Design, synthesis and biological evaluation of novel 4-phenoxyquinoline derivatives containing 3-oxo-3,4-dihydroquinoxaline moiety as c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.06.037

A series of novel 4-phenoxyquinoline derivatives containing 3-oxo-3,4-dihydroquinoxaline moiety were synthesized and evaluated for their c-Met kinase inhibitory activity and antiproliferative activity against five cancer cell lines (HT-29, H460, A549, MKN-45 and U87MG) in vitro. Most of the compounds exhibited moderate-to-significant cytotoxicity as compared with foretinib. The most promising compound 41 (with c-Met IC₅₀ value of 0.90?nM) showed remarkable cytotoxicity against HT-29, H460, A549, MKN-45 and U87MG cell lines with IC₅₀ values of 0.06?μM, 0.05?μM, 0.18?μM, 0.023?μM and 0.66?μM, respectively, and thus it was 1.22- to 3.50-fold more potent than foretinib. Their preliminary structure-activity relationships (SARs) studies indicate that electron-withdrawing groups on the terminal phenyl rings are beneficial for improving the antitumor activity.

Full text of DOI:10.1016/j.bmc.2017.06.037

Accepted Manuscript
Design, synthesis and biological evaluation of novel 4-phenoxyquinoline de-
rivatives containing 3-oxo-3,4-dihydroquinoxaline moiety as c-Met kinase in-
hibitors
Ju Liu, Di Yang, Xiuxiu Yang, Minhua Nie, Guodong Wu, Zhunchao Wang,
Wei Li, Yajing Liu, Ping Gong
PII:
S0968-0896(17)30649-1
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.06.037
BMC 13820
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
28 March 2017
31 May 2017
24 June 2017
Please cite this article as: Liu, J., Yang, D., Yang, X., Nie, M., Wu, G., Wang, Z., Li, W., Liu, Y., Gong, P., Design,
synthesis and biological evaluation of novel 4-phenoxyquinoline derivatives containing 3-oxo-3,4-
dihydroquinoxaline moiety as c-Met kinase inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: http://
dx.doi.org/10.1016/j.bmc.2017.06.037
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Design, synthesis and biological evaluation of novel 4-phenoxyquinoline derivatives containing
3-oxo-3,4-dihydroquinoxaline moiety as c-Met kinase inhibitors
Ju Liua,b, Di Yanga, Xiuxiu Yanga, Minhua Niea, Guodong Wua, Zhunchao Wanga, Wei Lic, Yajing Liua*, Ping
Gonga*1
a Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103
Wenhua Road, Shenhe District, Shenyang 110016, PR China
b
College of Pharmacy of Liaoning University, Key Laboratory of New Drug Research and Development of Liaoning Province, 66
Chongshan Road, Huanggu District, Shenyang 10036, P. R. China.
c
Department of Pharmaceutical Engineering, Shenyang University of Chemical Technology, 11 street, Jingjijishukaifa District,
Shenyang 110142, P. R. China.
Abstract
A series of novel 4-phenoxyquinoline derivatives containing 3-oxo-3,4-dihydroquinoxaline moiety were
synthesized and evaluated for their c-Met kinase inhibitory activity and antiproliferative activity against five cancer
cell lines (HT-29, H460, A549, MKN-45 and U87MG) in vitro. Most of the compounds exhibited
moderate-to-significant cytotoxicity as compared with foretinib. The most promising compound 41 (with c-Met
IC50 value of 0.90 nM) showed remarkable cytotoxicity against HT-29, H460, A549, MKN-45 and U87MG cell
lines with IC50 values of 0.06µM, 0.05µM, 0.18µM, 0.023µM and 0.66 µM, respectively, and thus it was 1.22- to
3.50- fold more potent than foretinib. Their preliminary structure-activity relationships (SARs) studies indicate that
electron-withdrawing groups on the terminal phenyl rings are beneficial for improving the antitumor activity.
Keywords: Synthesis; 4-phenoxyquinoline derivatives; c-Met inhibitors; Antitumor activity.
1. Introduction
Receptor tyrosine kinases (RTKs) play crucial roles in numerous signal transduction pathways and cellular
processes. Many are implicated in cancer, 1 including the Hepatocyte Growth Factor Receptor (HGFR) c-Met, 2
which is over-expressed and/or mutated in various human tumor types. Dysregulation of c-Met/HGF signaling
pathway affects cell proliferation, survival and motility, leading to tumor growth, angiogenesis, and metastasis.
Therefore, c-Met has become an attractive target for cancer therapy. 3, 4
Recently, a series of 4-phenoxyquinoline derivatives have been reported as class II c-Met inhibitors, such as
Cabozantinib (1), Foretinib (2), Kirin (3), AM7 (4), MG10 (5) and Amgen (6)(Fig. 1).5-10 Among them,
Cabozantinib was approved as a novel oral multi-kinase inhibitor for the treatment of patients with progressive
metastatic medullary thyroid cancer (MTC).11 Foretinib (Fig. 1) which is currently undergoing phase III studies for
different cancer types, is also a multikinase inhibitor targeting c-Met, VEGFR-2, RON and Flt-1.12 Meanwhile,
other derivatives have been progressed into clinical or preclinical studies as c-Met kinase inhibitors. However, it
was reported that the more potent VEGFR activity in some class II c-Met inhibitors may lead to suboptimal dosing
for c-Met inhibition in clinical applications because of VEGFR-related side effects.13, 14 Moreover，it is important
that compounds designed as kinase inhibitors have a good kinase selectivity profile because inhibiting particular
kinases carries the risk of adverse effects.15 Thus far, a majority of the class II c-Met molecules are multikinase
inhibitors. Above all, improving the selectivity of class II c-Met inhibitors has been a significant challenge.
During the course of analyzing the SAR of these analogues (Fig. 1), 16-18 most of them may be disconnected
* Corresponding author. Tel./fax: +86 24 2398 6429.
E-mail address: lyjpharm@126.com (Y. Liu), gongpinggp@126.com (P. Gong).

into three units according to their structures and subunit functions. We found that modification of moiety A usually
occurs at position 7 of quinoline, where the side chain is replaced by different water-soluble fragment, such as
3-morpholinopropoxy, 3-piperidinylpropoxy and 3-(4-methylpiperazinyl) propoxy group. Furthermore, there is
little change to B moiety except for a phenyl ring or a substituted phenyl ring. We found that moiety A and moiety
B appears to be critical for kinase activity. In contrast, the structure of moiety C is alterable, and various linear
chains and heterocyclic rings can be introduced to the main chain of 5-atom linker (i.e., six chemical bonds distance
between moiety A and B, as summarized recently by our team.19), containing hydrogen-bond donors and acceptors.
These structural characteristics suggest that exploring a satisfactory linker is a practicable way of designing this
series of quinoline derivatives and could improve the kinase selectivity of the molecules. In the light of the results
mentioned above, our research group has introduced different 5-atom linkers such as imidazole,
pyrimidine-2,4,6-trione and 1,2,4-triazolone into the moiety C and the resulting derivatives 7-9 (Fig. 1) showed
excellent potency and kinase selectivity. 20-22
Figure 1. Structures of small-molecule c-Met inhibitors
To our knowledge, compounds bearing quinoxalinone fragment have been reported to exhibit a large field of
biological activities, including antitumor, antibacterial, and anti-inflammatory activities, etc (10-13, Fig. 2).23-26
Remarkably, the 3-oxo-3,4-dihydroquinoxaline-2-carboxamide framework conforms to “5 atoms regulation”, and
contains both hydrogen-bond donor and acceptor, which makes it a satisfactory linker. So we replaced the
cyclopropane-1,1-dicarboxamide fragment of foretinib with 3-oxo-3,4-dihydroquinoxaline-2-carboxamide scaffold
to obtain a series of novel 4-(2-fluorophenoxy)quinoline derivatives (Fig. 3). Meanwhile, the morpholinyl group at
7-position of quinoline scaffold was replaced by several water-soluble groups including piperidinyl, pyrrolidinyl
and 4-methyl piperidinyl groups. Additionally, various substituents (R2) were introduced into the terminal phenyl
ring (moiety B) to investigate their effects on activity.

Figure 2. Anticancer agents bearing quinoxalinone moiety
Figure 3. Design strategy and general structure of the target compounds
In the current study, all target compounds were evaluated for their antiproliferative activity in vitro against
five cancer cell lines: human colon cancer cell line (HT-29), human lung cancer cell lines (H460), human lung
adenocarcinoma cell line (A549), human gastric cancer cell line (MKN-45) and human glioblastoma cell line
(U87MG), and their structure-activity relationships (SARs) were further explored. Additionally, to determine c-Met
kinase inhibition, the enzymatic assays of several potent compounds were evaluated, and most of them showed
promising inhibition. Furthermore, the inhibitory activities of the most potent compound 41 against seven other
RTK kinases were also investigated, and a docking analysis was also performed to elucidate the binding mode of
the target compound 41 with c-Met kinase.
2. Chemistry
The synthesis of target compounds 19–46 is described in Scheme 1. The key intermediates
6,7-disubstituted-4-phenoxyquinolines 14a–14e were synthesized using a convenient eight-step procedure starting
from 1-(4-hydroxy-3-methoxyphenyl)ethanone, which was illustrated in detail in our previous study.19,27 The
condensation of commercially available 1-fluoro-2-nitrobenzene with different substituted amines in the presence
of sodium hydride in N,N-dimethylformamide afforded intermediates diphenyl amines 15 as yellow solids.
Reduction of the nitro group of 15 with iron powder provided compounds 16. The cyclization of 16 with diethyl
ketomalonate afforded 3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid esters 17, which were converted to acids 18
using lithium hydroxide monohydrate in THF/water at RT for 2 hours. Finally, acids 18 were refluxed in toluene
and SOCl2 for 5 h to afford acyl chlorides which were condensed with intermediates 14a–14e in the presence of
sodium carbonate in dichloromethane at room temperature overnight to afford the target compounds 19–46 having
a 3-oxo-4-aryl -3,4-dihydroquinoxaline-2-carboxamide group.

