The design, synthesis and evaluation of selenium-containing 4-anilinoquinazoline hybrids as anticancer agents and a study of their mechanism

Article abstract of DOI:10.1039/c8ob00875b

Inhibition of tubulin polymerization is one of the significant strategies in the treatment of cancer. Inspired by the excellent antitumor activity of EP128495 and the beneficial biological activities of selenium compounds, a series of new selenium-containing 4-anilinoquinazoline hybrids were synthesized and evaluated as tubulin polymerization inhibitors. An anti-proliferative activity assay showed that most of the compounds inhibited human sensitive cancer cells at low nanomolar concentrations. A mechanism study revealed that the optimal compound 5a disrupted microtubule dynamics, decreased the mitochondrial membrane potential and arrested HeLa cells in the G2/M phase, finally resulting in cellular apoptosis.

Full text of DOI:10.1039/c8ob00875b

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Design, synthesis and evaluation of seleniumꢀcontaining 4ꢀanilinoquinazoline hybrids
as anticancer agents and the mechanism study
Baijiao An1, Shun Zhang1, Jinhui Hu1, Tingting Pan1, Ling Huang1, Johnny Cheukꢀon
TANG2,*, Xingshu Li1,* and Albert S. C. Chan1
School of Pharmaceutical Sciences, Sun Yatꢀsen University, Guangzhou 510006, China
State Key Laboratory of Chirosciences, Department of Applied Biology and Chemical
Technology, The Hong Kong Polytechnic University, Hong Kong, P.R. China.
Abstract：
Inhibition of the tubulin polymerization represents one of the significant strategies in the
treatment of cancer. Inspired by the excellent antitumor activity of EP128495 and the
beneficial biological activities of selenium compounds, a series of new seleniumꢀ
containing 4ꢀanilinoquinazoline hybrids were synthesized and evaluated as tubulin
polymerization inhibitors. The antiproliferative activities assay showed that most of
compounds inhibited human sensitive cancer cells at low nanomolar concentrations.
Mechanism study revealed that the optimal compound 5a disrupted microtubule dynamics,
decreased mitochondrial membrane potential and arrested Hela cells in the G2/M phase
that finally resulted cellular apoptosis.
Introduction
Cancer is a major disease that seriously endangers human life and health.1 With the rapid
increase in the incidence of cancers worldwide, the existing drugs are far from to meet the
needs. In cancer treatment, many singleꢀtarget drugs play a crucial role in cancer therapy
such as cisplatin, paclitaxel, and the kinase inhibitors. However, the complexity of
carcinogenic mechanism, tumor heterogeneity and the emergence of drug resistance make
1

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the treatment of cancer still a long way to go.2ꢀ3 Among the various targets for the
development of anticancer agents, tubulin polymerization or deꢀpolymerization inhibitors
has attracted a lot of attention as tubulinꢀmicrotubule systems play a critical role for a wide
range of cellular processes.4ꢀ8 Taxane and vinca alkaloids are the typical examples for the
treatment of variety human cancer in the past decades.9ꢀ10 Recetly, Cai et al. reported the
discovery and evaluation of 2ꢀchloroꢀNꢀ(4ꢀmethoxyphenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine
as a tubulin polymerization inhibitor, which exhibited highly activity in inducing of
apoptosis and cell proliferation inhibition.11
Selenium (Se) is an essential trace mineral nutrient with multiple roles in human health,12ꢀ13
People living in low selenium areas are susceptible to seleniumꢀrelated diseases such as
Keshan disease,14 KashinꢀBeck disease,15 and male infertility.16 Thus, selenium compounds
have attracted a vast interest in the last decades as promising chemoꢀpreventive agents.17ꢀ18
There are twentyꢀfive selenoproteins in the human body that exert specific biological
functions.19 Selenoꢀcysteine (SeCys), which confers important functions in selenoprotein,
may be replaced by cysteine (Cys) in the conditions of low Se status,20 resulting in
seleniumꢀrelated diseases. One of the important functions of selenium compounds is
antioxidant. It has been shown that selenium compounds exhibit antineoplastic properties
by inhibiting the development of reactive oxygen species (ROS), thus protecting cells
against oxidative DNA damage.21 Because of the potential cancer therapeutic effects of Se,
several organoselenium compounds have been developed for the inhibition of cancer cell
growth in various xenograft rodent models for different cancers types, as well as to have
synergistic effects in combination with chemotherapeutic drugs. 22ꢀ29
In our previous work, we have reported the synthesis and evaluation of the seleniumꢀ
containing compound, N,2ꢀdimethylꢀNꢀ(4ꢀ(methylselanyl)phenyl)quinazolinꢀ4ꢀamine, and
its analogues as anticancer agents.30 It is well known that the minor modification of
structure often leads to great changes in biological activity. In this paper, we report the
design,
synthesis
and
evaluation
of
2ꢀchloroꢀNꢀmethylꢀNꢀ(4ꢀ
selenocyanatophenyl)quinazolinꢀ4ꢀamine and its analogues as anticancer agents and the
mechanism study.
2

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Se
R2
Se
O
SeCN
N
N
N
N
N
N
N
R2
R1
Cl
R1
Cl
Se
N
N
N
O
O
EP128495
5a-5f
6a-6i
10
Figure 1. Design strategy of seleniumꢀcontaining 4ꢀanilinoquinazoline derivatives.
Results and discussion
Chemistry
The synthetic routes target compounds 5a, 5b, 5d, 5f, 6, 7a, 10 were outlined in Schemes
1ꢀ2. Commercially available starting materials substituted anilines (1) were reacted with
paraformaldehyde in the solution CH3ONa−CH3OH and sequentially NaBH4 to afford Nꢀ
methylated compounds 2. Treatment of 2 with malononitrile and SeO2 in the solution of
DMSO provided phenyl selenocyanate compounds 3, which was reacted with NaBH4 and
CH3I to afford the key intermediates 4a, 4c, 4e, 4g, 4h and 4i. Intermediates 3 were reacted
with 2,4ꢀdichloroquinazoline in the presence of hydrogen chloride in isopropanol to afford
the target compounds 5a, 5b, 5d, 5f, which were further Seꢀmethylated to give the target
compounds 6a, 6b, 6d, 6f. Compounds 4 were reacted with 2, 4ꢀdichloroquinazoline in the
presence of NaOAc or NaH to give the target compounds 6a, 6c, 6e, 6g, 6h and 6i. The
chloride on the 2ꢀposition of quinazoline moiety of 6a was substituted by OCH3 in the
presence of NaOH and CH3OH to afford 7a. Target compounds 10 was synthesized by the
reaction of 4ꢀhydroxy coumarins (8) with P2O5 and Bu4NBr in toluene to get intermediate
9, which was coupled with 2, 4ꢀdichloroquinazoline in the presence of DIEPA in DMF at
110°C for 12 hours.
3

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Scheme 1. The synthesis of intermediates 3 and 4. Reagents and conditions: (a) i: Na, paraformaldehyde,
CH3OH, 12h, 0°C. ii: NaBH4, 2h, 60°C. (b) Malononitrile, SeO2, DMSO, 2h, r.t. (c) NaBH4, CH3I,
CH3OH, 5min, r.t.
Scheme 2. The synthesis of target compounds 5, 6, 7a and 10 Reagents and conditions: (d) 2, 4ꢀ
dichloroquinazoline, HCl, IPA, 12h, r.t. (e) NaBH4, CH3I, CH3OH, 5min, r.t. (f) 4c or 4g, 2, 4ꢀ
dichloroquinazoline, NaOAc, THF/H2O=1:1, 2h, 70°C; 4e, 4h or 4i, 2,4ꢀdichloroquinazoline, NaH,
4

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DMF, 8h, r.t. (g) NaOH, CH3OH, H2O, 3h, 50°C. (h) Bu4NBr, P2O5, Toluene, 1h, 120oC. (i) 2, 4ꢀ
dichloroquinazoline, DIEPA, DMF, H2O, 12h, 110oC.
In vitro human cancer cell lines growth inhibition and the SARs
To evaluate the antiproliferative activities of the seleniumꢀcontaining 4ꢀanilinoquinazoline
hybrids, all the synthesized compounds were screened against a panel of six human tumor
cell lines (RKO, human colon cancer cell line; HEPG2, Human hepatoma carcinoma cells;
MCFꢀ7, breast adenocarcinoma; HeLa, human epithelial cervical cancer cell line; HCTꢀ
116, human colon cancer cell line; MGC803, humaan gastric cancer cell line). The results
summarized in Table 1 indicated that all the target compounds possess excellent
antiproliferative activities with the IC50 at the nM level. Among them, compound 5a, 2ꢀ
chloroꢀNꢀmethylꢀNꢀ(4ꢀselenocyanatophenyl)quinazolinꢀ4ꢀamine exhibited the best results
with the IC50 values ranged from 2.09 to 9.98 nM against for the six human tumor cell
lines. The study of the structure and activity relationship shows that the substituent R1 and
R2 in the aniline moiety have a great influence on activity. The methyl on R1 or R2 position
is obviously not favorable for the activity. For example, 5b, with the methyl on 3ꢀposition,
exhibited 6.01ꢀ22.7 nM of the IC50 values, while 5f, with the methyl on 2ꢀposition,
provided even weaker (37.9ꢀ79.4 nM). Compound series 6, with 4ꢀmethylselanyl on aniline
moiety, also provided very potent activities and exhibited the similar tendency of
relationship between structure and activity. However, in some case, this series gave
relatively slightly weaker activities than their 4ꢀselenocyanatophenyl analogues
(comparison the activity between 5a and 6a; 5b and 6b). This result is slightly different
from our previous study of N,2ꢀdimethylꢀNꢀphenylquinazolinꢀ4ꢀamine seleniumꢀcontaining
derivatives as anticancer agents, which the 4ꢀmethylselanyl (on aniline moiety) derivatives
gave the best results. Compound 10, seleniumꢀcoumarin derivatives, exhibited 17.4ꢀ89.5
nM of the IC50 values indicating that the quinazolin structure is a better pharmacophore
than coumarin in these compounds.
