Synthesis and antiproliferative activity of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing the 2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety

Article abstract of DOI:10.1016/j.bmcl.2016.02.037

A series of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing the 2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety were synthesized, and evaluated for their antiproliferative activity against 5 cancer cell lines (H460, HT-29, MKN-45, A549, and U87MG). Most compounds showed moderate to excellent potency, and compared to foretinib, the most promising analog 42 (c-Met/Flt-3 IC₅₀= 1.21/2.15 nM) showed a 6.1-fold increase in activity against H460 cell line in vitro. The enzymatic assays (c-Met, VEGFR-2, Flt-3, PDGFR-β, c-Kit, and EGFR) of compound 42 were evaluated in vitro. Docking analysis showed that compound 42 could form three hydrogen bonds with c-Met. Structure–activity relationship studies indicated that a more water-soluble cyclic tertiary amine and electron-withdrawing groups at 4-position of the phenyl ring contribute to the antitumour activity.

Full text of DOI:10.1016/j.bmcl.2016.02.037

Accepted Manuscript
Synthesis and antiproliferative activity of 6,7-disubstituted-4-phenoxyquinoline
derivatives bearing the 2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide
moiety
Qidong Tang, Xin Zhai, Yayi Tu, Ping Wang, Linxiao Wang, Chunjiang Wu,
Wenhui Wang, Hongbo Xie, Ping Gong, Pengwu Zheng
PII:
S0960-894X(16)30149-4
DOI:
http://dx.doi.org/10.1016/j.bmcl.2016.02.037
BMCL 23591
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
20 January 2016
10 February 2016
15 February 2016
Please cite this article as: Tang, Q., Zhai, X., Tu, Y., Wang, P., Wang, L., Wu, C., Wang, W., Xie, H., Gong, P.,
Zheng, P., Synthesis and antiproliferative activity of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing the
2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety, Bioorganic & Medicinal Chemistry Letters (2016),
doi: http://dx.doi.org/10.1016/j.bmcl.2016.02.037
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Synthesis and antiproliferative activity of 6,7-disubstituted-4-phenoxyquinoline derivatives bearing
the 2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety
Qidong Tang a,b,,†, Xin Zhai b,†, Yayi Tu a, Ping Wang a, Linxiao Wang a, Chunjiang Wu a, Wenhui Wang a,
Hongbo Xie c, Ping Gong b, Pengwu Zheng a,*
a School of Pharmacy, Jiangxi Science and Technology Normal University, Nanchang, Jiangxi 330013, PR
China
b Key Laboratory of Structure-Based Drug Design and Discovery of Ministry of Education, School of
Pharmaceutical Engineering, Shenyang Pharmaceutical University, Shenyang 110016, PR China
c College of Bioinformatics Science and Technology, Harbin Medical University, Harbin 150081, PR China
Abstract
A
series
of
6,7-disubstituted-4-phenoxyquinoline
derivatives
bearing
the
2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety were synthesized, and evaluated for their
antiproliferative activity against 5 cancer cell lines (H460, HT-29, MKN-45, A549, and U87MG). Most
compounds showed moderate to excellent potency, and compared to foretinib, the most promising analogue
42 (c-Met/Flt-3 IC50 = 1.21/2.15 nM) showed a 6.1-fold increase in activity against H460 cell line in vitro.
The enzymatic assays (c-Met, VEGFR-2, Flt-3, PDGFR-β, c-Kit, and EGFR) of compound 42 were
evaluated in vitro. Docking analysis showed that compound 42 could form three hydrogen bonds with
c-Met. Structure-activity relationship studies indicated that a more water-soluble cyclic tertiary amine and
electron-withdrawing groups at 4-position of the phenyl ring contribute to the antitumour activity.
Keywords: Synthesis; Quinoline derivatives; 2-oxo-4-chloro-1,2-dihydroquinoline; c-Met; antiproliferative
activity
Cancer is a widespread, complex, and lethal disease. There were 14.1 million new cancer cases, 8.2
million deaths and 32.6 million people living with cancer (within 5 years of diagnosis) in 2012 worldwide. 1
2–5
Despite the efforts to discover and develop small molecule anticancer drugs in the last decade,
development of new antitumor agents with improved tumor selectivity, efficiency, and safety remains
desirable.
Recently, a number of new quinoline derivatives with excellent antitumor activity have been reported.
6–14
Among them, 6,7-disubstituted(or 7-substituted)-4-aryloxyquinoline derivatives, which could inhibit
 Corresponding authors. Tel./fax: +86 791 83802393.
E-mail addresses: tangqidongcn@126.com (Q. Tang), zhengpw@126.com (P. Zheng).
† These authors contribute equally to this work.

