Design, synthesis and evaluation of azaacridine derivatives as dual-target EGFR and Src kinase inhibitors for antitumor treatment

Article abstract of DOI:10.1016/j.ejmech.2017.05.006

Overexpression of EGFR is often associated with advanced stage disease and poor prognosis. In certain cancers, Src works synergistically with EGFR to promote proliferation, survival, invasion and metastasis. Development of dual-target drugs against EGFR and Src is of therapeutic advantage against these cancers. Based on molecular docking and our previous studies, we rationally designed a new series of azaacridine derivatives as potent EGFR and Src dual inhibitors. Most of the synthesized azaacridines displayed good antiproliferative activity against K562 and A549?cells. The representative compound 13b showed nM IC₅₀ values against K562 and A549?cells, and inhibited EGFR at inhibition rate of 33.53% at 10?μM and Src at inhibition rate of 72.12% at 1?μM. Furthermore, compound 13b could inhibit the expression of EGFR, p-EGFR, Src and p-Src. Moreover, 13b efficiently inhibited the invasion of tumor cells and induced cancer cells apoptosis. Our study suggested that azaacridine scaffold can be developed as novel multi-target kinase inhibitors for cancer therapy.

Full text of DOI:10.1016/j.ejmech.2017.05.006

Accepted Manuscript
Design, synthesis and evaluation of azaacridine derivatives as dual-target EGFR and
Src kinase inhibitors for antitumor treatment
Zhishan Cui, Shaopeng Chen, Yanwei Wang, Chunmei Gao, Yuzong Chen, Chunyan
Tan, Yuyang Jiang
PII:
S0223-5234(17)30367-7
10.1016/j.ejmech.2017.05.006
EJMECH 9436
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 December 2016
Revised Date: 28 March 2017
Accepted Date: 2 May 2017
Please cite this article as: Z. Cui, S. Chen, Y. Wang, C. Gao, Y. Chen, C. Tan, Y. Jiang, Design,
synthesis and evaluation of azaacridine derivatives as dual-target EGFR and Src kinase
inhibitors for antitumor treatment, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.05.006.
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Design, Synthesis and Evaluation of Azaacridine Derivatives as Dual-target EGFR
and Src Kinase Inhibitors for Antitumor Treatment
A series of azaacridine compounds were rationally designed and synthesized as EGFR
and Src dual inhibitors. The typical compound 13b could inhibit the invasion of tumor
cells and induce cancer cells apoptosis in the nano-molar range.

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Design, Synthesis and Evaluation of Azaacridine Derivatives as Dual-target
EGFR and Src Kinase Inhibitors for Antitumor Treatment
†
†
Zhishan Cuia, b , Shaopeng Chena , Yanwei Wanga, b, Chunmei Gaoa, b, *, Yuzong
Chena, b, c, Chunyan Tana, b, Yuyang Jianga, b, d, *
a The Guangdong Province Key Laboratory of Chemical Biology, the Graduate School at Shenzhen,
Tsinghua University, Shenzhen 518055, PR China.
b National & Local United Engineering Lab for Personalized antitumor drugs, the Graduate School at
Shenzhen, Tsinghua University, Shenzhen 518055, PR China.
c Bioinformatics and Drug Design Group, Department of Pharmacy, Centre for Computational Science
and Engineering, 117543, Singapore.
d School of Medicine, Tsinghua University, Beijing 100084, PR China.
Keywords
EGFR; Src; Azaacridine derivatives; Antitumor; Invasion; Apoptosis.
* Corresponding authors. Tel./Fax: +86 (755) 26036533
E-mail: chunmeigao@sz.tsinghua.edu.cn, jiangyy@sz.tsinghua.edu.cn
†Authors with equal contributions.

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Abstract
Overexpression of EGFR is often associated with advanced stage disease and poor
prognosis. In certain cancers, Src works synergistically with EGFR to promote
proliferation, survival, invasion and metastasis. Development of dual-target drugs
against EGFR and Src is of therapeutic advantage against these cancers. Based on
molecular docking and our previous studies, we rationally designed a new series of
azaacridine derivatives as potent EGFR and Src dual inhibitors. Most of the
synthesized azaacridines displayed good antiproliferative activity against K562 and
A549 cells. The representative compound 13b showed nM IC50 values against K562
and A549 cells, and inhibited EGFR at inhibition rate of 33.53% at 10 µM and Src at
inhibition rate of 72.12% at 1 µM. Furthermore, compound 13b could inhibit the
expression of EGFR, p-EGFR, Src and p-Src. Moreover, 13b efficiently inhibited the
invasion of tumor cells and induced cancer cells apoptosis. Our study suggested that
azaacridine scaffold can be developed as novel multi-target kinase inhibitors for
cancer therapy.

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1. Introduction
EGFR (Epidermal Growth Factor Receptor, a class of membrane receptor tyrosine
kinases) plays an indispensable role in cancer cell proliferation, survival, adhesion,
migration, and differentiation [1-5]. Overexpression of EGFR have been associated
with a variety of cancers [6-8]. Development of EGFR inhibitors has become a main
focus in antitumor drug campaigns. At present, a series of EGFR inhibitors have been
developed, some of which have been approved or entered into clinical phases, such as
Gefitinib [9], Erlotinib [10], Icotinib [11], and Lapatinib [12]. However, EGFR
signalling pathways involve a complicated network of interconnected circuits. When
only EGFR is targeted, redundancy and crosstalk between these pathways will cause
drug resistance [13, 14]. Consequently, a huge effort has been poured in the
exploration of escape pathways activated by EGFR drugs, which will give more clues
for cancer therapy.
Src (sarcoma) is a type of non-receptor tyrosine kinases, which functions in diverse
cellular processes [15, 16]. It is an important anticancer target [17], because of its key
roles in signalling pathways including apoptosis [18], migration [19] and invasion
[20]. Researches showed that the disruption of EGFR and Src interactions could
effectively overcome the resistance to EGFR inhibitors in certain cancers [21, 22], for
example the combination of Lapatinib and Saracatinib could induce the
Lapatinib-resistant cells apoptosis [23]. Therefore, development of drugs targeted
both EGFR and Src might offer a better therapeutic advantage. However, to the best
of our knowledge, there is no report on the rational design and synthesis of molecules
acted as both EGFR and Src dual kinase inhibitors, expect that some drugs targeted
EGFR were later found to have Src kinase inhibition activity [24, 25].
Our group has been involved for several years in the design and synthesis of
antitumor agents [26-47], including multi-target kinase inhibitors [43-47]. Based on
our group previous research and molecular docking methods, we rationally designed a
new series of azaacridine derivatives, which can be efficiently bind with the key
residues of EGFR and Src. Preliminary in vitro antiproliferative ability of synthetic
compounds against K562 (a human erythroleukemic cell line) and A549 (a human
lung carcinoma cell line) cells were studied, and the structure–activity relationship
was disclosed. The mechanism of action of the representative compound 13b was also
studied.