Table 1. Antiꢀproliferative activities of synthesized seleniumꢀcontaining 4ꢀanilinoquinazoline derivatives
against human cancer cell lines
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IC50 (nΜ)b
Comp.
RKO
HEPG2
MCF7
HELA
HCT116
MGC803
3.39±0.002
5.24±0.003
9.98±0.007
2.09±0.003
4.97±0.004
3.54±0.005
5a
5b
5d
5f
6.01±0.002
3.42±0.002
47.1±0.011
4.61±0.003
15.0±0.005
4.55±0.001
33.1±0.009
7.87±0.002
14.9±0.009
12.9± 0.007
43.5±0.011
76.8± 0.09
7.85±0.004
6.79±0.004
6.78±0.003
37.9±0.009
6.81±0.004
17.9±0.003
4.79±0.004
19.5±0.003
13.2±0.009
8.90±0.007
52.6±0.009
50.8± 0.008
58.8± 0.012
8.92±0.002
18.9±0.002
10.6±0.009
78.9±0.013
14.7±0.010
36.1±0.008
14.3±0.011
91.3±0.016
22.5±0.017
32.8±0.008
96.8±0.013
95.7± 0.032
53.5± 0.007
22.7±0.010
8.73±0.004
6.78±0.005
38.9±0.009
2.77±0.002
12.6±0.003
6.93±0.001
57.8±0.007
9.28±0.09
22.7±0.009
9.17±0.006
79.4±0.015
8.99±0.003
45.9±0.019
11.7±0.004
78.4±0.013
12.3±0.06
16.5±0.001
9.67±0.004
55.7±0.011
5.46±0.002
9.96±0.004
9.93±0.002
44.5±0.008
14.8±0.011
38.3±0.012
55.6±0.015
53.8±0.090
48.9± 0.007
4.78±0.004
6a
6b
6d
6f
6c
6e
6g
6h
6i
16.9±0.010
11.1±0.004
29.5± 0.008
49.8± 0.008
9.15±0.002
34.3±0.009
45.6±0.010
48.9±0.024
63.4± 0.016
11.4±0.010
7a
17.4±0.005
4.22±0.001
89.5±0.015
6.47±0.001
74.9±0.022
5.61±0.001
84.1±0.007
5.11±0.002
63.4±0.016
8.27±0.001
44.8±0.018
6.23±0.008
10
EP128495
a Cell lines were treated with compounds for 48 h. Cell viability was measured by MTT assay as described in the Experimental Section. b IC50
values are indicated as the mean ± SD (standard error) of at least three independent experiments.
Selectivity of 5a towards normal cells.
As compound 5a exhibited excellent antiproliferative activity in the initial cytotoxicity
screening, we further evaluated their activities against human normal cells (LO2, human
normal liver cells; BJ, human dermal fibroblast cells; HLF, human embryonic lung
fibroblast cells). It can be seen from table 2 that 5a exhibited no obvious cytotoxicity
against human normal cells in a broad concentration range (14.5ꢀ17.2 nM) for 48 h, the
selectivity factor values (RF: 7.2, 6.9 and 8.3) displayed 5a possess less potent antiꢀ
proliferative activities towards human normal cell lines which deserve further study.
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Table 3. Antiꢀproliferative activities of compound 5a towards human normal cell lines
Cell lines
LO2
BJ
HLF
HELA
2.09±0.003
IC50 (nM)a
15.1±0.003
14.5±0.005
6.9
17.2±0.006
8.3
Selectivity ratiob 7.2
a
b
Data are presented as the mean ± SE from the doseꢀresponse cures of at least three independent experiments.
Selectivity ratio = (IC50 human normal cells) / (IC50 HELA).
Inhibition of tubulin polymerization in vitro
Cai et al. have confirmed that EP128495 could inhibit41 tubulin polymerization. To
determine whether the synthesized seleniumꢀcontaining compounds could target the tubulin
microtubule system, we used compound 5a, which exhibited relatively better antiproliferative
activities in the initial cytotoxicity screening among all the tested compounds, for the in vitro
tubulin polymerization inhibition activity assay. After the purified and unpolymerized tubulin
was incubated with 5a in the concentrations of 0.75, 1.25, 2.5, 5.0 and 10 ꢁM, the
polymerization was measured by the method of fluorescence intensity assay. As shown in Fig.
2, the fluorescence intensity of the control samples increased with time at 37°C indicating the
tubulin polymerization had occurred. When tubulin was incubated with the 5a at increased
concentrations, the increase of the fluorescence intensity was obviously slowed down. The
inhibitory concentrations that reduced the polymerized tubulin by 50% (IC50) of compound 5a
was 1.88 ± 0.11 ꢁM, being stronger than the reference compound EP128495 (IC50: 2.74±0.09
ꢁM).
Fig.2 Effects of compounds 5a compared to EP128495 on microtubule dynamics. Purified tubulin
protein at 10 mM in a reaction buffer was incubated at 37 °C in the absence (control) or presence of 5a
at the indicated concentrations (ranging from 0.75 to 10 ꢁM). Polymerizations are followed by an increase
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in fluorescence emission at 410 nM over a 90 min period at 37 °C (excitation wavelength was 340 nM). The
experiments were performed three times, and the results of representative experiments are shown.
Detection of microtubule immunofluorescence
Tubulinꢀmicrotubule systems are very important in the maintenance of cellular
morphology. To reveal if compound 5a could affect microtubule dynamics in living cells,
immunofluorescence assay in human lung epithelium cancer cells (A549) and human
epithelial cervical cancer (Hela) cells were performed (Fig. 3). The control group was
observed to be predominantly in interphase of the cell cycle, which was characterized by
uncondensed chromosomes and the regularly assembled, slim, fibrous microtubules
wrapped around the cell nucleus. In contrast, the microtubule network became disorganized
with the treatment of compound 5a at 1 nM of the concentration. When the concentration
was increased to 5 nM, the microtubule network was completely disrupted. These
morphological changes of microtubules suggest that compound 5a effectively disrupts the
microtubule dynamics, which might leads to cell death eventually.
Fig.3 Effect of hybrid compounds 5a on the microtubule network and the nucleus morphology. Cultured
A549 and HeLa cells were incubated in the presence of varying concentrations of compound 5a for 8 h.
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The samples were stained for immunofluorescence using AlexaꢀFluor 488 antibody (green) and
corresponding Hoechst 33342 conjugated (blue). Images of the spindle microtubule were taken under
LSM 570 laser confocal microscope. The results represent the best of data collected from three
experiments with similar results (n = 3).
Induction of mitotic arrest
As we know, tubulinꢀdestabilizing agents could block the cell cycle in the G2/M phase
because of microtubule depolymerization and cytoskeleton disruption.31 On the base of the
in vitro tubulin polymerization inhibition activity and detection of morphological
aberrations assay, we then evaluated the effects on the cell cycle progression of compound
5a by flow cytometry method. After treatment with increasing concentrations of 5a for 12
h or 24 h, cells were harvested, fixed with 70% ethanol, and stained with propidium iodide
(PI) for the assay. The results (Fig.4) indicated that treatment with 5a results a doseꢀ
dependent accumulation of HELA cells in the G2/M phase with concomitant losses in G1
phase. The changes of S phase were not observed during the entire 12 or 24 h treatments.
On the other hand, subꢀG phase, which indicates apoptotic cells, was obviously found as
the concentration of 5a increased, especially after the treatment for 24 hours. Altogether,
the above results demonstrated that 5a induces cellꢀcycle progression arrest in G2/M phase,
which is in agreement with the activity of most antimitotic agents.
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Fig. 4 Effect of 5a on cell cycle progression. HELA cells (3 × 105 cells/sample) were treated with
increasing concentrations of 5a for 12 h or 24 h. (A) 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. The experiments were performed for three times, and the results of
representative experiments are shown.