c-Met kinase, have attracted our attention. Many of these derivatives are already being marketed or are
under clinical/preclinical studies, such as cabozantinib, foretinib, AMG458, MG10, Amgen, and AM7 (1–6,
Fig. 1). The main modification of these quinoline derivatives was focused on the 5-atom linker between
moiety A and moiety B, which was characterized by the illustrated‘5 atoms regulation’ or ‘hydrogen-bond
donor or acceptor’ in our previous work. 15-16
Figure 1. The representative small-molecule c-Met kinase inhibitors.
In our previous study, we introduced 1,4-dihydrocinnoline and quinoline fragment as part of the
5-atom
N-(3-fluoro-4-(6,7-disubstitutedquinolin-4-yloxy)phenyl)-4-oxo-1-(substitutedphenyl)-1,4-dihydrocinnolin
e-3-carboxamides (7, Fig. 2) and
linker,
and
the
resulting
derivatives
N-(3-fluoro-4-(6,7-disubstitutedquinolin-4-yloxy)phenyl)-2-substitutedphenylquinoline-4-carboxamides (8,
15-16
Fig. 2) showed excellent potency.
2-oxo-1,2-dihydroquinoline fragment is widely used as a building
block in drug design because of its ability to form hydrogen-bonding interactions with drug targets, such as
H2L (9, Fig. 2) 17 which could interact with CT-DNA through intercalation.
Figure 2. Our previous work on antiproliferative agents bearing quinoline scaffolds and H2L.
Taking
foretinib
as
the
leading
compound,
we
introduced
the
2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety to foretinib as the 5-atom linker (Fig. 3) based
on the illustrated ‘5 atoms regulation’ and ‘hydrogen-bond donor or acceptor’ to make further study. The

3-carbon tether at the 7 position of quinoline was reserved, while the morpholinyl group was replaced by
four other water-soluble substituents based on our previous study, 18 including pyrrolidinyl, piperidinyl,
4-methyl piperidinyl, and 4-methyl piperazinyl groups, to observe the effects of the different cyclic tertiary
amino groups on activity of the new compounds. Furthermore, various substituents were introduced at the
phenyl ring (moiety B) to investigate their effects on activity. Accordingly, we designed a novel series of
6,7-disubstituted-4-phenoxyquinoline
derivatives
bearing
2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety.
Figure 3. Design strategy for the quinoline derivatives bearing 2-oxo-4-chloro-1,2-dihydroquinoline
moiety
The antiproliferative effect of all the 23 newly synthesized compounds on the growth of five cell lines,
namely human lung cancer (H460), human colon cancer (HT-29), human lung adenocarcinoma (A549),
human gastric cancer (MKN-45), and human glioblastoma (U87MG) overexpression), was evaluated in
vitro. 19-21 Apparent growth inhibition was observed for most of the compounds, and 12 of these compounds
were more potent than foretinib against one or more cell lines. Furthermore, eight compounds were chosen
for further evaluation of c-Met kinase inhibitory activity in vitro. To examine the selectivity of compound
42 on c-Met over other kinases, this compound was screened against 5 other tyrosine kinases. To further
elucidate the binding mode of compounds with c-Met kinase, docking analysis was performed using
compound 42.
The synthesis of the key intermediates of 6,7-disubstituted-4-phenoxyquinolines 17a – e was achieved
in 8 steps from commercially available 1-(4-hydroxy-3-methoxyphenyl)ethanone as shown in Scheme 1,
which has been illustrated in detail in our previous study.15

