (125 MHz, CDCl3)
d
: 162.0, 151.1, 148.8, 147.3, 138.4, 137.2,
dium was replaced by solutions (200 mL/well) containing various
134.3,133.5,130.0,127.3,127.0,126.9,124.0,122.0,122.0,120.8, 43.4,
concentrations (ranging from 0.1 to 20
(dissolved in DMSO) for 48 h. Cell proliferation was monitored
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
mM) of compounds 4e8
31.4, 20.2, 13.8. APCI-MS m/z: 350.16 (Mþ1)þ. Anal. Calcd for
C
20H19N3O3: C, 68.75; H, 5.48; N, 12.03. Found: C, 68.78; H, 5.56; N,
12.13.
8g: pale yellow solid, yield 21%, m.p. 122e126 ꢁC, 1H NMR
(500 MHz, CDCl3)
diphenyltetrazolium bromide) assay. MTT (5 mg/mL) was added
to the wells and the plates were incubated for 4 h at 37 ꢁC. The MTT
reaction was stopped by adding dimethyl sulfoxide (150 mL per
well) and mixed for 10 min vigorously before measuring absor-
bance at 490 nm in a multi-well plate reader. Cell viability was
d
: 8.27e8.25 (m, 1H, ArH), 7.96 (d, J ¼ 15.2 Hz,
1H, ¼CH), 7.75e7.67 (m, 2H, ArH), 7.45e7.39 (m, 1H, ArH), 7.21 (dd,
J ¼ 8.3, 1.9 Hz, 1H, ArH), 7.10 (d, J ¼ 1.9 Hz, 1H, ArH), 6.96 (d,
J ¼ 15.2 Hz, 1H, ¼CH), 6.91 (d, J ¼ 8.3 Hz, 1H, ArH), 4.26 (t, J ¼ 7.8 Hz,
2H, NCH2), 3.94 (s, 3H, OCH3), 3.93 (s, 3H, OCH3), 1.83e1.77 (m, 2H,
CH2), 1.53e1.48 (m, 2H, CH2), 1.03 (t, J ¼ 7.4 Hz, 3H, CH3); 13C NMR
calculated using the following formula: cell viability (%) ¼ (A490e
/
A
490c) ꢀ 100%, A490e and A490c represented the absorbance values
from the experimental and control groups, respectively.
(125 MHz, CDCl3) d: 162.2, 152.2, 150.7, 149.2, 147.6, 141.1, 134.2,
128.5, 127.1, 126.9, 126.2, 121.6, 120.4, 116.7, 111.2, 110.1, 56.1, 55.9,
4.5. Analyses of cell cycle and cell size
43.3, 31.1, 20.2, 13.8. APCI-MS m/z: 365.16 (Mþ1)þ. Anal. Calcd for
C
22H24N2O3: C, 72.50; H, 6.64; N, 7.69. Found: C, 72.48; H, 6.56; N,
Cell cycle and cell size analyses were carried out by flow
cytometry (FCM). MGC-803 cells were harvested from adherent
cultures by trypsinization. Following centrifugation at 1000 rpm for
5 min, cells were washed with PBS and then fixed with 70% ethanol
in PBS. Fixed cells were collected by centrifugation at 2500 rpm for
3 min, washed with PBS and centrifuged as before. Cellular pellets
7.73.
8h: pale yellow solid, yield 40%, m.p. 164e166 ꢁC, 1H NMR
(500 MHz, CDCl3)
d
: 8.26 (dd, J ¼ 8.0, 1.0 Hz, 1H, ArH), 7.92 (d,
J ¼ 15.2 Hz, 1H, ¼CH), 7.74e7.67 (m, 2H, ArH), 7.44e7.40 (m, 1H,
ArH), 7.10e7.08 (m, 2H, ArH), 6.92 (d, J ¼ 15.2 Hz, 1H, ¼CH), 6.85 (d,
J ¼ 7.9 Hz, 1H, ArH), 6.03 (s, 2H, 6H, OCH2O), 4.24 (t, J ¼ 7.8 Hz, 2H,
NCH2), 1.82e1.75 (m, 2H, CH2), 1.54e1.43 (m, 2H, CH2), 1.02 (t,
were re-suspended in 50
mg/mL propidium iodide (Sigma) in PBS
for nucleic acids staining and treated with 100
mg/mL RNaseA
J ¼ 7.4 Hz, 3H, CH3); 13C NMR (125 MHz, CDCl3)
d
: 162.2, 152.2,
(Sigma). Cell cycle distribution was analyzed in a Becton Dickinson
FACSAriaII flow cytometer (San Jose, CA) using ModFit LT software,
and mean FSC-H population was used to determine relative cell
size.