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2. Results and discussion
2.1. Rationale: based on the previous lead and molecular docking method
In the previous paper [43], we reported that the acridine compound (Fig. 1)
containing anilinoacridine and phenyl-urea moieties can inhibit the activity of Src,
however the inhibition activity need to be improved. We thought that the moderate
inhibition activity might be due to the formation of only one hydrogen bond between
the acridine compound and the hinge region of Src. Therefore, we converted the
scaffold of the acridine compound (the previous lead) to azaacridine, which might
form more hydrogen bonds with the hinge region of Src. In addition, we found that
azaacridine ring could also bind with the active site of EGFR efficiently. Moreover,
amido and urea linkages were also introduced to the azaacridine ring to bind with the
DFG-out conformation of Src and the hydrophobic pocket of EGFR, respectively.
Therefore, the azaacridine derivatives (Fig. 1) can be potent dual-target EGFR and Src
kinase inhibitors.
Fig. 1. Rational design from the acridine compound to azaacridine derivatives.
Molecular docking confirmed that the azaacridine derivatives had higher binding
affinity with EGFR and Src than the arcidine compound. The molecular docking study
of the representative compound 13b with EGFR and Src was showed in Fig. 2. For
EGFR, the azaacridine ring can form three hydrogen bonds with Thr 766, Gln 767 and
Lys 721 in the hinge region area, and the amido linkage can get deep into the
hydrophobic pocket region to improve the binding ability. For Src, -NH group and
two nitro atoms in the azaacridine ring could form three hydrogen bonds with Met
340, Met 341 and Lys 295. Meanwhile, as we expected, the amido moiety formed two

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hydrogen bonds with Glu310 and Asp 404 in the DFG-out pocket. In addition, three
π-π interaction bonds and one cation–π bond were also formed between compound
13b and Leu 273, Phe 405, Tyr 382, and Lys 295 respectively. The binding energy
(CDOCKER Energy and CDOCKER Interaction Energy) had been calculated and
showed in Table 1. All the results revealed that the azaacridine derivatives we
rationally designed could be potential dual inhibitors of EGFR and Src.
Fig. 2. Molecular docking study of 13b with EGFR and Src. (A) 13b with EGFR
(PDB code 1M17); (B) 13b with Src(PDB code 3G6H).
Table 1. Molecular docking data of the acridine compound and 13b.
Src
EGFR
Energya（kcal/mol）
-Interaction -CDOCKER
58.43 18.16
62.84 32.27
-CDOCKER
16.96
-Interaction
51.34
Acridine
compound
13b
50.32
63.20
Energya: CDOCKER Energy and CDOCKER Interaction Energy were calculated
using Discovery Studio.3.1/CDOCKER Model (EGFR: PDB code 1M17; Src: PDB
code 3G6H).

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2.2. Chemistry
Firstly, a plausible pathway for the synthesis of the intermediate compound 5 was
proposed in Scheme 1. The reduction reaction of nitrobenzaldehyde 1 with Fe and
NH4Cl was carried out in EtOH/H2O, which was further reacted with malononitrile to
obtain compound 2 in good yield. Hydrolysis of 2 gave compound 3. Cyclization of
compound 3 with formamide got compound 4. As compound 4 was important, the
reaction conditions were optimized, and the yield reached up to 85% (Table 1s).
Chlorination of compound 4 by POCl3 obtained compound 5 in good yield (80%), the
1
1
structure of which was confirmed by H NMR, 13C NMR, DEPT135, H-1H COSY
and HSQC (Fig. 1s). .
Secondly, the side chains were synthesized. The synthesis of amide linkers was
illustrated in Scheme 1. Different substituents of benzoic acid were treated with
SOCl2 to get compounds 6 (a-f), which were than reacted with 3-nitroaniline or
p-nitroaniline to obtain compounds 7 (a-f) and 9 (a-f), respectively. Subsequent
reduction of 7 (a-f) and 9 (a-f) in the presence of Fe and NH4Cl led to the expected
amide derivatives 8 (a-f) and 10 (a-f). The phenyl-urea linker 11 (a-f) were
synthesized according to our early paper[43].
Lastly, the nucleophilic substitution reaction was carried out between compound 5
and the side chains 8 (a-f), 10 (a-f) and 11 (a-f) to obtain the desired compounds 12
(a-f), 13 (a-f), and 14 (a-f) in moderate to good yields.

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Scheme 1. Synthetic pathway to prepare the target compounds 12 (a-f), 13 (a-f), 14
(a-f). Reagents and conditions: (i) Fe, NH4Cl, reflux, 2 h; (ii) malononitrile, 85 °C,
3 h; (iii) conc. H2SO4, r.t., 24 h; (iv) formamide, 160 °C 12 h; (v) POCl3, 100 °C, 3 h;
(vi) SOCl2, 80 °C, 2-3 h; (vii) 3-nitroaniline, CH2Cl2, -20 °C, r.t., 2 h; (viii)
p-nitroaniline, CH2Cl2, -20 °C, r.t., 2 h; (ix) isopropanol, Et3N, reflux, overnight.
2.3. In vitro cell growth inhibition assay
The antiproliferative activity of the desired azaacridine derivatives was tested
against K562 and A549 cells by MTT assay, the acridine compound and Imatinib
were used as the positive controls. As shown in Table 2, most of the compounds
displayed good antiproliferative activity against K562 with low micromolar IC50
values, and both the position of the amide-/urea linker in the phenyl ring and the
substituents affected the antiproliferative activity significantly. As the synthesized
compounds were more sensitive to K562 cells than A549 cells, K562 cells were
selected to study the structure-activity relationship (Fig. 3).