Scheme 1. Reagents and conditions: (ⅰ) Br(CH2)3Cl, acetone, 0 °C, 30 min, rt., 12 h; (ⅱ) 98%HNO3, CH2Cl2, 0 °C, 4 h;
(ⅲ) DMF-DMA, toluene, 110 °C, 10 h; (ⅳ) Fe powder, AcOH, rt., 30 min, 80 °C, 2 h; (ⅴ) secondary amines, CH3CN,
85 °C, 10 h; (ⅵ) POCl3, 85 °C, 6 h; (ⅶ) 2-fluoro-4-nitrophenol, PhCl, 140 °C, 30 h; (ⅷ) Fe powder, NH4Cl (cat.),
EtOH/H2O, reflux, 5 h.
The target compounds 23 – 45 were prepared as illustrated in Scheme 2. Using Cu and Cu2O as
catalyst, Ullmann reaction of commercially 2-chlorobenzoic acid with substituted phenylamines in ethylene
glycol at 170 °C yielded intermediates 18a – g. 22 Acylation of 18a – g with triphosgene in the presence of
potassium carbonate (K2CO3) in ethyl acetate resulted in high yield of intermediates 19a – g, which were
condensed with dimethyl malonate in the presence of sodium methylate (NaOCH3) in hot dimethyl
23
formamide (DMF) to afford intermediates 20a – g.
Simple procedures such as hydrolysis and acyl
chlorination were used to convert 20a – g to the corresponding acyl chlorides 22a – g; the reactions
proceeded with AcOH/HCl (conc.) and thionyl chloride, respectively. Reaction of amides 17a – e with acyl
chlorides 22a – g promoted by DIPEA in dichloromethane at room temperature yielded the target
compounds 23 – 45. 15
Scheme 2. Reagents and conditions: (ⅰ) appropriate aniline, Cu, Cu2O, K3CO3, Ethylene glycol, 170 °C, 24 – 30 h; (ⅱ)
Triphosgene, K3CO3, ethyl acetate, rt., 6 – 10 h; (ⅲ) Dimethyl malonate, CH3ONa, DMF, N2, 50 °C, 0.5 h, 85 °C, 30 min;
(ⅳ) AcOH, HCl (conc.), 80 °C, 3 h; (ⅴ) SOCl2, reflux, 6 h; (ⅵ) appropriate aniline, DIPEA, CH2Cl2, 0 °C, 1 h, rt, 15 – 20
h.
The cytotoxic activity of targeted compounds was listed in Table 1. 24 All the 23 newly synthesized