Induction of mitotic cell apoptosis
Cell apoptosis is a process that organisms eliminate excessive cells and control cell numbers.32
Promoting apoptosis has been suggested as a promising strategy for cancer drug discovery.33 To
determine whether compound 5a could induce cell apoptosis, we carried out the cell apoptosis
assay. After 5aꢀtreated HELA cells (24 or 48 h) were stained with Annexin VꢀFITC and PI, the
samples were analyzed by flow cytometry. As shown in Fig. 5A, 18.55% of the early and late
apoptosis were obtained in HELA cells treated with 5a for 24 h at 1.0 nM of the concentration. The
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numbers increased to 34.55% at 5.0 nM of the concentration and arrived to 51.15% at 20 nM. After
incubation for 48 h, the total percentage of early and late apoptotic cells was 24.73%, 50.30%, and
65.63% in the cases of 1, 5 and 10 nM of 5a, respectively. In contrast, only 1.11% and 1.28% of the
early and late apoptotic cells were detected in the control cells. To further determine whether 5aꢀ
induced cell death is concerned with Bclꢀ2 family, we treated hela cells with 5a for 48 h, and
detected the level of proteins Bax, Bad and Bcl2. As shown in Fig. 5C, the proapoptotic protein
increased significantly with the increase of drug concentration (e.g., Bax and Bad), and
accompanied the antiꢀapoptotic protein Bclꢀ2 reduction significantly. Overall, the results above
suggest that 5a could efficiently induced cell apoptosis in a doseꢀdependent and timeꢀdependent
manner.
Fig. 5 Inhibition of proliferation and induction of apoptosis in HELA cells treated with 5a. (A) Treated
with 5a (1, 5 and 10 nM) for 24 h or 48h. (B) Total apoptosis cells percentage (obtained by EXPO32
ADC analysis software). (C) The expression of proteins (Bax, Bad and Bcl2) by the western blot assay.
The experiments were performed for three times.
Loss of Mitochondrial Potential and ROS Generation
Studies have shown that progression of apoptosis is closely related to mitochondria.34 It is
thought a signal of apoptosis to be the decreased mitochondrial membrane potential (MMP,
ꢂΨ m). To evaluate whether the target compounds were associated with apoptosis, we used
flow cytometry analysis and a laser scanning confocal microscope to detect the MMP. Fig.
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6A showed that after cells exposed to 5a at the concentration of 1.0 nM, the red
fluorescence intensity (JCꢀ1 aggregates) decreased from 98.28% (control) to 91.88%;
correspondingly, the green fluorescence intensity (JCꢀ1 monomers) increase from 1.72% to
8.12%. When the concentration of 5a increased from 5.0 to 10 nM, JCꢀ1 monomers
increased from 31.12% to 71.48%, which also showed a doseꢀdependent manner. Fig. 6B
exhibited the results of confocal microscopy. With the concentration of 5a increased, there
is a shift from aggregates (red fluorescence) toward the form of the monomers (green
fluorescence), which is consistent with the results of the flow cytometry analysis, and
demonstrated that compound 5a lead to mitochondrial dysfunction.
Reactive oxygen species (ROS) are closely related to tumorigenesis and the apoptosis of
cancer cells through DNA damage in the cell nucleus.35 In cancer cells, growth and
proliferation are encouraged with a condition of a modest rise in intracellular ROS, and on
the other hand, apoptosis is also induced by a higher levels of ROS. To determine whether
compound 5a could induce ROS accumulation, the laser scanning confocal microscope
assay was performed to evaluate the generation of intracellular ROS in Hela cells. The
green fluorescence in Fig 6D, which reflects the level of intracellular ROS, was more and
more to observe after compound 5a was added to the cells at the concentration from 1.0 to
10 nM for 12 h. In contrast, green fluorescence was difficulty to be observed in the control.
Fig. 6C indicated the results of quantitative analysis, which also showed that compound 5a
induced mitochondrial depolarization and thus resulted in the intracellular ROS production.
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Fig. 6 Decreased the mitochondrial membrane potential and increased ROS levels by 5a of Hela cells.
The Hela cells were treated with 5a at different concentrations (1, 5, and 10 nM) or DMSO (0.01%) for
24 h, followed by incubation with the fluorescent probe JCꢀ1 for 30 min. Then, the cells were analyzed
by flow cytometry (A) or fluorescence microscopy (B). (D)The fluorescence images demonstrate the
motivation of 5aꢀinduced ROS. The experiments were performed at least three times, and the results of
the representative experiments are shown. *P<0.05, ***P<0.001 vs. the control group.
Conclusions
We have developed a series of new seleniumꢀcontaining 4ꢀanilinoquinazoline derivatives
by inducing the selenium the molecular, which the element is an essential trace mineral
nutrient with multiple roles in human health, to obtain a series of new tubulin
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polymerization inhibitors. The cytotoxic activities of all these compounds and the
interactions of some of them with tubulin have been investigated. Among them, compound
5a, (2ꢀchloroꢀNꢀ(4ꢀmethoxyphenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine), possessed potent
antiproliferative abilities,with IC50 values ranging from 3.39 to 9.98 nM against all the
tested tumor cell lines. Preliminary mode of action studies demonstrated that 5a arrested
most cells in the G2/M phase of the cell and disrupted cellular microtubules and induced
apoptosis in HELA cells. In addition, mechanism considerations suggested that the
induction of apoptosis was related to loss of mitochondrial potential and ROS generation.
In a summary, these newly developed compounds showed marked biological activity and
have potential for further development as a novel class of anticancer agents.
Experimental
Unless otherwise noted, all commercially available solvents and reagents were analytically
1
pure used without further purification. H NMR and 13C NMR were recorded on a Bruker
Avance 400 spectrometer (Bruker Company, Germany) in CDCl3 using TMS as an internal
standard. Chemical shifts are quoted in ppm (parts per million). Highꢀresolution mass
spectra (HRMS) were determined using a Shimadzu LCMSꢀITTOF mass spectrometer.
Melting points (MP) of all the final solid compounds were determined on SRSꢀOptiMelt
automated melting point instrument. The purity of all the final synthesized compounds
screened in biological assays were determined to be >95% by HPLC analysis with an
Agilent LC–MS 6120 instrument (column, Eclipse Plus C8 column 4.6 × 150 mm, 5 ꢁm;
mobile phase, acetonitrile(90%)/ water (10%); flow rate, 0.20 mL/min, injection volume, 2
ꢁL; UV wavelength, 214−400 nm).
General procedure for the preparation of 2aꢀ2i
Sodium (2.3 g, 100 mmol) was dissolved in methanol (200 mL) at 0ꢀ . Paraformaldehyde
(0.84g, 28 mmol) was added to the above solution and then stirred overnight at room
temperature. Substituted phenylamine 1 (20 mmol) was added and then NaBH4 (0.76g, 20.0
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mmol). After the reaction was constituted at 60 for 2ꢀ4 hours, the solvent was evaporated,
and water was added. The mixture was extracted with ethyl acetate and then washed
(brine), dried (Na2SO4). Evaporation of the solvents to afford the crude product which was
purified by silica gel chromatography (petroleum ether: ethyl acetate=20:1) to yield the
intermediate compounds 2aꢀ2i.
Nꢀmethylaniline (2a)
1
Colorless oil, yield: 85%. H NMR (400 MHz, CDCl3) δ 7.16 (dd,
J
= 8.5, 7.4 Hz, 2H),
6.68 (t,
J
= 7.8 Hz, 1H), 6.55 (dd,
J
= 8.6, 0.9 Hz, 2H), 3.57 (s, 1H), 2.73 (s, 3H). 13C NMR
(101 MHz, CDCl3) δ 149.55 (s), 129.36 (s), 117.34 (s), 112.58 (s), 30.82 (s).
N,3ꢀdimethylaniline (2b)
Colorless oil, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 6.99 (t,
= 7.3 Hz, 1H), 6.34 (d,
= 6.4 Hz, 2H), 3.40 (s, 1H), 2.72 (s, 3H), 2.20 (s, 3H). 13C NMR
J = 8.1 Hz, 1H), 6.45 (d, J
J
(101 MHz, CDCl3) δ 149.48 (s), 139.01 (s), 129.13 (s), 118.26 (s), 113.25 (s), 109.70 (s),
30.82 (s), 21.69 (s).
3ꢀfluoroꢀNꢀmethylaniline (2c)
1
Colorless oil, yield: 70%. H NMR (400 MHz, CDCl3) δ 7.07 (dd,
J
= 14.9, 8.1 Hz, 1H),
= 11.7, 2.3 Hz, 1H), 3.75 (s, 1H), 2.77 (s,
= 242.3 Hz), 151.22 (d, = 10.7 Hz),
= 2.2 Hz), 103.51 (d, = 21.6 Hz), 98.92 (d, = 25.3
6.35 (ddd,
3H). 13C NMR (101 MHz, CDCl3) δ 164.27 (d,
130.24 (d, = 10.3 Hz), 108.39 (d,
J = 10.1, 8.5, 2.6 Hz, 2H), 6.26 (dt, J
J
J
J
J
J
J
Hz), 30.57 (s).
3ꢀchloroꢀNꢀmethylaniline (2d)
Colorless oil, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.05 (t,
= 7.8 Hz, 1H), 6.53 (s, 1H), 6.42 (dd,
= 8.1, 1.6 Hz, 1H), 3.73 (s, 1H), 2.75 (s, 3H). 13C
J = 8.0 Hz, 1H), 6.64 (d, J
J
NMR (101 MHz, CDCl3) δ 150.53 (s), 135.05 (s), 130.20 (s), 116.99 (s), 111.92 (s), 110.89
(s), 30.55 (s).
