Synthesis and Biological Evaluation of Selenium-Containing 4-Anilinoquinazoline Derivatives as Novel Antimitotic Agents

Article abstract of DOI:10.1021/acs.jmedchem.8b00128

Twenty-eight novel selenium-containing 4-anilinoquinazoline derivatives were designed, synthesized, and evaluated as antiproliferative agents. Most of them had significant in vitro activities, particularly for compounds 23a, 25a, and 25d, which also exhibited the most potent antitumor activities against cisplatin-resistant cell lines and the doxorubicin-resistant cell lines, good selectivity toward normal cells, and obvious inhibitory effect on migration of A549 cell lines. Further mechanistic studies revealed that 23a, 25a, and 25d induce G2/M phase arrest and apoptosis in A549 cells, which was associated with a collapse of the mitochondrial membrane potential, alterations in the expression of some cell cycle-related and apoptosis-related proteins, and increasing the intracellular ROS level. Finally, compounds 23a, 25a, and 25d also effectively inhibited the tumor growth in the A549 xenograft model without obvious hints of toxicity. Taken together, these in vitro and in vivo results suggest that 23a, 25a, and 25d may be promising microtubule-stabilizing agents and can be used as a promising lead for the development of new antitumor agents.

Full text of DOI:10.1021/acs.jmedchem.8b00128

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Synthesis and Biological Evaluation of Selenium-Containing
4‑Anilinoquinazoline Derivatives as Novel Antimitotic Agents
Baijiao An, Bo Wang, Jinhui Hu, Shaoyu Xu, Ling Huang,* Xingshu Li,* and Albert S. C. Chan
School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, China
S
* Supporting Information
ABSTRACT: Twenty-eight novel selenium-containing 4-
anilinoquinazoline derivatives were designed, synthesized,
and evaluated as antiproliferative agents. Most of them had
significant in vitro activities, particularly for compounds 23a,
25a, and 25d, which also exhibited the most potent antitumor
activities against cisplatin-resistant cell lines and the doxor-
ubicin-resistant cell lines, good selectivity toward normal cells,
and obvious inhibitory effect on migration of A549 cell lines.
Further mechanistic studies revealed that 23a, 25a, and 25d induce G2/M phase arrest and apoptosis in A549 cells, which was
associated with a collapse of the mitochondrial membrane potential, alterations in the expression of some cell cycle-related and
apoptosis-related proteins, and increasing the intracellular ROS level. Finally, compounds 23a, 25a, and 25d also effectively
inhibited the tumor growth in the A549 xenograft model without obvious hints of toxicity. Taken together, these in vitro and in
vivo results suggest that 23a, 25a, and 25d may be promising microtubule-stabilizing agents and can be used as a promising lead
for the development of new antitumor agents.
In recent years, the potential therapeutic effects of selenium
for cancer treatment have attracted great interest. Some
selenium compounds, including selenocyclodextrin, phenyl-
butyl isoselenocyanate, selenomethionine, sodium selenite,
diaryl selenide, 3,4-diaryl-1,2,5-selenadiazol analogues, and
selenoaspirin analogues, have been shown to inhibit cancer
cell growth in vitro or in various xenograft rodent models for
different cancers types.14−21 Additionally, selenium has been
reported to have a protective effect against some types of cancer
and might also enhance male fertility in patients undergoing
chemotherapy.22 Recently, reports indicated that supplemental
selenium may decrease ovarian cancer risk in African-American
women, and selenium reduced doxorubicin gonadotoxicity in
male rats.23
Inspired by the synergistic effects of organoselenium
compounds in combination with chemotherapeutic
drugs,24−26 herein we report the modification of EP128495
by introducing selenocyanato or methylseleno at various
positions of the anilino moiety of 4-anilinoquinazoline to
obtain diverse derivatives. These derivatives were evaluated for
antitumor activities in vitro and in vivo, and their underlying
cytotoxic mechanisms were also elucidated
INTRODUCTION
■
The high incidence of cancer and the high cost of its treatment
are important factors driving the search for new and effective
chemotherapeutic substances with multitarget activity and no
toxicity to normal cells. Among the variety of cancer drug
targets, tubulin polymerization is an excellent choice as the
microtubule system plays a critical role in essential cellular
processes including the movement of organelles, intracellular
transportation, and the formation and maintenance of cell
shape.1−4 In addition, several drugs (e.g., paclitaxel, docetaxel,
and vinblastine) act on this target and have been used
clinically,5,6 and additional candidates are also in clinical trials.
Among them, EP128495 (Figure 1), developed by Cai et.al.,
shows potent and broad-spectrum in vitro and in vivo cytotoxic
activities and has reached a phase II clinical trial for the
treatment of recurrent glioblastoma.7,8
Selenium (Se) is an essential trace mineral nutrient with
multiple roles in human health.9 It is known that 25
selenoproteins in the human body exert specific biological
functions; for example, glutathione peroxidase, one of the
selenoproteins, efficiently metabolizes cellular peroxides and
functions as an antioxidant defense mechanism that protects
against reactive oxygen and nitrogen species.10 Selenium is also
known to provide protection from free-radical-induced cell
damage.11 Numerous studies reported that the levels of
selenium in humans are correlated with the incidence of
many diseases. Selenium compounds have been used as
promising chemopreventive agents for some diseases in the
past few decades, including Keshan disease and Kashin−Beck
disease.12,13
RESULTS AND DISCUSSION
■
Chemistry. The synthesis of new selenium-containing 4-
anilinoquinazoline derivatives 7a−f is shown in Scheme 1. First,
2-hydroxy-5-nitrobenzoic acid reacted with dimethyl sulfate and
then was hydrogenated in the presence of Pd/C to give
Received: January 24, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 1. Design strategy of selenium-containing 4-anilinoquinazoline derivatives.
a
Scheme 1
a
Reagents and conditions: (a) K2CO3, dimethyl sulfate, acetone, 60 °C. (b) Pd/C, H2, MeOH. (c) HAc, (CH2O)n, NaBH3CN, rt. (d) 4-Chloro-2-
methylquinazoline, HCl, IPA, rt. (e) NaOH, H2O, MeOH, rt. (f) (1) Br(CH2)nBr, K2CO3, CH3CN, reflux. (2) SOCl2, NH2CH2CH2Br, DCM. (g)
KSeCN, CH3CN, reflux.
a
Scheme 2
a
Reagents and conditions: (a) Na, MeOH, reflux. (b) (CH2O)n, Na, NaBH4, MeOH, 60 °C. (c) 4-Chloro-2-methylquinazoline, HCl, IPA. (d) Pd/C,
H2, MeOH. (e) HCl, NaNO2, KSeCN.
intermediate 2. The reactions of 2 with paraformaldehyde and
sodium cyanoborohydride provided the secondary amine
intermediate 3. The reaction of 3 with 4-chloro-2-methyl-
quinazoline and the following hydrolyzation produced the acid
intermediate 5. Finally, the esterification of intermediate 5 with
the appropriate dibromoalkanes afforded compound 6 which
was reacted with KSeCN to give the target products 7a−f.
The synthesis of the target compounds 10, 18, and 21, which
contain 4-methoxy-3-selenocyanato at the anilino moiety or its
analogues, is summarized in Schemes 2, 3, and 4. The reaction
of 4-fluoro-3-nitroaniline with sodium methoxide in reflux gave
4-methoxy-3-nitroaniline, and the following alkylation afforded
the key intermediate 8. Compound 8 was reacted with 4-
chloro-2-methylquinazoline and then hydrogenated in the
presence of Pd/C to provide the intermediate 9. Finally, the
diazotization of 9 and the subsequently reaction with potassium
selenocyanate afforded the target product 10 (Scheme 2).
The selenocyanato-containing compounds 16a−e were
synthesized as shown in Scheme 3. The hydrogenation of
MOM-protected 2-methoxy-5-nitrophenol in the presence of
Pd/C provided compound 12, and subsequent alkylation gave
the key intermediate 13. By the reaction of 13 with 4-chloro-2-
B
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Scheme 3
a
Reagents and conditions: (a) MOMCl, DIPEA, DCM, 0 °C. (b) Pd/C, H2, MeOH. (c) (CH2O)n, Na, NaBH4, MeOH, 60 °C. (d) 4-Chloro-2-
methylquinazoline, HCl, IPA. (e) K2CO3, BrCH2CH2Br, CH3CN, reflux. (f) KSeCN, CH3CN, reflux.
a
Scheme 4
a
Reagents and conditions: (a) LiAlH4, THF, rt. (b) SOCl2, reflux. (c) KSeCN, CH3CN, reflux.
a
methylquinazoline, compound 14 was obtained. Finally, the
reaction of 14 with the appropriate dibromoalkane and then
with potassium selenocyanate afforded the target products.
Compound 19 was synthesized by the route shown in
Scheme 4. The ester group of intermediate 4 was reduced with
aluminum lithium hydride and then reacted with thionyl
chloride to give compound 17, which was converted to 18 by
the same method above.
Scheme 6
To investigate the role of selenium on the proliferative
activity of human cancer cells, compound 20 was also prepared
by the reaction of intermediate 15 with ammonium hydroxide
(Scheme 5).
a
Reagents and conditions: (a) (CH2O)n, Na, NaBH4, MeOH, 60 °C.
(b) Malononitrile, SeO2, DMSO, rt. (c) 4-Chloro-2-methylquinazoline
or its anlogues, HCl, IPA, rt.
a
Scheme 5
a
Scheme 7
a
Reagents and conditions: (a) NH4OH, rt.
a
Reagents and conditions: (a) MeI, NaBH4, rt.
Compounds 23a−f, for which selenocyanato is at the 4-
position of the anilino moiety, were synthesized as Scheme 6.
The alkylation of meta-substituted anilines by paraformalde-
hyde and sodium borohydride created compound 21, which
reacted with selenium dioxide and malononitrile to give 22.
Finally, compound 22 reacted with 4-chloroquinazoline to
provide target compounds 23a−f.
Compounds 25a−f, in which the methylseleno is at the 4-
position of the anilino moiety, were prepared by the reaction of
23 with sodium borohydride and methyl iodide (Scheme 7).
In Vitro Human Cancer Cell Line Growth Inhibition
and the SARs. Recently, Sharma group reported the design
and evaluation of Se-aspirin analogues as an effective agent in
reducing the viability of different cancer cell lines, and among
them, selenocyanato containing compounds exhibited the most
antiproliferative activity.21 Inspired by their work, we
synthesized compounds 7a−f and evaluated their antiprolifer-
ative activities against A549 (non-small-cell lung carcinoma),
MDAMB-231 (human breast carcinoma), HepG2 (liver
hepatocellular), HELA (human epithelial cervical cancer cell
line), HCT116, LOVO, and RKO (human colorectal
carcinoma) cell lines using the MTT (3-(4,5- dimethylthiazol-
2-yl)-2,5-diphenyltetrazolium bromide) assay. As shown in
Table 1, most of the compounds of this series exhibited good
C
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 1. Antiproliferative Activity of Target Compounds against Six Human Cancer Cell Lines
b
IC50 (μM)
compd
7a
A549
HCT116
HEPG2
MDAMB-231
LOVO
RKO
0.187 0.009
0.353 0.013
0.250 0.044
0.155 0.009
0.592 0.007
0.488 0.018
0.049 0.014
0.013 0.001
0.382 0.004
1.20 0.053
2.44 0.033
5.52 0.042
0.098 0.003
0.122 0.031
0.003 0.002
0.313 0.013
0.951 0.051
0.720 0.015
0.662 0.040
0.551 0.005
1.99 0.017
0.034 0.008
0.018 0.004
0.852 0.021
0.512 0.009
0.256 0.007
6.18 0.023
0.113 0.011
0.198 0.004
0.004 0.001
0.179 0.005
0.890 0.012
0.34 0.004
0.975 0.008
0.667 0.017
2.65 0.097
0.059 0.005
0.014 0.001
1.04 0.012
6.46 0.113
5.21 0.024
4.89 0.092
1.11 0.065
0.343 0.013
0.005 0.002
1.25 0.025
1.48 0.016
1.18 0.013
0.554 0.023
0.851 0.018
2.21 0.074
0.052 0.018
0.013 0.005
0.815 0.007
1.35 0.024
4.41 0.035
3.55 0.016
0.153 0.011
0.682 0.003
0.008 0.001
0.561 0.015
2.96 0.087
0.854 0.006
1.08 0.060
0.714 0.005
1.05 0.092
0.073 0.006
0.019 0.003
0.921 0.015
0.882 0.067
5.21 0.006
8.58 0.153
0.277 0.005
0.421 0.006
0.008 0.0001
1.17 0.036
2.39 0.045
1.14 0.034
1.02 0.112
0.356 0.011
1.99 0.036
0.047 0.004
0.027 0.067
0.799 0.035
1.360 0.059
3.56 0.035
1.54 0.062
0.316 0.031
0.343 0.005
7b
7c
7d
7e
7f
10
16a
16b
16c
16d
16e
19
20
EP128495
0.003 0.0002
a
b
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the Experimental Section. IC50 values
are indicated as the mean SD (standard error) of at least three independent experiments.
a
Table 2. Antiproliferative Activity of Target Compounds against Six Human Cancer Cell Lines
b
IC50 (μM)
compd
23a
A549
HCT116
HEPG2
MDAMB-231
LOVO
RKO
0.011 0.001
0.008 0.005
0.007 0.002
0.004 0.001
0.007 0.001
0.012 0.007
0.893 0.031
0.002 0.003
0.002 0.001
0.002 0.0004
0.002 0.0001
0.003 0.0001
0.013 0.001
0.011 0.001
0.003 0.002
0.015 0.001
0.013 0.004
0.012 0.001
0.020 0.002
0.010 0.004
0.062 0.007
2.29 0.015
0.003 0.002
0.003 0.001
0.005 0.001
0.006 0.0001
0.003 0.001
0.015 0.006
0.028 0.004
0.004 0.001
0.009 0.001
0.009 0.001
0.012 0.001
0.037 0.005
0.021 0.002
0.041 0.002
1.68 0.098
0.004 0.001
0.006 0.001
0.004 0.001
0.004 0.0002
0.005 0.0002
0.032 0.006
0.017 0.002
0.005 0.002
0.020 0.0001
0.016 0.003
0.009 0.001
0.006 0.0002
0.011 0.001
0.029 0.001
0.317 0.004
0.004 0.001
0.007 0.001
0.007 0.002
0.005 0.001
0.009 0.001
0.018 0.001
0.010 0.001
0.008 0.001
0.032 0.003
0.019 0.003
0.012 0.003
0.013 0.003
0.014 0.003
0.011 0.003
0.547 0.006
0.009 0.001
0.009 0.001
0.003 0.001
0.009 0.0001
0.005 0.007
0.011 0.003
0.003 0.005
0.008 0.0001
0.009 0.006
0.017 0.002
0.011 0.001
0.008 0.004
0.011 0.004
0.051 0.007
0.652 0.014
0.004 0.001
0.008 0.001
0.004 0.001
0.007 0.0001
0.008 0.001
0.021 0.004
0.011 0.001
23b
23c
23d
23e
23f
24
25a
25b
25c
25d
25e
25f
26
EP128495
0.003 0.0002
a
b
Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the Experimental Section. IC50 values
are indicated as the mean SD (standard error) of at least three independent experiments.
antiproliferative activity with IC50 values at submicromolar
level. However, reference compound EP128495 exhibits a more
excellent antiproliferative activity, which urged us to search for
potential compounds with better activities. We then evaluated
D
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
a
Table 3. Antiproliferative Activity of Optimal Compounds against Other Human Cancer Cell Lines
IC50 (μM)
compd
23a
25a
25d
EP128495
MCF7
0.019 0.006
0.009 0.002
0.012 0.001
0.010 0.003
0.003 0.001
0.007 0.002
0.009 0.004
0.005 0.001
0.007 0.002
0.011 0.002
0.005 0.003
0.008 0.001
HELA
MGC803
a
Data are presented as the mean SE from the dose−response curves of at least three independent experiments.