target compounds (23-45) showed moderate to excellent cytotoxic activity against the five tested cancer
cells with potencies in the single digit μM range, which suggested that replacement of
N-(4-fluorophenyl)cyclopropane-1,1-dicarboxamide
framework
of
foretinib
with
2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety maintained the potent cytotoxic activity. The
IC50 values of the most promising compound 42 25 were 0.031 μM, 0.068 μM, and 0.52 μM against H460,
HT-29, and U87MG cell lines, respectively; these values indicated that this compound was 6.1, 2.4, and 2.1
times more active than foretinib (IC50 values: 0.19 μM, 0.16 μM, and 1.1 μM, respectively). In addition,
most of the compounds displayed higher selectivity against H460 and HT-29 cells than against the other
three cell lines.
Table 1
Cytotoxic activities of the target compounds 23–45 against the H460, HT-29, MKN-45, A549, and U87MG
cancer cell lines in vitro
IC50 (μM)
Compd.
R1
R2
H460
HT-29
MKN-45
A549
U87MG
4.8 ± 0.26
8.5 ± 0.50
ND
23
H
0.26 ± 0.063
0.061 ± 0.0050a
0.58 ± 0.032
0.85 ± 0.026
1.2 ± 0.26
0.46 ± 0.025
0.14 ± 0.016
1.21 ± 0.33
0.39 ± 0.014
1.4 ± 0.19
0.31 ± 0.052
0.21 ± 0.040
0.74 ± 0.063
0.78 ± 0.080
1.26 ± 0.081
0.21 ± 0.030
0.57 ± 0.042
0.90 ± 0.014
1.2 ± 0.26
0.93 ± 0.064
0.11 ± 0.030
NDb
24
4-F
25
2-F
26
4-CH3
2-OCH3
H
0.83 ± 0.041
ND
5.16 ± 0.23
ND
27
28
0.21 ± 0.022
0.12 ± 0.080
0.23 ± 0.031
0.62 ± 0.015
0.36 ± 0.031
0.14 ± 0.07
0.39 ± 0.028
0.15 ± 0.021
0.48 ± 0.036
0.30 ± 0.029
1.5 ± 0.16
0.81 ± 0.032
0.72 ± 0.060
0.23 ± 0.057
ND
1.6 ± 0.18
0.64 ± 0.045
3.24 ± 0.16
ND
29
4-F
30
4-CH3
3,5-(CH3)2
2-OCF3
H
31
32
1.4 ± 0.20
ND
ND
33
41.6 ± 0.23
0.29 ± 0.051
0.52 ± 0.062
12.6 ± 0.31
0.35 ± 0.048
0.11 ± 0.037
0.19 ± 0.039
0.57 ± 0.062
0.091 ± 0.0035
0.068 ± 0.0082
0.17 ± 0.034
0.15 ± 0.025
0.53 ± 0.084
0.16 ± 0.015
0.28 ± 0.063
0.31 ± 0.032
0.48 ± 0.020
0.42 ± 0.025
0.46 ± 0.053
0.16 ± 0.046
0.12 ± 0.082
0.31 ± 0.032
0.12 ± 0.087
0.16 ± 0.037
0.41 ± 0.021
0.12 ± 0.015
0.12 ± 0.015
0.032 ± 0.0052
0.51 ± 0.040
0.16 ± 0.025
ND
1.3 ± 0.10
1.6 ± 0.18
ND
34
4-F
0.11 ± 0.058
0.17 ± 0.030
0.58 ± 0.04
35
4-CH3
2-OCH3
H
36
ND
ND
37
0.15 ± 0.075
0.089 ± 0.0046
0.18 ± 0.0070
0.38 ± 0.030
0.061 ± 0.052
0.031 ± 0.0053
0.27 ± 0.058
0.12 ± 0.041
0.15 ± 0.041
0.19 ± 0.036
0.39 ± 0.022
0.99 ± 0.024
0.35 ± 0.036
ND
1.42 ± 0.43
1.92 ± 0.72
3.2 ± 0.37
ND
38
4-F
39
4-CH3
2-OCF3
H
40
41
ND
ND
42
4-F
0.11 ± 0.024
0.29 ± 0.036
0.31 ± 0.060
ND
0.52 ± 0.31
1.32 ± 0.25
1.06 ± 0.27
ND
43
2-F
44
4-CH3
2-OCH3
45
Foretinibc
0.13 ±0.010
1.1±0.12
a Bold values show the IC50 values of the target compounds lower than the values of the positive control.
b ND: Not determined.

c Used as the positive control.
The cell lines data revealed a clear preference for activity when the R1 group was 4-methyl piperazinyl
group, which indicated that a more water-soluble cyclic tertiary amino group at the 7 position of quinoline
contributed to the potency of the target compounds. For example, the IC50 value of compound 41 (R1 =
4-methyl piperazinyl), 0.061μM, was clearly lower than that of 23, 28, 33, and 37, 0.26 μM, 0.21 μM, 0.14
μM, and 0.15 μM, respectively, against H460 cells.
Further analysis clearly revealed that different cytotoxic activities were observed when various R2
groups were introduced into the phenyl ring (moiety B). Compound 23, with no substituent on the phenyl
ring, displayed strong cytotoxicity with an IC50 of 0.26 μM against H460 cells. The introduction of
electron-withdrawing groups at 4-position of the phenyl ring (24, R2 = 4-F, IC50 = 0.061 μM) led to an
obvious improvement in antitumour activity, which could be further confirmed by compounds 29, 34, 38,
and 42. However, the introduction of electron-withdrawing (25, 2-F, IC50 = 0.82 μM) or electron-donating
groups (27, 2-OCH3, IC50 = 1.2 μM) at other position reduced the activities.
The c-Met enzymatic assays of eight compounds were evaluated using homogeneous time-resolved
fluorescence (HTRF) assay and showed excellent result, 26 which suggested that the inhibition of c-Met
may be one mechanism of the antiproliferative effect of these derivatives (Table 2). Compound 42 showed
the most potent activity with an IC50 value of 1.21 nM, which was superior to that of the positive control
foretinib (IC50 = 1.84 nM), and this compound should be studied further.
Table 2
c-Met kinase activity of selected compounds 24, 29, 33, 34, 37, 38, 41, 42 and foretinib in vitro.
Compd.
IC50 on c-Met (nM)
24
5.32
12.51
18.39
12.18
6.84
29
33
34
37
38
3.26
41
1.75
42
1.21
Foretinib
1.84
Compound 42 were chosen for further evaluation the selectivity on c-Met over other tyrosine
kinases.16 As shown in Table 3, compared with its high potency against c-Met (IC50 = 1.21 nM), 42 also
exhibited high inhibitory effects against Flt-3 (IC50 = 2.15 nM). While, 42 showed weak potency on c-Kit,
VEGFR-2, PDGFR-β, and EGFR. These data suggested that compound 42 had a selectivity on c-Met and