Scheme 1. Reagents and conditions: (a) amines, NaH, DMF, RT, 16 h; (b) Fe powder, HOAc, CH3COOEt/H2O,
reflux, 6 h; (c) diethyl ketomalonate, toluene, reflux, 12 h; (d) LiOH, THF/ H2O, RT, 1.5 h, then 6 N HCl; (e)
toluene, SOCl2, reflux, 5 h; Na2CO3, CH2Cl2, 25 °C, 3 h.
3. Results and discussion
3.1. In vitro cytotoxicity and structure–activity relationships
All the 28 newly synthesized compounds (19-46) were screened for their in vitro cytotoxic activity by the
MTT-based assay using Foretinib as a positive control. They were tested against five c-Met overexpresed human
cancer cell lines, namely human colon cancer cell line (HT-29), human lung cancer cell lines (H460), human lung
adenocarcinoma cell line (A549), human gastric cancer cell line (MKN-45) and human glioblastoma cell line
(U87MG). The results were expressed as IC50 values and summarized in Table 1. The IC50 values were the average
of at least three independent experiments.
As illustrated in Table 1, all the target compounds showed excellent cytotoxic activity against different cancer
cells and some exhibited more or similar potent activities against certain cancer lines in comparison with foretinib,
which suggested that replacement of cyclopropane-1,1-dicarboxamide framework of foretinib with 3-oxo-4-aryl-3,
4-dihydroquinoxaline-2-carboxamide moiety as the linker maintained the potent cytotoxic activity. It is worth
pointing out that 23 compounds were more potent than foretinib against one or more cell lines. The most significant
cytotoxic activity was achieved for compounds 25, 41 and 44 with IC50 values ranging from 0.023 µM to 0.94 µM
against all tested cell lines, which were better than that of foretinib (IC50 = 0.030 to 0.96 µM). More importantly,
most of the compounds were more potent against A549 and MKN-45 cell lines with potencies in the double-digit
nM range than other three cell lines. These results revealed that this series of compounds possessed selectivity for
A549 and MKN-45 cancer cell lines, and had the makings of good drugs for lung and gastric cancer.
Table 1
Structures and cytotoxicity of compounds 19-46.
Compd.
R1
R2
IC50 (µmol/L) SDa
HT-29
A549
H460
MKN-45
U87MG
Morpholinyl
Morpholinyl
H
0.41 0.05
1.72 0.02
1.04 0.01
1.85 0.03
0.082 0.008
0.66 0.05
1.72 0.07
>32 0.07
19
20
0.20 0.03
4-OCH3
0.69 0.01

Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Morpholinyl
Piperidinyl
4-CH3
1.03 0.01
2.16 0.01
5.11 0.03
0.33 0.05
1.12 0.07
2.05 0.03
0.58 0.02
0.86 0.07
4.17 0.01
16.25 0.07
1.22 0.09
1.78 0.07
0.81 0.21
1.06 0.02
1.17 0.02
0.65 0.02
1.50 0.02
1.85 0.31
0.98 0.05
5.04 0.21
1.61 0.02
1.31 0.02
1.17 0.22
1.23 0.01
0.87 0.22
1.41 0.04
1.12 0.01
2.17 0.02
0.66 0.22
1.21 0.04
1.10 0.01
0.94 0.22
1.52 0.21
1.03 0.10
21
2,4-(OCH3)2 1.29 0.03
22
23
4-OCF3
2-CF3
4-F
0.20 0.003
0.29 0.002 0.26 0.01
0.22 0.003 0.28 0.002 0.56 0.001
24
0.19 0.003
25
0.11 0.01
0.030 0.05 0.14 0.05
0.026 0.03
0.029 0.08
0.11 0.03
0.029 0.01
4-Cl
4-Br
2-F
0.28 0.06
0.21 0.01
0.23 0.002
26
0.16 0.003 0.042 0.04
0.32 0.002
0.17 0.012 0.033 0.03
27
0.17 0.04
28
2-Cl
3-F
29
0.15 0.004 0.050 0.03 0.14 0.002 0.025 0.01
0.31 0.01
0.24 0.05
0.17 0.06
0.40 0.01
0.53 0.02
0.64 0.08
0.39 0.03
30
0.20 0.09
0.33 0.04
0.59 0.01
0.16 0.04
0.24 0.04
0.22 0.01
0.15 0.03
0.16 0.01
0.24 0.05
0.19 0.03
0.17 0.04
0.06 0.03
0.20 0.08
0.16 0.01
0.13 0.03
0.25 0.11
0.35 0.03
0.21 0.01
3-Cl
2,4-(Cl)2
4-F
0.34 0.02
31
0.075 0.02
0.020 0.08
0.053 0.08
0.034 0.05
0.060 0.11
0.028 0.08
0.098 0.07
0.040 0.01
0.075 0.03
0.023 0.02
0.043 0.07
0.039 0.02
0.024 0.02
0.056 0.14
0.048 0.11
32
33
0.055 0.02 0.23 0.03
0.31 0.03
0.084 0.02 0.14 0.01
Piperidinyl
4-Cl
2-F
34
0.09 0.02
Piperidinyl
35
Piperidinyl
2-Cl
0.20 0.01
0.052 0.08
0.14 0.012
0.10 0.01
0.35 0.12
0.31 0.22
0.28 0.06
0.16 0.03
36
4-Methylpiperidinyl 4-F
4-Methylpiperidinyl 4-Cl
4-Methylpiperidinyl 2-F
4-Methylpiperidinyl 2-Cl
4-Methylpiperazinyl 4-F
4-Methylpiperazinyl 4-Cl
4-Methylpiperazinyl 2-F
4-Methylpiperazinyl 2-Cl
37
38
39
0.39 0.002 0.53 0.01
40
41
0.050 0.20 0.18 0.01
0.25 0.02
42
0.11 0.09
43
0.049 0.03 0.25 0.05
0.025 0.20 0.12 0.01
0.090 0.02 0.21 0.21
44
Pyrrolidinyl
Pyrrolidinyl
4-F
45
2-Cl
46
0.12 0.01
0.19 0.09
Foretinibb
0.15 0.03
0.22 0.01
0.030 0.005 0.96 0.12
Bold values show the IC50 values of target compounds lower than the values of the positive control.
IC50: concentration of the compound (mM) producing 50% cell growth inhibition after 72 h of drug exposure, as determined by the MTT assay. Each
a
experiment was carried out in triplicate.
b Used as a positive control.
As show in Table 1, the SARs based on IC50 values of cytotoxic activity revealed that variations in the R2
groups on the phenyl ring (moiety B) markedly affect their activities. Primarily, mono-electron-donating groups
(mono-EDGs) such as 4-methoxy and 4-methyl analogues (20, 21) exhibited a negative effect compared to no
substituent on the phenyl ring (19: R2 = H). A dramatic drop in antiproliferative activity was seen as we introduced
double-EDGs (22: R2 = 2, 4-dimethoxy) at this region of the molecule. Gratifyingly, the introduction of
mono-electron-withdrawing groups (mono-EWGs) and double-EWGs exhibited a positive effect on the cytotoxic
activity. In particular, incorporation of the halogen atom (R2 = F, Cl, Br, respectively) to the 2- or 4- position of
phenyl ring showed a higher potency, such as compound 25 (R2 = 4-F, IC50 = 0.026 to 0.81 µM against all tested
cell lines). However, moving the halogen atom from 2-or 4-position to 3-position (30, 31) led to decrease in
cytotoxicity against all cancer cells. Moreover, incorporation of another halogen atom in the form of 2, 4-dichloro
compound (32: R2 = 2, 4-dichloro, IC50 = 0.59 µM against HT-29 ) showed somewhat a decreased cellular activity
compared with 4-chloro analogue (26: R2 = 4-Cl, IC50 = 0.16 µM against HT-29) and 2-chloro analogue (29: R2 =
2-Cl, IC50 = 0.15 µM against HT-29), which indicates monosubstitution of phenyl is more preferred. Meanwhile,
compared with halogen analogs 28 (R2 = 2-F) and 29 (R2 = 2-Cl), incorporation of strong electron-withdrawing
groups (EWGs) such as trifluoromethyl group (24) on the phenyl ring (moiety B) reduced the antitumor activity