Figure 2. The human cancer cell lines were treated with 25a at the indicated concentration or DMSO (0.01%) for 48 h, and the detection of the
fixed cells morphology was performed with microscope. All images were collected with a 20× objective.
the antiproliferative activity of compounds 10, 16, and 19, for
which the selenocyanato is linked at the 3-position of the
anilino moiety either directly or by a linker. The results in
Table 1 showed that the position of the selenocyanato was
important for the antiproliferative activity. Compound 10,
which has the selenocyanato linked directly at the 3-position of
anilino moiety, exhibited very good activity with IC50 values
that ranged from 34 to 73 nM against the six human cancer cell
lines. The activity increased obviously when the selenocyanato
was linked by a two-carbon ether chain (16a, IC50 values
ranged from 13 to 27 nM) but decreased with an increase in
the chain length (16b, n = 3, IC50 values ranged from 0.382 to
1.038 μM; 16e, n = 5, IC50 values ranged from 1.541 to 8.583
μM), which revealed that the length of the linkage has a great
effect on the inhibition of cancer cell proliferation. Compound
19, in which the selenocyanato is linked at the 3-position of the
anilino moiety via a methylene, had IC50 values that ranged
from 0.098 to 1.105 μM, which is lower than that of
compounds 10 and 16a, suggesting that the ether chain is
necessary in this series. Finally, by comparison of the IC50
values of 20 and 16a, a difference of nearly 5 times the
antiproliferative activity indicated that selenocyanato is indeed
beneficial for the activity.
The antiproliferative activities of compounds 23a−f, with
selenocyanato at the 4-position of the anilino moiety with
different substitutes at the 3-position, are listed in Table 2. All
the members of this series of analogues exhibited excellent
activities with IC50 values that ranged from 4 to 62 nM. There
was not much activity difference against the six cancer cell lines,
regardless of whether the substitutions at 3-position were
hydrogen, methyl group, or a halogen. Similarly, the
introduction of a CD3 group at the R1 position instead of a
methyl group also gave nearly the same activities. As the
presence of a cyclopropyl group is very crucial in many drugs,
we introduced this group at the R1 position to obtain
compound 23f. Compared with 23a, we found the anti-
proliferative activities of 23f were slightly decreased for some
cancer cell lines. Compound 24, in which the selenocyanato
was linked via an ethyoxyl chain at the 4-position of the anilino
moiety, had relatively poor antiproliferative activities which
indicated that selenocyanato linked directly at the 4-position of
the anilino moiety is necessary.
Considering the bioelectronic isostatic effect of selenium and
oxygen, we introduced methylseleno at the 4-position of the
anilino moiety to give compounds 25a−f. The antiproliferative
activity results summarized in Table 2 showed that most of the
4-methylseleno derivatives exhibited very good potencies
compared to that of series of 7, 16, and 23. Compounds
25a−d, for which R1 was a methyl group and the R2 position
contained H, CH3, Cl, or F, exhibited very good antiprolifer-
ative activities that were not weaker than that of the reference
compound EP128495. In contrast to the excellent activity of
25a−d, compound 25f provided relatively weaker potencies
with IC50 values that ranged from 11 to 32 nM against the six
human cancer cell lines, which indicated that the cyclopropyl
group is not beneficial for antiproliferation activities at this
position. Compound 26, with the methoxyl group at the 4-
position and methylseleno at the 3-position of the anilino
moiety, gave slightly weaker antiproliferative activity compared
to 25a−d and showed that large groups at the 3-position might
be not beneficial for potency.
23a, 25a, and 25d Inhibit Another Three Human
Cancer Cell Lines and Alter Cell Morphology. As
compounds 23a, 25a, and 25d exhibited excellent antiprolifer-
ative activity in the initial cytotoxicity screening, we further
evaluated their activities against another three different cancer
cell lines [MCF7 (human breast carcinoma), HELA (human
cervical carcinoma), MGC803 (human gastric adenocarcino-
ma)]. The results shown in Table 3 indicated that 23a, 25a, and
25d also exhibited excellent antiproliferative activity with IC50
values in the low nanomolar range (IC50 = 3−19 nM).
Additionally, the confocal image assays revealed that the
compounds can highly alter cell morphology (Figure 2). All
these data suggested that 23a, 25a, and 25d were worthy of
further study.
Antiproliferative Activity of 23a, 25a, and 25d against
Drug-Resistant Cancer Cell Lines. Previous studies have
shown that overexpression of P-glycoprotein (Pgp) in cancer
cells can export anticancer drugs out and often cause clinical
chemotherapy failure for anticancer drugs. To evaluate the anti-
MDR potential of selenium-containing compounds 23a, 25a,
E
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Table 4. Antiproliferative Activities of Optimal Compounds on the Cisplatin-Resistant Cell Line A549 and the Doxorubicin-
Resistant Cell Line HEPG2
a
a
IC50 (μM)
IC50 (μM)
b
b
compd
23a
A549
A549/CDDP
resistant factor
HEPG2
HEPG2/DOX
resistant factor
0.009 0.001
0.002 0.002
0.003 0.001
0.004 0.001
0.003 0.001
8.917 0.013
2.905 0.009
0.032 0.009
0.009 0.003
0.011 0.001
0.017 0.003
0.009 0.001
>10
3.56
4.09
3.67
4.25
3.00
nd
0.006 0.002
0.004 0.001
0.004 0.002
0.009 0.003
0.005 0.002
>10
0.029 0.004
0.008 0.001111
0.005 0.0012
0.027 0.006
0.011 0.002
>10
4.83
2.00
1.25
3.00
2.21
nd
25a
25d
CA4
EP128495
CDDP
DOX
4.59 0.063
1.58
1.89 0.08
>10
nd
a
b
Data are presented as the mean SE from the dose−response curves of at least three independent experiments. Selectivity ratio = (IC50 human
resistance cells)/(IC50 human cancer sensitive cells).
Figure 3. Effects on microtubule dynamics and microtubule network. Purified tubulin protein at 10 mM in a reaction buffer was incubated at 37 °C
in the absence (control) or presence of 23a (A), 25a (B), 25d (C), and EP128495 (D) at the indicated concentrations (ranging from 0.1 to 10 μM).
And cultured A549 and HeLa cells were incubated in the presence of varying concentrations of compound 25a for 12 h. The samples were stained
for immunofluorescence using Alexa-Fluor 488 antibody (green) and corresponding Hoechst 33342 conjugated (blue). Images of the spindle
microtubule in the control and treated A549 cells (E) and HeLa cells (F) were taken under LSM 570 laser confocal microscope. The results
represent the best of data collected from three experiments with similar results (n = 3). Scale bars: 10 μm.
and 25d, we used the cisplatin-resistant cell line A549/CDDP
and the doxorubicin-resistant cell line HEPG2/DOX for an
anti-MDR potential study, with EP128495 as the reference
compound. The results in Table 4 indicated that 23a, 25a, and
25d could effectively inhibit the proliferation of the cell lines
A549/CDDP (IC50 values of 32, 9, and 11 nM; RF of 3.56,
4.09, and 3.67, respectively) and HEGP2/DOX (IC50 values of
29, 8, and 5 nM, RF of 4.83, 2.00, and 1.25, respectively) with
very small resistant factor values. From the data it can be
concluded that these selenium-containing compounds possess
potent anti-MDR potential and deserve further study.
Inhibition of Microtubule Assembly and Induction of
Morphological Aberrations. Tubulin is an essential
eukaryotic protein that plays critical roles in cell division.27
To investigate whether the activities of these selenium-
containing 4-anilinoquinazoline derivatives were related to the
interactions with microtubule systems, the most cytotoxic
compounds 23a, 25a, 25d and reference compounds EP128495
F
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 4. Effect of 23a, 25a, and 25d on cell cycle progression. (A) A549 cells (3 × 105 cells/sample) were treated with increasing concentrations of
23a, 25a, and 25d for 12 or 24 h. Cells were harvested, fixed with 70% ethanol, and stained with propidium iodide (PI). The cellular DNA content
was then determined by flow cytometry analysis. (B) Quantitative analysis of the percentage of cells in each cell cycle phase was analyzed by
EXPO32 ADC analysis software. (C) A549 cells were treated with 23a, 25a, and 25d for various lengths of time, and whole-cell lysates were
subjected to Western blotting using antibodies against the indicated proteins. The experiments were performed three times, and the results of
representative experiments are shown: (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001 vs the control group.
were evaluated as tubulin polymerization inhibitors. The results
indicated that 23a, 25a, and 25d produced a concentration-
dependent inhibition of tubulin polymerization, with calculated
IC50 values of 3.12 0.11 μM, 1.89 0.05 μM, and 1.27
0.04 μM, respectively (Figure 3A−C). Interestingly, 25d
exhibited the best tubulin polymerization inhibitory activity
(90.46%) at a concentration of 7.5 μM, compared with 77.63%
for the reference compound EP128495 (7.5 μM). Thus, the
results indicated that compounds 23a, 25a, and 25d might
inhibit the cellular proliferation activity by direct inhibition of
tubulin polymerization. Therefore, next we determined the
effect of optimal compounds on the microtubule cytoskeleton
of A549 and HeLa cells. After incubation for 12 h in the
presence of 25a or DMSO (0.01%), cells were surveyed with an
LSM 570 laser confocal microscope. As shown in Figure 3, in
the control, the integrity of the mitotic spindle and cellular
microtubule network of the two cells were observed. After
treatment with 25a at a concentration of 2 nM, the spindle
microtubule organization was not significantly deranged, but at
the concentrations of 4 nM, the nucleus of the cells narrowed
sharply and the spindle microtubules were completely
disorganized. Furthermore, treatment with 8 nM compound
25a led to a significant decrease in the polymeric tubulin
fraction (Figure 3E,F).
accumulation of cells in the G2/M phase of the cell cycle in a
concentration-dependent manner, whereas the vehicle cells
were primarily in the G1 phase (Figure 4A). Compound 23a
led to an accumulation of cells in the G2/M phase, from
10.13% (0.01% DMSO) to 69.69% (4 nM), with a concomitant
decrease of cells in the G1 and S phase cells. This phenomenon
is more obvious for compounds 25a and 25d at 4 nM for 12 h,
which arrested 67.1% and 60.89% of the cells respectively in the
G2/M phase, and when exposed to 4 nM for 24 h, the
percentage of cells in the G2/M phase increased to 87.16% and
79.71%, respectively. These results implied that these
compounds induce cell cycle arrest at the G2/M phase,
probably through induction of apoptosis.
As karyokinesis, promoted by the factors Cdc25C, Cdc2, and
cyclin B1, is an important event for eukaryotic cell entry into
mitosis, we then investigated the correlation of 23a and 25a,
which induced G2/M phase arrest, with alterations in the
expression of proteins that regulate cell division. As depicted in
Figure 4C, treatment with 23a and 25a resulted in dose- and
time-dependent decreased expression of the Cdc25C, Cdc2,
and cyclin B1 proteins. These results, which fit perfectly along
with the results of cell cycle analysis described in Figure 4A,
revealed that compounds 23a and 25a can convincingly induce
G2/M phase arrest in A549 cells.
Detection of Cell Cycle and Related Protein Ex-
pressions. In the following study, the most valid compounds
23a, 25a, and 25d were examined for its effect on cell cycle
progression of A549 cells by flow cytometry. Treatment of 23a,
25a, and 25d (2 and 4 nM) for 12 or 24 h resulted in a gradual
Detection of Apoptosis and Related Protein Expres-
sion. Mitotic arrest of tumor cells by tubulin-directed agents is
generally associated with cellular apoptosis. To evaluate the
capacity of compounds 23a, 25a, and 25d to induce apoptosis
in human cancer cell lines, a FITC conjugated annexin-V/PI
G
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 5. Inhibition of proliferation and induction of apoptosis in the cultured A549 cells upon treatment with compound 23a, 25a, and 25d. (A)
Dot plot representation of annexin-V-FITC fluorescence (x-axis) vs PI fluorescence (y-axis) of the apoptotic A549 (annexin-V positive) cells, treated
with 23a, 25a, and 25d (15, 10 nM) for 24 h and 48h. (B) Total apoptosis cells percentage were obtained by EXPO32 ADC analysis software. (C)
Analysis of the expression of proteins cyclin B1, Cdc25c, and Cdc2 by Western blot. The experiments were performed three times, and the results of
representative experiments are shown: (∗) P < 0.05, (∗∗) P < 0.01, (∗∗∗) P < 0.001 vs the control group.
assay was performed with A549 cells. As exhibited in Figure 5A,
after A549 cells were exposed to 2 or 4 nM of the compounds
23a, 25a, and 25d for 24 h, the total fractions of early apoptotic
cells (annexin-V+/PI−) and late apoptotic cells (annexin-V
+/PI+) were 15.93%, 20.17%, and 18.49%, respectively, for the
concentration of 2 nM and were 33.04%, 37.17%, and 38.95%
for 4 nM, compared with 1.06% for the control. After 48 h
incubation, the percentages of early and late apoptotic cells
strikingly increased to 22.34%, 47.86%, 39.97% (2 nM) and
57.86%, 72.89%, 64.71% (4 nM), respectively.
Increasing evidence has indicated that the regulation of the
Bcl- 2 family of proteins is involved in the signaling pathways
that are induced by antimicrotubule agents and are classified as
either activators (e.g., Bax and Bad) or inhibitors (e.g., Bcl-2).
As shown in Figure 5C, after treatment with compounds 23a
and 25a, the level of Bax and Bad was efficiently up-regulated,
whereas the antiapoptotic protein Bcl-2 was down-regulated
significantly. Thus, as described above, compounds 23a, 25a,
and 25d exert excellent antiproliferative activity, display tubulin
polymerization inhibition, and induce significant cell cycle
arrest and cell apoptosis.