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3ꢀbromoꢀNꢀmethylaniline (2e)
Colorless oil, yield: 93%. 1H NMR (400 MHz, CDCl3) δ 6.98 (t,
= 7.8 Hz, 1H), 6.68 (s, 1H), 6.45 (dd,
= 8.2, 2.2 Hz, 1H), 3.71 (s, 1H), 2.73 (s, 3H). 13C
J
= 8.0 Hz, 1H), 6.78 (d, J
J
NMR (101 MHz, CDCl3) δ 150.67 (s), 130.52 (s), 123.34 (s), 119.87 (s), 114.82 (s), 111.30
(s), 30.56 (s).
N,2ꢀdimethylaniline (2f)
Colorless oil, yield: 83%. 1H NMR (400 MHz, CDCl3) δ 7.08 (t,
J
= 7.7 Hz, 1H), 6.97 (d,
J
= 7.2 Hz, 1H), 6.59 (t, J = 7.3 Hz, 1H), 6.53 (d, J = 8.0 Hz, 1H), 3.46 (s, 1H), 2.80 (s, 3H),
13
2.05 (s, 3H). C NMR (101 MHz, CDCl3) δ 146.20 (s), 128.86 (s), 126.15 (s), 120.86 (s),
115.81 (s), 108.09 (s), 29.73 (s), 16.35 (s).
2ꢀfluoroꢀNꢀmethylaniline (2g)
Colorless oil, yield: 65%. 1H NMR (400 MHz, CDCl3) δ 6.94 (t,
= 11.9, 8.0 Hz, 1H), 6.60 (t, = 8.4 Hz, 1H), 6.57 – 6.50 (m, 1H), 3.84 (s, 1H), 2.78 (s,
3H). 13C NMR (101 MHz, CDCl3) δ 151.65 (d,
= 238.3 Hz), 137.84 (d, = 11.5 Hz),
124.64 (d, = 3.5 Hz), 116.39 (d, = 6.9 Hz), 114.19 (d, = 18.2 Hz), 111.53 (d, = 3.4
J = 7.7 Hz, 1H), 6.87 (dd,
J
J
J
J
J
J
J
J
Hz), 30.25 (s).
2ꢀchloroꢀNꢀmethylaniline (2h)
Colorless oil, yield: 84%. 1H NMR (400 MHz, CDCl3) δ 7.17 (d,
= 7.7 Hz, 1H), 6.56 (t, = 8.3 Hz, 2H), 4.26 (s, 1H), 2.82 (d,
J
= 7.1 Hz, 1H), 7.09 (t,
J
J
J
= 5.0 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 145.03 (s), 128.98 (s), 127.86 (s), 119.07 (s), 117.03 (s), 110.64 (s),
30.41 (s).
2ꢀbromoꢀNꢀmethylaniline (2i)
1
Colorless oil, yield: 77%. H NMR (400 MHz, CDCl3) δ 7.36 (dd,
J = 7.8, 1.4 Hz, 1H),
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7.19 – 7.09 (m, 1H), 6.56 – 6.46 (m, 2H), 4.26 (s, 1H), 2.74 (d,
J
= 5.2 Hz, 3H). 13C NMR
(101 MHz, CDCl3) δ 146.13 (s), 132.43 (s), 128.77 (s), 117.75 (s), 110.92 (s), 109.77 (s),
30.69 (s).
General procedure for the preparation of 3aꢀ3i
SeO2 (1.33 g, 12 mmol) was added to the solution of malononitrile (396 mg, 6 mmol) in
DMSO. After the mixture was stirred at room temperature for 1 hours, compounds 2 (10
mmol) was added, and the reaction was stirred for 4 hours until conversion was completed.
Water was added to the mixture and then ethyl acetate. The organic phase was separated,
washed (brine), dried (Na2SO4) and evaporated to afford crude product 3, which was
purified by silica gel chromatography (petroleum ether: ethyl acetate=10:1).
Nꢀmethylꢀ4ꢀselenocyanatoaniline (3a)
White soild, yield: 75%. 1H NMR (400 MHz, CDCl3) δ 7.45 (d,
= 8.8 Hz, 2H), 4.10 (s, 1H), 2.82 (d,
= 4.3 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
J = 8.8 Hz, 2H), 6.54 (d, J
J
151.11 (s), 136.63 (s), 113.55 (s), 105.18 (s), 102.99 (s), 30.23 (s).
N,3ꢀdimethylꢀ4ꢀselenocyanatoaniline (3b)
White soild, yield: 70%. 1H NMR (400 MHz, CDCl3) δ 7.47 (d,
= 2.5 Hz, 1H), 6.37 (dd,
= 8.5, 2.7 Hz, 1H), 4.00 (s, 1H), 2.82 (s, 3H), 2.49 (s, 3H). 13C
J = 8.5 Hz, 1H), 6.51 (d, J
J
NMR (101 MHz, CDCl3) δ 151.71 (s), 143.27 (s), 137.90 (s), 114.39 (s), 111.13 (s), 107.05
(s), 102.49 (s), 30.24 (s), 23.36 (s).
3ꢀfluoroꢀNꢀmethylꢀ4ꢀselenocyanatoaniline (3c)
White soild, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.41 (t,
= 9.4 Hz, 2H), 4.25 (s, 1H), 2.84 (d,
= 5.1 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ
163.29 (d, = 245.8 Hz), 153.83 (d, = 10.9 Hz), 137.36 (d, = 2.6 Hz), 109.90 (d, = 2.1
Hz), 101.55 (s), 99.13 (d, = 22.5 Hz), 30.22 (s).
J = 7.8 Hz, 1H), 6.35 (d, J
J
J
J
J
J
J
= 26.8 Hz), 91.65 (d, J
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3ꢀchloroꢀNꢀmethylꢀ4ꢀselenocyanatoaniline (3d)
White soild, yield: 79%. 1H NMR (400 MHz, CDCl3) δ 7.49 (d,
= 2.6 Hz, 1H), 6.47 (dd, = 8.7, 2.6 Hz, 1H), 4.14 (s, 1H), 2.83 (d,
J
= 8.7 Hz, 1H), 6.66 (d,
J
J
J
= 5.1 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 151.84 (s), 137.52 (s), 135.69 (s), 112.88 (s), 112.63 (s), 106.07
(s), 101.92 (s), 30.24 (s).
3ꢀbromoꢀNꢀmethylꢀ4ꢀselenocyanatoaniline (3e)
White soild, yield: 87%. 1H NMR (400 MHz, CDCl3) δ 7.50 (d,
= 2.6 Hz, 1H), 6.52 (dd, = 8.7, 2.6 Hz, 1H), 4.10 (s, 1H), 2.82 (d,
J
= 8.7 Hz, 1H), 6.82 (d,
J
J
J
= 5.2 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 151.54 (s), 135.09 (s), 127.11 (s), 116.08 (s), 113.28 (s), 108.78
(s), 102.42 (s), 30.26 (s).
N,2ꢀdimethylꢀ4ꢀselenocyanatoaniline (3f)
White soild, yield: 77%. 1H NMR (400 MHz, CDCl3) δ 7.43 (dd,
(s, 1H), 6.52 (d, = 8.5 Hz, 1H), 3.89 (s, 1H), 2.89 (d,
J
= 8.4, 1.9 Hz, 1H), 7.34
13
J
J
= 4.8 Hz, 3H), 2.09 (s, 3H). C
NMR (101 MHz, CDCl3) δ 149.16 (s), 136.47 (s), 134.75 (s), 123.72 (s), 110.07 (s), 104.85
(s), 103.05 (s), 30.41 (s), 17.17 (s).
2ꢀfluoroꢀNꢀmethylꢀ4ꢀselenocyanatoaniline (3g)
White soild, yield: 74%. 1H NMR (400 MHz, CDCl3) δ 7.32 (d,
= 10.6 Hz, 1H), 6.60 (t, = 8.6 Hz, 1H), 4.30 (s, 1H), 2.86 (d,
(101 MHz, CDCl3) δ 150.89 (d, = 244.9 Hz), 140.11 (d,
= 20.0 Hz), 111.96 (d, = 3.9 Hz), 103.67 (d, J
J
= 8.5 Hz, 1H), 7.27 (d, J
13
J
J
= 5.2 Hz, 3H). C NMR
J
J
= 11.4 Hz), 132.47 (d,
J = 3.1
Hz), 120.75 (d,
29.75 (s).
J
J
= 7.1 Hz), 102.38 (s),
2ꢀchloroꢀNꢀmethylꢀ4ꢀselenocyanatoaniline (3h)
White soild, yield: 82%. 1H NMR (400 MHz, CDCl3) δ 7.57 (d,
J = 2.1 Hz, 1H), 7.46 (dd,
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J
= 8.5, 2.0 Hz, 1H), 6.59 (d, J = 8.6 Hz, 1H), 4.68 (s, 1H), 2.91 (d, J
= 5.1 Hz, 3H). 13C
NMR (101 MHz, CDCl3) δ 146.84 (s), 135.32 (s), 135.30 (s), 119.63 (s), 111.37 (s), 104.68
(s), 102.28 (s), 30.10 (s).