slightly against HT-29, H460 and U87MG, but lowered the efficiency even further against A549 and MKN-45. It
suggested that the moiety B probably need medium electron density. Accordingly, compounds possessing chloro
and fluoro groups at the 2- and 4-position of phenyl ring were further studied in our work.
To understanding the SAR deeply, we examined the effects of the R1 substituent on the three carbon ether
connected to the quinoline at the 7-position by replacing the morpholinyl group with piperidinyl,
4-methylpiperidinyl, 4-methylpiperazinyl and pyrrolidinyl groups. As show in Table 1, the results indicated that the
introduction of different water-soluble R1 groups had a slight influence on activity.Taking R2=2-Cl series
compounds for example, compounds 29 (R1 = Morpholinyl), 36 (R1 = Piperidinyl), 40 (4-Methylpiperidinyl), 44
(4-Methylpiperazinyl) and 46 (R1 = Pyrrolidinyl), exhibited comparable cytotoxic activities against the different
cancer cells.
3.2. In vitro enzymatic assays
Based on the cellular assay results, we selected nine compounds which were more potent than foretinib against
three or more cell lines for c-Met kinase activity determination using homogenous time-resolved fluorescence
(HTRF) assays and the results were outlined in Table 2. As shown in Table 2, all the ten tested compounds
exhibited excellent c-Met enzymatic potency with IC50 values ranging from 0.90 to 13.14 nM. These data suggest
that c-Met inhibition may be a mechanism for the antitumor effect of these derivatives. Compounds 41 and 44
showed the most potent activity with an IC50 values of 0.90 nM and 1.22 nM, respectively, which were better than
that of foretinib (IC50 = 1.41 nM), indicating that these compounds deserve further study with regard to its
application in the treatment of cancer.
Table 2
c-Met kinase activity of selected compounds 25, 26, 28, 29, 33, 37, 39, 41, 44 and Foretinib in vitro.
Compd.
IC50 on c-Met (nM)a
2.35
6.76
4.52
3.23
8.03
3.57
13.14
0.90
1.22
1.41
25
26
28
29
33
37
39
41
44
Foretinibb
a The values are an average of two separate determinations.
b Used as a positive control.
The inhibitory activity of the most potent compound 41 against c-Kit, PDGFRα, Ron, VEGFR-2, Flt-3, EGFR
and ALK kinase was also assayed using HTRF method (Table 3). In contrast to its high potency against c-Met (IC50
= 0.90 nM), 41 also exhibited high inhibitory effects against c-Kit (IC50= 2.45 nM) and PDGFRα (IC50= 19.13 nM).
Moreover, 41 demonstrated moderate-to-excellent selectivity against Ron, VEGFR-2, Flt-3, EGFR and ALK kinase
with IC50 values of 82.56 nM, 151.47 nM, 268.81 nM, 980.83 nM and 2840.72 nM, respectively.

Table 3
Inhibition of tyrosine kinases by compound 41 and foretinib
Kinase
IC50(nM)
foretinib
6.83
41
2.45
c-Kit
19.13
6.82
PDGFRα
Ron
82.56
3.48
151.52
268.81
980.83
2840.72
4.79
VEGFR-2
Flt-3
5.58
2990
>3000
EGFR
ALK
4. Binding model analysis
To further elucidate the binding mode of compounds, a detail docking analysis was performed. In our study,
the co-crystal structure of foretinib (GSK1363089) with c-Met was selected as the docking model (PDB ID code:
3LQ8). The docking simulation was conducted using Glide XP (Schrödinger 2014), since Glide uses a hierarchical
series of filters to search for possible locations of the ligand in the active-site region of the receptor. 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 image files were generated using Accelrys DS visualizer 4.0 system.
The binding model was exemplified by the interaction of compound 41 with c-Met. As shown in Figure 4A, the
nitrogen atom of the quinoline, the NH and the oxygen of amide formed three hydrogen-bonding interactions with
Met1160, Asp1222 and Lys1110, respectively. In the meantime, two Pi-Sigma interaction between quinoline with
ILE1084 and MET1211 had beenformed. All these interactions played an important role in stabilizing the
conformation of ligand-protein complex. Docking structure of compound 41 overlayed with foretinib in c-Met
showed that the conformation and binding mode of compound 41 were well consistent with foretinib, which
suggested that a 3-oxo-3,4-dihydroquinoxaline-2-carboxamide moiety could serve as a scaffold from which to build
a novel series of c-Met inhibitors (Figure 4B).

Figure 4. A：The c-Met active site in complex with compound 41, the proteins were displayed by silver ribbon. Compound 41 was
shown in colored sticks (cyan:carbon atom, blue: nitrogen atom, red: oxygen atom, light cyan: fluorine atom). H-bonding interactions
between the 41 and c-Met were indicated with dotted lines in green, and Pi-Sigma interaction was shown in purple dotted lines. B：
Superposed docking poses of foretinib (purple) and compound 41 (blue) in c-Met.
5. Conclusion
In summary, on the basis of our previous work, a series of 6, 7-disubstituted-4-phenoxyquinoline derivatives
bearing 3-oxo-3,4-dihydroquinoxaline moiety scaffold were designed and synthesized. The entire target compounds
were investigated for their in vitro antiproliferative activity using the MTT-based assay against five human cancer
cell lines (HT-29, H460, A549, MKN-45 and U87MG). Most compounds showed significant antiproliferative
activities against all cancer cell lines. In particular, the most promising compound 41 (c-Met IC50= 0.90 nM)
showed antitumor activities with IC50 values of 0.06µM, 0.05µM, 0.18µM, 0.023µM and 0.66 µM against HT-29,
H460, A549, MKN-45 and U87MG cell lines, respectively. The preliminary studies on enzymatic selectivity also
revealed that compound 41 showed moderate-to-excellent selectivity against Ron, VEGFR-2, Flt-3, EGFR and
ALK kinase, but it is still a multitarget inhibitor of tyrosine kinases. The structure-activity relationships (SARs)
analyses indicated that the replacement of the cyclopropane-1,1-dicarboxamide framework of foretinib with the
3-oxo-3,4-dihydroquinoxaline moiety maintained the potent cytotoxicity. Meanwhile, compounds with
mono-EWGs, especially chloro, fluoro group at 2- or 4-position on the phenyl ring (moiety B) were more active
than those without substituents, double-EWGs or EDGs. Further studies on structural optimization and biological
activities about these derivatives are still underway in our laboratory and will be reported in the future.

6. Experimental
6.1. Chemistry
Unless otherwise specified, all melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
Labortechnik, Flawil, Switzerland) and were uncorrected. Mass spectra (MS) were taken in ESI mode on Agilent
1100 LC-MS (Agilent, Palo Alto, CA, U.S.A.). 1H NMR and 13C NMR spectra were recorded on Bruker ARX-400,
400MHz or Bruker ARX-600, 600MHz spectrometers (Bruker Bioscience, Billerica, MA, USA) with TMS as an
internal standard. All materials were obtained from commercial suppliers and were used without further purification.
Reactions’ time and purity of the products were monitored by TLC on FLUKA silica gel aluminum cards(0.2 mm
thickness) with fluorescent indicator 254 nm. Column chromatography was run on silica gel (200–300 mesh) from
Qingdao Ocean Chemicals (Qingdao, Shandong, China). The IR spectra were recorded by means of the KBr pellet
technique on a Bruker FTS 135 spectrometer. The elemental analysis of the compounds was performed on a Perkin
Elmer 2400 Elemental Analyser (In the mode of measurement C, H, and N, the sample into the combustion tube in
pure oxygen atmosphere static combustion and products by a specific reagent after formation of CO2, H2O, N2 and
nitrogen oxides, uniform mixing under the atmospheric pressure. The thermal conductivity detector is used for
determining the content of C, H and N from mixed gases.).
6.2. General procedure for preparation intermediates of N-substituted-2-nitroanilines (15a–15n)
To the suspension of NaH (75 mol) in DMF (60 mL), substituted amines (50 mmol) was added at 0 °C. The
mixture was stirred for 30 min at the same temperature, and then 2-fluoronitrobenzene (60 mmol) diluted in DMF
(30 mL) was added slowly. The mixture was warmed to room temperature and stirred for 16 hours. The reaction
mixture was carefully poured into stirring saturated NH4Cl (500 mL), then filtered. The filter cake was washed with
water, and recrystallized from methanol to afford corresponding N-aryl-2-nitroanilines 15a–15n.
6.2.1.
2-nitro-N-phenylaniline (15a)
Red solid; Yield: 79.4%; M.p.: 74.2–76.1 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.52 (s, 1H), 8.23 (dd, J = 8.4
Hz, 1.6 Hz, 1H), 7.43 (m, 3H), 7.30 (m, 2H), 7.25 (m, 2H), 6.79 (m, 1H); MS (ESI) m/z(%): 213.3 [M-H]—.
6.2.2.
N-(4-methoxyphenyl)-2-nitroaniline (15b)
Red solid; Yield: 79.7%; M.p.: 88.1–89.7 °C; MS (ESI) m/z(%): 243.3 [M-H]—.
6.2.3.
2-nitro-N-(p-tolyl)aniline (15c)
Orange solid; Yield: 81.0%; M.p.: 69.3–70.9 °C; MS (ESI) m/z(%): 227.2 [M-H]—.
6.2.4.
2,4-dimethoxy-N-(2-nitrophenyl)aniline (15d)
Red solid; Yield: 81.7%; M.p.: 100.0–101.8 °C; MS (ESI) m/z(%): 273.3 [M-H]—.
6.2.5.
2-nitro-N-(4-(trifluoromethoxy)phenyl)aniline (15e)
Red solid; Yield: 65.9%; M.p.: 67.8–69.6 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.34 (s, 1H), 8.12 (d, J = 8.4,
1H), 7.54(t, J = 8.0 Hz, 1H), 7.42 (m, 4H), 7.24 (d, J = 8.4 Hz, 1H), 6.95 (t, J = 8.0 Hz, 1H); MS (ESI) m/z(%):
297.0 [M-H]—.
6.2.6.
2-nitro-N-(2-(trifluoromethyl)phenyl)aniline (15f)
Red solid; Yield: 61.6%; M.p.: 98.1–99.7 °C; MS (ESI) m/z(%): 281.1 [M-H]—.
6.2.7.
N-(4-fluorophenyl)-2-nitroaniline (15g)
Red solid; Yield: 71.4%; M.p.: 82.5–83.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.42 (s, 1H), 8.23 (dd, J = 8.4
Hz, 1.6 Hz, 1H), 7.38 (m, 1H), 7.28 (m, 2H), 7.15 (m, 2H), 7.07 (m, 1H), 6.79 (m, 1H); MS (ESI) m/z(%): 230.8
[M-H]—.
6.2.8.
N-(4-chlorophenyl)-2-nitroaniline (15h)
Orange solid; Yield: 72.3%; M.p.: 135.1–137.0 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.31 (s, 1H), 8.12 (d, J =
8.0, 1H), 7.53(t, J = 7.6 Hz, 1H), 7.44 (d, J = 8.8 Hz, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.22 (d, J = 8.4 Hz, 1H), 6.93 (t,