Flt-3.
Table 3
Inhibition of tyrosine kinases by compound 42
Kinase
Flt-3
Enzyme IC50 (nM)
2.15
c-Kit
362.8
PDGFR-β
VEGFR-2
EGFR
526.5
542.6
>100,000
To further elucidate the binding mode of compounds, docking analysis was performed. In our study,
the co-crystal structure of foretinib with c-Met kinase was selected as the docking model (PDB ID: 3LQ8).
The docking simulation was conducted using SURFLEX-DOCK module of SYBYL 6.9.1 package version.
As depicted in Fig. 4, the shape and properties of the receptor are represented on a grid by several different
sets of fields that provide progressively more accurate scoring of the ligand poses. The binding model was
exemplified by the interaction of compound 42 with c-Met. As shown in Fig. 4, the nitrogen atom of the
quinoline ring in 42 formed hydrogen-bonding interactions with MET1160. The hydrogen atom of the
amido linkage and the oxygen atom of the 2-oxo-4-chloro-1,2-dihydroquinoline formed two hydrogen
bonds with ASP1222. The result demonstrated that compound 42 could form three hydrogen bonds with
c-Met.
Figure 4. Binding poses of compound 42 with c-Met. The proteins were displayed by grid ribbon.
Compound 42 were displayed by multicolor sticks. H-bonding interactions between the 42 and c-Met were
indicated with dashed lines in yellow.
In summary, we designed and synthesized 23 quinoline derivatives bearing
a
2-oxo-4-chloro-1,2-dihydroquinoline-3-carboxamide moiety. Five human cell lines were used to evaluate
the potency of the synthesized compounds, and 12 of these derivatives were more potent than foretinib.