149.2, 148.4, 147.6, 140.9, 134.2, 129.9, 127.1, 126.9, 126.2, 124.1,
120.5, 116.9, 108.7, 106.2, 101.6, 43.4, 31.2, 20.2, 13.8. APCI-MS m/z:
349.15 (Mþ1)þ. Anal. Calcd for C21H20N2O3: C, 72.40; H, 5.79; N,
8.04. Found: C, 72.48; H, 5.96; N, 7.95.
8i: pale yellow solid, yield 31%, m.p. 163e165 ꢁC, 1H NMR
4.6. Fluorescence confocal microscopy
(500 MHz, DMSO-d6)
d
: 9.59 (s, 1H, OH), 8.12 (d, J ¼ 8.0 Hz,1H, ArH),
7.84 (d, J ¼ 15.3 Hz, 1H, ¼CH), 7.81e7.78 (m, 1H, ArH), 7.68 (d,
J ¼ 8.0 Hz, 1H, ArH), 7.49e7.46 (m, 1H, ArH), 7.30 (d, J ¼ 15.3 Hz,
1H, ¼CH), 7.27e7.20 (m, 2H, ArH), 7.16 (s, 1H, ArH), 6.84 (dd, J ¼ 8.0,
1.2 Hz, 1H, ArH), 4.29 (t, J ¼ 7.5 Hz, 2H, NCH2), 1.68e1.62 (m, 2H,
CH2), 1.42e1.35 (m, 2H, CH2), 0.93 (t, J ¼ 7.4 Hz, 3H, CH3); 13C NMR
MGC-803 cells were cultured on glass slide treated with com-
pound 6c. Forty-eight hours after treatment, the cells were washed
with PBS, fixed in 4% paraformaldehyde, and permeablized with 1%
Triton X-100. The cells were washed for four times, and a final
concentration of 0.5 mg/mL Hoechst 33258 (Sigma) was included in
(125 MHz, DMSO-d6)
d
: 161.6, 158.3, 152.5, 147.6, 140.9, 137.0, 134.8,
the final wash to stain the nuclei. The images were visualized with a
Zeiss 710 fluorescence confocal microscope and either Plan-
130.4, 127.5, 126.8, 126.7, 120.4, 120.0, 119.7, 117.5, 114.8, 42.7, 31.3,

G.-H. Zhang et al. / European Journal of Medicinal Chemistry 95 (2015) 377e387
387
N. Lukashchuk, D.A. Gillespie, R.L. Ludwig, P. Gosselin, A. Cromer, J.S. Brugge,
O.J. Sansom, J.C. Norman, K.H. Vousden, Cell 139 (2009) 1327e1341.
[7] T. Cooks, I.S. Pateras, O. Tarcic, H. Solomon, A.J. Schetter, S. Wilder, G. Lozano,
E. Pikarsky, T. Forshew, N. Rosenfeld, N. Harpaz, S. Itzkowitz, C.C. Harris,
V. Rotter, V.G. Gorgoulis, M. Oren, Cancer Cell 23 (2013) 634e646.
[8] P.A. Muller, A.G. Trinidad, P. Timpson, J.P. Morton, S. Zanivan, P.V. van-den
Berghe, C. Nixon, S.A. Karim, P.T. Caswell, J.E. Noll, C.R. Coffill, D.P. Lane,
O.J. Sansom, P.M. Neilsen, J.C. Norman, K.H. Vousden, Oncogene 32 (2013)
1252e1265.
Neofluar 20 ꢀ or 40 ꢀ objectives, appropriate filters for Alexa 488,
546, or Hoechst and Zeiss AxioCam HRc color camera. For immu-
nostaining, 48 h after treatment, the cells were washed with PBS,
fixed with 4% paraformaldehyde, and incubated with appropriate
primary antibodies followed by the addition of Cy3-conjugated
secondary antibodies.
[9] P.A. Muller, A.G. Trinidad, P.T. Caswell, J.C. Norman, K.H. Vousden, J. Biol.
Chem. 289 (2014) 122e132.
4.7. In vitro assay of caspase-3 activity
[10] C. Kandoth, M.D. McLellan, F. Vandin, K. Ye, B. Niu, C. Lu, M. Xie, Q. Zhang,
J.F. McMichael, M.A. Wyczalkowski, M.D. Leiserson, C.A. Miller, J.S. Welch,
M.J. Walter, M.C. Wendl, T.J. Ley, R.K. Wilson, B.J. Raphael, L. Ding, Nature 502
(2013) 333e339.
[11] B. Leroy, J.L. Fournier, C. Ishioka, P. Monti, A. Inga, G. Fronza, T. Soussi, Nucleic
Acids Res. 41 (2013) 962e969.