J = 7.6 Hz, 1H); MS (ESI) m/z(%): 247.2 [M-H]—.
6.2.9. N-(4-bromophenyl)-2-nitroaniline (15i)
Orange solid; Yield: 74.8%; M.p.: 168.7–170.2 °C; MS (ESI) m/z(%): 292.1[M-H]—.
6.2.10. 2-fluoro-N-(2-nitrophenyl)aniline (15j)
Red solid; Yield: 74.1%; M.p.: 74.4–75.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 9.28 (s, 1H), 8.14 (d, J = 8.0
Hz, 1H), 7.51 (m, 2H), 7.35 (m, 3H), 6.90 (m, 2H); MS (ESI) m/z(%): 231.1 [M-H]—.
6.2.11. 2-chloro-N-(2-nitrophenyl)aniline (15k)
Red solid; Yield: 77.3%; M.p.: 110.1–111.8 °C; MS (ESI) m/z(%):247.2 [M-H]—.
6.2.12. N-(3-fluorophenyl)-2-nitroaniline (15l)
Red solid; Yield: 72.2%; M.p.: 63.5–65.1 °C; MS (ESI) m/z(%):230.8 [M-H]—.
6.2.13. N-(3-chlorophenyl)-2-nitroaniline (15m)
Red solid; Yield: 74.5%; M.p.: 93.5–95.2 °C; MS (ESI) m/z(%):247.1 [M-H]—.
6.2.14. 2,4-dichloro-N-(2-nitrophenyl)aniline (15n)
Red solid; Yield: 69.5%; M.p.: 137.7–138.8 °C; MS (ESI) m/z(%): 282.0 [M-H]—.
6.3. General procedure for preparation of N1- substituted-benzene-1,2-diamine (16a–16n)
A mixture of N-substituted-2-nitroanilines (50 mmol), iron powder (250 mmol), acetic acid (500 mmol), water
(35 ml) and ethyl acetate (80 ml) was heated to reflux for 6 hours. After completion of the reaction as indicated by
TLC, the mixture was filtered immediately. The organic layer of the filtrate was separated, washed with water, dried
over anhydrous Na2SO4 and concentrated under reduced pressure to obtain diamines(16a–16n) as a brown solid.
6.4. General procedure for preparation of ethyl 4-substituted-3-oxo-3,4-dihydroquinoxaline-2-carboxylate
(17a–17n)
21 mmol of corresponding diamine and 23 mmol of diethyl ketomalonate were dissolved in toluene (60 ml)
and the solution was heated to reflux for 12 hours while removing water using a Dean-Stark apparatus. After
cooling to room temperature, the reaction mixture was concentrated under reduced pressure and then partitioned
between water (50 mL) and ethyl acetate (50 mL). The organic layer was separated and the aqueous layer was then
extracted with ethyl acetate (50 mL × 3). The combined organic extracts were sequentially washed with water (50
mL × 3), brine (80 mL × 3) and dried over anhydrous Na2SO4. After evaporation of the organic solvent, the residue
was purified by column chromatography to afford 17a–17n.
6.4.1.
Ethyl 3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-carboxylate (17a)
White solid; Yield: 78.2%; M.p.: 146.0–147.6 °C; 1H NMR (400 MHz, CDCl3) δ 8.01 (dd, J = 7.6 Hz, 1.6 Hz,
1H), 7.63 (m, 3H), 7.46 (m, 1H), 7.38 (m, 1H), 7.34 (m, 2H), 6.75 (m, 1H), 4.53 (q, J = 6.8 Hz, 2H), 1.46 (t, J = 6.8
Hz, 3H); MS (ESI) m/z(%): 295.0 [M+H]+.
6.4.2.
Ethyl 4-(4-methoxyphenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17b)
Light yellow solid; Yield: 82.1%; M.p.: 140.8–142.3 °C; MS (ESI) m/z(%): 325.0 [M+H]+, 347.1 [M+Na]+.
6.4.3.
Ethyl 3-oxo-4-(p-tolyl)-3,4-dihydroquinoxaline-2-carboxylate (17c)
White solid; Yield: 80.1%; M.p.: 98.8–100.9 °C; MS (ESI) m/z(%): 309.2 [M+H]+.
6.4.4.
Ethyl 4-(2,4-dimethoxyphenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17d)
Yellow solid; Yield: 79.5%; M.p.: 87.0–89.3 °C; MS (ESI) m/z(%): 355.1 [M+H]+.
6.4.5.
Ethyl 3-oxo-4-(4-(trifluoromethoxy)phenyl)-3,4-dihydroquinoxaline-2-carboxylate (17e)
White solid;Yield: 69.6%; M.p.: 135.3–137.2 °C; 1H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 8.0 Hz, 1H), 7.51
(m, 3H), 7.41 (m, 3H), 7.41 (m, 1H), 6.75 (d, J = 8.0 Hz, 1H), 4.53 (q, J = 6.8 Hz, 2H), 1.46 (t, J = 6.8 Hz, 3H); MS
(ESI) m/z(%): 379.1 [M+H]+.
6.4.6.
Ethyl 3-oxo-4-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinoxaline-2-carboxylate (17f)
White solid; Yield: 61.5%; M.p.: 107.0–108.8 °C; MS (ESI) m/z(%): 363.1 [M+H]+.

6.4.7.
Ethyl 4-(4-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17g)
White solid; Yield: 69.2%; M.p.: 132–133.8 °C; 1H NMR (400 MHz, CDCl3) δ 8.01 (d, J = 7.6 Hz, 1H), 7.40
(m, 6H), 6.75 (d, J = 8.0 Hz, 1H), 4.53 (q, J = 6.8 Hz, 2H), 1.46 (t, J = 6.8 Hz, 3H); MS (ESI) m/z(%): 313.1
[M+H]+, 647.1 [2M+Na]+.
6.4.8.
Ethyl 4-(4-chlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17h)
White solid; Yield: 69.6%; M.p.: 124.7–125.9 °C; 1H NMR (400 MHz, CDCl3) δ 8.01 (dd, J = 8.0 Hz, 1.6 Hz,
1H), 7.62 (m, 2H), 7.48 (m, 1H), 7.41 (m, 1H), 7.29 (m, 2H), 6.75 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 4.53 (q, J = 6.8 Hz,
2H), 1.46 (t, J = 6.8 Hz, 3H); MS (ESI) m/z(%): 329.1 [M+H]+.
6.4.9.
Ethyl 4-(4-bromophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17i)
Light yellow solid; Yield: 61.3%; M.p.: 140–142 °C; MS (ESI) m/z(%): 373.0 [M+H]+.
6.4.10. Ethyl 4-(2-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17j)
White solid; Yield: 62.5%; M.p.: 143.3–145 °C; 1H NMR (400 MHz, CDCl3) δ 8.03 (d, J = 8.0 Hz, 1H), 7.61
(m, 1H), 7.51 (m, 1H), 7.40 (m, 4H), 4.53 (q, J = 7.2 Hz, 2H), 1.46 (t, J = 7.2 Hz, 3H); MS (ESI) m/z(%): 313.0
[M+H]+;
6.4.11. Ethyl 4-(2-chlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17k)
White solid; Yield: 68.5%; M.p.: 132.4–134.8 °C; MS (ESI) m/z(%): 329.1 [M+H]+.
6.4.12. Ethyl 4-(3-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17l)
White solid; Yield: 60.9%; M.p.: 128.8–130.9 °C; MS (ESI) m/z(%): 313.1 [M+H]+.
6.4.13. Ethyl 4-(3-chlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17m)
White solid; Yield: 59.6%; M.p.: 114.1–115.9 °C; MS (ESI) m/z(%): 329.1 [M+H]+.
6.4.14. Ethyl 4-(2,4-dichlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylate (17n)
Yellow solid; Yield: 63.4%; M.p.: 144.0–145.8 °C; MS (ESI) m/z(%): 363.1 [M+H]+.
6.5. General procedure for preparation of 4-subistituted-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid(18a–18n)
To a solution of an appropriate intermediate (17a–17n) (7.56 mmol) dissolved in THF/H2O (40 mL, 4:1), was
added 2 eq of lithium hydroxide monohydrate, and the resulting suspension was stirred for 2 h at room temperature.
Then, most of the solvent was evaporated, and the residue was poured into H2O (50 mL) and acidified by addition
of 6 N HCl solution until pH 2. A solid precipitated therefrom was separated by filtration, washed with water and
then dried to obtain substituted acids (18a–18n) as pale yellow powder.
6.5.1. 3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-carboxylic acid (18a)
Yield: 88.1%; M.p.: 172–174 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.08 (br, 1H), 7.95 (dd, J = 8.0 Hz, 1.2
Hz, 1H), 7.62 (m, 4H), 7.47 (m, 3H), 6.62 (m, 1H); MS (ESI) m/z(%): 265.1 [M-H]—.
6.5.2. 4-(4-methoxyphenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18b)
1
Yield: 83.8%; M.p.: 194.5–195.8 °C; H NMR (400 MHz, DMSO-d6) δ 14.05 (br, 1H), 7.93 (d, J = 7.2 Hz,
1H), 7.56 (t, J = 7.6 Hz, 1H), 7.41 (m, 3H), 7.19 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.8 Hz, 1H), 3.87 (s, 3H); MS
(ESI) m/z(%): 295.1 [M-H]—.
6.5.3. 3-oxo-4-(p-tolyl)-3,4-dihydroquinoxaline-2-carboxylic acid (18c)
Yield: 80.9%; M.p.: 177.4–179 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.03 (br, 1H), 7.94 (dd, J = 8.0 Hz, 1.2
Hz, 1H), 7.55 (m, 1H), 7.46 (m, 3H), 7.33 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 8.4 Hz, 1H), 2.45(s, 3H); MS (ESI)
m/z(%): 281.4 [M+H]+.
6.5.4. 4-(2,4-dimethoxyphenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18d)
Yield: 81.2%; M.p.: 180.4–182 °C; MS (ESI) m/z(%): 325.0 [M-H]—.
6.5.5. 3-oxo-4-(4-(trifluoromethoxy)phenyl)-3,4-dihydroquinoxaline-2-carboxylic acid (18e)
Yield: 91.3%; M.p.: 184–186 °C; MS (ESI) m/z(%): 349.0 [M-H]—.
6.5.6. 3-oxo-4-(2-(trifluoromethyl)phenyl)-3,4-dihydroquinoxaline-2-carboxylic acid (18f)