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Fig. 3. The structure of azaacridine derivatives.
Firstly, the substituents (R) played a significate role in the antiproliferative activity.
As showed in Table 2, the introduction of fluorine group can increase the
antiproliferative activity greatly (12b : 0.72 µM vs 12a : 3.11 µM, 13b : 0.22 µM vs
13a : 2.52 µM, 14b : 3.14 µM vs 14a : 5.08 µM), and compound 13b exhibited the
most cytotoxic activity with an IC50 value of 0.22 µM, which was better than the
acridine compound and Imatinib. When the substituent was a chlorine group, the
cytotoxicity was also increased (12c : 1.21 µM vs 12a : 3.11 µM, 13c : 0.41 µM vs
13a : 2.52 µM, 14c : 3.08 µM vs 14a : 5.08 µM), however, they were less potent than
fluorine substituted compounds (except 14c : 3.08 µM). The tri-fluoromethyl group
was also a good choice. Compounds 12f (1.89 µM), 13f (1.32 µM) and 14f (3.35 µM)
also displayed better antiproliferative activity than compounds 12a (3.11 µM), 13a
(2.52 µM) and 14a (5.08 µM), respectively.
Table 2. Anti-proliferation activities against K562 and A549 cells of the designed
compounds 12 (a-f), 13 (a-f) and 14 (a-f).
IC50(µM)
Entry
Compound
R
K562
A549
1
2
H
F
3.11±0.39
0.72±0.16
18.29±0.22
9.05±0.51
12a
12b

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3
4
Cl
Me
OMe
CF3
H
1.21±0.32
4.90±0.12
3.08±0.57
1.89±0.38
2.52±0.14
0.22±0.03
0.41±0.16
3.62±0.24
2.16±0.01
1.32±0.01
5.08±0.11
3.14±0.06
3.08±0.29
6. 97±0.37
3.62±0.35
3.35±0.09
4.08±0.14
0.53±0.01
12.32±0.19
>25
12c
12d
5
13.46±1.23
9.58±0.67
14.29±0.72
0.253±0.16
20.69±1.29
24.77±1.08
14.07±0.67
12.25±0.03
18.01±1.27
6.82±1.42
17.93±0.79
>25
12e
6
12f
7
13a
8
F
13b
9
Cl
13c
10
11
12
13
14
15
16
17
18
19
20
Me
OMe
CF3
H
13d
13e
13f
14a
F
14b
Cl
14c
Me
OMe
CF3
14d
13.42±0.2
9.26±0.67
9.03±0.11
4.56±0.23
14e
14f
Acridine compound
Imatinib
-
The introduction of methoxyl group almost had no effect on the antiproliferative
activity compared to the corresponding compounds with no substituents (12e : 3.08
µM vs 12a : 3.11 µM, 13e : 2.16 µM vs 13a : 2.52 µM, 14e : 3.62 µM vs 14a : 5.08
µM). However, when changing methoxyl to methyl group, compounds showed
decreasing inhibitory effect (12d : 4.90 µM vs 12a : 3.11 µM, 13d : 3.62 µM vs 13a :
2.52 µM, 14d : 6.97 µM vs 14a : 5.08 µM).
Secondly, the position of the amide linker had some effect on the antiproliferative
activity. Compared with the data in Table 2, compounds 13 with amide bond linker at
para-position had higher cytotoxicity activity than compounds 12 generally. Thirdly,
the influence of linkers on the antiproliferation activity was also discussed. As amide

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bond and urea are the most commonly used linkers, compounds 13 (a-f) and 14 (a-f)
bearing amide and urea linkers at para-position in phenyl ring were synthesized. In
Table 2, compounds 13 almost had better inhibitory effect than compounds 14, which
indicated the amide linker was better than the corresponding urea linker. These results
above suggested that the substituents, the linker and its position in phenyl ring could
change the cytotoxic profile.
2.4. Inhibition assay
As the designed compounds had good antiproliferative activity, in vitro
EGFR and Src kinase inhibition capability was firstly evaluated to see whether
their cytotoxicity was due to kinase inhibition. As shown in Table 3, almost all
the compounds displayed better Src inhibition activity than the acridine
compound, except 14c and 14f, which showed comparable Src inhibition
activity with the acridine compound at 10 µM. In addition, compound 13b
inhibited 72.12% of Src activity at 1 µM. At the same condition, 17 compounds
exhibited moderate activity against EGFR at 10 µM (Table 3), except that
compound 13f had 95.41% and 52.38% inhibition rates against EGFR at 10 µM
and 1 µM, respectively. As compound 13b displayed the lowest IC50 values
against both K562 and A549 cells, as well as good Src and moederate EGFR
inhibition capability, it was selected as a representative compound to do further
studies.
To further evaluate whether EGFR and Sr are the targets, we evaluated the
influences of 13b on the expression levels of EGFR, p-EGFR, Src and p-Src in
A549 cells. After a 48 h treatment with 13b at four concentrations (0, 0.1, 0.5,
and 2.5 µM), A549 cells were harvested and lysed for an IP/wt assay. As shown
in Fig. 4, compound 13b inhibited the protein expression levels of EGFR,
p-EGFR, Src and p-Src in a dose-dependent manner. Compared with the
control, the expression level of EGFR and p-EGFR experienced a sharp decline
from 48.02% to 3.03% and 50.21% to 3.11% with the concentration of 13b
increased from 0.1 µM to 2.5 µM, respectively. The same trend was also

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observed in the expression of Src and p-Src. Therefore, it is reasonable to
conclude that inhibition of EGFR and Src is the main cause for the high
antiproliferation activity of compound 13b
.
Table 3. Inhibition test of 18 compounds against EGFR and Src.
Inhibition rate (%)
Entry
Compound
R
EGFR
Src
1
2
H
F
30.70
24.50
30.25
52.99
31.85
16.31
12.56
33.15
30.93
51.07
35.38
95.41
32.01
55.36
42.11
65.39
24.89
35.38
21.39
-
75.63
69.91
79.23
72.03
86.86
72.88
66.31
75.42
77.54
60.38
88.77
76.69
73.09
79.87
59.11
66.10
86.01
53.38
59.67
97.12
12a
12b
3
Cl
12c
4
Me
OMe
CF3
H
12d
5
12e
6
12f
7
13a
8
F
13b
9
Cl
13c
10
11
12
13
14
15
16
17
18
19
20
Me
OMe
CF3
H
13d
13e
13f
14a
F
14b
Cl
14c
Me
OMe
CF3
-
14d
14e
14f
Acridine compound
Imatinib
-