2ꢀbromoꢀNꢀmethylꢀ4ꢀselenocyanatoaniline (3i)
White soild, yield: 75%. 1H NMR (400 MHz, CDCl3) δ 7.73 (d,
= 8.6, 2.0 Hz, 1H), 6.57 (d, = 8.6 Hz, 1H), 4.69 (s, 1H), 2.91 (d, J
J
= 2.1 Hz, 1H), 7.50 (dd,
= 5.1 Hz, 3H). 13C
J
J
NMR (101 MHz, CDCl3) δ 147.74 (s), 138.47 (s), 135.90 (s), 111.41 (s), 109.79 (s), 105.21
(s), 102.27 (s), 30.34 (s).
General procedure for the preparation of 5a, 5b, 5d, 5f
To a solution containing 3 (3a, 3b, 3d, 3f, 1 mmol, respectively) and 2, 4ꢀ
dichloroquinazoline (199 mg, 1 mmol) in 30 mL IPA, HCl (12N, 0.5 mL) was added. The
mixture was stirred for 5ꢀ8 hours and then filtered. The precipitate was purified by silica
gel chromatography with petroleum ether/ethyl acetate (5: 1) to get the title compounds 5a,
5b, 5d and 5f.
2ꢀchloroꢀNꢀmethylꢀNꢀ(4ꢀselenocyanatophenyl)quinazolinꢀ4ꢀamine (5a)
1
White soild, yield: 70%. mp: 142.2°C ꢀ143.2°C. H NMR (400 MHz, CDCl3) δ 7.79 (dd,
J
J
= 8.3, 3.7 Hz, 1H), 7.66 (dd,
J
= 8.5, 1.9 Hz, 2H), 7.62 (dd,
J
= 8.4, 1.7 Hz, 1H), 7.22 (d,
= 1.7 Hz, 1H), 7.20 (s, 1H), 7.14 – 7.08 (m, 1H), 6.99 (dd,
J
= 8.5, 1.4 Hz, 1H), 3.66 (d,
J
=
3.0 Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 163.11 (s), 156.54 (s), 153.17 (s), 149.19 (s),
134.70 (s), 133.29 (s), 128.11 (s), 127.35 (s), 126.02 (s), 125.75 (s), 119.45 (s), 114.83 (s),
+
+
101.01 (s), 42.66 (s). ESIꢀMS (m/z) 375.0 [M+H] ; (HRMS (ESI) (m/z) [M+Na] calcd
for C16H11ClN4Se, 396.9728; found, 396.9726. Purity: 96.2% (by HPLC).
2ꢀchloroꢀNꢀmethylꢀNꢀ(3ꢀmethylꢀ4ꢀselenocyanatophenyl)quinazolinꢀ4ꢀamine (5b)
1
White soild, yield: 39%. mp: 164.7°C ꢀ165.4°C. H NMR (400 MHz, CDCl3) δ 7.77 (dd,
J
= 21.0, 8.3 Hz, 2H), 7.61 (s, 1H), 7.15 (s, 1H), 7.11 (d, J = 7.2 Hz, 1H), 7.02 (d, J = 8.9 Hz,
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2H), 3.62 (s, 3H), 2.46 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.98 (s), 156.50 (s),
153.09 (s), 149.50 (s), 142.42 (s), 135.85 (s), 133.22 (s), 128.06 (s), 128.01 (s), 126.09 (s),
125.66 (s), 124.90 (s), 120.91 (s), 114.86 (s), 100.72 (s), 42.73 (s), 22.59 (s). ESIꢀMS (m/z
)
+
+
389.0 [M+H] ; HRMS (ESI) (m/z) [M+H] calcd for C17H13ClN4Se, 389.0065; found,
389.0063. Purity: 97.9% (by HPLC).
2ꢀchloroꢀNꢀ(3ꢀchloroꢀ4ꢀselenocyanatophenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine (5d)
White soild, yield: 44%. mp: 207.7°C ꢀ208.6°C. 1H NMR (400 MHz, CDCl3) δ 7.83 (d,
8.2 Hz, 1H), 7.76 (d, = 8.5 Hz, 1H), 7.68 (t, = 7.4 Hz, 1H), 7.27 (s, 1H), 7.23 – 7.17 (m,
1H), 7.15 (d, = 8.4 Hz, 1H), 7.08 (d,
= 8.4 Hz, 1H), 3.67 (s, 3H). 13C NMR (101 MHz,
J =
J
J
J
J
CDCl3) δ 163.02 (s), 156.51 (s), 153.28 (s), 149.36 (s), 134.04 (s), 133.51 (s), 132.42 (s),
128.30 (s), 126.53 (s), 126.06 (s), 125.80 (s), 125.74 (s), 121.66 (s), 114.83 (s), 100.44 (s),
42.65 (s). ESIꢀMS (m/z) 409.0 [M+H] +; HRMS (ESI) (m/z) [M+Na]+ calcd for
C16H10Cl2N4Se, 430.9336; found, 430.9333. Purity: 95.6% (by HPLC).
2ꢀchloroꢀNꢀmethylꢀNꢀ(2ꢀmethylꢀ4ꢀselenocyanatophenyl)quinazolinꢀ4ꢀamine (5f)
White soild, yield: 38%. mp: 153.8°C ꢀ154.3°C. 1H NMR (400 MHz, CDCl3) δ 7.77 (d,
J =
7.3 Hz, 1H), 7.65 (s, 1H), 7.61 (t,
J
= 7.5 Hz, 1H), 7.53 (d,
J
= 7.9 Hz, 1H), 7.19 (d,
J
= 8.2
13
Hz, 1H), 7.05 (t, = 7.6 Hz, 1H), 6.75 (d,
J
J
= 8.5 Hz, 1H), 3.54 (s, 3H), 2.22 (s, 3H). C
NMR (101 MHz, CDCl3) δ 162.15 (s), 156.58 (s), 152.93 (s), 147.16 (s), 137.87 (s), 135.99
(s), 133.08 (s), 132.08 (s), 129.47 (s), 128.08 (s), 125.76 (s), 124.64 (s), 121.65 (s), 114.60
+
(s), 100.96 (s), 41.76 (s), 17.93 (s). ESIꢀMS (m/z) 389.0 [M+H] ; HRMS (ESI) (m/z
)
[M+Na]+ calcd for C17H13ClN4Se, 410.9884; found, 410.9890. Purity: 96.4% (by HPLC).
General procedure for the preparation of 4a, 4c, 4e, 4g, 4h, 4i, 6a, 6b, 6d, 6f
To a solution of 3 (3a, 3c, 3e, 3g, 3h, 3i, 10 mmol, respectively) or 5 (5a, 5b, 5d, 5f, 10
mmol, respectively) in CH3OH (10 mL), NaBH4 (380 mg, 10 mmol) and CH3I (920 ꢁL, 15
mmol) were added in sequence. After the mixture was stirred at room temperature for 2
minutes, saturated NaHSO4 solution was added and then ethyl acetate. The organic layer
was separated, washed (brine), and dried (Na2SO4). Evaporation of the solvents to afford
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the crude product which was purified by silica gel chromatography (petroleum ether/ethyl
acetate, 5: 1) to obtain the title compounds 4a, 4c, 4e, 4g, 4h, 4i, 6a, 6b, 6d and 6f.
Nꢀmethylꢀ4ꢀ(methylselanyl)aniline (4a)
Colorless oil, yield: 75%. 1H NMR (400 MHz, CDCl3) δ 7.28 (d,
= 8.6 Hz, 2H), 3.66 (s, 1H), 2.74 (s, 3H), 2.19 (s, 3H). 13C NMR (101 MHz, CDCl3) δ
J = 8.7 Hz, 2H), 6.45 (d,
J
148.67 (s), 134.31 (s), 116.87 (s), 113.16 (s), 30.66 (s), 9.34 (s).
3ꢀfluoroꢀNꢀmethylꢀ4ꢀ(methylselanyl)aniline (4c)
1
Colorless oil, yield: 66%. H NMR (400 MHz, CDCl3) δ 7.27 (t,
J = 8.2 Hz, 1H), 6.31 (s,
1H), 6.28 (s, 1H), 3.90 (s, 1H), 2.79 (d,
CDCl3) δ 163.55 (d, = 240.4 Hz), 151.20 (d,
(d, = 2.4 Hz), 102.33 (d, = 23.3 Hz), 99.16 (d,
Hz).