Yield: 93.1%; M.p.: 189–190.2 °C; MS (ESI) m/z(%): 335.4 [M+H]+.
6.5.7. 4-(4-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18g)
Yield: 86.4%; M.p.:288–290 °C; 1H NMR (400 MHz, DMSO-d6) δ 7.74 (dd, J = 7.6 Hz, 1.6 Hz, 1H), 7.45 (m,
4H), 7.33 (m, 2H), 6.53 (d, J = 7.2 Hz, 1H); MS (ESI) m/z(%): 282.7 [M-H]—.
6.5.8. 4-(4-chlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18h)
1
Yield: 89.9%; M.p.: 186.7–188.6 °C; H NMR (400 MHz, DMSO-d6) δ 14.01 (br, 1H), 7.94 (d, J = 7.2 Hz,
1H), 7.50 (d, J = 8.4 Hz, 2H), 7.54 (m, 3H), 7.44 (t, J = 7.2 Hz, 1H), 6.67 (d, J = 8.0 Hz, 1H); MS (ESI) m/z(%):
299.0[M-H]—.
6.5.9. 4-(4-bromophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18i)
Yield: 88.6%; M.p.: 190–192 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.04 (br, 1H), 7.95 (dd, J = 8.0 Hz, 1.2
Hz, 1H), 7.88 (d, J = 8.4 Hz, 2H), 7.56 (m, 1H), 7.56 (m, 3H), 6.67 (d, J = 7.6 Hz, 1H); MS (ESI) m/z(%): 345.3
[M+H]+.
6.5.10. 4-(2-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18j)
1
Yield: 89.8%; M.p.: 184.4–186.5 °C; H NMR (400 MHz, DMSO-d6) δ 14.14 (br, 1H), 7.98 (d, J = 7.6 Hz,
1H), 7.67 (m, 4H), 7.50 (m, 2H), 6.72 (d, J = 8.8 Hz, 1H); MS (ESI) m/z(%):283.1 [M-H]—.
6.5.11. 4-(2-chlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18k)
Yield: 90.4%; M.p.: 155.4–157 °C; MS (ESI) m/z(%): 298.9[M-H]—.
6.5.12. 4-(3-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18l)
Yield: 87.9%; M.p.: 146.4–149 °C; 1H NMR (400 MHz, DMSO-d6) δ 14.03 (br, 1H), 7.95 (dd, J = 8.0 Hz, 1.2
Hz, 1H), 7.73 (m, 1H), 7.52 (m, 4H), 7.36 (d, J = 7.6 Hz, 1H), 6.67 (d, J = 7.6 Hz, 1H); MS (ESI) m/z(%): 283.2
[M-H]—.
6.5.13. 4-(3-chlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18m)
Yield: 88.4%; M.p.: 174.5–175.8 °C; MS (ESI) m/z(%): 299.1[M-H]—.
6.5.14. 4-(2,4-dichlorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxylic acid (18n)
Yield: 89.8%; M.p.: 154.8–157 °C; MS (ESI) m/z(%): 334.1 [M-H]—.
6.6. General procedure for Preparation of the target Compounds (19–46)
A mixture of the corresponding acids (18a–18n) (1.30 mmol), toluene (10 mL), and SOCl2 (8 mL) was heated
at 85 °C for 5 h. Upon cooling to room temperature, the solvent was evaporated in vacuum. The residue was
dissolved in dried CH2Cl2 (10 mL) and drop-wise added to a mixture of the corresponding anilines (14a–14e) (1.00
mmol), Na2CO3 (3.90 mmol) and CH2Cl2 (10 mL) in an ice bath, which was then removed to raise the temperature
to room temperature and stirred for 3 h. The resulting mixture was sequentially washed with 20% K2CO3 (20 mL ×
3) and brine (20 mL × 3), and the organic phase was separated, dried, and evaporated. The crude product obtained
was purified by silica gel chromatography to afford 19–46 as white solids.
6.6.1.
N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-4-phenyl-3,4-dihydr
oquinoxaline-2-carboxamide (19)
Yield: 72.1%; M.p.: 205.3–207.2 °C; IR (KBr) cm-1: 3434.6, 2956.1, 2917.6, 2846.2, 1703.7, 1637.9, 1597.0,
1554.9, 1508.2, 1477.8, 1430.0, 1349.5, 1304.8, 1247.9, 1212.2, 1166.0, 1113.2, 852.4, 743.1; 1H NMR (400 MHz,
CDCl3) δ 12.06 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H), 8.31 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.03 (dd, J = 12.0 Hz, 2.4 Hz,
1H), 7.70 (m, 3H), 7.52 (m, 5H), 7.36 (d, J = 7.2 Hz, 2H), 7.23 (d, J = 8.4 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.44 (d,
J = 5.2 Hz, 1H), 4.27 (t, J = 6.4 Hz, 2H), 4.03 (s, 3H), 3.73 (t, J = 4.4 Hz, 4H), 2.59 (t, J = 6.8 Hz, 2H), 2.51 (br,
4H), 2.14 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 160.16, 158.96, 155.68, 153.14, 152.32, 149.89, 148.53, 146.52,
144.28, 137.57, 136.63, 134.92, 134.47, 133.55, 133.05, 132.21, 130.77, 130.36, 127.72, 125.51, 123.74, 116.72,
115.88, 115.54, 109.92, 108.52, 102.36, 99.60, 67.14, 66.70, 56.16, 55.42, 53.57, 25.75; MS (ESI) m/z(%): 676.3
[M+H]+; Anal. calcd. for C38H34FN5O6 (%): C, 67.55; H, 5.07; N, 10.36. Found (%): C, 67.74; H, 5.00; N, 10.43.