Compound 42 (c-Met/Flt-3 IC50 = 1.21/2.15 nM) showed the strongest cytotoxic activities against H460,
HT-29, and U87MG cell lines which was 6.1, 2.4, and 2.1 times more active than foretinib against the 3
cell lines, respectively. Docking analysis showed that compound 42 could form three hydrogen bonds with
c-Met. The analysis of SARs indicated that a more water soluble cyclic tertiary amine (R1 = 4-methyl
piperazinyl) contributed to the potency. Electron-withdrawing groups at 4-position (R2 = 4-F) of the phenyl
ring contribute to the antiproliferative activity. Further studies on SARs and mechanism of action of these
compounds are in progress, and their results will be reported in the future.
Acknowledgments
This work was supported by National Natural Science Foundation of China (NSFC No. 81573295).
References and notes
1. http://www.who.int/mediacentre/factsheets/fs297/en/ (WHO Media Centre, Cancer, Updated Feb, 2015,
last accessed: 10/30/2015)
2. Samadi, A. K.; Bazzill, J.; Zhang, X.; Gallagher, R.; Zhang, H. P.; Gollapudi, R.; Kindscher, K.;
Timmermann, B.; Cohen, M. S. Surgery 2012, 152, 1238.
3. You, W. K.; Sennino, B.; Williamson, C. W.; Falcón, B.; Hashizume, H.; Yao, L. C.; Aftab, D. T.;
McDonald, D. M. Cancer Res. 2011, 71, 4758.
4. Dayyani, F.; Gallick, G. E.; Christopher, C. L.; Corn, P. G. J. Natl. Cancer Inst. 2011, 103, 1665.
5. Hoelder, S.; Clarke, P. A.; Workman, P. Molecular Oncology 2012, 6, 155.
6. Kung, P. P.; Funk, L.; Meng, J.; Alton, G.; Padrique, E.; Mroczkowski B. Eur. J. Med. Chem. 2008, 43,
1321.
7. Yakes, F. M.; Chen, J.; Tan, J.; Yamaguchi, K.; Shi, Y. C.; Yu, P. W.; Qian, F.; Chu, F.; Bentzien, F.;
Cancilla, B.; Orf, J.; You, A.; Laird, A. D.; Engst, S.; Lee, L.; Lesch, J.; Chou, Y. C.; Joly, A. H. Mol.
Cancer Ther. 2011, 10, 2298.
8. Norman, M. H.; Liu, L. B.; Lee, M.; Xi, N.; Fellows, I.; D’Angelo, N. D.; Dominguez, C.; Rex, K.;
Bellon, S. F.; Kim, T. S.; Dussault, I. J. Med. Chem. 2012, 55, 1858.
9. Cecchi, F.; Rabe, D. C.; Bottaro, D. P. Eur. J. Cancer 2010, 46, 1260.
10. Liu, X. D.; Newton, R. C.; Scherle, P. A. Trends in Mol. Med. 2009, 16, 1471.
11. Kataoka, Y.; Mukohara, T.; Tomioka, H.; Funakoshi, Y.; Kiyota, N.; Fujiwara, Y.; Yashiro, M.;
Hirakawa, K.; Hirai, M.; Minami, H. Invest New Drugs 2012, 30, 1352.
12. González-Sánchez, I.; Solano, J. D.; Loza-Mejía, M. A.; Olvera-Vázquez, S.; Rodríguez-Sotres, R.;
Morán, J.; Lira-Rocha, A.; Cerbón, M. A. Eur. J. Med. Chem. 2011, 46, 2102.
13. Ai, Y.; Liang, Y. J.; Liu, J. C.; He, H. W.; Chen, Y.; Tang, C.; Yang, G. Z.; Fu, L. W. Eur. J. Med.
Chem. 2012, 47, 206.
14. Abonia, R.; Insuasty, D.; Castillo, J.; Insuasty, B.; Quiroga, J.; Nogueras, M.; Cobo, J. Eur. J. Med.
Chem. 2012, 57, 29.
15. Li, S.; Huang, Q.; Liu, Y. J.; Zhang, X. L.; Liu, S.; He, C.; Gong, P. Eur. J. Med. Chem. 2013, 64, 62.
16. Li, S.; Zhao, Y. F.; Wang, K. W.; Gao. Y. L.; Han, J. M.; Cui, B. B.; Gong, P. Bioorg. Med. Chem. 2013,
21, 2843.
17. Duraisamy, S. R.; Eswaran, R.; Nattamai, S. P.; Karuppannan, N. Eur. J. Med. Chem. 2013, 64, 148.