Effects on Mitochondrial Dysfunction and ROS
Generation. Decreased mitochondrial membrane potential
(MMP, ΔΨm) has been implicated as an early event in
apoptotic cells. To confirm this property, we utilized a
quantitative MMP assay to measure the activities of JC-1
stained mitochondria. As presented in Figure 6A, with the
concentration of 25a increasing from 2 nM to 8 nM, there is an
obvious shift in JC-1 aggregates to increased forms of JC-1
monomers (the red fluorescence intensity decreased from
83.86% to 36.52%, and the green fluorescence intensity
accordingly increased from 15.85% to 63.13%), which indicated
that compound 25a decreased the MMP of A549 cells during
the apoptosis process. To further illustrate this phenomenon, a
confocal microscopy assay was performed. The results in Figure
6B were consisted with that of flow cytometry and showed a
shift toward the monomer form and a decrease of the
aggregates.
Mitochondria are an important intracellular source of
reactive oxygen species (ROS).28−30 To further explore the
mechanism of compound 25a for inhibiting tumor cells, we
measured the ability of compound 25a to generate ROS in
A549 cells using an oxidation-sensitive fluorescent probe
(DCFH-DA). As the results illustrated in Figure 6D, the
green fluorescence, which reflects the level of intracellular ROS,
was difficult to observe in the control group. In contrast, after
compound 25a was added for 12 h, the intensity of the green
fluorescence was brighter than that of the control group.
Furthermore, the intensity of the green fluorescence became
progressively brighter when the concentration of compound
25a was increased. Altogether, according to the observed loss of
mitochondrial potential, compound 25a was able to increase
ROS levels and eventually triggered apoptotic cell death.
Inhibition of A549 Cells Migration. Tumor cell migration
is one of the important causes leading to the death of tumor
patients. To determine the inhibitory ability of 23a, 25a, and
25d to cell motility, A549 cells or PC9 cells (5 × 104 cells per
well) were seeded in six-well plates and cultured as confluent
monolayers. Figure 7 showed the typical images of the wound
at the beginning of the experiment (0 h) and after 24 h of the
control and the tested compounds (4 nM). It is obvious that
the wound closure of the migration was significantly suppressed
H
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 6. Decreased the mitochondrial membrane potential and increased ROS levels by 25a of A549 cells. The A549 cells were treated with 25a at
different concentrations (2, 4, and 8 nM) or DMSO (0.01%) for 48 h, followed by incubation with the fluorescent probe JC-1 for 30 min. Then, the
cells were analyzed by flow cytometry (A) or fluorescence microscopy (B). (D) The fluorescence images (magnification, 10× objective) demonstrate
the motivation of 25a-induced ROS. The experiments were performed at least three times, and the results of the representative experiments are
shown.
for 23a, 25a, and 25d treated groups compared to the control.
Accordingly, the inhibition rate of migration was in the order of
25a > 25d > 23a > EP128495 > control. Together these results
indicated that 23a, 25a, and 25d displayed an obvious
inhibitory effect on migration of NSCLC cell and may be a
promising candidate for chemotherapy of metastatic cancer.
Selectivity of 23a, 25a, and 25d toward Normal Cells.
To study the selectivity of 23a, 25a, and 25d for tumor cells
and normal cells, four types of nontumorigenic cell lines from
different origins, including human normal liver cells (LO2),
human embryonic lung fibroblast cells (HLF), and human
dermal fibroblast cells (BJ), were used for the test. The results
in Table 5 showed that 23a, 25a, and 25d are associated with a
relatively low toxicity in nontumorigenic cell lines, which
exhibited a 4.1-fold to 10-fold selectivity ratio for human
nontumorigenic cells vs tumorigenic cells.
Antitumor Activity of Compounds 23a·HCl, 25a·HCl,
and 25d·HCl in A549 Xenografts. To evaluate the
antitumor potency of these selenium-containing 4-anilinoqui-
nazoline derivatives in vivo, we established xenograft models by
subcutaneously injecting A549 cells at the logarithmic growth
phase into the right armpit of mice. Compounds 23a, 25a, and
25d were selected for an in vivo xenografts mouse study in light
of its nanomolar potency in vitro in the NCI cancer cell line
panel and its potent depolymerization activity. EP128495·HCl
and CA-4P were used as reference compounds. As the
bioavailability of pharmacological agents largely depends on
their water solubility, 23a, 25a, 25d, and EP128495 were used
in their hydrochloride forms. When the tumor sizes of well-
established A549 xenografts were about 100 mm3, the mice
were randomly allocated to six groups (vehicle-treated, CA-4P-
treated, EP128495·HCl-treated, 23a·HCl-treated, 25a·HCl-
treated, and 25d·HCl-treated groups), with 5 mice per group.
After the acute toxicity experiment was performed, the safe
dosage was determined. Then, the mice were intraperitoneally
injected with CA-4P at a dose of 30 mg/kg and with
compounds 23a·HCl, 25a·HCl, 25d·HCl, and EP128495·HCl
at a dose of 2.5 mg/kg every 2 days for the entire observation
period. As shown in Figure 8A, treatment with compounds 23a·
HCl, 25a·HCl, 25d·HCl, EP128495·HCl, and CA-4P resulted
in significant reductions, 69.85%, 75.69%, 77.23%, 68.62%, and
56.62%, respectively, in the tumor volume compared with the
vehicle group.
CONCLUSION
■
Selenium is an essential trace mineral nutrient with multiple
roles in the growth and function of living animal cells, and it
effectively inhibits tumorigenesis in both animal models and
epidemiological studies. In the present study, 28 novel
selenium-containing 4-anilinoquinazoline derivatives were
synthesized and evaluated for antiproliferative activity. The
SAR indicated that 4-methylseleno derivatives exhibited greater
potencies compared to that of other series (compounds 7, 16,
and 23). Especially, compounds 25a and 25d exhibited the
most potent antiproliferative activities against six human cancer
cell lines with IC50 values of 0.004−0.022 μΜ. Moreover,
compounds 25a and 25d inhibited tubulin polymerization
effectively and concentration-dependently with IC50 values of
1.89 0.05 and 1.27 0.04, respectively, which indicated that
compounds 25a and 25d might inhibit cellular proliferative
activity by direct inhibition of tubulin polymerization.
Interestingly, compounds 25a and 25d also displayed equally
I
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 7. Effect of 23a, 25a, 25d, and EP128495 on the migration of A549 or PC9 cells. A549 or PC9 cells (5× 104 cells) suspended in free serum
DMEM containing 23a, 25a, 25d, and EP128495 (4 nM) for 24 h were photographed under a phase contrast microscopy (magnification, 4×
objective). All results were expressed as the mean SD of at least three independent experiments.
Table 5. Antiproliferative Activity of Compounds 23a, 25a, and 25d against Human Normal Cells
compd
23a
25a
25d
EP128495
a
Lo2, IC50 (μM)
0.037 0.009
0.041 0.011
0.033 0.009
0.009 0.001
4.1/4.6/3.7
0.019 0.003
0.020 0.001
0.019 0.001
0.002 0.002
9.5/10/9.5
0.016 0.001
0.019 0.001
0.019 0.001
0.002 0.001
8/9.5/9.5
0.013 0.002
0.018 0.001
0.015 0.002
0.003 0.001
6.5/9/7.5
a
HLF, IC50 (μM)
a
BJ, IC50 (μM)
a
A549, IC50 (μM)
b
resistant
a
b
Data are presented as the mean SE from the dose−response cures of at least three independent experiments. Selectivity ratio = (IC50 human
normal cells)/(IC50 A549).
potent cytotoxicity against several drug-resistant cell lines and
showed high selectivity for normal human cells. Further flow
cytometry analysis showed that 25a and 25d induce G2/M
phase arrest and apoptosis in A549 cells. Cellular studies
revealed that the induction of apoptosis by 25a and 25d was
associated with a collapse of the mitochondrial membrane
potential and alterations in the expression of some cell-cycle-
related proteins (e.g., cyclin B1, Cdc25c, Cdc2) and apoptosis-
related proteins (e.g., Bax, Bad, Bcl-2, Bcl-xl). In addition to
these mechanism studies, we also evaluated the antitumor
potency of compounds 23a, 25a, and 25d in vivo. Treatment
with these compounds resulted in a significant reduction in the
tumor volume compared with the vehicle group, with 69.85%,
75.69%, and 77.23% tumor volume reductions observed,
respectively. Taken together, these in vitro and in vivo results
suggest that 25a and 25d may be promising lead compounds
for the development of new anticancer drugs.
EXPERIMENTAL SECTION
■
General Methods (Chemistry). All reagents used in the synthesis
were obtained commercially and used without further purification
1
unless otherwise specified. The H NMR and 13C NMR spectra were
recorded using TMS as the internal standard on a Bruker BioSpin
GmbH spectrometer at 400 and 101 MHz, respectively. High-
resolution mass spectra (HR-MS) were obtained using a Shimadzu
LCMS-ITTOF mass spectrometer. The melting points were
determined using an SRS-OptiMelt automated melting point instru-
ment. The reactions were monitored by thin layer chromatography
(TLC) on glass-packed precoated silica gel plates and visualized in an
iodine chamber or with a UV lamp. Flash column chromatography was
performed using silica gel (200−300 mesh) purchased from Qingdao
Haiyang Chemical Co. Ltd. Chemicals and solvents were of reagent
grade and used as obtained from Alfa Aesar (Ward Hill, MS) and
Sigma-Aldrich (St. Louis, MO) without further purification. The purity
of all the final synthesized compounds was ≥95% as determined by
high-performance liquid chromatography (HPLC) with a TC-C18
column (4.6 mm × 250 mm, 5 μm).
J
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Figure 8. Antitumor activity in vivo. A549 cells were injected into the flanks of nude mice. When the tumor volume reached about 100 mm3, the
mice were sorted into six groups (n = 5) and administration started. (A) Images of sacrificed mice and excised tumors in each group. (B) Growth
●
difference of tumor volumes. (C) At the end of experiment, tumors were resected and weighed. indicates the weight value of each tumor; the black
line indicates the average value of the tumor weights.
Preparation of Intermediate 2. To a solution of 2-hydroxy-5-
nitrobenzoic acid (5 mmol, 0.91 g) in 50 mL of acetone, K2CO3 (15
mmol, 2.07 g) was added. When the mixture was warmed to 60 °C,
dimethyl sulfate (5 mmol, 0.63 g) was added in dropwise. The reaction
was continued at the same temperature until the material disappeared
as monitored by TLC. Water was added to the solution, and the
mixture was then placed at room temperature for 2 h. The precipitates
were collected to obtain the methyl 2-hydroxy-5-nitrobenzoate (91%
yield). 1H NMR (400 MHz, chloroform-d) δ 8.71 (d, J = 2.8 Hz, 1H),
8.37 (dd, J = 9.2, 2.9 Hz, 1H), 7.08 (d, J = 9.2 Hz, 1H), 4.03 (s, 4H),
3.94 (d, J = 1.1 Hz, 3H).
To a solution of methyl 2-hydroxy-5-nitrobenzoate (5 mmol, 0.985
g) in 20 mL of methanol, 100 mg of 10% Pd/C was added in batches.
The reaction system was replaced by nitrogen three times and then
hydrogenated with H2 at room temperature and monitored by TLC.
The catalyst was filtered and the solvent was removed in vacuum to
afford an oily product for the use of next step without further
purification (100%yield). MS (ESI) (m/z) [M + H]+: 182.2.
Preparation of Intermediate 3. To a solution of intermediate 2
(2 mmol, 0.385 g) in 20 mL of water-free ethanol, paraformaldehyde
(2 mmol, 0.18 g) and a drop of acetic acid as the catalyst were added.
After the reaction was carried out for 2 h at room temperature, sodium
cyanoborohydride (2 mmol, 0.126 g) was added and the reaction was
continued until the material disappeared as monitored by TLC. The
solvent was removed in vacuum, and 50 mL of ethanol was added to
the mixture. The resulting mixture was washed by saturated sodium
carbohydrate and water subsequently, dried over sodium sulfate,
concentrated in vacuum to afford pale yellow oily intermediate 3 (78%
yield). MS (ESI) (m/z) [M + H]+: 196.1.
Preparation of Intermediate 5. To a solution of 4 (2 mmol,
0.675 g) in methanol and water (v/v = 1:1), sodium hydroxide (2
mmol, 0.08 g) was added. The reaction was constituted at room
temperature until the materials disappeared as monitored by TLC.
Methanol was removed in vacuum, and the solution was adjusted to
pH 7 by saturated ammonium chloride, extracted with ethyl acetate for
three times. The organic phase was washed with water, dried over
sodium sulfate, concentrated to afford intermediate 5 (91.0% yield).
MS (ESI) (m/z) [M + H]+: 324.1.
Preparation of Intermediate 6a. Intermediate 5 (2 mmol, 0.646
g) was dissolved in 10 mL of SOCl2, and the reaction system was
heated at 80 °C for 2 h. The excess thionyl chloride was removed in
vacuum, and the resulting acyl chloride was dissolved in 10 mL of
dichloromethane. The acyl chloride solution obtained above was
added dropwise to a solution of 2-bromoethan-1-amine (3 mmol) in
10 mL of dichloromethane at 0 °C and then reacted at room
temperature. After the reaction finished (monitored by TLC), the
reaction mixture was washed with saturated sodium carbonate and
water, dried over sodium sulfate, concentrated, and purified by column
chromatography to afford oily compound 6a (75% yield). MS (ESI)
(m/z) [M + H]+: 429.2.
General Preparation of Intermediate 6b−f. To a solution of
compound 5 (2 mmol, 0.646 g) in 20 mL of ancetonitrile, potassium
carbonate (2 mmol) was added. After the mixture was stirred at room
temperature for 15 min, dibromoethane or corresponding dibromoal-
kane (2 mmol) was added, and the reaction was stirred at reflux until
the material disappeared. The solvent was removed in vacuum and the
residue was dissolved in 20 mL of ethyl acetate, washed with water,
dried over sodium sulfate, concentrated, and purified by column
chromatography to afford oily compounds 6b−f.
Preparation of Intermediate 4. To a solution of 3 (2 mmol,
0.392 g) in 20 mL of 2-propanol, 4-chloro-2-methylquinazoline (2
mmol, 0.357 g) was added at room temperature. After the reaction
finished, 2-propanol was removed in vacuum and water was added to
the residue. The resulting solution was adjusted to pH 9 by sodium
carbonate and then extracted with ethyl acetate. The organic phase was
washed with water, dried over sodium sulfate, concentrated, and
purified by column chromatography to afford compound 4 (89%
2-Bromoethyl 2-Methoxy-5-(methyl(2-methylquinazolin-4-
1
yl)amino)benzoate (6b). Yellow oily liquid, 76.0% yield. H NMR
(400 MHz, chloroform-d) δ 7.76 (dt, J = 8.4, 0.9 Hz, 1H), 7.73 (d, J =
2.9 Hz, 1H), 7.55 (ddd, J = 8.4, 6.6, 1.7 Hz, 1H), 7.20 (dd, J = 8.9, 2.9
Hz, 1H), 7.08−6.98 (m, 2H), 6.94 (d, J = 8.9 Hz, 1H), 4.58 (t, J = 6.2
Hz, 2H), 3.93 (s, 3H), 3.60 (m, 5H), 2.74 (s, 3H).