6.6.2.
N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-4-(4-methoxyphenyl)-3-ox
o-3,4-dihydroquinoxaline-2-carboxamide (20)
1
Yield: 74.5%; M.p.: 164.3–165.7 °C; H NMR (400 MHz, CDCl3) δ 12.17 (s, 1H), 8.51 (d, J = 5.4 Hz, 1H),
8.32 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.08 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.58 (m, 5H), 7.29 (m, 3H), 7.22 (m, 2H),
6.89 (d, J = 7.6 Hz, 1H), 6.56 (d, J = 5.6 Hz, 1H), 4.34 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.99 (br, 4H), 3.95 (s, 3H),
2.97 (m, 6H), 2.43 (m, 2H); MS (ESI) m/z(%): 704.9 [M-H]—; Anal. calcd. for C39H36FN5O7 (%): C, 66.37; H, 5.14;
N, 9.92. Found (%): C, 66.59; H, 5.28; N, 10.02.
6.6.3.
N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-4-(p-tolyl)-3,4-dihy
droquinoxaline-2-carboxamide (21)
1
Yield: 70.3%; M.p.: 122.5–124.1 °C; H NMR (400 MHz, CDCl3) δ 12.13 (s, 1H), 8.50 (d, J = 5.6 Hz, 1H),
8.32 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.05 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.54 (m, 7H), 7.26 (m, 3H), 6.86 (d, J = 8.4
Hz, 1H), 6.48 (d, J = 5.2 Hz, 1H), 4.30 (t, J = 6.4 Hz, 2H), 4.05 (s, 3H), 3.82 (t, J = 4.4 Hz, 4H), 2.72 (t, J = 7.2 Hz,
2H), 2.63 (br, 4H), 2.54 (s, 3H), 2.23 (m, 2H); MS (ESI) m/z(%): 690.1 [M+H]+; Anal. calcd. for C39H36FN5O6 (%):
C, 67.91; H, 5.26; N, 10.15. Found (%): C, 68.12; H, 5.38; N, 10.00.
6.6.4.
4-(2,4-dimethoxyphenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-
3-oxo-3,4-dihydroquinoxaline-2-carboxamide (22)
1
Yield: 66.3%; M.p.: 149.7–151.4 °C; H NMR (400 MHz, CDCl3) δ 12.21 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H),
8.31 (d, J = 7.6 Hz, 1H), 8.07 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.55(m, 5H), 7.22 (m, 2H), 6.86 (d, J = 8.4 Hz, 1H),
6.76 (m, 2H), 6.50 (d, J = 5.2 Hz, 1H), 4.31 (t, J = 6.8 Hz, 2H), 4.06 (s, 3H), 3.95 (s, 3H), 3.86 (br, 4H), 3.77 (s,
3H), 2.75 (m, 6H), 2.28 (m, 2H); MS (ESI) m/z(%): 736.4 [M+H]+, 758.9 [M+Na]+; Anal. calcd. for C40H38FN5O8
(%): C, 65.30; H, 5.21; N, 9.52. Found (%): C, 65.51; H, 5.31; N, 9.63.
6.6.5.
N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-4-(4-(trifluorometh
oxy)phenyl)-3,4-dihydroquinoxaline-2-carboxamide (23)
Yield: 88.9%; M.p.: 202.5–204.7 °C; 1H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.51 (br, 1H), 8.34 (d, J =
8.0 Hz, 1H), 8.06 (dd, J = 12.0 Hz, 2.0Hz, 1H), 7.55 (m, 9H), 7.28 (t, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.52 (d, J = 5.2
Hz, 1H), 4.32 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.92 (m, 4H), 2.84 (m, 6H), 2.34 (m, 2H); MS (ESI) m/z(%):
760.20[M+H]+; Anal. calcd. for C39H33F4N5O7 (%): C, 61.66; H, 4.38; N, 9.22. Found (%): C, 61.93; H, 4.45; N,
9.41.
6.6.6.
N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-4-(2-(trifluoromethy
l)phenyl)-3,4-dihydroquinoxaline-2-carboxamide (24)
1
Yield: 79.9%; M.p.: 131.1–132.8 °C; H NMR (400 MHz, CDCl3) δ 11.91 (s, 1H), 8.51 (d, J = 5.4 Hz, 1H),
8.33 (d, J = 8.0 Hz, 1H), 8.06 (m, 2H), 7.95 (m, 1H), 7.86 (m, 1H), 7.70 (br, 1H), 7.53 (m, 5H), 7.28 (t, J = 8.8 Hz,
1H), 6.62 (d, J = 8.0 Hz, 1H), 6.56 (d, J = 5.4 Hz, 1H), 4.34 (t, J = 5.6 Hz, 2H), 4.06 (s, 3H), 4.02 (m, 4H), 3.02 (m,
6H), 2.45 (br, 2H); MS (ESI) m/z(%): 744.9 [M+H]+, 766.4 [M+Na]+; Anal. calcd. for C39H33F4N5O6 (%): C, 62.98;
H, 4.47; N, 9.42. Found (%): C, 62.81; H, 4.59; N, 9.66.
6.6.7.
N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (25)
Yield: 66.2%; M.p.: 202.1–203.1 °C; IR (KBr) cm-1: 3428.5, 3065.9, 2950.5, 2851.6, 1698.1, 1618.7, 1596.9,
1542.6, 1507.0, 1479.2, 1430.6, 1349.9, 1305.7, 1251.9, 1208.9, 1171.6, 1116.1, 846.0, 761.1; 1H NMR (400 MHz,
CDCl3) δ 11.98 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.32 (d, J = 7.6 Hz, 1H), 8.04 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.57
(m, 3H), 7.42 (m, 6H), 7.26 (t, J = 8.8 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 5.2 Hz, 1H), 4.29 (t, J = 6.4
Hz, 2H), 4.05 (s, 3H), 3.78 (t, J = 4.4 Hz, 4H), 2.66 (t, J = 7.2 Hz, 2H), 2.57 (br, 4H), 2.19 (m, 2H); 13C NMR (100
MHz, CDCl3) δ 164.52, 162.02, 160.13, 158.81, 155.65, 153.14, 152.34, 149.92, 148.50, 146.56, 144.30, 137.70,
136.50, 134.45, 133.63, 133.00, 132.30, 130.68, 129.82, 125.61, 123.75, 117.94, 116.69, 115.60, 109.99, 108.56,

102.36, 99.60, 67.14, 66.69, 56.16, 55.41, 53.57, 25.76; MS (ESI) m/z(%): 694.4 [M+H]+; Anal. calcd. for
C38H33F2N5O6 (%): C, 65.79; H, 4.79; N, 10.10. Found (%): C, 65.68; H, 4.82; N, 10.20.
6.6.8.
4-(4-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (26)
Yield: 82.6%; M.p.: 136.5–138.6 °C; 1H NMR (400 MHz, CDCl3) δ 11.98 (s, 1H), 8.51 (br, 1H), 8.32 (d, J =
8.0 Hz, 1H), 8.05 (dd, J = 12.0 Hz, 2.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.53 (m, 5H), 7.34 (d, J = 8.4 Hz, 2H),
7.27 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.49 (d, J = 4.8 Hz, 1H), 4.30 (t, J = 6.4 Hz, 2H), 4.05 (s, 3H),
3.85 (m, 4H), 2.74 (m, 6H), 2.27 (m, 2H); MS (ESI) m/z(%): 710.1 [M+H]+; Anal. calcd. for C38H33ClFN5O6 (%):
C, 64.27; H, 4.68; N, 9.86. Found (%): C, 64.49; H, 4.79; N, 9.76.
6.6.9.
4-(4-bromophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (27)
Yield: 85.9%; M.p.: 159.0–161.0 °C; 1H NMR (400 MHz, CDCl3) δ 12.00 (s, 1H), 8.51 (br, 1H), 8.32 (d, J =
8.0 Hz, 1H), 8.06 (d, J = 12.0 Hz, 1H), 7.87 (d, J = 8.0 Hz, 2H), 7.57 (m, 5H), 7.27 (m, 3H), 6.83 (d, J = 8.4 Hz,
1H), 6.56 (br, 1H), 4.33 (t, J = 6.4 Hz, 2H), 4.05 (s, 3H), 3.98 (m, 4H), 2.96 (m, 6H), 2.41 (m, 2H); MS (ESI)
m/z(%): 754.3 [M+H]+, 776.2 [M+Na]+; Anal. calcd. for C38H33BrFN5O6 (%): C, 60.48; H, 4.41; N, 9.28. Found
(%): C, 60.32; H, 4.60; N, 9.34.
6.6.10. N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-4-(2-fluorophenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (28)
1
Yield: 77.8%; M.p.: 120.3–121.8 °C; H NMR (400 MHz, CDCl3) δ 11.91 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H),
8.33 (d, J = 6.8 Hz, 1H), 8.03 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.52 (m, 9H), 7.23 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4
Hz, 1H), 6.44 (d, J = 5.2 Hz, 1H), 4.27 (t, J = 6.4 Hz, 2H), 4.04 (s, 3H), 3.73 (t, J = 4.4 Hz, 4H), 2.58 (t, J = 7.2 Hz,
2H), 2.49 (br, 4H), 2.14 (m, 2H); MS (ESI) m/z(%): 695.1 [M+H]+; Anal. calcd. for C38H33F2N5O6 (%): C, 65.79; H,
4.79; N, 10.10. Found (%): C, 65.93; H, 4.85; N, 10.03.
6.6.11. 4-(2-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (29)
Yield: 80.2%; M.p.: 192.3–193.9 °C; IR (KBr) cm-1: 3434.7, 2926.7, 1698.2, 1621.2, 1599.5, 1508.0, 1479.7,
1431.1, 1349.6, 1305.9, 1251.2, 1210.8, 1168.8, 1115.3, 855.1, 749.9; 1H NMR (400 MHz, CDCl3) δ 12.03 (s, 1H),
8.51 (d, J = 6.0 Hz, 1H), 8.35 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.11 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.91 (br, 1H), 7.78
(m, 1H), 7.65 (m, 4H), 7.55 (m, 2H), 7.44 (m, 1H), 7.32 (t, J = 8.8 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 6.64 (d, J =
6.0 Hz, 1H), 4.38 (t, J = 6.0 Hz, 2H), 4.08 (m, 7H), 3.14 (m, 6H), 2.54 (br, 2H); 13C NMR (100 MHz, CDCl3) δ
160.77, 158.86, 155.51, 154.97, 153.03, 152.61, 150.11, 147.53, 145.33, 144.34, 137.43, 136.72, 133.53, 133.06, 132.56,
132.39, 132.33, 131.85, 131.46, 129.55, 129.02, 125.77, 123.69, 116.84, 115.67, 115.11, 109.99, 107.67, 102.36, 99.71,
66.88, 65.79, 56.19, 55.42, 53.13, 24.97; MS (ESI) m/z(%): 710.1 [M+H]+; Anal. calcd. for C38H33ClFN5O6 (%): C,
64.27; H, 4.68; N, 9.86. Found (%): C, 64.46; H, 4.81; N, 9.78.
6.6.12. N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-4-(3-fluorophenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (30)
1
Yield: 65.4%; M.p.: 214.2–215.4 °C; H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.51 (d, J = 5.6 Hz, 1H),
8.34 (d, J = 6.8 Hz, 1H), 8.05 (dd, J = 12.0 Hz, 2.4Hz, 1H), 7.72 (m, 1H), 7.55 (m, 5H), 7.42 (m, 1H), 7.27 (d, J =
8.8 Hz, 1H), 7.27 (d, J = 8.0 Hz, 1H), 7.15 (m, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.49 (d, J = 5.2 Hz, 1H), 4.31 (t, J =
6.4 Hz, 2H), 4.06 (s, 3H), 3.85 (m, 4H), 2.70 (m, 6H), 2.27 (m, 2H); MS (ESI) m/z(%): 694.3 [M+H]+; Anal. calcd.
for C38H33F2N5O6 (%): C, 65.79; H, 4.79; N, 10.10. Found (%): C, 65.91; H, 4.88; N, 10.35.
6.6.13. 4-(3-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-oxo-
3,4-dihydroquinoxaline-2-carboxamide (31)
1
Yield: 76.8%; M.p.: 135.2–136.5 °C; H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H),