[12] P.M. Chandrika, T. Yakaiah, A.R. Rao, B. Narsaiah, N.C. Reddy, V. Sridhar,
J.V. Rao, Eur. J. Med. Chem. 43 (2008) 846e852.
[13] X. Wang, C.X. Zhou, J.W. Yan, J.Q. Hou, S.B. Chen, T.M. Ou, L.Q. Gu, Z.S. Huang,
J.H. Tan, ACS Med. Chem. Lett. 4 (2013) 909e914.
[14] B. Maggio, G. Daidone, D. Raffa, S. Plescia, L. Mantione, V.M. Catena Cutuli,
N.G. Mangano, A. Caruso, Eur. J. Med. Chem. 36 (2001) 737e742.
[15] G. Grover, S.G. Kini, Eur. J. Med. Chem. 41 (2006) 256e262.
[16] M. Sharma, K. Chauhan, R. Shivahare, P. Vishwakarma, M.K. Suthar, A. Sharma,
S. Gupta, J.K. Saxena, J. Lal, P. Chandra, B. Kumar, P.M. Chauhan, J. Med. Chem.
56 (2013) 4374e4392.
[17] M. Casagrande, A. Barteselli, N. Basilico, S. Parapini, D. Taramelli, A. Sparatore,
Bioorg. Med. Chem. 20 (2012) 5965e5979.
[18] P. Verhaeghe, N. Azas, M. Gasquet, S. Hutter, C. Ducros, M. Laget, S. Rault,
P. Rathelot, P. Vanelle, Bioorg. Med. Chem. Lett. 18 (2008) 396e401.
[19] R.S. Giri, H.M. Thaker, T. Giordano, J. Williams, D. Rogers, V. Sudersanam,
K.K. Vasu, Eur. J. Med. Chem. 44 (2009) 2184e2189.
[20] A. Kumar, C.S. Rajput, S.K. Bhati, Bioorg. Med. Chem. 15 (2007) 3089e3096.
[21] R.A. Smits, M. Adami, E.P. Istyastono, O.P. Zuiderveld, C.M. van-Dam, F.J. de-
Kanter, A. Jongejan, G. Coruzzi, R. Leurs, I.J. de-Esch, J. Med. Chem. 53 (2010)
2390e2400.
[22] V. Alagarsamy, V. Raja-Solomon, M. Murugan, K. Dhanabal, P. Parthiban,
G.V. Anjana, J. Enzyme Inhib, Med. Chem. 23 (2008) 839e847.
[23] M.A. Ismail, S. Barker, D.A. Abou el-Ella, K.A. Abouzid, R.A. Toubar, M.H. Todd,
J. Med. Chem. 49 (2006) 1526e1535.
[24] K.S. Jain, J.B. Bariwal, M.K. Kathiravan, M.S. Phoujdar, R.S. Sahne, B.S. Chauhan,
A.K. Shah, M.R. Yadav, Bioorg. Med. Chem. 16 (2008) 4759e4800.
[25] S.K. Kashaw, V. Kashaw, P. Mishra, N.K. Jain, J.P. Stables, Eur. J. Med. Chem. 44
(2009) 4335e4343.
[26] V. Jatav, P. Mishra, S. Kashaw, J.P. Stables, Eur. J. Med. Chem. 43 (2008)
135e141.
The assay was performed based on the ability of the active
enzyme to cleave the chromophore from the enzyme substrate
FITC-DEVD-FMK (for caspase-3). The inhibitor-treated cells and
control cells were harvested at a density of 1 ꢀ 106 cells/mL in RPMI
1640 medium supplemented with 10% FCS. 300
m
L each of the
L of FITC-
induced and control cultures were incubated with 1
m
DEVD-FMK for 1 h in a 37 ꢁC incubator with 5% CO2. Flow cyto-
metric analysis was performed using a FACS AriaII flow cytometer
equipped with a 488 nm argon laser. Results are represented as the
percent change of the activity compared to the untreated control.
4.8. Western blotting
MGC-803 cells (3 ꢀ 105) were cultured in each well of 6-well
plates. The cells were exposed to 6b or 6c (0e10 mM) for 24 h,
and then washed once with ice-cold PBS and extracted with the
sample buffer. The cell extracts were separated on polyacrylamide-
SDS gels, transferred to nitrocellulose membrane and probed with
primary antibodies as indicated. The membrane was incubated
with anti-rabbit IgG (AP-linked) and developed by an ECL Western
blot system (Kodak, USA).