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Moreover, DNA binding assay was also evaluated, as acridine derivatives
generally have good DNA binding ability. UV-vis spectrophotometric titration
experiments indicated that the DNA binding ability of 13b was very weak
compared with our synthesized DNA binding agents [48, 49], which indicated
that DNA binding was not the main reason for the antiproliferative activity of
compound 13b (Fig. 2s). The spectrofluorimetric study was also performed to
further evaluate the DNA binding ability of 13b, and the quenching constant
was 973.23 M-1(Fig. 2s), which was about 100-fold lower than the DNA
binding agents with similar antiproliferative activity published by our group[26,
31, 32, 36]. All the results indicated that DNA was not contribute to the
cytotoxicity, and EGFR and Src were the targets of compound 13b. Although
the kinases activities of synthesized compounds were weaker than the
antiproliferative activity, the synergistic effects of multi-target agents of
moderate activities might contribute to the cytotoxicity [50].
Fig. 4. Western blot analysis of 13b. (A)The inhibition effects of compound 13b (0.1
µM, 0.5 µM and 2.5 µM) on the expression of EGFR, p-EGFR, Src and p-Src in A549
cells are depicted. Equal loading was testified by the detection of β-actin. (B) The
percentages of protein expressions in the different concentrations of compound 13b.

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2.5. Effects of compound 13b on invasion
As Src plays an important role in cancer invasion, the effects of compound 13b on
A549 cell invasion were assessed using transwell assays, in which the number of
invading cells reflected the invasive capacity of the cells. Results in Fig. 5A showed
after treatment of compound 13b, the invasion of A549 cells was inhibited. As
depicted in Fig. 5B, compared with the control, compound 13b at 0.1 µM and 1 µM
potently produced rates of inhibition of 52.50% and 82.81%, respectively. These
results demonstrated that 13b could efficiently inhibit the invasion of tumor cells.
Fig. 5. Transwell assay of 13b. (A) Images depicting the penetration of A549 cells
through the filter membrane, 200 µM bar. (B) The number of A549 cells that
penetrated the membrane was quantified by Image-Pro-Plus 6.0 of Media Cybernetics
(MD, USA). Data are represented as the mean ± SEM of five different experiments:
**(p < 0.01) versus control.
2.6. Apoptosis induced by compound 13b
EGFR and Src inhibitors can induce cancer cell apoptosis. Due to 13b could inhibit
EGFR and Src activity and displayed good antiproliferative activity, it was selected to

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test its impact on apoptosis. Compound 13b induced K562 cells apoptosis in a
concentration-dependent manner (Fig. 6). The early apoptosis ratio increased from
4.68% to 18.82% with increasing 13b concentration from 0 µM to 0.1 µM, when
compound 13b was added in K562 cells for 48 h. As the concentration of 13b
increased from 0.1 µM to 1 µM, more and more apoptotic cells progressed from the
early stage to the late stage resulting in either death or secondary necrosis. The results
indicated that compound 13b could effectively induce K562 cells apoptosis.
Fig. 6. Apoptosis assay of 13b. Analysis was carried out on a flow cytometer using an
Annexin V-FITC/PI apoptosis staining kit according to the manufacturer’s
instructions.
3. Conclusion
To summarize, we designed and synthesized a new series of azaacridine derivatives
as potent EGFR and Src dual inhibitors. The results showed that most of the
synthesized azaacridine compounds possessed good antiproliferative activity against
K562 and A549 cells. The representative 13b could inhibit the activity of EGFR and
Src, inhibit the invasion of tumor cells and induce tumor cell apoptosis. Further

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optimization of this structure for improved the kinase inhibition activity and
antiproliferative activity is ongoing.
4. Experimental section
4.1. Chemistry
Generally, the procedure for the synthesis of 5 was as follows. Nitrobenzaldehyde 1
(10 mmol) was added to EtOH/H2O (25 mL/5 mL) at room temperature, followed by
Fe (3.0 equiv) and NH4Cl (1.0 equiv). The resultant mixture was heated to reflux and
stirred for 2 h. And then, the hot liquid was filtrated rapidly through celite. The
appropriate malononitrile (1.0 equiv) and EtOH (10 mL) were added to the filtrate and
stirred at 80 °C for 4 h. Then, the mixture was cooled to room temperature and the
formed precipitates were filtered off. Washed the precipitates with diethyl ether, we
obtained the pure compound 2. At room temperature, compound 2 (10 mmol) was
dissolved in conc. H2SO4 (8 mL) and then stirred for 24 h. The liquid was poured into
ice water (80 mL), and NaOH (2 mol/L, aq) as the neutralizer was used to deal with
the redundant conc. H2SO4. Filtered the precipitates, the filter cake was washed with
water and dried under vacuum to obtain 3 (yield, 95%). Cyclization of compound 3
(10 mmol) with formamide (10 mL) at 160 °C for 12 h under air, we got compound 4
through filteration and washing with MeOH (15 mL) (yield, 85%). POCl3 (5 mL) was
added to compound 4 (10 mmol) and stirred at 80 °C for 3 h. Cooled the solution and
poured into ice water (100 mL), we titrated with NaOH (2 mol/L, aq) in the mixture
solution till pH = 8. Filtered the precipitates, the filter cake was washed with water
and dried under vacuum to obtain the compound 5 in high yield (80%).
Generally, the procedure for the synthesis of 12-14 (a-f) was as follows.
Compounds 8 (a-f) (0.41 mmol) and compound 5 (0.4 mmol) were dissolved in
isopropanol (10 mL) in the presence of Et3N (1 mL). The mixture was stirred for
overnight at 80 °C. The raw products were filtered off, washed with diethyl ether to
obtain the desired pure compounds 12 (a-f) in moderate to good yields. Those crude
products were purified by column chromatography with CH2Cl2 and MeOH (50:1