3-Bromopropyl 2-Methoxy-5-(methyl(2-methylquinazolin-
4-yl)amino)benzoate (6c). Yellow oily liquid, 74.0% yield. 1H
NMR (400 MHz, chloroform-d) δ 7.76 (d, J = 8.4 Hz, 1H), 7.66 (d, J
= 2.8 Hz, 1H), 7.58−7.52 (m, 1H), 7.20 (dt, J = 8.8, 3.2 Hz, 1H),
7.08−6.97 (m, 2H), 6.94 (d, J = 8.9 Hz, 1H), 4.42 (t, J = 6.0 Hz, 2H),
3.92 (s, 3H), 3.60 (s, 3H), 2.73 (s, 3H), 2.26 (p, J = 6.2 Hz, 2H),
1.89−1.78 (m, 2H).
1
yield). H NMR (400 MHz, DMSO-d6) δ 7.68 (dd, J = 8.4, 1.3 Hz,
1H), 7.61 (ddd, J = 8.3, 6.7, 1.4 Hz, 1H), 7.52 (d, J = 2.8 Hz, 1H), 7.42
(dd, J = 8.9, 2.9 Hz, 1H), 7.19 (d, J = 8.9 Hz, 1H), 7.11 (ddd, J = 8.3,
6.7, 1.5 Hz, 1H), 7.00 (dd, J = 8.6, 1.3 Hz, 1H), 3.85 (s, 3H), 3.74 (s,
3H), 3.50 (s, 3H), 2.60 (s, 3H).
K
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
4-Bromobutyl 2-Methoxy-5-(methyl(2-methylquinazolin-4-
141.18, 131.86, 130.87, 128.90, 127.78, 125.97, 124.26, 121.30, 114.54,
113.40, 101.07, 63.96, 56.31, 42.47, 28.87, 28.09, 27.67, 26.40. HRMS
(ESI) m/z [M + H]+ for C23H24N4O3Se pred 485.1088, meas
485.1090.
1
yl)amino)benzoate (6d). Yellow oily liquid, 76.0% yield. H NMR
(400 MHz, chloroform-d) δ 7.80−7.73 (m, 1H), 7.66 (d, J = 2.9 Hz,
1H), 7.55 (ddd, J = 8.4, 6.5, 1.7 Hz, 1H), 7.20 (dd, J = 8.8, 2.9 Hz,
1H), 7.07−6.97 (m, 2H), 6.94 (d, J = 8.9 Hz, 1H), 4.31 (t, J = 6.1 Hz,
2H), 3.92 (s, 3H), 3.60 (s, 3H), 3.44 (t, J = 6.5 Hz, 2H), 2.73 (s, 3H),
2.08−1.81 (m, 4H).
5-Selenocyanatopentyl 2-Methoxy-5-(methyl(2-methyl-
quinazolin-4-yl)amino)benzoate (7e). Yellow oily liquid, 71.0%
yield. 1H NMR (400 MHz, chloroform-d) δ 7.76 (d, J = 8.4 Hz, 1H),
7.64 (d, J = 2.7 Hz, 1H), 7.55 (ddd, J = 8.3, 6.6, 1.7 Hz, 1H), 7.23 (dd,
J = 9.0, 2.8 Hz, 1H), 7.07−7.00 (m, 2H), 6.96 (d, J = 8.8 Hz, 1H), 4.29
(t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.60 (s, 3H), 3.03 (t, J = 7.4 Hz, 2H),
2.73 (s, 3H), 1.94 (p, J = 7.3 Hz, 2H), 1.76 (h, J = 8.0, 7.4 Hz, 2H),
1.53 (ddd, J = 15.0, 8.8, 6.5 Hz, 2H). 13C NMR (101 MHz,
chloroform-d) δ 165.30, 163.37, 161.60, 157.24, 152.11, 141.08,
131.88, 130.82, 128.88, 127.76, 126.00, 124.27, 121.41, 114.53, 113.39,
101.33, 64.58, 56.31, 42.51, 30.39, 29.24, 27.88, 26.45, 25.58.
6-Selenocyanatohexyl 2-Methoxy-5-(methyl(2-methyl-
quinazolin-4-yl)amino)benzoate (7f). Yellow oily liquid, 71.0%
5-Bromopentyl 2-Methoxy-5-(methyl(2-methylquinazolin-4-
1
yl)amino)benzoate (6e). Yellow oily liquid, 73.0% yield. H NMR
(400 MHz, chloroform-d) δ 7.76 (d, J = 8.4 Hz, 1H), 7.67 (d, J = 2.8
Hz, 1H), 7.55 (ddd, J = 8.2, 6.5, 1.7 Hz, 1H), 7.20 (dd, J = 8.9, 2.8 Hz,
1H), 7.08−6.97 (m, 2H), 6.94 (d, J = 8.8 Hz, 1H), 4.29 (t, J = 6.5 Hz,
2H), 3.93 (s, 3H), 3.60 (s, 3H), 3.42 (q, J = 6.5 Hz, 2H), 2.73 (s, 3H),
1.88 (ddd, J = 18.3, 12.8, 6.9 Hz, 2H), 1.74 (h, J = 8.2, 7.3 Hz, 2H),
1.56 (ddt, J = 15.1, 10.4, 6.3 Hz, 2H).
6-Bromohexyl 2-Methoxy-5-(methyl(2-methylquinazolin-4-
1
yl)amino)benzoate (6f). Yellow oily liquid, 75.0% yield. H NMR
1
yield. H NMR (400 MHz, chloroform-d) δ 7.78−7.73 (m, 1H), 7.64
(400 MHz, chloroform-d) δ 7.76 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 2.8
Hz, 1H), 7.55 (ddd, J = 8.4, 6.5, 1.7 Hz, 1H), 7.19 (dd, J = 8.8, 2.9 Hz,
1H), 7.07−6.96 (m, 2H), 6.94 (d, J = 8.9 Hz, 1H), 4.28 (t, J = 6.6 Hz,
2H), 3.92 (s, 3H), 3.60 (s, 3H), 3.40 (t, J = 6.7 Hz, 2H), 2.73 (s, 3H),
1.86 (p, J = 6.9 Hz, 2H), 1.75 (dq, J = 14.1, 7.2 Hz, 2H), 1.46 (dp, J =
23.0, 7.7, 7.3 Hz, 4H).
(d, J = 2.8 Hz, 1H), 7.54 (ddd, J = 8.4, 6.6, 1.7 Hz, 1H), 7.21 (dd, J =
8.8, 2.9 Hz, 1H), 7.06−6.97 (m, 2H), 6.95 (d, J = 8.9 Hz, 1H), 4.27 (t,
J = 6.6 Hz, 2H), 3.92 (s, 3H), 3.60 (s, 3H), 3.03 (t, J = 7.3 Hz, 2H),
2.73 (s, 3H), 1.90 (p, J = 7.3 Hz, 2H), 1.80−1.68 (m, 2H), 1.55−1.37
(m, 4H). 13C NMR (101 MHz, chloroform-d) δ 165.37, 163.34,
161.59, 157.20, 152.14, 141.09, 131.83, 130.70, 128.82, 127.78, 125.98,
124.23, 121.65, 114.54, 113.42, 101.35, 64.82, 56.30, 42.47, 30.70,
29.34, 28.65, 28.36, 26.42, 25.23. HRMS (ESI) m/z [M + H]+ for
C25H28N4O3Se pred 513.1401, meas 513.1420.
General Synthesis of Target Compounds 7a−f. To a solution
of 6a (or 6b−f) (1 mmol) in 5 mL of acetonitrile, KSeCN (1.5 mmol)
was added, and the mixture was stirred at reflux until the reaction
finished. Ethyl acetate 20 mL of was added and the mixture was
washed with saturated sodium carbonate and water, dried over sodium
sulfate, concentrated, and purified by column chromatography to
afford oily compounds 7a−f.
Preparation of the Intermediate 8. Sodium (25 mmol) was
added to 100 mL of methanol at 0 °C. After the reaction finished, 4-
fluoro-3-nitroaniline (5 mmol, 0.78 g) was added, and then the
mixture was refluxed until the reaction finished. Paraformaldehyde (5
mmol, 0.45 g) was added when reaction system was cooled to room
temperature. After the mixture was stirred at room temperature for 2
h, sodium borohydride (5 mmol, 0.185 g) was added, and the mixture
was stirred at reflux. After the reaction finished (monitored by TLC),
the solvent was removed in vacuum and water was added. The mixture
was extracted three times with ethyl acetate, washed with ammonium
chloride and then water, dried over sodium sulfate, concentrated, and
purified by column chromatography to give compound 8 (77% yield).
Yellow solid. 1H NMR (400 MHz, DMSO-d6) δ 8.08 (s, 1H), 7.50 (s,
1H), 7.30 (d, J = 9.4 Hz, 1H), 7.00 (d, J = 9.4 Hz, 1H), 3.75 (s, 3H),
2.95 (s, 3H).
2-Methoxy-5-(methyl(2-methylquinazolin-4-yl)amino)-N-(2-
selenocyanatoethyl)benzamide (7a). Yellow oily liquid, 76.0%
1
yield. H NMR (400 MHz, chloroform-d) δ 8.45 (t, J = 6.0 Hz, 1H),
8.17 (d, J = 3.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.52 (ddd, J = 8.4,
6.5, 1.7 Hz, 1H), 7.11 (dd, J = 8.8, 3.0 Hz, 1H), 7.04−6.94 (m, 2H),
6.92 (d, J = 8.8 Hz, 1H), 3.99 (s, 3H), 3.92 (q, J = 6.3 Hz, 2H), 3.59
(s, 3H), 3.35 (t, J = 6.3 Hz, 2H), 2.72 (s, 3H). 13C NMR (101 MHz,
chloroform-d) δ 165.05, 163.28, 161.72, 155.63, 151.89, 142.37,
131.87, 130.41, 129.40, 127.61, 126.01, 124.25, 122.13, 114.57, 112.82,
101.42, 56.50, 42.44, 40.33, 29.13, 26.31. HRMS (ESI) m/z [M + H]+
for C21H21N5O2Se pred 456.0934, meas 456.0948.
2-Selenocyanatoethyl 2-Methoxy-5-(methyl(2-methyl-
quinazolin-4-yl)amino)benzoate (7b). Yellow oily liquid, 75.0%
yield. 1H NMR (400 MHz, chloroform-d) δ 7.80−7.71 (m, 2H), 7.55
(ddd, J = 8.4, 6.5, 1.7 Hz, 1H), 7.23 (dd, J = 8.9, 2.8 Hz, 1H), 7.11−
6.98 (m, 2H), 6.95 (d, J = 8.9 Hz, 1H), 4.66 (t, J = 6.1 Hz, 2H), 3.94
(s, 3H), 3.60 (s, 3H), 3.38 (t, J = 6.2 Hz, 2H), 2.73 (s, 3H). 13C NMR
(101 MHz, chloroform-d) δ 164.66, 163.39, 161.64, 157.53, 152.18,
141.24, 131.90, 131.48, 129.21, 127.84, 125.98, 124.31, 120.00, 114.54,
113.41, 100.76, 63.26, 56.32, 42.45, 27.31, 26.47. HRMS (ESI) m/z
[M + H]+ for C21H21N4O3Se pred 457.0775, meas 457.0781.
3-Selenocyanatopropyl 2-Methoxy-5-(methyl(2-methyl-
quinazolin-4-yl)amino)benzoate (7c). Yellow oily liquid, 72.0%
yield. 1H NMR (400 MHz, chloroform-d) δ 7.79−7.72 (m, 1H), 7.63
(d, J = 2.8 Hz, 1H), 7.55 (ddd, J = 8.4, 6.4, 1.9 Hz, 1H), 7.25 (dd, J =
8.9, 2.8 Hz, 1H), 7.06−6.99 (m, 2H), 6.96 (d, J = 8.9 Hz, 1H), 4.41 (t,
J = 5.8 Hz, 2H), 3.92 (s, 3H), 3.60 (s, 3H), 3.12 (t, J = 7.1 Hz, 2H),
2.73 (s, 3H), 2.37−2.27 (m, 2H). 13C NMR (101 MHz, chloroform-d)
δ 165.21, 163.39, 161.63, 157.17, 152.15, 141.23, 131.88, 131.02,
128.87, 127.84, 125.95, 124.25, 120.86, 114.53, 113.43, 101.17, 63.15,
56.31, 42.43, 29.83, 26.41, 25.96. HRMS (ESI) m/z [M + H]+ for
C22H22N4O3Se pred 471.0931, meas 471.0930.
Preparation of the Intermediate 9. To a solution of 8 (2 mmol,
0.364 g) in 20 mL of 2-propanol, 4-chloro-2-methylquinazoline (2
mmol, 0.358 g) was added, and the mixture was stirred at room
temperature. After the reaction finished (monitored by TLC), 2-
propanol was removed in vacuum and the resulting solid was dissolved
in 20 mL of methanol. To the solution above, 40 mg of 10% Pd/C was
added in batches. The reaction system was replaced by nitrogen three
times and then hydrogenated with H2 at room temperature and
monitored by TLC. The catalyst were filtered and the solvent was
removed in vacuum to afford the intermediate 9 (100% yield). Yellow
1
solid. H NMR (400 MHz, chloroform-d) δ 7.74−7.68 (m, 1H), 7.51
(ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 7.14 (dd, J = 8.6, 1.4 Hz, 1H), 6.98
(ddd, J = 8.5, 6.9, 1.3 Hz, 1H), 6.73 (d, J = 8.4 Hz, 1H), 6.57 (d, J =
2.5 Hz, 1H), 6.51 (dd, J = 8.4, 2.6 Hz, 1H), 3.88 (s, 3H), 3.87 (s, 2H),
3.56 (s, 3H), 2.70 (s, 3H).
Preparation of Target Compound 10. To a solution of
intermediate 9 (2 mmol, 0.689 g) in 25 mL of water containing 0.5
mL of concentrated hydrogen chloride at 0 °C, sodium nitrite (2
mmol) was added slowly. After the reaction was continued for 30 min,
the solution was adjusted to pH 6 by sodium acetate and then KSeCN
(2 mmol) was added in batches. The mixture was stirred at room
temperature until the reaction finished monitored by TLC. Ethyl
acetate (50 mL) was added and the organic phase was washed with
sodium carbonate and water subsequently, dried over sodium sulfate,
concentrated, and purified by column chromatography to give
compound 10 (50% yield). Yellow solid. 1H NMR (400 MHz,
chloroform-d) δ 7.79 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 2.5 Hz, 1H),
7.59−7.52 (m, 1H), 7.04 (d, J = 3.6 Hz, 2H), 6.99 (dd, J = 8.7, 2.5 Hz,
4-Selenocyanatobutyl 2-Methoxy-5-(methyl(2-methyl-
quinazolin-4-yl)amino)benzoate (7d). Yellow oily liquid, 73.0%
1
yield. H NMR (400 MHz, chloroform-d) δ 7.76 (dt, J = 8.4, 1.0 Hz,
1H), 7.65 (d, J = 2.9 Hz, 1H), 7.54 (ddd, J = 8.4, 6.5, 1.8 Hz, 1H), 7.22
(dd, J = 8.8, 2.9 Hz, 1H), 7.08−6.97 (m, 2H), 6.95 (d, J = 8.9 Hz, 1H),
4.32 (t, J = 6.2 Hz, 2H), 3.93 (s, 3H), 3.60 (s, 3H), 3.08 (t, J = 7.2 Hz,
2H), 2.73 (s, 3H), 2.10−1.99 (m, 2H), 1.95−1.83 (m, 2H). 13C NMR
(101 MHz, chloroform-d) δ 165.39, 163.37, 161.62, 157.18, 152.13,
L
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1H), 6.81 (d, J = 8.7 Hz, 1H), 3.91 (s, 3H), 3.61 (s, 3H), 2.74 (s, 3H).