8.34 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.06 (dd, J = 12.0 Hz, 2.0Hz, 1H), 7.60 (m, 6H), 7.49 (d, J = 9.6 Hz, 1H), 7.42
(m, 1H), 7.29 (m, 2H), 6.83 (d, J = 8.0 Hz, 1H), 6.51 (d, J = 5.2 Hz, 1H), 4.31 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.89
(m, 4H), 2.77 (m, 6H), 2.32 (m, 2H); MS (ESI) m/z(%): 710.4 [M+H]+; Anal. calcd. for C38H33ClFN5O6 (%): C,
64.27; H, 4.68; N, 9.86. Found (%): C, 64.44; H, 4.81; N, 10.02.
6.6.14. 4-(2,4-dichlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-
oxo-3,4-dihydroquinoxaline-2-carboxamide (32 )
Yield: 67.9%; M.p.: 140.1–141.3 °C; IR (KBr) cm-1: 3429.9, 2921.9, 2846.2, 1695.5, 1618.7, 1598.3, 1508.1,
1479.7, 1431.3, 1384.3, 1350.1, 1251.9, 1211.2, 1113.9, 855.0, 761.4; 1H NMR (400 MHz, CDCl3) δ 11.85 (s, 1H),
8.51 (d, J = 5.2 Hz, 1H), 8.35 (d, J = 8.0 Hz, 1H), 8.05 (dd, J = 12.0 Hz, 2.0 Hz, 1H), 7.79 (d, J = 2.0 Hz, 1H), 7.61
(m, 4H), 7.48 (m, 2H), 7.39 (d, J = 8.4 Hz, 1H), 7.26 (t, J = 8.8 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 5.2
Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.77 (t, J = 4.4 Hz, 4H), 2.65 (t, J = 7.2 Hz, 2H), 2.56 (br, 4H), 2.18
(m, 2H); MS (ESI) m/z(%): 741.7 [M-H]—, 778.4 [M+Cl]—; Anal. calcd. for C38H32Cl2FN5O6 (%): C, 61.30; H,
4.33; N, 9.41. Found (%): C, 61.01; H, 4.41; N, 9.56.
6.6.15. N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)-3-o
xo-3,4-dihydroquinoxaline-2-carboxamide (33)
1
Yield: 62.2%; M.p.: 234.9–236.8 °C; H NMR (400 MHz, CDCl3) δ 11.98 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H),
8.33 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.04 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.58(m, 3H), 7.42 (m, 6H), 7.26 (t, J = 8.8 Hz,
1H), 6.83 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 5.2 Hz, 1H), 4.27 (t, J = 6.8 Hz, 2H), 4.05 (s, 3H) , 2.54 (m, 6H), 2.16 (m,
2H), 1.61 (m, 4H), 1.46 (br, 2H); MS (ESI) m/z(%): 692.4 [M+H]+; Anal. calcd. for C39H35F2N5O5 (%): C, 67.72; H,
5.10; N, 10.12. Found (%): C, 67.51; H, 5.02; N, 10.24.
6.6.16. 4-(4-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-3-
oxo-3,4-dihydroquinoxaline-2-carboxamide (34)
1
Yield: 84.1%; M.p.: 149.3–151.4 °C; H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H),
8.32 (d, J = 8.0 Hz, 1H), 8.04 (d, J = 12.0 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.56 (m, 3H), 7.45 (m, 2H), 7.34 (d, J
= 8.4 Hz, 2H), 7.26 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 4.8 Hz, 1H), 4.27 (t, J = 6.0 Hz, 2H),
4.05 (s, 3H), 2.66 (m, 6H), 2.29 (m, 2H), 1.77 (m, 4H), 1.53 (m, 2H); MS (ESI) m/z(%): 708.3 [M+H]+; Anal. calcd.
for C39H35ClFN5O5 (%): C, 66.14; H, 4.98; N, 9.89. Found (%): C, 66.38; H, 5.09; N, 9.79.
6.6.17. N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(2-fluorophenyl)-3-o
xo-3,4-dihydroquinoxaline-2-carboxamide (35)
1
Yield: 73.7%; M.p.: 131.2–132.1 °C; H NMR (400 MHz, CDCl3) δ 11.91 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H),
8.33 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.03 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.54(m, 9H), 7.23 (t, J = 8.4 Hz, 1H), 6.83 (d,
J = 8.4 Hz, 1H), 6.44 (d, J = 5.2 Hz, 1H), 4.26 (t, J = 6.8 Hz, 2H), 4.02 (s, 3H), 2.89 (m, 6H), 2.42 (m, 2H), 1.91 (m,
4H), 1.60 (br, 2H); MS (ESI) m/z(%): 692.1 [M+H]+; Anal. calcd. for C39H35F2N5O5 (%): C, 67.72; H, 5.10; N,
10.12. Found (%): C, 67.88; H, 5.21; N, 10.26.
6.6.18. 4-(2-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(piperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-3-
oxo-3,4-dihydroquinoxaline-2-carboxamide (36 )
1
Yield: 77.5%; M.p.: 165.0–166.7 °C; H NMR (400 MHz, CDCl3) δ 12.08 (s, 1H), 8.51 (d, J = 6.4 Hz, 1H),
8.34 (d, J = 8.0 Hz, 1H), 8.29 (br, 1H), 8.15 (d, J = 12.4 Hz, 1H), 7.79 (m, 1H), 7.62 (m, 6H), 7.45 (m, 1H), 7.34 (t,
J = 8.4 Hz, 1H), 6.78 (d, J = 6.4 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 4.44 (t, J = 6.4 Hz, 2H), 4.10 (s, 3H), 3.62 (br,
2H), 3.31 (br, 2H), 2.84 (br, 2H), 2.63 (br, 2H), 2.33 (br, 3H), 1.89 (br, 3H); MS (ESI) m/z(%): 708.5 [M+H]+; Anal.
calcd. for C39H35ClFN5O5 (%): C, 66.14; H, 4.98; N, 9.89. Found (%): C, 66.25; H, 5.06; N, 9.94.
6.6.19. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(4-fluoroph
enyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (37)
Yield: 65.4%; M.p.: 232.7–233.8 °C; IR (KBr) cm-1: 3433.9, 3071.4, 2923.4, 1702.1, 1597.2, 1552.4, 1508.2,