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v/v). The same processes were used to get the compound 13 (a-f) and compound 14
(a-f).
4.1.1. N-(3-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-benzamide
1
(12a). Yellow solid; yield, 83.7%; mp, 218.2-219.0 °C; H NMR (400 MHz,
DMSO-d6) δ 10.19 (s, 1H), 9.76 (s, 1H), 8.25 (s, 1H), 7.76-7.71 (m, 4H), 7.29 (d, J =
4 Hz, 2H), 7.16-7.12 (m, 2H), 7.04 (s, 1H), 6.92 (m, 2H), 6.60 (d, J = 8 Hz, 2H), 5.76
(s, 1H), 4.11 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 159.67, 158.65, 157.53,
156.87, 149.37, 140.13, 137.40, 137.27, 129.81, 129.51. 129.25, 128.17, 123.08,
120.35, 19.28, 117.59, 113.42, 110.34, 109.94, 109.02, 106.77, 27.74. HRMS (ESI)
m/z calculated for [M+H]+: 394.1668; found: 394.1656.
4.1.2. N-(3-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-fluoroben
1
-amide (12b). Yellow solid; yield, 86.7%; mp, 232.7-233.2 °C; H NMR (400 MHz,
DMSO- d6) δ 10.18 (s, 1H), 9.94 (s, 1H), 8.25 (s, 1H), 7.92 (d, J = 8 Hz, 2H),
7.57-7.49 (m, 2H), 7.16-7.11 (m, 3H), 6.96-6.92 (m, 3H), 6.91 (d, J = 8 Hz, 1H),
5.07(s, 1H), 4.11 (s, 2H); 13C NMR (100 MHz, DMSO- d6) δ 164.67, 158.86, 157.53,
156.69, 149.47, 139.92, 137.79, 133.65, 131.65, 130.82, 129.51, 129.33, 128.12,
127.88, 126.93, 123.08, 119.28, 115.89, 110.53, 109.94, 108.94, 106.71, 27.48.
HRMS (ESI) m/z calculated for [M+H]+: 412.1574; found: 412.1560.
4.1.3. 3-chloro-N-(3-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)benza
1
-mide(12c). Yellow solid; yield, 87.3%; mp, 239.1-240.5 °C; H NMR (400 MHz,
DMSO-d6) δ 10.20 (s, 1H), 10.06 (s, 1H), 8.26 (s, 1H), 7.96 (s, 1H), 7.88 (d, J = 8 Hz,
1H), 7.65 (d, J = 8 Hz, 1H), 7.55 (dd, J1 = J2 = 8 Hz, 1H), 7.16-7.12 (m, 4H),
6.94-6.92 (m, 4H), 5.12 (s, 1H), 4.12 (s, 2H); 13C NMR (100 MHz, DMSO- d6) δ
164.28, 158.67, 157.53, 156.86, 149.48, 139.79, 137.40, 134.15, 132.65, 130.82,
129.51, 128.12, 127.82, 126.93, 123.08, 119.28, 115.81, 110.53, 109.94, 108.94,
106.69, 27.74. HRMS (ESI) m/z calculated for [M+H]+: 428.1278; found: 428.1257.
4.1.4. N-(3-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-methylbenz

ACCEPTED MANUSCRIPT
-amide(12d). Yellow solid; yield, 83.1%; mp, 212.6-213.2 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.218 (s, 1H), 10.01 (s, 1H), 8.25 (s, 1H), 7.79-7.73 (m, 3H), 7.57 (s, J
= 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 7.14-7.09 (m, 3H), 6.97-6.87 (m, 2H), 6.34 (m,
13
2H), 5.10 (s, 1H), 4.11 (s, 2H); C NMR (100 MHz, DMSO- d6) δ 164.28, 158.67,
157.53, 156.86, 149.48, 139.97, 139.71, 137.42, 133.65, 131.65, 130.85, 129.56,
128.28, 127.88, 126.93, 123.18, 119.43, 115.81, 110.69, 109.94, 108.94, 106.56,
27.74, 19.07. HRMS (ESI) m/z calculated for [M+H]+: 408.1824; found: 408.1813.
4.1.5. N-(3-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)3-methoxyben-
1
zamide(12e). Yellow solid; yield, 80.3%; mp, 216.2-216.9 °C; H NMR (400 MHz,
DMSO-d6) δ 10.21 (s, 1H), 9.93 (s, 1H), 8.26 (s, 1H), 7.50 (d, J = 8 Hz, 1H),
7.44-7.40 (m, 2H), 7.16-7.10 (m, 3H), 7.08 (s, 1H), 6.98-6.90 (m, 3H), 6.85 (d, J =
13
8Hz, 1H), 6.31 (d, J =8 Hz, 1H), 5.11 (s, 1H), 4.11 (s, 2H), 3.83 (s, 3H); C NMR
(100 MHz, DMSO-d6) δ 159.65, 158.67, 157.53, 156.87, 149.42, 140.17, 137.40,
137.25, 129.96, 129.51, 129.27, 128.13, 123.08, 120.35, 119.28, 117.59, 115.81,
113.37, 110.34, 109.94, 109.02, 106.77, 55.84, 27.74. HRMS (ESI) m/z calculated for
[M+H]+: 424.1773: found: 424.1760.
4.1.6. N-(3-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-(trifluorome
-thyl)benzamide(12f). Yellow solid; yield, 78.3%; mp, 242.8-243.6 °C; 1H NMR (400
MHz, DMSO-d6) δ 10.21 (s, 1H), 10.17 (s, 1H), 8.21 (s, 1H), 7.65 (m, 3H), 7.60 (d, J
= 8 Hz, 1H), 7.54 (d, J = 8 Hz, 1H), 7.50 (d, J = 8 Hz, 1H), 7.43-7.41 (m, 2H),
7.33-7.31 (m, 2H), 7.15 (d, J = 8 Hz, 2H), 4.11 (s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 164.64, 164.59, 158.61, 157.50, 156.79, 150.55, 147.47, 137.87,
136.10 ,132.30, 130.25, 129.43, 128.07, 125.32, 124.81, 123.06, 122.35, 122.06,
121.24, 119.20, 115.79, 109.82, 27.72. HRMS (ESI) m/z calculated for [M+H]+:
462.1542; found: 462.1520.
4.1.7. N-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)benzamide
1
(13a). Yellow solid; yield, 88.3%; mp, 201.4-202.6 °C; H NMR (400 MHz,