J
= 3.7 Hz, 3H), 2.23 (s, 3H). 13C NMR (101 MHz,
= 10.7 Hz), 135.82 (d, = 4.7 Hz), 109.13
= 28.1 Hz), 30.53 (s), 8.35 (d, = 2.1
J
J
J
J
J
J
J
3ꢀbromoꢀNꢀmethylꢀ4ꢀ(methylselanyl)aniline (4e)
Colorless oil, yield: 79%. 1H NMR (400 MHz, CDCl3) δ 7.18 (d,
= 2.5 Hz, 1H), 6.46 (dd,
= 8.5, 2.6 Hz, 1H), 3.76 (s, 1H), 2.76 (s, 3H), 2.26 (s, 3H). 13C
J
= 8.5 Hz, 1H), 6.79 (d,
J
J
NMR (101 MHz, CDCl3) δ 149.30 (s), 132.87 (s), 127.63 (s), 118.80 (s), 116.13 (s), 112.72
(s), 30.60 (s), 8.47 (s).
2ꢀfluoroꢀNꢀmethylꢀ4ꢀ(methylselanyl)aniline (4g)
Colorless oil, yield: 72%. 1H NMR (400 MHz, CDCl3) δ 7.09 (d,
= 11.4, 1.8 Hz, 1H), 6.49 (t,
NMR (101 MHz, CDCl3) δ 150.15 (d,
J
= 8.2 Hz, 1H), 7.05 (dd,
13
J
J
= 8.6 Hz, 1H), 3.87 (s, 1H), 2.76 (s, 3H), 2.19 (s, 3H). C
J
= 242.3 Hz), 136.05 (d,
J
= 11.7 Hz), 128.30 (d,
J
= 3.2 Hz), 117.71 (d,
8.17 (s).
J
= 18.8 Hz), 114.97 (d, = 6.0 Hz), 110.87 (d, J
J
= 3.8 Hz), 29.12 (s),
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2ꢀchloroꢀNꢀmethylꢀ4ꢀ(methylselanyl)aniline (4h)
Colorless oil, yield: 74%. 1H NMR (400 MHz, CDCl3) δ 7.41 (d,
= 8.4, 1.9 Hz, 1H), 6.52 (d, = 8.4 Hz, 1H), 4.34 (s, 1H), 2.85 (d, J
J
= 2.0 Hz, 1H), 7.30 (dd,
= 5.1 Hz, 3H), 2.25
J
J
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 144.31 (s), 133.32 (s), 132.89 (s), 119.21 (s),
116.59 (s), 111.17 (s), 30.39 (s), 9.50 (s).
2ꢀbromoꢀNꢀmethylꢀ4ꢀ(methylselanyl)aniline (4i)
Colorless oil, yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.51 (d,
= 8.4, 1.8 Hz, 1H), 6.43 (d, = 8.4 Hz, 1H), 4.28 (s, 1H), 2.78 (d, J
J
= 1.9 Hz, 1H), 7.27 (dd,
= 5.2 Hz, 3H), 2.18
J
J
(s, 3H). 13C NMR (101 MHz, CDCl3) δ 145.24 (s), 136.47 (d,
J
= 4.9 Hz), 133.58 (d,
J
=
4.9 Hz), 117.12 (s), 111.22 (s), 109.66 (s), 30.59 (s), 9.60 (s).
2ꢀchloroꢀNꢀmethylꢀNꢀ(4ꢀ(methylselanyl)phenyl)quinazolinꢀ4ꢀamine (6a)
Green soild, yield: 68%. mp: 123.9°C ꢀ124.6°C. 1H NMR (400 MHz, CDCl3) δ 7.74 (d,
J
=
8.3 Hz, 1H), 7.62 – 7.52 (m, 1H), 7.43 (d,
7.01 (m, 1H), 6.98 (d,
= 9.3 Hz, 1H), 3.62 (s, 3H), 2.39 (s, 3H). 13C NMR (101 MHz,
CDCl3) δ 162.72 (s), 156.57 (s), 153.02 (s), 145.32 (s), 132.81 (s), 131.58 (s), 131.26 (s),
127.78 (s), 126.66 (s), 126.35 (s), 125.21 (s), 114.81 (s), 42.95 (s), 7.29 (s). ESIꢀMS (m/z
J = 8.5 Hz, 2H), 7.10 (d, J = 8.5 Hz, 2H), 7.07 –
J
)
+
+
364.0 [M+H] ; HRMS (ESI) (m/z) [M+H] calcd for C16H14ClN3Se, 364.0112; found,
364.0115. Purity: 99.3% (by HPLC).
2ꢀchloroꢀNꢀmethylꢀNꢀ(3ꢀmethylꢀ4ꢀ(methylselanyl)phenyl)quinazolinꢀ4ꢀamine (6b)
White soild, yield: 81%. mp: 164.3°C ꢀ165.3°C. 1H NMR (400 MHz, CDCl3) δ 7.74 (d,
8.3 Hz, 1H), 7.62 – 7.53 (m, 1H), 7.28 (d, = 8.3 Hz, 1H), 7.06 – 7.01 (m, 2H), 7.00 (d,
1.1 Hz, 1H), 6.97 (dd,
J
J
=
=
J
J
= 8.4, 2.3 Hz, 1H), 3.61 (s, 3H), 2.36 (s, 3H), 2.34 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 162.65 (s), 156.61 (s), 152.99 (s), 144.92 (s), 139.81 (s), 132.75
(s), 132.40 (s), 129.44 (s), 127.72 (s), 127.18 (s), 126.45 (s), 125.13 (s), 124.15 (s), 114.91
(s), 43.07 (s), 21.74 (s), 6.40 (s). ESIꢀMS (m/z) 378.0 [M+H] +; HRMS (ESI) (m/z) [M+H]+
calcd for C17H16ClN3Se, 378.0269; found, 378.0269. Purity: 98.8% (by HPLC).
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2ꢀchloroꢀNꢀ(3ꢀchloroꢀ4ꢀ(methylselanyl)phenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine (6d)
White soild, yield: 50%. mp: 164.3°C ꢀ165.3°C. 1H NMR (400 MHz, CDCl3) δ 7.77 (d,
J
J
=
=
6.6 Hz, 1H), 7.61 (s, 1H), 7.24 (d,
J = 12.6 Hz, 2H), 7.10 (d, J = 6.2 Hz, 1H), 7.03 (dd,
15.4, 8.0 Hz, 2H), 3.62 (s, 3H), 2.37 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.71 (s),
156.52 (s), 153.08 (s), 145.70 (s), 134.84 (s), 133.06 (s), 132.06 (s), 129.49 (s), 127.96 (s),
126.61 (s), 126.15 (s), 125.56 (s), 124.90 (s), 114.76 (s), 42.94 (s), 6.58 (s). ESIꢀMS (m/z
)
+
398.0 [M+H] ; HRMS (ESI) (m/z) [M+H]+ calcd for C16H13Cl2N3Se, 397.9720; found,
397.9728. Purity: 95.6% (by HPLC).
2ꢀchloroꢀNꢀmethylꢀNꢀ(2ꢀmethylꢀ4ꢀ(methylselanyl)phenyl)quinazolinꢀ4ꢀamine (6f)
White soild, yield: 59%. mp: 104.6°C ꢀ105.3°C. 1H NMR (400 MHz, CDCl3) δ 7.66 (d, J
= 8.3 Hz, 1H), 7.49 (t, J = 7.6 Hz, 1H), 7.30 (s, 1H), 7.21 (d, J = 9.9 Hz, 1H), 6.96 (d, J =
8.3 Hz, 1H), 6.92 (d, J = 7.6 Hz, 1H), 6.72 (d, J = 8.6 Hz, 1H), 3.45 (s, 3H), 2.33 (s, 3H),
2.07 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 160.94 (s), 155.60 (s), 151.81 (s), 142.31 (s),
134.74 (s), 131.98 (s), 131.73 (s), 131.59 (s), 128.19 (s), 126.95 (s), 126.76 (s), 124.37 (s),
+
124.05 (s), 113.72 (s), 40.88 (s), 16.66 (s), 6.11 (s). ESIꢀMS (m/z) 378.0 [M+H] ; HRMS
+
(ESI) (m/z) [M+H] calcd for C17H16ClN3Se, 378.0271; found, 378.0269. Purity: 95.4%
(by HPLC).
General procedure for the preparation of 6c, 6e, 6g, 6h, 6i
For the preparation of 6c and 6g, intermediates 4c or 4g (218 mg, 1 mmol), 2, 4ꢀ
dichloroquinazoline (199 mg, 1 mmol) and NaAc (246 mg 3 mmol) were added to the
solution of THF/H2O (1/1, 18ml), and stirred at 60 for 3 h. The mixture was diluted with
H2O and ethyl acetate after the reaction completed. The organic layer was separated,
washed with brine, dried over Na2SO4, and evaporated to give an amber residue, which was
subjected to silica gel chromatography (petroleum ether: ethyl acetate=10:1) to obtain 6c
and 6g. For the preparation of 6e, 6h and 6i, the mixture of 4e or 4h, 4i (1 mmol), 2, 4ꢀ
dichloroquinazoline (199 mg, 1 mmol) and NaH (60%, 60 mg, 1.5 mmol) in DMF (15ml)
was stirred at 0 for 4 h.