18. Tang, Q. D.; Zhao, Y. F.; Du, X. M.; Chong, L. E.; Gong, P.; Guo, C. Euro. J. Med. Chem. 2013, 69, 77.
19. Tiedt, R.; Degenkolbe, E.; Furet, P.; Appleton, B. A.; Wagner, S.; Schoepfer, J.; Buck, E.; Ruddy, D. A.;
Monahan, J. E.; Jones, M. D.; Blank, J.; Haasen, D.; Drueckes, P.; Wartmann, M.; McCarthy, C.;
Sellers, W. R.; Hofmann, F. Cancer Res. 2011, 71, 5255.
20. Ted, L. U.; Torsten, H.; Sheila, J. M. Anticancer Agent Med. 2010, 10, 7.
21. Takayuki, N.; Osamu, T.; Atsumi, Y.; Tomohiro, M.; Keiko, T.; Setsuo, F.; Shuji, S.; Makoto, A.;
Hiroshi, O. Cancer Sci. 2010, 101, 210.
22. Martín, A.; Mesa, M.; Docampo, M. L. Synthetic communications 2006, 36, 271.
23. Jönsson, S.; Andersson, G.; Fex, T. J. Med. Chem. 2004, 47, 2075.
24. Cytotoxicity assay: A standard MTT assay was used to measure cell growth. The cancer cell lines were
cultured in minimum essential medium (MEM) supplemented with 10% fetal bovine serum (FBS).
Approximate 4 × 103 cells, suspended in MEM medium, were plated onto each well of a 96-well plate
and incubated in 5% CO2 at 37 °C for 24 h. The compounds tested at the indicated final concentrations
were added to the culture medium and the cell cultures were continued for 72 h. Fresh MTT was added
to each well at a terminal concentration of 5 μg/mL, and incubated with cells at 37 °C for 4 h. The
formazan crystals were dissolved in 100 mL DMSO of each well, and the absorbency at 492 nm (for
absorbance of MTT formazan) and 630 nm (for the reference wavelength) was measured with an
ELISA reader. All of the compounds were tested in triplicate in each cell line. The results expressed as
IC50 (inhibitory concentration 50%) were the averages of three determinations and calculated by using
the Bacus Laboratories Incorporated Slide Scanner (Bliss) software.
25. Analytic data of potent inhibitor 42: 1H NMR (300MHz, CDCl3) δ 11.12(S), 8.63(d, J = 5.2, 1H),
8.06(dd, J = 12.1, 2.4 Hz, 1H), 7.87(d, J = 8.4, 1H), 7.81-7.75 (m, 2H), 7.59(S), 7.48(d, J = 8.4, 1H),
7.43(S), 7.41-7.25(m, 6H), 6.41(d, J = 5.4, 1H), 4.26 (t, J = 6.8 Hz, 2H), 4.03 (s, 3H), 2.66-2.52 (m,
4H), 2.47 (br, 6H), 2.27 (s, 3H), 2.15-2.03 (m, 2H); 13C NMR (150MHz, CDCl3) 164.89, 161.32,
159.52, 158.17,155.36, 153.26, 152.11, 150.12, 149.16, 147.83, 146.12, 142.48, 139.35, 136.91, 136.29,
134.82, 133.84, 131.14, 129.28 (2C), 128.64, 125.95, 124.26, 123.91, 116.45, 116.21, 110.35, 109.73,
108.62, 102.84, 99.65, 67.54, 56.22, 55.25 (2C), 54.82, 53.09 (2C), 46.36, 26.42; MS m/z (ESI): 740.4
[M+H]+; Anal. calcd. for C40H36F2ClN5O5 (%): C, 64.91; H, 4.90; N, 9.46. Found (%): C, 64.89; H,
4.88; N, 9.41.
26. In vitro kinase assay: The kinase assays were performed by homogeneous time-resolved fluorescence
(HTRF) assay as previously reported protocol. Briefly, 20 μg/mL poly (Glu, Tyr) 4:1 (Sigma) was
preloaded as a substrate in 384-well plates. Then 50 μL of 10 mM ATP (Invitrogen) solution diluted in
kinase reaction buffer (50 mM HEPES, pH 7.0, 1 M DTT, 1 M MgCl2, 1 M MnCl2, and 0.1% NaN3)
was added to each well. Various concentrations of compounds diluted in 10 μL of 1% DMSO (v/v)
were used as the negative control. The kinase reaction was initiated by the addition of purified tyrosine
kinase proteins diluted in 39 μL of kinase reaction buffer solution. The incubation time for the reactions
was 30 min at 25 °C, and the reactions were stopped by the addition of 5 μL of Streptavidin-XL665 and
5 μL Tk Antibody Cryptate working solution to all of wells. The plates were read using Envision
(PerkinElmer) at 320 nm and 615 nm. The inhibition rate (%) was calculated using the following
equation: % inhibition = 100 - [(Activity of enzyme with tested compounds - Min)/ (Max - Min)] × 100
(Max: the observed enzyme activity measured in the presence of enzyme, substrates, and cofactors;
Min: the observed enzyme activity in the presence of substrates, cofactors and in the absence of
enzyme). IC50 values were calculated from the inhibition curves.

Graphical abstract