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DMSO-d6) δ 10.21 (s, 1H), 9.96 (s, 1H), 8.26 (s, 1H), 7.78 (dd, J1 =8 Hz, J2 = 4 Hz,
2H), 7.56-7.47 (d, J = 8 Hz, 1H), 7.41-7.35 (m, 3H), 7.16-7.12 (m, 2H), 6.93-6.92 (m,
13
2H), 6.54 (d, J = 8 Hz, 2H), 6.37 (s, 1H) 5.01 (s, 1H), 4.11 (s, 2H); C NMR (100
MHz, DMSO-d6) δ 158.67, 158.42, 157.15, 149.13, 141.53, 140.89, 140.16, 1400.00,
136.43, 135.45, 134.65, 129.25, 127.29. 125.60, 122.89, 122.36, 18.99, 118.60, 12.95,
110.99, 106.05, 27.62. HRMS (ESI) m/z calculated for [M+H]+: 394.1668; found:
394.1650.
4.1.8. N-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-fluorobenza
1
-mide(13b). Yellow solid; yield, 89.1%; mp, 213.3-214.4 °C; H NMR (400 MHz,
DMSO-d6) δ 10.21 (s, 1H), 9.96 (s, 1H), 8.26(s, 1H), 7.79-7.77 (m, 2H), 7.74 (d, J =
8.0Hz, 1H), 7.60-7.54 (m, 1H), 7.41-7.35 (m, 3H), 7.16-7.12 (m, 2H), 6.92 (d, J = 8
Hz, 2H), 6.52 (d, J = 8 Hz, 1H), 5.01 (s, 1H), 4.12(s, 2H); 13C NMR (100 MHz,
DMSO-d6) δ 158.42, 157.15, 156.86, 150.13, 141.53, 140.89, 140.16, 140.03, 136.45,
135.44, 134.63, 129.29, 127.25, 125.60, 122.86, 122.33, 118.99, 118.62, 112.95,
110.99, 106.00, 27.65. HRMS (ESI) m/z calculated for [M+H]+: 412.1574; found:
412.1549.
4.1.9. 3-chloro-N-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-benz
1
-amide(13c). Yellow solid; yield, 90.6%; mp, 220.1-221.3 °C; H NMR (400 MHz,
DMSO-d6) δ 10.21 (s, 1H), 9.96 (s, 1H), 8.26 (s, 1H), 7.78 (d, J = 8 Hz, 1H), 7.76 (d,
J = 8 Hz, 1H), 7.56 (d, J = 8 Hz, 1H), 7.39 (d, J =8 Hz, 1H), 7.38-7.35 (m, 2H),
7.16-7.12(m, 2H), 6.92 (d, J = 8 Hz, 2H), 6.54 (d, J = 8 Hz, 2H), 5.01 (s, 1H), 4.12 (s,
2H); 13C NMR (100 MHz, DMSO-d6) δ 163.61, 158.67, 15.53, 156.86, 145.94,
137.83, 137.40, 133.66, 131.43, 130.79, 129.50, 128.32, 128.12, 127.71, 126.74,
123.07, 122.75, 119.27, 115.81, 114.18, 109.93, 27.74. HRMS (ESI) m/z calculated
for [M+H]+: 428.1278; found: 428.1256.
4.1.10. N-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-methylben
1
-zamide(13d). Yellow solid; yield, 80.5%; mp, 214.8-215.6 °C; H NMR (400 MHz,

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DMSO-d6) δ 10.21 (s, 1H), 9.90 (s, 1H), 8.26 (s, 1H), 7.92 (d, J = 8 Hz, 2H),
7.53-7.48 (m, 2H), 7.38(d, J = 8 Hz, 2H), 7.16-7.10 (m, 2H), 6.93-6.90 (m, 2H), 6.55
(d, J = 8 Hz, 2H), 5.23 (s, 1H), 4.09 (s, 2H), 2.09 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 164.61, 158.54, 157.51, 156.79, 139.07, 138.06, 137.35, 136.85, 136.09,
134.39, 132.31, 129.42, 128.22, 125.25, 123.06, 122.30, 122.06, 121.24, 119.20,
115.82, 109.87, 27.72, 19.04. HRMS (ESI) m/z calculated for [M+H]+: 408.1824;
found: 408.1809.
4.1.11. N-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-methoxy-b
-enzamide(13e). Yellow solid; yield, 78.6%; mp, 228.8-229.6 °C; 1H NMR (400 MHz,
DMSO-d6) δ 10.21 (s, 1H), 9.87 (s, 1H), 8.27 (s, 1H), 7.51 (d, J = 8 Hz, 1H), 7.46 (s,
J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 7.37 (d, J = 8 Hz, 2H), 7.17-7.11 (m, 3H), 6.94
13
(d, J = 8 Hz, 2H), 6.55 (d, J = 8 Hz, 2H), 4.13 (s, 2H), 3.84 (s, 3H); C NMR (100
MHz, DMSO-d6) δ 158.67, 157.53, 156.86, 153.13, 141.53, 140.89, 136.45, 135.44,
134.63, 128.27, 126.29, 125.20, 122.69, 121.86, 118.29, 117.90, 112.95, 110.96,
106.95, 104.03, 56.62, 27.52. HRMS (ESI) m/z calculated for [M+H]+: 424.1773;
found: 424.1765.
4.1.12. N-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-(trifluoro-
1
methyl)benzamide(13f). Yellow solid; yield, 70.5%; mp, 240.6-241.9 °C; H NMR
(400 MHz, DMSO-d6) δ 10.18 (s, 1H), 9.94 (s, 1H), 8.25 (s, 1H), 7.92 (d, J = 8 Hz,
2H), 7.57-7.49 (m, 2H), 7.16-7.11 (m, 3H), 6.96-6.92 (m, 3H), 6.31 (d, J = 8 Hz, 2H),
5.07 (s, 1H), 4.11 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 164.79, 164.49, 158.54,
157.51, 156.82, 142.13, 139.07, 138.06, 137.35, 136.85, 136.09, 134.39, 129.42,
128.22, 125.25, 123.06, 122.30, 122.06, 121.24, 119.20, 115.87, 109.82, 27.72.
HRMS (ESI) m/z calculated for [M+H]+: 462.1542; found: 462.1528.
4.1.13. 1-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-phenylurea
1
(14a). Yellow solid; yield, 67.5%; mp, 251.6-252.8 °C; H NMR (400 MHz,
DMSO-d6) δ 10.58 (s, 1H), 10.27 (s, 1H), 9.34 (s, 1H), 8.20 (s, 1H), 7.55-7.51 (m,