4.9. Statistical analysis
All data are shown as mean
standard deviation (SD) using
two-tailed Student t tests and one-way ANOVA with Bonferroni
multiple comparison post-test. P less than 0.05 was considered as
significant difference.
[27] M. Decker, Eur. J. Med. Chem. 40 (2005) 305e313.
[28] J.E. Girardini, C. Marotta, G. Del-Sal, Pharmacol. Res. 79 (2014) 75e87.
[29] M. Napoli, J.E. Girardini, S. Piazza, G. Del-Sal, Oncotarget 2 (2011) 654e656.
[30] C.L. Brooks, W. Gu, Curr. Opin. Cell Biol. 15 (2003) 167e171.
[31] K.L. Harms, X. Chen, Cancer Res. 67 (2007) 3145e3152.
[32] S. Minucci, P.G. Pelicci, Nat. Rev. Cancer 6 (2006) 38e51.
[33] Y. Tang, W. Zhao, Y. Chen, Y. Zhao, W. Gu, Cell 133 (2008) 612e626.
[34] S. Kumar, H. Kaur, I. Singh, M. Sharma, P. Vishwakarma, K.K. Saxena, A. Kumar,
World J. Chem. 4 (2009) 195e200.
[35] A. Kamal, E. Vijaya-Bharathi, M. Janaki-Ramaiah, D. Dastagiri,
J. Surendranadha-Reddy, A. Viswanath, F. Sultana, S.N. Pushpavalli, M. Pal-
Bhadra, H.K. Srivastava, G. Narahari-Sastry, A. Juvekar, S. Sen, S. Zingde, Bio-
org. Med. Chem. 18 (2010) 526e542.
[36] D. Wang, J. Liang, Y. Zhang, B. Gui, F. Wang, X. Yi, L. Sun, Z. Yao, Y. Shang,
J. Biol, Chem. 287 (2012) 12612e12621.
[37] S.Y. Xu, D.J. Xiao, Y.J. Luan, Y.S. Wang, L. Wang, K. Shi, J. Shandong Univ.
(Health Sci.) 46 (2008) 478e480 (Chinese).
[38] C.M. Fenoglio-Preiser, J. Wang, G.N. Stemmermann, A. Noffsinger, Hum.
Mutat. 21 (2003) 258e270.
Acknowledgments
This work was supported by the National Natural Science
Foundation of China (21262007, 21462008, 21162002 and
81401912), Ministry of Education of China (IRT1225), Guangxi Sci-
entific Research and Technology Development Project (13349002),
Natural Science Foundation of Guangxi (2011GXNSFA018042), State
Key Laboratory Cultivation Base for the Chemistry and Molecular
Engineering of Medicinal Resources, Ministry of Science and
Technology of China (CMEMR2014-A01, CMEMR2012-A04 and
CMEMR 2011-17), Guilin Science and Technology Development
Fund (20110331).
[39] T. Wang, J. Xu, N.S. Zhong, Zhonghua Jie He He Hu Xi Za Zhi 28 (2005)
102e107 (Chinese).
[40] L. Yang, Y. Zhou, Y. Li, J. Zhou, Y. Wu, Y. Cui, G. Yang, Y. Hong, Cancer Lett. 357
(2015) 520e526.
[41] T. Zheng, D. Yin, Z. Lu, J. Wang, Y. Li, X. Chen, Y. Liang, X. Song, S. Qi, B. Sun,
C. Xie, X. Meng, S. Pan, J. Liu, H. Jiang, L. Liu, Mol. Cancer 13 (2014) 1e12.
[42] A. Petitjean, E. Mathe, S. Kato, C. Ishioka, S.V. Tavtigian, P. Hainaut, M. Olivier,
Hum. Mutat. 28 (2007) 622e629.
[43] X.L. Li, M.F. Jones, M. Subramanian, A. La, FEBS Lett. 5793 (2014) 273e277.
[44] R.M. Golsteyn, Cancer Lett. 217 (2005) 129e138.
References
[1] J. Bai, M. Ma, M. Cai, F. Xu, J. Chen, G. Wang, X. Shuai, K. Tao, Cell Prolif. 47
(2014) 211e218.
[2] P.A. Muller, K.H. Vousden, Cancer Cell. 25 (2014) 304e317.
[3] D. Li, N.D. Marchenko, U.M. Moll, Cell Death Differ. 18 (2011) 1904e1913.
[4] N. McCarthy, Nat. Rev. Cancer 14 (2014) 385.
[5] P.A. Muller, K.H. Vousden, J.C. Norman, J. Cell Biol. 192 (2011) 209e218.
[6] P.A. Muller, P.T. Caswell, B. Doyle, M.P. Iwanicki, E.H. Tan, S. Karim,