13C NMR (101 MHz, chloroform-d) δ 163.31, 161.60, 153.91, 151.86,
143.64, 132.02, 127.67, 127.40, 127.25, 125.82, 124.42, 114.49, 114.39,
111.60, 100.86, 56.63, 42.70, 26.25. HRMS (ESI) m/z [M + H]+ for
C18H16N4OSe pred 385.0563, meas 385.0566.
Preparation of the Intermediate 12. To a solution of 2-
methoxy-5-nitrophenol (5 mmol, 0.85 g) in 50 mL of dry
dichloromethane, DIPEA (7.5 mmol) was added at 0 °C. After the
mixture was stirred for 5 min, methyl chloromethyl ether (6 mmol)
was added slowly and the reaction was continued at the same
temperature until the starting materials disappeared. The mixture was
washed with saturated ammonium chloride and water subsequently,
dried over sodium sulfate, concentrated, and purified by column
chromatography to give the intermediate 1-methoxy-2-(methoxyme-
thoxy)-4-nitrobenzene as yellow solid (92% yield). 1H NMR (400
MHz, DMSO-d6) δ 7.97 (d, J = 8.9 Hz, 1H), 7.90 (s, 1H), 7.23 (d, J =
9.0 Hz, 1H), 5.30 (s, 2H), 3.93 (s, 3H), 3.41 (s, 3H).
Intermediate 1-methoxy-2-(methoxymethoxy)-4-nitrobenzene (4
mmol, 0.85 g) was dissolved in 20 mL of methanol, and 80 mg of
10% Pd/C was added in batches. The reaction system was replaced by
nitrogen three times and then hydrogenated with H2 at room
temperature and monitored by TLC. The catalyst was filtered and the
solvent was removed in vacuum to afford the intermediate 12 (100%
yield) as pale yellow liquid. 1H NMR (400 MHz, chloroform-d) δ 6.73
(d, J = 8.5 Hz, 1H), 6.60 (d, J = 2.6 Hz, 1H), 6.31 (dd, J = 8.5, 2.7 Hz,
1H), 5.19 (s, 2H), 3.80 (s, 3H), 3.51 (s, 3H).
Preparation of the Intermediate 13. Sodium (10 mmol, 0.23 g)
was added in 50 mL of methanol in 0 °C. After the metal dissolved
completely, intermediate 12 (2 mmol, 0.43 g) and paraformaldehyde
(2 mmol) were added. After the mixture was stirred at room
temperature for two hours, sodium borohydride (2 mmol, 0.076 g)
was added and the reaction was stirred at 60 °C until it finished. The
solvent was removed in vacuum, and then water and ethyl acetate were
added to the residue. The organic phase was washed with saturated
ammonium chloride and water subsequently, dried over sodium
sulfate, concentrated, and purified by column chromatography to give
the intermediate 13 (77% yield) as yellow liquid. 1H NMR (400 MHz,
chloroform-d) δ 6.79 (d, J = 8.6 Hz, 1H), 6.53 (d, J = 2.6 Hz, 1H),
6.23 (dd, J = 8.7, 2.7 Hz, 1H), 5.20 (s, 2H), 3.80 (s, 3H), 3.51 (s, 3H),
2.79 (s, 3H).
N-(3-(3-Bromopropoxy)-4-methoxyphenyl)-N,2-dimethyl-
1
quinazolin-4-amine (15b). H NMR (400 MHz, chloroform-d) δ
7.73 (d, J = 8.3 Hz, 1H), 7.52 (ddd, J = 8.3, 6.8, 1.5 Hz, 1H), 7.04 (dd,
J = 8.5, 1.3 Hz, 1H), 6.98 (ddd, J = 8.5, 6.8, 1.3 Hz, 1H), 6.85 (d, J =
8.5 Hz, 1H), 6.79−6.71 (m, 2H), 4.02 (t, J = 5.9 Hz, 2H), 3.89 (s,
3H), 3.60 (s, 3H), 3.56 (t, J = 6.4 Hz, 2H), 2.72 (s, 3H), 2.27 (p, J =
6.2 Hz, 2H).
N-(3-(4-Bromobutoxy)-4-methoxyphenyl)-N,2-dimethyl-
1
quinazolin-4-amine (15c). H NMR (400 MHz, chloroform-d) δ
7.73 (d, J = 8.3 Hz, 1H), 7.58−7.45 (m, 1H), 7.04 (d, J = 9.0 Hz, 1H),
7.00−6.93 (m, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.76 (dd, J = 8.5, 2.4 Hz,
1H), 6.68 (d, J = 2.4 Hz, 1H), 3.90 (d, J = 7.9 Hz, 5H), 3.59 (s, 3H),
3.44 (t, J = 6.6 Hz, 2H), 2.72 (s, 3H), 2.00 (q, J = 6.9 Hz, 2H), 1.94−
1.87 (m, 2H).
N-(3-(5-Bromopentyloxy)-4-methoxyphenyl)-N,2-dimethyl-
1
quinazolin-4-amine (15d). H NMR (400 MHz, chloroform-d) δ
7.73 (d, J = 8.4 Hz, 1H), 7.53 (ddd, J = 8.3, 6.7, 1.5 Hz, 1H), 7.03 (dd,
J = 8.5, 1.4 Hz, 1H), 6.97 (ddd, J = 8.4, 6.7, 1.3 Hz, 1H), 6.85 (d, J =
8.5 Hz, 1H), 6.75 (dd, J = 8.5, 2.4 Hz, 1H), 6.67 (d, J = 2.5 Hz, 1H),
3.89 (d, J = 5.1 Hz, 5H), 3.60 (s, 3H), 3.40 (t, J = 6.8 Hz, 2H), 2.72 (s,
3H), 1.88 (p, J = 6.9 Hz, 2H), 1.76 (q, J = 6.9 Hz, 2H), 1.56 (tt, J =
9.8, 6.1 Hz, 2H).
N-(3-(6-Bromohexyloxy)-4-methoxyphenyl)-N,2-dimethyl-
1
quinazolin-4-amine (15e). H NMR (400 MHz, chloroform-d) δ
7.73 (d, J = 8.3 Hz, 1H), 7.52 (t, J = 6.7 Hz, 1H), 7.03 (d, J = 8.5 Hz,
1H), 7.00−6.93 (m, 1H), 6.85 (d, J = 8.5 Hz, 1H), 6.75 (d, J = 8.6 Hz,
1H), 6.67 (s, 1H), 3.95−3.81 (m, 5H), 3.59 (d, J = 1.5 Hz, 3H), 3.40
(d, J = 6.8 Hz, 2H), 2.72 (d, J = 1.4 Hz, 3H), 1.85 (t, J = 6.9 Hz, 2H),
1.74 (d, J = 7.6 Hz, 2H), 1.50−1.38 (m, 4H).
General Preparation of the Target Products 16a−e. To a
solution of 15a−e (1 mmol) in 5 mL of acetonitrile, KSeCN (1.5
mmol) was added, and the mixture was stirred at reflux. After the
reaction finished (monitored by TLC), ethyl acetate (20 mL) was
added. The mixture was washed with saturated sodium carbonate and
water, dried over sodium sulfate, concentrated, and purified by column
chromatography to give the target products 16a−e.
N-(4-Methoxy-3-(2-selenocyanatoethoxy)phenyl)-N,2-
1
dimethylquinazolin-4-amine (16a). H NMR (400 MHz, chloro-
form-d) δ 7.74 (d, J = 6.9 Hz, 1H), 7.53 (ddd, J = 8.3, 6.5, 1.8 Hz, 1H),
7.06−6.95 (m, 2H), 6.88 (d, J = 8.6 Hz, 1H), 6.83 (dd, J = 8.6, 2.5 Hz,
1H), 6.72 (d, J = 2.5 Hz, 1H), 4.29 (t, J = 6.2 Hz, 2H), 3.89 (s, 3H),
3.59 (s, 3H), 3.32 (t, J = 6.1 Hz, 2H), 2.72 (s, 3H). 13C NMR (101
MHz, chloroform-d) δ 163.37, 161.55, 152.12, 148.50, 147.90, 141.75,
131.78, 127.73, 126.02, 124.05, 120.09, 114.65, 113.57, 112.95, 101.01,
68.27, 56.13, 42.52, 27.85, 26.45. HRMS (ESI) m/z [M + H]+ for
C20H20N4O2Se pred 429.0825, meas 429.0834.
Preparation of the Intermediate 14. To a solution of 4-chloro-
2-methylquinazoline in 20 mL of 2-propanol, intermediate 13 (2
mmol, 0.366 g) was added, and the mixture was stirred at room
temperature until the reaction finished. Solvent was removed in
vacuum, and the residue was dissolved in 50 mL of saturated HCl
methanol. After the mixture was stirred at room temperature for 0.5 h,
the solution was adjusted to pH 9, extracted with ethyl acetate three
times, washed with water, dried over sodium sulfate, concentrated, and
purified by column chromatography to give the intermediate 14 (89%
yield) as white liquid. 1H NMR (400 MHz, DMSO-d6) δ 9.74 (s, 1H),
7.92 (d, J = 8.4 Hz, 1H), 7.86 (t, J = 7.7 Hz, 1H), 7.31 (t, J = 7.9 Hz,
1H), 7.09 (d, J = 8.5 Hz, 1H), 6.94 (s, 1H), 6.90 (d, J = 8.6 Hz, 1H),
6.86 (d, J = 8.8 Hz, 1H), 3.85 (s, 3H), 3.71 (s, 3H), 2.75 (s, 3H).
General Preparation of the Intermediates 15a−e. To a
solution of intermediate 14 (2 mmol, 0.59 g) in 20 mL of acetonitrile,
potassium carbonate (4 mmol) was added. After the mixture was
stirred for 15 min at room temperature, dibromoethane or
corresponding dibromoalkane (2 mmol) was added and the reaction
was stirred at reflux until the material disappeared. The solvent was
removed in vacuum and the residue was dissolved in 20 mL of ethyl
acetate, washed with water, dried over sodium sulfate, concentrated,
and purified by column chromatography to afford the intermeadiates
15a−e.
N-(4-Methoxy-3-(3-selenocyanatopropoxy)phenyl)-N,2-
1
dimethylquinazolin-4-amine (16b). H NMR (400 MHz, chloro-
form-d) δ 7.74 (d, J = 8.4 Hz, 1H), 7.53 (ddd, J = 8.4, 6.6, 1.7 Hz, 1H),
7.03 (ddd, J = 8.5, 1.6, 0.6 Hz, 1H), 7.01−6.96 (m, 1H), 6.86 (d, J =
8.6 Hz, 1H), 6.79 (dd, J = 8.5, 2.4 Hz, 1H), 6.69 (d, J = 2.5 Hz, 1H),
4.00 (t, J = 5.6 Hz, 2H), 3.88 (s, 3H), 3.59 (s, 3H), 3.25 (t, J = 6.8 Hz,
2H), 2.72 (s, 3H), 2.32 (p, J = 6.8 Hz, 2H). 13C NMR (101 MHz,
chloroform-d) δ 163.33, 161.51, 152.04, 148.61, 148.19, 141.64,
131.74, 127.64, 126.06, 124.01, 119.19, 114.66, 112.51, 112.15, 101.80,
67.53, 56.03, 42.57, 29.98, 26.43, 26.26. HRMS (ESI) m/z [M + H]+
for C21H22N4O2Se pred 443.0982, meas 443.0975.
N-(4-Methoxy-3-(4-selenocyanatobutoxy)phenyl)-N,2-
1
dimethylquinazolin-4-amine (16c). H NMR (400 MHz, chloro-
form-d) δ 7.73 (d, J = 8.3 Hz, 1H), 7.52 (ddd, J = 8.3, 6.6, 1.5 Hz, 1H),
7.03 (dd, J = 8.5, 1.4 Hz, 1H), 7.01−6.94 (m, 1H), 6.85 (d, J = 8.5 Hz,
1H), 6.76 (dd, J = 8.5, 2.4 Hz, 1H), 6.67 (d, J = 2.4 Hz, 1H), 3.92 (t, J
= 6.0 Hz, 2H), 3.87 (s, 3H), 3.59 (s, 3H), 3.11 (t, J = 7.2 Hz, 2H), 2.71
(s, 3H), 2.05 (q, J = 7.2 Hz, 2H), 1.89 (q, J = 6.1 Hz, 2H). 13C NMR
(101 MHz, chloroform-d) δ 163.24, 161.43, 151.90, 148.90, 148.02,
141.48, 131.72, 127.48, 126.14, 124.00, 118.61, 114.62, 112.29, 111.42,
101.63, 68.27, 56.03, 42.62, 29.44, 28.04, 27.81, 26.42. HRMS (ESI)
m/z [M + H] + for C22H24N4O2Se pred 457.1138, meas 457.1148.
N-(4-Methoxy-3-(5-selenocyanatopentyloxy)phenyl)-N,2-
N-(3-(2-Bromoethoxy)-4-methoxyphenyl)-N,2-dimethyl-
1
quinazolin-4-amine (15a). H NMR (400 MHz, chloroform-d) δ
7.78−7.71 (m, 1H), 7.53 (ddd, J = 8.4, 6.7, 1.6 Hz, 1H), 7.08−6.95
(m, 2H), 6.87 (d, J = 8.6 Hz, 1H), 6.80 (dd, J = 8.5, 2.5 Hz, 1H), 6.73
(d, J = 2.5 Hz, 1H), 4.21 (t, J = 6.5 Hz, 2H), 3.90 (s, 3H), 3.59 (s,
3H), 3.56 (t, J = 6.5 Hz, 2H), 2.72 (s, 3H).