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2ꢀchloroꢀNꢀ(3ꢀfluoroꢀ4ꢀ(methylselanyl)phenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine (6c)
White soild, yield: 74%. mp: 159.1°C ꢀ160.0°C. 1H NMR (400 MHz, CDCl3) δ 7.78 (d,
J =
8.2 Hz, 1H), 7.62 (t,
= 8.4 Hz, 1H), 6.92 (dd,
Hz, 3H). 13C NMR (101 MHz, CDCl3) δ 162.83 (d,
J
= 7.1 Hz, 1H), 7.35 (t,
= 11.4, 9.6 Hz, 2H), 3.62 (d,
= 11.0 Hz), 160.33 (s), 156.54 (s),
= 9.2 Hz), 133.09 (s), 132.25 (d, = 5.1 Hz), 127.99 (s), 126.11 (s),
J
= 6.6 Hz, 1H), 7.17 – 7.08 (m, 1H), 7.05 (d,
J
J
J
= 14.6 Hz, 3H), 2.36 (d, = 14.3
J
J
153.11 (s), 147.07 (d,
J
J
125.56 (s), 122.25 (d,
J
= 3.0 Hz), 117.78 (d,
J
= 22.3 Hz), 114.86 (s), 113.02 (d, J = 24.4
+
Hz), 42.80 (s), 6.29 (s). ESIꢀMS (m/z) 382.1 [M+H] ; HRMS (ESI) (m/z) [M+H]+ calcd
for C16H13ClFN3Se, 382.0018; found, 382.0026. Purity: 99.6% (by HPLC).
Nꢀ(3ꢀbromoꢀ4ꢀ(methylselanyl)phenyl)ꢀ2ꢀchloroꢀNꢀmethylquinazolinꢀ4ꢀamine (6e)
1
White soild, yield: 85%. mp: 195.6°C ꢀ196.3°C. H NMR (400 MHz, CDCl3) δ 7.77 (dd,
J
= 8.2, 3.8 Hz, 1H), 7.65 – 7.57 (m, 1H), 7.44 – 7.39 (m, 1H), 7.19 (d,
J = 8.4 Hz, 1H), 7.11
(ddd, = 6.9, 5.4, 1.2 Hz, 1H), 7.08 – 7.02 (m, 2H), 3.61 (dd, = 2.8, 1.0 Hz, 3H), 2.36 (s,
J
J
3H). 13C NMR (101 MHz, CDCl3) δ 162.69 (s), 156.52 (s), 153.10 (s), 145.64 (s), 134.49
(s), 133.06 (s), 129.83 (s), 129.32 (s), 127.97 (s), 126.16 (s), 125.57 (s), 125.44 (s), 124.58
+
(s), 114.76 (s), 42.98 (s), 7.48 (s). ESIꢀMS (m/z) 443.9 [M+H] ; HRMS (ESI) (m/z
)
[M+H]+ calcd for C16H13BrClN3Se, 441.9215; found, 441.9217. Purity: 99.0% (by HPLC).
2ꢀchloroꢀNꢀ(2ꢀfluoroꢀ4ꢀ(methylselanyl)phenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine (6g)
Yellow oil, yield: 45%. 1H NMR (400 MHz, CDCl3) δ 7.70 (d,
7.2 Hz, 1H), 7.16 – 7.12 (m, 1H), 7.11 (d, = 3.2 Hz, 1H), 7.04 (d,
(d, = 7.2 Hz, 1H), 6.96 (d,
= 8.3 Hz, 1H), 3.50 (s, 3H), 2.33 (s, 3H). 13C NMR (101
MHz, CDCl3) δ 162.84 (s), 158.68 (s), 156.28 (d, = 25.8 Hz), 152.90 (s), 134.01 (d,
6.6 Hz), 133.00 (s), 132.26 (d, = 11.9 Hz), 128.72 (d, = 25.6 Hz), 127.96 (s), 126.46 (d,
= 3.6 Hz), 125.60 (s), 124.89 (s), 118.17 (d, = 21.3 Hz), 114.76 (s), 41.93 (s), 7.24 (s).
J
= 8.3 Hz, 1H), 7.53 (t,
J =
J
J
= 7.8 Hz, 1H), 7.01
J
J
J
J =
J
J
J
J
+
ESIꢀMS (m/z) 382.0 [M+H] ; HRMS (ESI) (m/z) [M+H]+ calcd for C16H13ClFN3Se,
382.0018; found, 382.0010. Purity: 99.1% (by HPLC).
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2ꢀchloroꢀNꢀ(2ꢀchloroꢀ4ꢀ(methylselanyl)phenyl)ꢀNꢀmethylquinazolinꢀ4ꢀamine (6h)
White soild, yield: 44%. mp: 117.1°C ꢀ117.9°C. 1H NMR (400 MHz, CDCl3) δ 7.76 (d,
J =
8.3 Hz, 1H), 7.59 (t,
J
= 7.6 Hz, 1H), 7.53 (d,
J
= 1.6 Hz, 1H), 7.31 (dd,
J = 8.2, 1.7 Hz,
1H), 7.09 (d, = 8.1 Hz, 1H), 7.06 (d,
J
J
= 7.4 Hz, 1H), 6.87 (d, J
= 8.5 Hz, 1H), 3.54 (s,
3H), 2.42 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.50 (s), 156.48 (s), 152.87 (s), 141.78
(s), 134.23 (s), 132.93 (s), 132.69 (s), 131.47 (s), 129.67 (s), 129.24 (s), 127.92 (s), 125.68
(s), 124.85 (s), 114.82 (s), 41.53 (s), 29.72 (s). ESIꢀMS (m/z) 397.9 [M+H] +; HRMS (ESI)
(
m/z) [M+H]+ calcd for C16H13Cl2N3Se, 397.9720; found, 397.9721. Purity: 95.9% (by
HPLC).
Nꢀ(2ꢀbromoꢀ4ꢀ(methylselanyl)phenyl)ꢀ2ꢀchloroꢀNꢀmethylquinazolinꢀ4ꢀamine (6i)
White soild, yield: 70%. mp: 116.9°C ꢀ117.4°C. 1H NMR (400 MHz, CDCl3) δ 7.67 (d,
J =
8.3 Hz, 1H), 7.64 (s, 1H), 7.51 (t,
J
= 7.5 Hz, 1H), 7.28 (d,
J = 7.7 Hz, 1H), 7.02 (d, J = 8.0
Hz, 1H), 6.98 (d, = 7.6 Hz, 1H), 6.78 (d,
J
J
= 8.5 Hz, 1H), 3.45 (s, 3H), 2.34 (s, 3H). 13C
NMR (101 MHz, CDCl3) δ 162.28 (s), 156.46 (s), 152.86 (s), 143.25 (s), 134.57 (s), 134.55
(s), 132.91 (s), 130.38 (s), 129.38 (s), 127.88 (s), 125.70 (s), 124.98 (s), 123.02 (s), 114.80
(s), 41.70 (s), 7.35 (s). ESIꢀMS (m/z) 443.9 [M+H] +; HRMS (ESI) (m/z) [M+H]+ calcd for
C16H13BrClN3Se, 441.9215; found, 441.9213. Purity: 97.6% (by HPLC).
General procedure for the preparation of 7a
A solution of 6a (2ꢀchloroꢀNꢀmethylꢀNꢀ(4ꢀ(methylselanyl)phenyl)quinazolinꢀ4ꢀamine,
363 mg, 1 mmol) in CH3OH (20ml) containing of NaOH (120 mg, 3 mmol) was heated at
70
ꢀ for 3 h. The solvent was evaporated to give an amber residue, which was further
purify by silica gel chromatography (petroleum ether: ethyl acetate=10:1) to give 7a.
2ꢀmethoxyꢀNꢀmethylꢀNꢀ(4ꢀ(methylselanyl)phenyl)quinazolinꢀ4ꢀamine (7a)
Yellow oil, yield: 52%. 1H NMR (400 MHz, CDCl3) δ 7.65 (d,
7.6 Hz, 1H), 7.40 (s, 1H), 7.38 (s, 1H), 7.08 (s, 1H), 7.05 (s, 1H), 7.02 (d,
6.88 (t,
= 7.7 Hz, 1H), 4.10 (s, 3H), 3.59 (s, 3H), 2.37 (s, 3H). 13C NMR (101 MHz,
J
= 8.3 Hz, 1H), 7.48 (t,
J =
J
= 8.4 Hz, 1H),
J
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CDCl3) δ 164.16 (s), 162.15 (s), 153.49 (s), 146.46 (s), 132.24 (s), 131.58 (s), 129.96 (s),
126.98 (s), 126.56 (s), 122.52 (s), 113.93 (s), 54.40 (s), 42.67 (s), 7.36 (s). ESIꢀMS (m/z
)
+
360.1 [M+H] ; HRMS (ESI) (m/z) [M+H]+ calcd for C17H17N3OSe, 360.0610; found,
360.0609. Purity: 99.8% (by HPLC).
General procedure for the preparation of 9
4ꢀbromoꢀ2Hꢀchromenꢀ2ꢀone (9) was synthesized according to literature method.1
4ꢀbromoꢀ2Hꢀchromenꢀ2ꢀone (9)
White soild, yield: 80%. 1H NMR (400 MHz, CDCl3) δ 7.83 (dt,
(dt, = 8.4, 4.2 Hz, 1H), 7.42 – 7.29 (m, 2H), 6.85 (d,
= 8.0 Hz, 1H). 13C NMR (101
J = 8.0, 4.1 Hz, 1H), 7.60
J
J
MHz, CDCl3) δ 158.61 (s), 152.44 (s), 141.44 (s), 133.21 (s), 128.01 (s), 124.96 (s), 119.54
(s), 118.89 (s), 116.97 (s).