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3H), 7.48-7.39 (m, 3H), 7.26-7.21 (m, 3H), 7.19-7.11 (m, 2H), 7.07-7.01 (m, 2H),
4.10 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 158.35, 157.12, 156.13, 149.23,
140.17, 139.37, 136.00, 134.32, 129.26, 128.68, 128.27, 126.14, 122.35, 121.38,
119.32, 118.58, 115.27, 111.83, 108.03, 28.00. HRMS (ESI) m/z calculated for
[M+H]+: 409.1777; found 409.1753.
4.1.14. 1-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-(3-fluoro-
1
phenyl)urea(14b). Yellow solid; yield, 77.5%; mp, 261.6-262.7 °C; H NMR (400
MHz, DMSO-d6) δ 10.58 (s, 1H), 10.27 (s, 1H), 9.49 (s, 1H), 8.68 (s, 1H), 8.40 (s,
1H), 7.60 (d, J = 8 Hz, 2H), 7.51 (d, J = 8 Hz, 1H), 7.39 (d, J =8 Hz, 1H), 7.34-7.28
(m, 3H), 7.12(d, J = 8 Hz, 1H), 7.06 (d, J = 8 Hz, 2H), 7.01 (d, J = 8 Hz, 2H), 4.11 (s,
2H); 13C NMR (100 MHz, DMSO-d6) δ 161.71, 158.67, 157.53, 156.86, 152.95,
150.21, 141.99, 141.61, 140.39, 139.06, 130.75, 128.94, 126.61, 124.54, 124.33,
119.72, 116.23, 115.02, 114.31, 108.73, 108.52, 105.35, 105.08, 27.61. HRMS (ESI)
m/z calculated for [M+H]+: 427.1683; found: 427.1661.
4.1.15. 1-(3-chlorophenyl)-3-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)-
1
phenyl)urea(14c). Yellow solid; yield, 87.6%; mp, 269.8-270.6 °C; H NMR (400
MHz, DMSO-d6) δ 10.27 (s, 1H), 9.88 (s, 1H), 9.52 (s, 1H), 8.67 (s, 1H), 8.39 (s, 1H),
7.69 (s, 1H), 7.65 (d, J = 8 Hz, 1H), 7.60 (d, J =8 Hz, 2H), 7.38 (s, 1H), 7.31-7.28 (m,
13
4H), 7.00 (d, J = 8 Hz, 2H), 6.97-6.94 (m, 2H), 6.92-6.84 (m, 2H), 4.11 (s, 2H); C
NMR (100 MHz, DMSO-d6) δ 158.80, 156.99, 155.59, 150.50, 141.00, 140.16,
136.44, 130.25, 128.97, 126.05, 125.67, 124.86, 123.35, 122.16, 119.73, 118.64,
116.62, 115.20, 114.47, 27.43. HRMS (ESI) m/z calculated for [M+H]+: 423.1387;
found: 423.1367.
4.1.16. 1-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-(m-tolyl)-
1
urea(14d). Yellow solid; yield, 75.5%; mp, 255.2-256.4 °C; H NMR (400 MHz,
DMSO-d6) δ 9.32 (s, 1H), 9.14 (s, 1H), 8.71 (s, 1H), 8.22 (s, 1H), 7.65-7.56 (m, 3H),
7.47 (d, J = 8 Hz, 2H), 7.29 (d, J =8 Hz, 1H), 7.26-7.23 (m, 2H), 7.06 (d, J =8 Hz,

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2H), 7.02 (d, J = 8 Hz, 2H), 4.21 (s, 2H), 2.02 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 158.35, 157.12, 155.20, 152.95, 150.10, 141.74, 141.58, 140.35, 135.75,
133.70, 130.87, 128.93, 126.60, 124.60, 124.36, 121,92, 119.71, 117.87, 116.94,
116.23, 115.03,27.28, 20.58. HRMS (ESI) m/z calculated for [M+H]+: 423.1933;
found: 423.1899.
4.1.17. 1-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-(3-methoxy
1
-phenyl)urea (14e). Yellow solid; yield, 76.5%; mp, 265.7-266.7 °C; H NMR (400
MHz, DMSO-d6) δ 9.32 (s, 1H), 9.14 (s, 1H), 8.71 (s, 1H), 8.23 (s, 1H), 7.64 (d, J = 8
Hz, 1H), 7.62-7.56 (m, 2H), 7.45 (dd, J1 =8 Hz, J2=4 Hz 2H), 7.29(d, J = 8 Hz, 1H),
7.25(dd, J1 =8 Hz, J2=4 Hz 2H), 7.16-7.12 (m, 2H), 6.93-6.91 (m, 2H), 4.14 (s, 2H),
3.73 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 158.35, 157.12, 155.86, 153.12,
140.15, 139.64, 134.75, 128.38, 126.65, 125.99, 125.90, 124.05, 122.77, 122.34,
121.79, 120.47, 119.89, 115.14, 113.79, 106.95, 56.19, 27.25. HRMS (ESI) m/z
calculated for [M+H]+: 439.1882; found: 439.1875.
4.1.18. 1-(4-((5,10-dihydropyrimido[4,5-b]quinolin-4-yl)amino)phenyl)-3-(3(trifluoro
1
-methyl)phenyl)urea(14f). Yellow solid; yield, 70.5%; mp, 275.4-276.8 °C; H NMR
(400 MHz, DMSO-d6) δ 10.21 (s, 1H), 9.74 (s, 1H), 9.67 (s, 1H), 8.81 (s, 1H), 8.47 (s,
1H), 7.65 (d, J = 8 Hz, 2H), 7.60 (d, J =8 Hz, 1H), 7.53 (d, J = 8 Hz, 1H), 7.50(d, J =
8 Hz, 1H), 7.42 (d, J =8 Hz, 2H), 7.32 (d, J = 8 Hz, 2H), 7.16-7.14 (m, 2H), 6.93-6.91
(m, 2H), 4.18 (s, 2H); 13C NMR (100 MHz, DMSO-d6) δ 164.23, 158.42, 157.15,
156.13, 148.23, 141.53, 140.89, 136.45, 135.44, 134.63, 129.27, 127.29, 125.60,
122.89, 122.36, 118.99, 118.60, 112.95, 112.03, 106.65, 27.62. HRMS (ESI) m/z
calculated for [M+H]+: 476.1572; found: 476.1552.
4.2. Molecular docking
The molecular modeling of compound 13b was performed with Discovery
Studio.3.1/CDOCKER protocol (Accelrys Software Inc.). The dimensional structure
of EGFR (PDB ID: 1M17) and Src (PDB ID: 3G6H) was downloaded from Protein