1
dimethylquinazolin-4-amine (16d). H NMR (400 MHz, chloro-
form-d) δ 7.73 (d, J = 8.3 Hz, 1H), 7.58−7.47 (m, 1H), 7.06−6.93 (m,
M
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
2H), 6.85 (d, J = 8.5 Hz, 1H), 6.75 (dd, J = 8.5, 2.4 Hz, 1H), 6.67 (d, J
= 2.5 Hz, 1H), 3.92−3.83 (m, 5H), 3.59 (s, 3H), 3.02 (t, J = 7.4 Hz,
2H), 2.71 (s, 3H), 1.91 (p, J = 7.4 Hz, 2H), 1.77 (p, J = 6.7 Hz, 2H),
1.56 (qd, J = 9.3, 8.8, 6.1 Hz, 2H). 13C NMR (101 MHz, chloroform-
d) δ 163.24, 161.42, 151.90, 149.10, 148.04, 141.46, 131.71, 127.46,
126.17, 123.98, 118.40, 114.62, 112.31, 111.37, 101.51, 68.53, 56.09,
42.62, 30.53, 29.25, 28.12, 26.44, 25.61. HRMS (ESI) m/z [M + H] +
for C23H26N4O2Se pred 471.1295, meas 471.1309.
concentrated, and purified by column chromatography to give the
intermediates 22a−d.
N-Methyl-4-selenocyanatoaniline (22a). Pale yellow solid
1
(76.0% yield). H NMR (400 MHz, chloroform-d) δ 7.47 (d, J =
8.5 Hz, 2H), 6.55 (d, J = 8.4 Hz, 2H), 2.84 (s, 3H).
N,3-Dimethyl-4-selenocyanatoaniline (22b). Pale yellow solid
1
(78.0% yield). H NMR (400 MHz, chloroform-d) δ 7.48 (d, J = 8.5
Hz, 1H), 6.52 (s, 1H), 6.38 (d, J = 8.5 Hz, 1H), 2.83 (s, 3H), 2.49 (s,
N-(4-Methoxy-3-(6-selenocyanatohexyloxy)phenyl)-N,2-
3H).
1
dimethylquinazolin-4-amine (16e). H NMR (400 MHz, chloro-
3-Chloro-N-methyl-4-selenocyanatoaniline (22c). Pale yellow
solid (75.0% yield). 1H NMR (400 MHz, chloroform-d) δ 7.49 (d, J =
8.0 Hz, 1H), 6.46 (m, 2H), 2.83 (s, 3H).
3-Fluoro-N-methyl-4-selenocyanatoaniline (22d). Pale yellow
solid (77.0% yield). 1H NMR (400 MHz, chloroform-d) δ 7.41 (d, J =
7.9 Hz, 1H), 6.36 (m, 2H), 2.84 (s, 3H).
form-d) δ 7.73 (d, J = 8.4 Hz, 1H), 7.52 (t, J = 7.2 Hz, 1H), 7.03 (d, J
= 8.5 Hz, 1H), 6.97 (t, J = 7.2 Hz, 1H), 6.86 (d, J = 8.5 Hz, 1H), 6.76
(dd, J = 8.5, 2.3 Hz, 1H), 6.67 (s, 1H), 3.94−3.83 (m, 5H), 3.60 (s,
3H), 3.04 (t, J = 7.4 Hz, 2H), 2.72 (s, 3H), 1.90 (p, J = 6.8 Hz, 2H),
1.76 (t, J = 6.8 Hz, 2H), 1.52−1.39 (m, 4H). 13C NMR (101 MHz,
chloroform-d) δ 163.31, 161.47, 151.92, 149.26, 148.05, 141.50,
131.74, 127.47, 126.19, 124.01, 118.30, 114.67, 112.27, 111.29, 101.49,
68.71, 56.13, 42.65, 30.66, 29.41, 28.75, 28.67, 26.43, 25.21. HRMS
(ESI) m/z [M + H]+ for C24H28N4O2Se pred 485.1452, meas
485.1452.
Preparation of the Intermediate 17. To a tetrahydrofuran
solution (25 mL), lithium aluminum hydride (10 mmol) was added at
0 °C, and then intermediate 4 (2 mmol, 0.675 g) in 10 mL of
tetrahydrofuran was added in dropwise. The reaction mixture was
warmed to room temperature and monitored by TLC. After the
reaction finished, ice−water was added to quench the reaction and
then sodium sulfate. After the mixture was stirred for 30 min, the
mixture was filtrated and the residue was washed with ethyl acetate for
three times. The combined organic phase was concentrated and
purified by column chromatography to give the intermediate 17
(45.0% yield). 1H NMR (400 MHz, DMSO-d6) δ 7.88−7.80 (m, 2H),
7.48 (d, J = 2.8 Hz, 1H), 7.35 (dd, J = 8.6, 2.8 Hz, 1H), 7.31−7.24 (m,
1H), 7.12 (d, J = 8.7 Hz, 1H), 6.80 (d, J = 8.7 Hz, 1H), 4.52 (s, 2H),
3.86 (s, 3H), 3.74 (s, 3H), 2.74 (s, 3H).
General Preparation of Target Compounds 23a−f. To a
solution of 4-chloro-2-methylquinazoline (2 mmol) or 4-chloro-2-
(methyl-d3)quinazolinein, 25 mL of 2-propanol, intermediate 22a, or
22b−d was added, and the mixture was stirred at room temperature.
After the reaction finished, 2-propanol was removed in vacuum, water
was added. The solution was adjusted to pH 9 by saturated sodium
carbonate, extracted with ethyl acetate three times. The combined
organic phase was washed with water, dried over sodium sulfate,
concentrated, and purified by column chromatography to give the
intermediates 23a−f.
N,2-Dimethyl-N-(4-selenocyanatophenyl)quinazolin-4-
amine (23a). Yellow solid (88% yield). 1H NMR (400 MHz,
chloroform-d) δ 7.81 (d, J = 8.4 Hz, 1H), 7.67−7.51 (m, 3H), 7.18−
7.04 (m, 4H), 3.65 (s, 3H), 2.76 (s, 3H). 13C NMR (101 MHz,
chloroform-d) δ 163.57, 162.13, 152.24, 150.39, 134.71, 132.40,
128.07, 125.91, 124.88, 116.93, 115.03, 101.38, 41.66, 26.42. HRMS
(ESI) m/z [M + H]+ for C17H14N4Se pred 355.0457, meas 355.0448.
N,2-Dimethyl-N-(3-methyl-4-selenocyanatophenyl)-
quinazolin-4-amine (23b). Yellow solid (88% yield). 1H NMR (400
MHz, chloroform-d) δ 7.80 (d, J = 8.4 Hz, 1H), 7.69−7.55 (m, 2H),
7.17−7.05 (m, 3H), 6.96−6.87 (m, 1H), 3.64 (s, 3H), 2.76 (s, 3H),
2.44 (s, 3H). 13C NMR (101 MHz, chloroform-d) δ 163.55, 162.10,
152.19, 150.88, 142.26, 135.93, 132.34, 127.99, 126.73, 125.81, 118.44,
115.08, 100.98, 41.76, 26.43, 22.69. HRMS (ESI) m/z [M + H]+ for
C18H16N4Se pred 369.0614, meas 369.0610.
N,2-Dimethyl-N-(4-selenocyanatophenyl)quinazolin-4-
amine (23c). Yellow solid (89% yield). 1H NMR (400 MHz,
chloroform-d) δ 7.82 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.2 Hz, 2H),
7.23−7.11 (m, 3H), 6.99 (d, J = 8.7, 1H), 3.63 (s, 3H), 2.75 (s, 3H).
13C NMR (101 MHz, chloroform-d) δ 163.68, 162.08, 152.38, 150.63,
134.20, 132.69, 132.50, 128.27, 125.49, 125.28, 124.85, 124.10, 118.59,
115.13, 100.68, 41.60, 26.40. HRMS (ESI) m/z [M + H]+ for
C17H13N4ClSe pred 389.0065, meas 389.0046.
N,2-Dimethyl-N-(4-selenocyanatophenyl)quinazolin-4-
amine (23d). Yellow solid (90% yield). 1H NMR (400 MHz,
chloroform-d) δ 7.92−7.79 (m, 1H), 7.68 (ddd, J = 8.4, 6.8, 1.5 Hz,
1H), 7.57 (dd, J = 8.5, 7.5 Hz, 1H), 7.28 (dd, J = 8.3, 1.6 Hz, 1H), 7.21
(ddd, J = 8.3, 6.8, 1.2 Hz, 1H), 6.87 (ddd, J = 17.0, 9.2, 2.4 Hz, 2H),
3.67 (s, 3H), 2.79 (s, 3H). 13C NMR (101 MHz, chloroform-d) δ
163.81, 162.39, 161.37 (d, J = 248.5 Hz), 152.49 (d, J = 9.4 Hz),
152.46, 135.35 (d, J = 2.8 Hz), 132.87, 128.32, 125.49, 125.42, 120.30
(d, J = 2.9 Hz), 115.52, 110.82 (d, J = 24.8 Hz), 102.80 (d, J = 22.5
Hz), 99.82 (d, J = 2.1 Hz), 41.27, 26.39. HRMS (ESI) m/z [M + H] +
for C17H13N4FSe pred 373.0363, meas 373.0354.
N,2-Dimethyl-N-(4-selenocyanatophenyl)quinazolin-4-
amine, 2-Methyl D3 (23e). Yellow solid (85% yield). 1H NMR (400
MHz, chloroform-d) δ 7.80 (dd, J = 8.6, 4.1 Hz, 1H), 7.58 (m, 3H),
7.20−6.99 (m, 4H), 3.70−3.57 (m, 3H). 13C NMR (101 MHz,
chloroform-d) δ 163.47, 162.09, 152.24, 150.35, 134.66, 132.35,
128.06, 125.87, 125.72, 124.85, 116.94, 115.03, 101.34, 41.62, 25.7 (m,
CD3). HRMS (ESI) m/z [M + H] + for C17H11D3N4Se Se pred
358.0650, meas 358.0635.
Preparation of the Target Compound 19. Intermediate 17 (2
mmol, 0.618 g) was dissolved in 10 mL of thionyl chloride, and the
mixture was stirred at reflux for 2 h. The excess thionyl chloride was
removed under vacuum, and the residue was dissolved in 10 mL of
acetonitrile. Potassium selenocyanate (3 mmol) was added, and then
the mixture was heated to reflux. After the reaction finished, ethyl
acetate (50 mL) was added and the organic phase was washed with
saturate sodium carbonate, dried over sodium sulfate, and purified by
1
column chromatography to give the 19 (40.0% yield). H NMR (400
MHz, chloroform-d) δ 7.75 (dd, J = 8.4, 1.3 Hz, 1H), 7.53 (ddd, J =
8.3, 6.5, 1.7 Hz, 1H), 7.18 (d, J = 2.7 Hz, 1H), 7.09−6.97 (m, 3H),
6.85 (d, J = 8.7 Hz, 1H), 4.21 (s, 2H), 3.91 (s, 3H), 3.59 (s, 3H), 2.73
(s, 3H). 13C NMR (101 MHz, chloroform-d) δ 163.25, 161.64, 155.39,
151.86, 141.48, 131.87, 127.81, 127.54, 126.23, 126.09, 124.28, 114.56,
111.85, 102.07, 55.83, 42.53, 27.61, 26.36.
Preparation of the Intermediate 21. Sodium metal (10 mmol,
0.23 g) was added to 50 mL of anhydrous methanol. After the sodium
dissolved completely, corresponding anilines (2 mmol) and
paraformaldehyde (2 mmol) were added. The mixture was stirred at
room temperature for 2 h, and then sodium borohydride (2 mmol)
was added. The reaction was heated at 60 °C until the anilines
disappeared. The solvent was removed, and water and ethyl acetate
were added to the residue. The organic phase was separated and the
water phase was extracted by ethyl acetate. The combined organic
phase was washed by saturated ammonium chloride and water, dried
over sodium sulfate, concentrated, and purified by column
chromatography to give the intermediatea 21a−d in 93−96% yields.
General Preparation of the Intermediates 22a−d. To a
solution of malononitrile (2 mmol) in 15 mL of dimethyl sulfoxide,
selenium dioxide (2 mmol) was added at 0 °C. After the mixture was
stirred for 5 min, the reaction was warmed to room temperature and
the stirred for another 15 min. Intermediate 21a or 21b−d were added
to the mixture, and the reaction was continued until finished
(monitored by TLC). Water was added to the reaction system and
extracted by ethyl acetate three times. The combined organic phase
was washed by brine and then water, dried over sodium sulfate,
2-Cyclopropyl-N-methyl-N-(4-selenocyanatophenyl)-
quinazolin-4-amine (23f). Yellow solid (82% yield). 1H NMR (400
MHz, chloroform-d) δ 7.78 (d, J = 8.4 Hz, 1H), 7.59−7.51 (m, 3H),
N
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
7.12−6.99 (m, 4H), 3.57 (d, J = 1.0 Hz, 3H), 2.35−2.21 (m, 1H),
1.30−1.21 (m, 2H), 1.05 (dq, J = 7.0, 3.8 Hz, 2H). 13C NMR (101
MHz, chloroform-d) δ 167.18, 162.03, 152.27, 150.37, 134.66, 132.32,
127.85, 126.06, 125.77, 124.29, 116.97, 115.29, 101.45, 41.57, 18.43,
9.92. HRMS (ESI) m/z [M + H]+ for C19H16N4Se pred 381.0614,
meas 381.0612.
General Preparation of Target Compounds 25a−f. Com-
pound 23a (1 mmol) or 23b−g were dissolved in 10 mL of absolute
ethyl alcohol. Sodium borohydride (1 mmol) was added in batches to
the solution and then methyl iodide (1.5 mmol). The reaction was
stirred at room temperature until the starting material disappeared.
Ammonium hydroxide (1 mL) was added to remove the excess methyl
iodide, and the solvents were removed in vacuum. Water was added to
the residue, and the mixture was extracted with ethyl acetate three
times. The combined organic phase was washed with water, dried over
sodium sulfate, concentrated, and purified by column chromatography
to give the intermediates 25a−f.
N,2-Dimethyl-N-(4-(methylselanyl)phenyl)quinazolin-4-
amine (25a). Yellow solid (88% yield). 1H NMR (400 MHz,
chloroform-d) δ 7.76 (d, J = 8.4 Hz, 1H), 7.53 (t, J = 8.2 Hz, 1H),
7.41−7.34 (m, 2H), 7.08 (d, J = 8.6 Hz, 1H), 7.06−6.97 (m, 3H), 3.59
(s, 3H), 2.73 (s, 3H), 2.35 (s, 3H). 13C NMR (101 MHz, chloroform-
d) δ 163.38, 161.76, 152.10, 146.86, 131.87, 131.56, 129.27, 127.75,
126.13 (d, J = 2.0 Hz), 124.22, 114.76, 42.20, 26.49, 7.40. HRMS
(ESI) m/z [M + H]+ for C17H17N3Se pred 344.0661, meas 344.0652.