General procedure for the preparation of 10
To a solution of 4ꢀbromoꢀ2Hꢀchromenꢀ2ꢀone (9, 224 mg, 1 mmol) and 2, 4ꢀ
dichloroquinazoline (199 mg, 1 mmol) in 20 mL DMF, DIPEA (193.5 mg, 1.5 mmol) was
added and the mixture was stirred at 110
ꢀ overnight. After cooled to the room temperature,
the reaction mixture was diluted with H2O and ethyl acetate. The organic layers was
filtered, washed with brine and dried over Na2SO4. Evaporation of the solvent to get the
crude product which was purified by silica gel chromatography (petroleum ether: ethyl
acetate=10:1) to afford 4ꢀ(methyl(4ꢀ(methylselanyl)phenyl)amino)ꢀ2Hꢀchromenꢀ2ꢀone
(10).
4ꢀ(methyl(4ꢀ(methylselanyl)phenyl)amino)ꢀ2Hꢀchromenꢀ2ꢀone (10)
Brown solid, yield: 49%. mp: 109.6°C ꢀ110.3°C
.
1H NMR (400 MHz, CDCl3) δ 7.36 (d,
J
= 7.9 Hz, 3H), 7.29 (s, 1H), 6.99 (d, = 7.9 Hz, 3H), 6.89 (t,
J
J
= 7.3 Hz, 1H), 5.90 (s, 1H),
3.38 (s, 3H), 2.35 (s, 3H). 13C NMR (101 MHz, CDCl3) δ 162.58 (s), 156.97 (s), 154.15 (s),
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146.48 (s), 131.61 (s), 131.05 (s), 129.13 (s), 126.63 (s), 125.29 (s), 122.94 (s), 117.58 (s),
+
115.94 (s), 97.71 (s), 43.64 (s), 7.44 (s). ESIꢀMS (m/z) 346 [M+H] ; HRMS (ESI) (m/z
)
[M+H]+ calcd. for C17H15NO2Se, 346.0341; found, 346.0338. Purity: 96.7% (by HPLC).
Cell Lines and Culture Conditions
The human cancer cell lines (A549, HeLa, HEPG2, RKO, LOVO, HCT116) used in this
study were purchased from the Laboratory Animal Service Center at Sun Yatꢀsen
University (Guangzhou, China). Human cancer cell lines A549, HeLa, HEPG2, RKO were
cultivated in DMEM containing 10% (v/v) heatꢀinactivated fetal bovine serum (FBS), 100
units/mL penicillin, and 100
μg/mL streptomycin, respectively. Human cancer cell lines
HCTꢀ116 were cultivated in RPMI 1640 medium containing 10% (v/v) heatꢀinactivated
FBS, 100 units/ mL penicillin, and 100 mg/mL streptomycin, respectively. The cells were
incubated at 37 oC under a 5% CO2 and 90% relative humidity (RH) atmosphere.
MTT assay. Cells grown to the logarithmic phase were seeded 5× ×
103 ꢀ10 103 cells/well
into 96ꢀwell plates (Nest Biotechnology Co.,Ltd.) 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 solubilized in dimethyl sulfoxide
(DMSO). The absorbance of the solution at 570 nm was measured using a multifunction
microplate reader (Molecular Devices, Flex Station 3), and each experiment was performed
at least in triplicate. IC50 values, which the drug concentrations required to cause 50%
cancer cell growth inhibition, were used to express the cytotoxic effects of each compound
and were calculated with GraphPad Prism Software version 5.02 (GraphPad Inc., La Jolla,
CA, USA).
In vitro tubulin polymerization assay. A tubulin polymerization assay was performed by
measuring the increase in fluorescence intensity, which can be easily recorded due to the
incorporation of a fluorescent reporter, DAPI (4
’,6ꢀdiamidinoꢀ2ꢀphenylindole), a
fluorophore that is known to be a DNA intercalator. A commercial kit (cytoskeleton, cat.
#BK011P) purchased from Cytoskeleton (Danvers, MA, USA) was used for the tubulin
polymerization. The final buffer used for tubulin polymerization contained 80.0 mM
piperazineꢀN, N
′
ꢀbis(2ꢀethanesulfonic acid) sequisodium salt (pH 6.9), 2.0 mM MgCl2,
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0.5 mM EGTA, 1 mM GTP, and 10.2% glycerol. First, 5
μ
L of the tested compounds at
C for 1 min;
L of the tubulin solution. The
the indicated concentrations was added, and the mixture was warmed to 37
then, the reaction was initiated by the addition of 55
°
μ
fluorescence intensity enhancement was recorded every 60 sec for 90 min in a
multifunction microplate reader (Molecular Devices, Flex Station 3) (emission wavelength
of 410 nm, excitation wavelength of 340 nm). The area under the curve was used to
determine the concentration that inhibited tubulin polymerization by 50% (IC50) and was
calculated using GraphPad Prism Software version 5.02 (GraphPad Inc., La Jolla, CA,
USA).
Immunofluorescence microscopy. In a 10 mm confocal culture dish, 1×
104 cells were
grown for 24 h and then incubated in the presence/absence of compound 5a at the indicated
concentrations for another 8 h. After being washed with phosphateꢀbuffered solution (PBS)
and fixed in 4% preꢀwarmed (37oC) paraformaldehyde for 15 min, the cells were
permeabilized with 0.5% Triton Xꢀ100 for 15 min and blocked for 30 min in 10% goat
serum. Then, the cells were incubated with mouse antiꢀtubulin antibody (CST, USA) at 4oC
overnight, washed with PBS three times, and incubated with goat antiꢀmouse IgG/Alexaꢀ
Fluor 488 antibody (Invitrogen, USA) for 1 h. The samples were immediately visualized on
a Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss, Germany) after the
nuclei were stained with Hoechst 33342 (Sigma, USA) in the dark at room temperature for
30 min.
Cell cycle analysis. HELA cells were seeded in 6ꢀwell plates (3
×
105 cells/well), incubated
in the presence/absence of compound 5a at the indicated concentrations for 12 or 24 h,
harvested by centrifugation, and then fixed in iceꢀcold 70% ethanol overnight. After the
ethanol was removed the next day, the cells were reꢀsuspended in iceꢀcold PBS, treated
with RNAse A (Keygen Biotech, China) at 37°C for 30 min, and then incubated with the
DNA staining solution propidium iodide (PI, Keygen Biotech, China) at 4°C for 30 min.
Approximately 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 analysed using EXPO32 ADC analysis software.
Apoptosis analysis. The preparation of the HELA cell sample was the same as for the cell
cycle analysis. After incubation, cells were harvested and incubated with 5
μL of Annexinꢀ
V/FITC (Keygen Biotech, China) in binding buffer (10 mM HEPES, 140 mM NaCl, and
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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 minꢀincubation. Almost 10,000 events were collected for each
sample and analysed by flow cytometry (Beckman Coulter, Epics XL). The percentage of
apoptotic cells was calculated using EXPO32 ADC Analysis software.
Mitochondrial membrane potential and ROS accumulation assay. A lipophilic cationic
dye, 5,5′,6,6′ꢀtetrachloroꢀ1,1′,3,3′ꢀtetraethylꢀbenzimidazolcarbocyanine (JCꢀ1, Beyotime,
China) was used to monitor the level of MMP in the cells. At normal state, the MMP is
high and JCꢀ1 appears as aggregates, which indicated by red fluorescence. However, when
apoptosis occurs, the MMP reduced and JCꢀ1 displayed as monomers, which indicated by
green fluorescence. We applied two methods which including flow cytometry and
fluorescence microscopy to detect the MMP. For flow cytometry analysis, HELA cells were
plated in 6ꢀwell plates (3×105 cells/well) and grown for 24 h, and treated with compounds
5a at the indicated concentrations for 24 h. Then the cells were harvested by centrifugation
and incubated with JCꢀ1 solution for 30 min. After briefly washing, the proportion of green
and red fluorescence intensity were immediately detected and analysed by flow cytometry.
For the fluorescence microscopy detection, HELA cells were plated in a confocal culture
dish at 5 × 104 cells/dish and grown for 24 h, and treated with compound 5a at the
indicated concentrations for another 24 h. Then the cells were stained with 2 ꢁM JCꢀ1 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 immediate detected by a
Zeiss LSM 570 laser scanning confocal microscope (Carl Zeiss, Germany).
Intracellular ROS accumulation assay was performed by fluorescence microscopy using the
Reactive Oxygen Species Assay Kit (ROS Assay Kit, Beyotime). HELA cells were cultured
in 6ꢀwell plates (3×105 cells/well) for 24 h. After adding compound 5a for 12 h, the culture
supernatants were removed, and the cells were then incubated with 10 mM DCFHꢀDA 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 multifunctional monochromatorꢀbased 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
29