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Data Bank (PDB). The following process was used to carry out molecular docking: (1)
deleting the water crystallization involved in protein kinase structure; (2) optimizing
protein structure and ligands; (3) defining receptor and ligand, finding the candidate
binding site; (4) deleting small molecular docking in candidate binding site; (5)
docking designed compounds into the candidate binding site on the target protein
kinase; (6) molecular modeling based on the above docking data.
4.3. Cell Antiproliferation Assay
Cell antiproliferation assay based on MTT were performed with 2 cancer cell lines.
Briefly, 100 µL of K562 cell solution with the concentration of 7×105 cells mL-1 was
seeded to each well of a 96-well plate and incubated for 12 h at 37 °C in a 5% CO2
incubator. The test compound solution was added to each well for the treatment of
maintained cells in triplicate per concentration, and incubated at 37 °C in a 5% CO2
incubator for 48 h. After this treatment, 10 µL MTT solution (5 mg mL-1) was then
added to each well and incubated for 4 h at 37 °C. The formazan precipitate was
dissolved in 100 µL DMSO and the absorbance at 490 nm was determined using
Multimode Detector DTX880 (Beckman Coulter).
A549 cells were seeded in 96-well plates at a density of 5-7 × 103 cells per well
with incubation overnight in a 5% CO2 incubator at 37 °C, the growth medium in
each well was then exchanged with 0.1 mL of fresh medium containing graded
concentrations of compounds to be tested or equal DMSO and incubated continuously
for 48 h. Then 10 µL MTT solution (5 mg mL-1) was added to each well, and the cells
were incubated for additional 4 h. The MTT - formazan crystals were dissolved in 100
µL of DMSO, the absorbance of each well was measured at 490 nm using
anautomatic ELISA reader system (TECAN, CHE). Etoposide and Doxorubicin was
used as positive controls.
4.4. Kinase Inhibition Assays
In vitro kinase assays were carried out by Medicilon Co., Ltd in Shanghai, China.
Kinases inhibitory activities of synthetic compounds against EGFR and Src. The

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general procedures were as follows: mix enzyme, substrate, ATP and compounds in a
buffer solution (pH = 7.0) of 50 mM HEPES/NaOH, 0.02% NaN3, 0.01% BSA, 0.1
mM Mortho-vanadate, 5 mM MgCl2, 1 mM DTT in an OptiPlate-384. The assay plate
was incubated at room temperature for 15 min and compounds in 10 µM dissolved in
2.5% DMSO. Then the mixture were added by Streptavidin-XL665 and TK antibody
europium cryptate (1:100) solution 10 µL (50 mM HEPES/NaOH pH = 7.0, 0.1%
BSA, 0.8 M KF, 20 mM EDTA). The Instrument (Perkinelmer) to detect the signal at
room temperature. The luminescence was read at envision. The signal was correlated
with the amount of ATP remaining in the reaction and was inversely correlated with
the kinase activity.
4.5. Western Blot Analysis
K562 cells were treated with vehicle or different concentration of 13b for 48 h,
whole cell proteins were extracted by RIPA lysis as described. The concentrations of
the cell lysates were measured by the DC Protein Assay Kit I from Bio-Rad
(California, USA). Equal amounts of protein were subjected to 10% SDS
polyacrylamide gel electrophoresis followed by transferring to PVDF membranes, and
were subsequently analyzed with EGFR, p- EGFR, Src and p-Src antibodies from Cell
Signalling (MA, USA). Specific protein signals from their respective horseradish
peroxidase linked secondary antibodies were detected and measured by the
Luminescence Image Analyzer Tanon 5200 (Shanghai, China). The density of the
bands were measured by Image Quant software (Molecular Dynamics, Sunnyvale, CA,
USA), and then expressed as the percentage of the density of the β-actin band.
4.6. Cell invasion assay
Cell invasion assay was performed as described [51]. Invasion inserts with 8 µm
pore membranes from Corning (New York, USA) were coated with fibronectin from
Sigma-Aldrich (Missouri, USA) as described [52]. A549 cells were pretreated with
different concentration of 13b for 6 h, respectively. The pretreated cells were seeded
on the inserts to reach confluence in 12 h, and then culture for another 24 h with drug

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treatment. After fixed with 4% formaldehyde, non-invading cells on the upper side of
the membranes were removed by cotton swab. The invading cells were stained with
0.08% trypan blue for 15 min as described [53] and were photographed by a
bright-field light microscope. Cell numbers of five random views were counted by
Image-Pro-Plus 6.0 of Media Cybernetics (MD, USA).
4.7. Cell Apoptosis Assays
A total of 1 × 105 cells mL-1 K562 cells were plated in a six-well plate and treated
with 13b for 48 h at 37 °C. After incubation, the cells were harvested and washed
with ice-cold PBS. The cell cycle progression was analyzed using an apoptosis
analysis kit (Beyotime), and the apoptosis ratio was performed with an annexin
V-FITC Apoptosis Detection Kit (keygentec).
Acknowledgments
The authors would like to thank the financial supports from Shenzhen Municipal
Development and Reform Commission (Disciplinary Development Program for
Chemical Biology), the financial supports from the Chinese National Natural Science
Foundation (21372141), and Shenzhen Sci & Tech Bureau (CXB201104210013A，
CXZZ20150529165045064 and JCYJ20150331151358131).
Conflict of Interest
The authors declare no conflicts of interest.
Appendix A. Supplementary material
13
Supplementary data (1H NMR, C NMR and 2D spectra of compound 4 and 5;the
1
13
UV-vis absorption spectra of compound 13b binding with ct DNA, H NMR and C
NMR spectra and high resolution mass spectrometry of 12-14 (a-f) )associated with
this article can be found in the online version.
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