N,2-Dimethyl-N-(3-methyl-4-(methylselanyl)phenyl)-
quinazolin-4-amine (25b). Yellow solid (90% yield). 1H NMR (400
MHz, chloroform-d) δ 7.75 (dd, J = 8.5, 1.3 Hz, 1H), 7.52 (d, J = 8.4
Hz, 1H), 7.23 (d, J = 8.3 Hz, 1H), 7.09 (dd, J = 8.6, 1.5 Hz, 1H),
7.02−6.94 (m, 2H), 6.89 (dd, J = 8.3, 2.5 Hz, 1H), 3.59 (s, 3H), 2.73
(s, 3H), 2.31 (d, J = 2.0 Hz, 6H). 13C NMR (101 MHz, chloroform-d)
δ 163.37, 161.71, 152.10, 146.54, 139.56, 131.78, 130.49, 129.65,
127.68, 126.82, 126.20, 124.11, 123.76, 114.86, 42.31, 26.48, 21.77,
6.43. HRMS (ESI) m/z [M + H]+ for C18H19N3Se pred 358.0818,
meas 358.0809.
N-(3-Chloro-4-(methylselanyl)phenyl)-N,2-dimethyl-
quinazolin-4-amine (25c). Yellow solid (85% yield). 1H NMR (400
MHz, chloroform-d) δ 7.78 (d, J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz,
1H), 7.22−7.12 (m, 3H), 7.07 (d, J = 8.3 Hz, 1H), 6.92 (dd, J = 8.5,
2.4 Hz, 1H), 3.59 (s, 3H), 2.74 (s, 3H), 2.33 (s, 3H). 13C NMR (101
MHz, chloroform-d) δ 163.44, 161.74, 152.19, 147.32, 134.78, 132.11,
129.75, 129.56, 127.93, 125.89, 124.59, 124.21, 114.81, 42.10, 26.44,
6.55. HRMS (ESI) m/z [M + H]+ for C17H16ClN3Se pred 378.0269,
meas 378.0264.
126.22, 126.11, 123.68, 115.11, 42.07, 18.40, 9.63, 7.40. HRMS (ESI)
m/z [M + H]+ for C19H19N3Se pred 370.0818, meas 370.0810.
Biology. Cell Lines and Culture. The human cancer cell lines
(A549, HCT116, HEPG2, MDAMB231, LOVO, RKO, HELA, MCF7,
MGC803, PC9, HLF, BJ, LO2, A549/CDDP, HEPG2/DOX) used in
this study were purchased from the Laboratory Animal Service Center
at Sun Yat-sen University (Guangzhou, China). Cell lines HELA,
HEPG2, RKO, LOVO, MGC803, MCF7, MDAMB231, HLF, BJ, and
LO2 were cultivated in DMEM containing 10% (v/v) heat-inactivated
fetal bovine serum (FBS), 100 units/mL penicillin, and 100 μg/mL
streptomycin. Cell lines A549, PC9, HCT116, HEPG2/DOX, and
A549/CDDP were cultivated in RPMI 1640 medium containing 10%
(v/v) heat-inactivated FBS, 100 units/mL penicillin, and 100 mg/mL
streptomycin.
Antibodies and Reagents. A commercial kit (Cytoskeleton,
catalog no. B011P) used for the tubulin polymerization assay was
purchased from Cytoskeleton (Danvers, MA, USA). The FITC-
conjugated mouse anti-tubulin antibody was purchased from Cell
Signaling Technology (Danvers, MA, USA). The goat anti-mouse
IgG/Alexa-Fluor 488 antibody was obtained from Invitrogen
(Camarillo, CA, USA). A purified brain tubulin polymerization kit
was purchased from Cytoskeleton (Danvers, MA, Q9 USA). Annexin-
V/FITC and the cell cycle analysis kit were purchased from Keygen
Biotech, China. MTT was purchased from Sigma, USA. A lipophilic
cationic dye, 5,50,6,60-tetrachloro-1,10,3,30-tetraethylbenzimidazol-
carbocyanine (JC-1), was obtained from Beyotime, China. The
mouse anti-Bcl-xl monoclonal antibody, mouse anti-α-tubulin
monoclonal antibody, rabbit anti-Bax, rabbit anti-Bad, rabbit anti-
Bcl-2, rabbit anti-cyclin B1, rabbit anti-Cdc2, rabbit anti-Cdc25C,
mouse anti-β-tubulin, and horseradish peroxidase-conjugated goat
anti-mouse IgG secondary antibody were all obtained from Cell
Signaling Technology (Danvers, MA, USA).
MTT Assay. For cytotoxicity assay, the cells grown in the
logarithmic phase were seeded into 96-well plates (5 × 103 cells/
well) for 24 h and then exposed to different concentrations of the test
compounds for 48 h. After attached cells were incubated with 5 mg/
mL MTT (Sigma, USA) for another 4 h, the suspension was discarded
and subsequently the dark blue crystals (formazan) were dissolved in
dimethyl sulfoxide (DMSO). Then the absorbance at 570 nm was
measured using a multifunction microplate reader (Molecular Devices,
Flex Station 3), and each experiment was performed at least in
triplicate. The cytotoxic effects of each compound were expressed as
IC50 values, which represent the drug concentrations required to cause
50% tumor cell growth inhibition, and calculated with GraphPad Prism
software version 5.02 (GraphPad Inc., La Jolla, CA, USA).
N-(3-Fluoro-4-(methylselanyl)phenyl)-N,2-dimethyl-
quinazolin-4-amine (25d). Yellow solid (87% yield). 1H NMR (400
MHz, chloroform-d) δ 7.79 (d, J = 8.5 Hz, 1H), 7.58 (d, J = 8.4 Hz,
1H), 7.35−7.24 (m, 1H), 7.17 (d, J = 8.6 Hz, 1H), 7.08 (d, J = 8.3 Hz,
1H), 6.86 (dd, J = 10.0, 2.3 Hz, 1H), 6.81 (dd, J = 8.3, 2.3 Hz, 1H),
3.61 (s, 3H), 2.75 (s, 3H), 2.34 (s, 3H). 13C NMR (101 MHz,
chloroform-d) δ 163.49, 161.93, 161.69 (d, J = 244.7 Hz), 152.23,
148.79 (d, J = 9.1 Hz), 132.48 (d, J = 5.0 Hz), 132.16, 127.97, 125.84,
124.62, 121.28 (d, J = 3.0 Hz), 115.25 (d, J = 22.7 Hz), 114.98, 112.08
(d, J = 24.8 Hz), 41.91, 26.44, 6.49 (d, J = 3.2 Hz). HRMS (ESI) m/z
[M + H]+ for C17H16FN3Se pred 362.0567, meas 362.0554.
N,2-Dimethyl-N-(4-(methylselanyl)phenyl)quinazolin-4-
amine, 2-Methyl D3 (25e). Yellow solid (89% yield). 1H NMR (400
MHz, chloroform-d) δ 7.75 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 6.9 Hz,
1H), 7.36 (d, J = 8.5 Hz, 2H), 7.08 (d, J = 8.6 Hz, 1H), 7.02 (m, 3H),
3.58 (d, J = 1.8 Hz, 3H), 2.34 (d, J = 1.8 Hz, 3H). 13C NMR (101
MHz, chloroform-d) δ 163.30, 16.74, 152.08, 146.86, 131.83, 131.60,
129.27, 127.74, 126.10 (d, J = 2.1 Hz), 124.19, 114.78, 42.16, 28.23−
24.09 (m,CD3), 7.38. HRMS (ESI) m/z [M + H]+ for C17H13D3N3Se
pred 347.0865, meas 347.0842.
In Vitro Tubulin Polymerization Assay and Immunofluor-
escence Microscopy. The tubulin polymerization assay was
performed according to the method described by Bonne et al. with
appropriate modification. The tubulin polymerization was monitored
by an increase in fluorescence intensity, which can be easily recorded
due to the incorporation of a fluorescent reporter, DAPI (4′,6-
diamidino-2-phenylindole), a fluorophore already known as a DNA
intercalator. We performed the tubulin polymerization assay using a
commercial kit (Cytoskeleton, catalog no. BK011P) purchased from
Cytoskeleton (Danvers, MA, USA). The final buffer concentration
used for tubulin polymerization contained 80.0 mM piperazine-N,N′-
bis(2-ethanesulfonic acid)sequisodium salt (pH 6.9), 2.0 mM MgCl2,
0.5 mM EGTA, 1 mM GTP, and 10.2% glycerol. After 5 μL of the
tested compounds at the indicated concentrations was added, the
mixture was warmed to 37 °C for 1 min, and the reaction was initiated
by the addition of 55 μL of the tubulin solution. The fluorescence
intensity enhancement was recorded every 60 s for 90 min in a
multifunction microplate reader (Molecular Devices, Flex Station 3)
(emission wavelength is 410 nm; excitation wavelength is 340 nm).
The area under the curve was used to determine the concentration
that inhibited tubulin polymerization by 50% (IC50), which was
calculated with GraphPad Prism software version 5.02 (GraphPad Inc.,
La Jolla, CA, USA). For the immunofluorescence microscopy, 3 × 105
cells were grown in a 10 mm3 confocal culture dish for 24 h and then
incubated in the presence or absence of compound 25a at the
2-Cyclopropyl-N-methyl-N-(4-(methylselanyl)phenyl)-
quinazolin-4-amine (25f). Yellow solid (91% yield). 1H NMR (400
MHz, chloroform-d) δ 7.73 (d, J = 8.4 Hz, 1H), 7.50 (t, J = 7.2 Hz,
1H), 7.35 (d, J = 10.3 Hz, 2H), 7.09−6.98 (m, 3H), 6.93 (d, J = 8.3
Hz, 1H), 3.52 (s, 3H), 2.34 (s, 3H). 13C NMR (101 MHz, chloroform-
d) δ 166.94, 161.69, 152.14, 146.86, 131.80, 131.54, 129.23, 127.56,
O
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
indicated concentrations for another 12 h. After being washed with
phosphate buffered solution (PBS) and fixed in 4% prewarmed (37
°C) paraformaldehyde for 15 min, the cells were permeabilized with
0.5% Triton X-100 for 15 min and blocked for 30 min in 10% goat
serum. Then, the cells were incubated with mouse anti-tubulin
antibody (CST, USA) at 4 °C overnight. On the next day, cells were
washed with PBS three times and incubated with goat anti-mouse
IgG/Alexa-Fluor 488 antibody (Invitrogen, USA) for 1 h. After the
nuclei were stained with Hoechst 33342 (Sigma, USA) in the dark at
room temperature for 30 min, the samples were immediately visualized
on a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss,
Germany).
Cell Cycle Analysis. A549 cells were seeded in six-well plates (3 ×
105 cells/well) and incubated in the presence or absence of
compounds 23a, 25a, and 25d at the indicated concentrations for
24 or 48 h. Then, cells were harvested by centrifugation and fixed in
ice-cold 70% ethanol overnight. After the ethanol was removed on the
next day, the cells were resuspended in the ice-cold PBS and treated
with RNase A (Keygen Biotech, China) at 37 °C for 30 min, followed
by incubation with the DNA staining solution propidium iodide (PI)
(Keygen Biotech, China) at 4 °C for 30 min. About 10 000 events
were detected by flow cytometry (Beckman Coulter, Epics XL) at 488
nm. The data regarding the number of cells in different phases of the
cell cycle were analyzed by EXPO32 ADC analysis software.
Apoptosis Analysis. A549 cells were seeded in six-well plates (3 ×
105 cells/well) and incubated in the presence or absence of
compounds 23a, 25a, and 25d at the indicated concentrations for
24 or 48 h to induce cell apoptosis. After incubation, cells were
harvested and incubated with 5 μL of annexin-V/FITC (Keygen
Biotech, China) in binding buffer (10 mM HEPES, 140 mM NaCl,
and 2.5 mM CaCl2 at pH 7.4) at room temperature for 15 min. PI
solution was then added to the medium for another 10 min incubation.
Almost 10 000 events were collected for each sample and analyzed by
flow cytometry (Beckman Coulter, Epics XL). The percentage of
apoptotic cells was calculated with EXPO32 ADC analysis software.
Western Blot Analysis. A549 cells seeded in 60 mm dishes at a
density of 5 × 105 cells/well were incubated with or without
compounds 23a, 25a, and 25d at indicated concentrations for 24 or 48
h. After incubation, the cells were washed twice with ice-cold PBS and
then lysed in RIPA lysis buffer containing 150 mM NaCl, 50 mM Tris
(pH 7.4), 1% (w/v) sodium deoxycholate, 1% (v/v) Triton X-100,
0.1% (w/v) SDS, and 1 mM EDTA (Beyotime, China). The lysates
were incubated at 0 °C for 30 min and vortexed every 10 min
intermittently, and then the total protein was harvested by centrifuging
at 12 500g for 15 min. After the protein concentrations were
determined by a BCA protein assay kit (Thermo Fisher Scientific,
Rockford, IL, USA), the protein extracts were reconstituted in loading
buffer containing 62 mM Tris-HCl, 2% SDS, 10% glycerol, and 5% β-
mercaptoethanol (Beyotime, China) and boiled at 100 °C for 3 min.
An equal amount of the proteins (40 μg) was separated by 8−12%
sodium dodecyl sulfate−polyacrylamide gel electrophoresis (SDS−
PAGE) and transferred to nitrocellulose membranes (Amersham
Biosciences, Little Chalfont, Buckinghamshire, U.K.). After blocked
with 5% nonfat dried milk in TBS containing 1% Tween-20 for 90 min
at room temperature, the membrane was incubated overnight with
specific primary antibodies (CST, USA) at 4 °C. After three washes in
TBST, the membranes were incubated with the appropriate HRP-
conjugated secondary antibodies at room temperature for 2 h. The
blots were developed with enhanced chemiluminescence (Pierce,
Rockford, IL, USA) and were detected by an LAS4000 imager (GE
Healthcare, Waukesha, WI, USA).
analysis, A549 cells were plated in six-well plates (3 × 105 cells/well)
and grown for 24 h and treated with compound 25a at the indicated
concentrations for 24 h. Then the cells were harvested by
centrifugation and incubated with JC-1 solution for 30 min. After
brief washing, the proportion of green and red fluorescence intensity
was immediately detected and analyzed by flow cytometry. For the
fluorescence microscopy detection, A549 cells were plated in a
confocal culture dish at 5 × 104 cells/dish and grown for 24 h and
treated with compound 25a at the indicated concentrations for
another 24 h. Then the cells were stained with 2 μM JC-1 at 37 °C for
30 min, washed with PBS, and then the cell nuclei were stained with
Hoechst 33342 (Sigma, USA) for 10 min in the dark. The cell images
were immediately detected by a Zeiss LSM 570 laser scanning confocal
microscope (Carl Zeiss, Germany). The intracellular ROS accumu-
lation assay was determined by fluorescence microscopy using the
reactive oxygen species assay kit (ROS assay kit, Beyotime). A549 cells
were cultured in six-well plates (3 × 105 cells/well) for 24 h. After
adding compound 25a for 12 h, the culture supernatants were
removed, and the cells were then incubated with 10 mM DCFH-DA in
fresh medium at 37 °C for 30 min. After incubation, the culture
medium was removed, and the cells were washed with PBS buffer
three times. The production of ROS could be measured by changes in
the fluorescence intensity due to the intracellular accumulation of
dichlorofluorescein (DCF) caused by the oxidation of DCFH. The
intracellular ROS level, as indicated by the DCF fluorescence, was
determined by measuring the fluorescence intensity on a multifunc-
tional monochromator-based microplate reader with the excitation and
emission wavelengths set at 488 and 525 nm, respectively. To observe
the intracellular ROS level visually, we used the Arrayscan VTI high
content analysis system to acquire cell images after the cell nuclei were
stained with Hoechst 33342 (Sigma) for 10 min in dark conditions.
Anti-Cell-Migration Study. A549 or PC9 cells were plated in a
six-well culture dish at 5 × 104 cells/dish and grown for 24 h, and the
nonmigrated cells were scraped off the upper surface of the membrane
with a 10 μL pipet. The medium was then replaced with serum-free
DMEM medium and treated with compound 23a, 25a, and 25d at the
indicated concentrations for another 24 h. After the samples were
washed with phosphate buffer solution (PBS), the cell images were
immediatelt detected by a Zeiss LSM 570 laser scanning confocal
microscope (Carl Zeiss, Germany).
Statistical Analysis. All data are presented as the mean
SD
(standard deviations). Statistical significance between the control and
test groups was evaluated by Student’s test. The differences were
considered statistically significant when P < 0.05.
Evaluation of in Vivo Antitumor Activity. Animals and
Implantation of Cancer Cells. Male BALB/c nude mice (5 weeks
old, 18−20 g) were purchased and housed at the Laboratory Animal
Service Center of Sun Yat-Sen University (Guangzhou, China) in
pathogen-free condition, maintained at constant room temperature,
and fed a standard rodent chow and water. 1 ×107 cells/mL A549 cells
grown in logarithmic phase were harvested and resuspended in FBS-
free DMEM medium. Then, 0.1 mL of the cell suspension was
subcutaneously injected into the right flank of each mouse. After
implantation, the tumor mass was measured with an electronic caliper
twice a week, and the tumor volume was calculated according to the
following formula: tumor volume (mm3) = 0.5 × length × width2.
Acute Toxicity Experiment. Male Kunming mice (5 weeks old,
18−24 g) were purchased and housed at the Laboratory Animal
Service Center of Sun Yat-Sen University (Guangzhou, China) in
pathogen-free condition, maintained at constant room temperature,
and fed a standard rodent chow and water. The mice were randomly
divided into experimental group and control group. The experimental
group was divided into 15 groups, each with 4 males, according to A−
D sequence number. Then, the mice were caudal vein injected with
CA-4P at a dose of 30/175/300 mg/kg, with compounds 23a·HCl,
25a·HCl, 25d·HCl, and EP128495·HCl at a dose of 3/17.5/300 mg/
kg.The control group was caudal vein injected with NaCl solution.
Drug Treatments and Evaluation of in Vivo Antitumor
Activity. When the tumor volume reached about 100 mm3, the
xenograft tumor-bearing nude mice were randomly allocated to six
Mitochondrial Membrane Potential and ROS Accumulation
Assay. A lipophilic cationic dye, 5,5′,6,6′-tetrachloro-1,1′,3,3′-
tetraethylbenzimidazolcarbocyanine (JC-1, Beyotime, China), was
used to monitor the level of MMP in the cells. At normal state, the
MMP is high and JC-1 appears as aggregates, which is indicated by red
fluorescence. However, when apoptosis occurs, the MMP reduced and
JC-1 displayed as monomers, which is indicated by green fluorescence.
We applied two methods which included flow cytometry and
fluorescence microscopy to detect the MMP. For flow cytometry
P
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
groups (vehicle-treated, CA-4P-treated, EP128495-treated, 23a-
treated, 25a-treated, and 25d-treated groups), with 5 mice per
group. Then, the mice were intraperitoneally injected with CA-4P at a
dose of 30 mg/kg, compounds 23a, 25a, 25d, and EP128495 at a dose
of 2.5 mg/kg every 2 days for the entire observation period, and the
control group was treated with an equivalent volume of vehicle.
Tumor volume and body weights were recorded every 2 days after
drug treatment. At the end of the observation period, the animals were
euthanized by cervical dislocation and the tumor bulks were peeled off,
conforming to the Guide for the Care and Use of Laboratory Animals
as published by the U.S. National Institutes of Health (NIH
Publication No. 85-23, revised 1996) and approved by the Institutional
Ethics Review Board of Sun Yat-Sen University.
REFERENCES
■
(1) Horio, T.; Murata, T. The role of dynamic instability in
microtubule organization. Front. Plant Sci. 2014, 5, 511.
(2) Pellegrini, F.; Budman, D. R. Review: tubulin function, action of
antitubulin drugs, and new drug development. Cancer Invest. 2005, 23,
264−273.
(3) Prota, A. E.; Bargsten, K.; Zurwerra, D.; Field, J. J.; Díaz, J. F.;
Altmann, K.-H.; Steinmetz, M. O. Molecular mechanism of action of
microtubule-stabilizing anticancer agents. Science 2013, 339, 587−590.
(4) Stanton, R. A.; Gernert, K. M.; Nettles, J. H.; Aneja, R. Drugs that
target dynamic microtubules: a new molecular perspective. Med. Res.
Rev. 2011, 31, 443−481.
(5) Dumontet, C.; Jordan, M. A. Microtubule-binding agents: a
dynamic field of cancer therapeutics. Nat. Rev. Drug Discovery 2010, 9,
790−803.
(6) Jordan, M. A.; Wilson, L. Microtubules as a target for anticancer
drugs. Nat. Rev. Cancer 2004, 4, 253−265.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jmed-
chem.8b00128.
(7) Kasibhatla, S.; Baichwal, V.; Cai, S. X.; Roth, B.; Skvortsova, I.;
Skvortsov, S.; Lukas, P.; English, N. M.; Sirisoma, N.; Drewe, J.;
Pervin, A.; Tseng, B.; Carlson, R. O.; Pleiman, C. M. MPC-6827: a
small-molecule inhibitor of microtubule formation that is not a
substrate for multidrug resistance pumps. Cancer Res. 2007, 67, 5865−
5871.
Molecular formula strings and some data (CSV)
(8) Chamberlain, M. C.; Grimm, S.; Phuphanich, S.; Recht, L.; Zhu,
J. Z.; Kim, L.; Rosenfeld, S.; Fadul, C. E. A phase 2 trial of verubulin
for recurrent glioblastoma: a prospective study by the brain tumor
investigational consortium (BTIC). J. Neuro-Oncol. 2014, 118, 335−
343.
(9) Prabhu, K. S.; Lei, X. G. Adv. Nutr. 2016, 7, 415−7.
(10) Kryukov, G. V.; Castellano, S.; Novoselov, S. V.; Lobanov, A. V.;
́
Zehtab, O.; Guigo, R.; Gladyshev, V. N. Characterization of
AUTHOR INFORMATION
■
Corresponding Authors
*L.H.: phone, +086-20-3994-3050; fax, +086-20-3994-3050; e-
mail, Huangl72@mail.sysu.edu.cn.
*X.L.: phone, +086-20-3994-3050; fax, +086-20-3994-3050; e-
mail, lixsh@mail.sysu.edu.cn.
ORCID
mammalian selenoproteomes. Science 2003, 300, 1439−1443.
(11) Allmang, C.; Wurth, L.; Krol, A. The selenium to selenoprotein
pathway in eukaryotes: more molecular partners than anticipated.
Biochim. Biophys. Acta, Gen. Subj. 2009, 1790, 1415−14123.
(12) Vinceti, M.; Crespi, C. M.; Malagoli, C.; Del Giovane, C.;
Krogh, V. Friend. The current epidemiologic evidence on selenium
and human cancer risk. J. Environ. Sci. Health C Environ. Carcinog
Ecotoxicol Rev. 2013, 31, 305−341.
(13) Sharma, A. K.; Amin, S. Post select: selenium on trial. Future
Med. Chem. 2013, 5, 163−174.
(14) Chung, C. Y.; Madhunapantula, S. V.; Desai, D.; Amin, S.;
Robertson, G. P. Melanoma prevention using topical PBISe. Cancer
Prev. Res. 2011, 4, 935−948.
(15) Lin, T.; Ding, Z.; Li, N.; Xu, J.; Luo, G.; Liu, J.; Shen, J. Seleno-
cyclodextrin sensitises human breast cancer cells to TRAIL-induced
apoptosis through DR5 induction and NF-kappaB suppression. Eur. J.
Cancer 2011, 47, 1890−1907.
(16) Nguyen, N.; Sharma, A.; Nguyen, N.; Sharma, A. K.; Desai, D.;
Huh, S. J.; Amin, S.; Meyers, C.; Robertson, G. P. Melanoma
chemoprevention in skin reconstructs and mouse xenografts using
isoselenocyanate-4. Cancer Prev. Res. 2011, 4, 248−258.
(17) Yang, Y.; Huang, F.; Ren, Y.; Xing, L.; Wu, Y.; Li, Z.; Pan, H.;
Xu, C. The anticancer effects of sodium selenite and selenomethionine
on human colorectal carcinoma cell lines in nude mice. Oncol. Res.
2009, 18, 1−8.
(18) Zeng, H.; Cheng, W. H.; Johnson, L. K. Methylselenol, a
selenium metabolite, modulates p53 pathway and inhibits the growth
of colon cancer xenografts in Balb/c mice. J. Nutr. Biochem. 2013, 24,
776−780.
(19) dos Santos, E. d. A.; Hamel, E.; Bai, R.; Burnett, J. C.; Tozatti, C.
S. S.; Bogo, D.; Perdomo, R. T.; Antunes, A. M. M.; Marques, M. M.;
Matos, M. F. C.; de Lima, D. P. Synthesis and evaluation of diaryl
sulfides and diaryl selenide compounds for antitubulin and cytotoxic
activity. Bioorg. Med. Chem. Lett. 2013, 23, 4669−4673.
(20) Guan, Q.; Yang, F.; Guo, D.; Xu, J.; Jiang, M.; Liu, C.; Bao, K.;
Wu, Y.; Zhang, W. Synthesis and biological evaluation of novel 3,4-
diaryl-1,2,5-selenadiazol analogues of combretastatin A-4. Eur. J. Med.
Chem. 2014, 87, 1−9.
Xingshu Li: 0000-0002-7433-739X
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the National Natural Science Foundation of China
(Grants 21772241 and 21302235), Guangdong Natural Science
Foundation (Grant 2014A030313124), Guangdong High-Level
Personnel of Special Support Program, Young Top-Notch
Talent Project (Grant 2015TQ01R244), Guangzhou Pearl
River New Star Fund Science and Technology Planning Project
(Grant 201610010111), and The Fundamental Research Funds
for the Central Universities (Grant 15ykpy04) for financial
support of this study.
ABBREVIATIONS USED
■
SAR, structure−activity relationship; TLC, thin-layer chroma-
tography; MOM, methyl−oxygen−methyl; IPA, isopropanol;
DIPEA, diisopropylethylamine; DMSO, dimethyl sulfoxide;
Pgp, P-glycoprotein; MDR, multidrug resistant; ROS, reactive
oxygen species; MMP, mitochondrial membrane potential;
NSCLC, non-small-cell lung carcinoma; CA-4P, combretastatin
A-4 phosphorus; FBS, fetal bovine serum; DAPI, 4′,6-
diamidino-2-phenylindole; PBS, phosphate buffer solution;
SDS−PAGE, sodium dodecyl sulfate−polyacrylamide gel
electrophoresis; DCFH-DA, 2′,7′-dichlorodihydrofluorescein
diacetate; DCF, dichlorofluorescein; FITC, fluorescein iso-
thiocyanate; PI, propidium iodide; DOX, doxorubicin; CDDP,
cis-platinum; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-
tetrazolium bromide; JC-1, 5,50,6,60-tetrachloro-1,10,3,30-
tetraethylbenzimidazolcarbocyanine; EGTA, ethylene glycol
tetraacetic acid; GTP, guanosine triphosphate
Q
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(21) Plano, D.; Karelia, D. N.; Pandey, M. K.; Spallholz, J. E.; Amin,
S.; Sharma, A. K. Design, synthesis, and biological evaluation of novel
selenium (Se-NSAID) molecules as anticancer agents. J. Med. Chem.
2016, 59, 1946−1959.
(22) Brown, K. M.; Arthur, J. R. Selenium, selenoproteins and
human. Public Health Nutr. 2001, 4, 593−599.
(23) Terry, P. D.; Qin, B.; Camacho, F.; Moorman, P. G.; Alberg, A.
J.; Barnholtz-Sloan, J. S.; Bondy, M.; Cote, M.L.; Funkhouser, E.;
Guertin, K. A.; Peters, E. S.; Schwartz, A. G.; Schildkraut, J. M.;
Bandera, E. V. Supplemental selenium may decrease ovarian cancer
risk in African-American women. J. Nutr. 2017, 147, 621−627.
(24) Rayman, M. P. Selenium in cancer prevention: a review of the
evidence and mechanism of action. Proc. Nutr. Soc. 2005, 64, 527−542.
(25) Boussada, M.; Ali, R. B.; Said, A. B.; Bokri, K.; Akacha, A. B.;
Dziri, C.; El May, M. V. Selenium and a newly synthesized
thiocyanoacetamide reduce doxorubicin gonadotoxicity in male rat.
Biomed. Pharmacother. 2017, 89, 1005−1017.
(26) Li, H.; Li, F.; Yang, F.; Fang, Y.; Xin, Z.; Zhao, Li.; Hu, Q. Size
effect of Se-enriched green tea particles on in vitro antioxidant and
antitumor activities. J. Agric. Food Chem. 2008, 56, 4529−4533.
(27) Aylett, C. H.; Lowe, J.; Amos, L. A. New insights into the
mechanisms of cytomotive actin and tubulin filaments. Int. Rev. Cell
Mol. Biol. 2011, 292, 1−71.
(28) Liu, C.; Fu, Y.; Li, C.; Chen, T.; Li, X. Phycocyanin-
functionalized selenium nanoparticles reverse palmitic acid-induced
pancreatic β cell apoptosis by enhancing cellular uptake and blocking
reactive oxygen species (ROS)-mediated mitochondria dysfunction. J.
Agric. Food Chem. 2017, 65, 4405−4413.
(29) Li, W. J.; Nie, S. P.; Yao, Y. F.; Liu, X. Z.; Shao, D. Y.; Gong, D.
M.; Cui, S. W.; Phillips, G. O.; He, M.; Xie, M. Y. Ganoderma atrum
polysaccharide ameliorates hyperglycemia-induced endothelial cell
death via a mitochondria-ROS pathway. J. Agric. Food Chem. 2015,
63, 8182−8191.
(30) Juan, M. E.; Wenzel, U.; Daniel, H.; Planas, J. M. Resveratrol
induces apoptosis through ROS-dependent mitochondria pathway in
HT-29 human colorectal carcinoma cells. J. Agric. Food Chem. 2008,
56, 4813−4818.
R
DOI: 10.1021/acs.jmedchem.8b00128
J. Med. Chem. XXXX, XXX, XXX−XXX