1476.2, 1430.5, 1349.7, 1307.2, 1252.2, 1209.6, 1175.9, 1091.7, 853.5, 766.0; 1H NMR (400 MHz, CDCl3) δ 11.98
(s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.33 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.04 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.59 (m,
3H), 7.41 (m, 6H), 7.26 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 5.2 Hz, 1H), 4.27 (t, J = 6.4 Hz,
2H), 4.05 (s, 3H), 3.14 (br, 2H), 2.82 (br, 2H), 2.25 (m, 4H), 1.72 (br, 2H), 1.48 (br, 3H), 0.98 (d, J = 5.6 Hz, 3H);
MS (ESI) m/z(%): 706.4 [M+H]+; Anal. calcd. for C40H37F2N5O5 (%): C, 68.07; H, 5.28; N, 9.92. Found (%): C,
68.18; H, 5.22; N, 9.99.
6.6.20. 4-(4-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)ph
enyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (38)
Yield: 83.8%; M.p.: 194.5–195.8 °C; 1H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.51 (d, J = 4.8 Hz, 1H), 8.33 (d,
J = 8.0 Hz, 1H), 8.04 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 2H), 7.58 (m, 3H), 7.46 (m, 2H), 7.34 (d, J
= 8.4 Hz, 2H), 7.26 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 4.8 Hz, 1H), 4.27 (t, J = 6.4 Hz, 2H),
4.06 (s, 3H), 2.94 (m, 2H), 2.57 (m, 2H), 2.16 (m, 2H), 1.98 (m, 2H), 1.65 (m, 2H), 1.27 (m, 3H), 0.94 (d, J = 6.4
Hz, 3H); 13C NMR (101 MHz, CDCl3) δ 159.93, 158.74, 155.51, 153.17, 152.33, 149.89, 148.72, 146.93, 144.28,
137.90, 137.77, 136.58, 136.42, 136.33, 134.21, 133.66, 133.26, 132.99, 132.34, 131.07, 129.26, 125.67, 123.79,
116.66, 115.59, 110.10, 109.87, 108.77, 102.33, 99.53, 67.62, 56.15, 55.44, 54.02, 34.31, 30.81, 26.51, 21.89; MS
(ESI) m/z(%): 722.4 [M+H]+; Anal. calcd. for C40H37ClFN5O5 (%): C, 66.52; H, 5.16; N, 9.70. Found (%): C, 66.78;
H, 5.29; N, 9.89.
6.6.21. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(2-fluoroph
enyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (39)
Yield: 64.9%; M.p.: 131.5–132.6 °C; IR (KBr) cm-1: 3409.3, 2917.6, 2796.7, 1704.9, 1625.7, 1557.6, 1499.2,
1476.8, 1432.1, 1349.9, 1307.0, 1248.2, 1214.7, 1175.9, 1089.2, 849.9, 762.9; 1H NMR (400 MHz, CDCl3) δ 11.91
(s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.32 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.54 (m, 9H), 7.23 (t,
J = 8.8 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.46 (d, J = 5.2 Hz, 1H), 4.26 (t, J = 6.4 Hz, 2H), 4.02 (s, 3H), 3.36 (br,
2H), 3.02 (br, 2H), 2.44 (m, 4H), 1.65 (m, 5H), 1.01 (d, J = 6.0 Hz, 3H); MS (ESI) m/z(%): 706.2 [M+H]+; Anal.
calcd. for C40H37F2N5O5 (%): C, 68.07; H, 5.28; N, 9.92. Found (%): C, 68.29; H, 5.35; N, 10.03.
6.6.22. 4-(2-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperidin-1-yl)propoxy)quinolin-4-yl)oxy)ph
enyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (40)
1
Yield: 71.2%; M.p.: 162.2–163.6 °C; H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.52 (d, J = 5.2 Hz, 1H),
8.35 (d, J = 8.0 Hz, 1H), 8.06 (dd, J = 12.0 Hz, 2.0 Hz, 1H), 7.79 (m, 1H), 7.54 (m, 8H), 7.26 (t, J = 8.4 Hz, 1H),
6.71 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 5.2 Hz, 1H), 4.29 (t, J = 6.8 Hz, 2H), 4.04 (s, 3H), 3.60 (br, 2H), 3.26 (m, 2H),
2.60 (m, 4H), 1.91 (br, 5H), 1.07 (d, J = 6.4 Hz, 3H); MS (ESI) m/z(%): 722.4 [M+H]+; Anal. calcd. for
C40H37ClFN5O5 (%): C, 66.52; H, 5.16; N, 9.70. Found (%): C, 66.75; H, 5.26; N, 9.82.
6.6.23. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(4-fluoroph
enyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (41)
Yield: 68.1%; M.p.: 125.0–126.8 °C; IR (KBr) cm-1: 3418.1, 2926.8, 1695.5, 1621.4, 1541.9, 1507.8, 1479.3,
1432.0, 1349.8, 1305.9, 1250.3, 1209.7, 1169.8, 1091.9, 854.3, 762.7; 1H NMR (400 MHz, CDCl3) δ 11.99 (s,
1H), 8.50 (d, J = 5.2 Hz, 1H), 8.33 (d, J = 8.0 Hz, 1H), 8.05 (dd, J = 12.0 Hz, 2.0 Hz, 1H), 7.59 (m, 3H), 7.43 (m,
6H), 7.26 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 5.2 Hz, 1H), 4.28 (t, J = 6.4 Hz, 2H), 4.05 (s,
3H), 3.20-2.61 (m, 10H), 2.54 (s, 3H), 2.17 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 164.51, 162.01, 160.00,
158.83, 155.65, 153.15, 152.22, 149.83, 148.72, 146.78, 144.28, 137.81, 136.48, 134.44, 133.66, 133.01, 132.31,
130.67, 129.84, 125.64, 123.78, 118.06, 117.83, 116.70, 115.65, 110.13, 109.90, 108.67, 102.35, 99.58, 67.01,
56.14, 54.55, 54.50, 51.94, 45.17, 26.13; MS (ESI) m/z(%): 707.2 [M+H]+; Anal. calcd. for C39H36F2N6O5 (%): C,
66.28; H, 5.13; N, 11.89. Found (%): C, 66.48; H, 5.23; N, 11.80.
6.6.24. 4-(4-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)p

henyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (42)
1
Yield: 78.9%; M.p.: 216.1–217.6 °C; H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H),
8.33 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.04 (dd, J = 12.0 Hz, 2.0 Hz, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.59 (m, 3H), 7.46
(m, 2H), 7.34 (d, J = 8.8 Hz, 2H), 7.26 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 5.2 Hz, 1H), 4.28 (t,
J = 6.4 Hz, 2H), 4.05 (s, 3H), 3.23-2.55 (m, 10H), 2.51 (s, 3H), 2.17 (m, 2H); MS (ESI) m/z(%): 723.4 [M+H]+;
Anal. calcd. for C39H36ClFN6O5 (%): C, 64.77; H, 5.02; N, 11.62. Found (%): C, 64.98; H, 4.89; N, 11.78.
6.6.25. N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(2-fluoroph
enyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (43)
1
Yield: 71.6%; M.p.: 123.9–125.3 °C; H NMR (400 MHz, CDCl3) δ 11.91 (s, 1H), 8.49 (d, J = 5.2 Hz, 1H),
8.33 (d, J = 8.0 Hz, 1H), 8.03 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.54 (m, 9H), 7.24 (t, J = 8.8 Hz, 1H), 6.83 (d, J = 8.4
Hz, 1H), 6.44 (d, J = 5.2 Hz, 1H), 4.26 (t, J = 6.4 Hz, 2H), 4.03 (s, 3H), 3.20-2.41 (m, 10H), 2.38 (s, 3H), 2.13 (m,
2H); MS (ESI) m/z(%): 707.8 [M+H]+; Anal. calcd. for C39H36F2N6O5 (%): C, 66.28; H, 5.13; N, 11.89. Found (%):
C, 66.15; H, 5.04; N, 11.70.
6.6.26. 4-(2-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(4-methylpiperazin-1-yl)propoxy)quinolin-4-yl)oxy)p
henyl)-3-oxo-3,4-dihydroquinoxaline-2-carboxamide (44)
Yield: 63.3%; M.p.: 170.5–171.8 °C; IR (KBr) cm-1: 3412.9, 2923.1, 2846.2, 1695.5, 1618.7, 1596.7, 1479.9,
1
1429.4, 1382.7, 1349.8, 1305.9, 1251.2, 1211.0; H NMR (400 MHz, CDCl3) δ 11.96 (s, 1H), 8.51 (d, J = 5.2 Hz,
1H), 8.35 (d, J = 8.0 Hz, 1H), 8.06 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.79 (m, 1H), 7.63 (m, 5H), 7.45 (m, 3H), 7.26 (t,
J = 8.8 Hz, 1H), 6.71 (d, J = 8.4 Hz, 1H), 6.46 (d, J = 5.2 Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 3.30-2.60
(m, 10H), 2.57 (s, 3H), 2.17 (m, 2H); 13C NMR (100 MHz, CDCl3) δ 160.00, 158.82, 155.63, 154.96, 153.15,
152.23, 149.83, 148.76, 146.81, 144.39, 137.80, 136.51, 133.52, 133.05, 132.58, 132.39, 132.35, 131.83, 131.45,
129.56, 129.01, 125.74, 123.75, 116.77, 115.51, 115.09, 109.95, 108.70, 102.35, 99.58, 67.08, 56.15, 54.63, 52.18,
45.34, 29.69, 26.17; MS (ESI) m/z(%): 724.3 [M+H]+; Anal. calcd. for C39H36ClFN6O5 (%): C, 64.77; H, 5.02; N,
11.62. Found (%): C, 64.91; H, 5.21; N, 11.78.
6.6.27. N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)-3-
oxo-3,4-dihydroquinoxaline-2-carboxamide (45)
1
Yield: 59.3%; M.p.: 218.2–219.5 °C; H NMR (400 MHz, CDCl3) δ 11.98 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H),
8.33 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 8.04 (dd, J = 12.0 Hz, 2.0Hz, 1H), 7.58 (m, 3H), 7.43 (m, 6H), 7.26 (t, J = 8.8 Hz,
1H), 6.82 (d, J = 8.4 Hz, 1H), 6.45 (d, J = 5.2 Hz, 1H), 4.29 (t, J = 6.4 Hz, 2H), 4.06 (s, 3H), 2.72 (t, J = 7.2 Hz,
2H), 2.60 (m, 4H), 2.20 (m, 2H), 1.83 (m, 4H); MS (ESI) m/z(%): 678.8 [M+H]+; Anal. calcd. for C38H33F2N5O5
(%): C, 67.35; H, 4.91; N, 10.33. Found (%): C, 67.22; H, 4.98; N, 10.44.
6.6.28. 4-(2-chlorophenyl)-N-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-yl)oxy)phenyl)-3-
oxo-3,4-dihydroquinoxaline-2-carboxamide (46)
1
Yield: 61.3%; M.p.: 197.3–197.9 °C; H NMR (400 MHz, CDCl3) δ 11.87 (s, 1H), 8.42 (d, J = 5.2 Hz, 1H),
8.27 (dd, J = 8.0 Hz, 1.6 Hz, 1H), 7.97 (dd, J = 12.0 Hz, 2.4 Hz, 1H), 7.69 (m, 1H), 7.52 (m, 5H), 7.37 (m, 3H),
7.17 (t, J = 8.8 Hz, 1H), 6.62 (d, J = 7.2 Hz, 1H), 6.38 (d, J = 5.2 Hz, 1H), 4.20 (t, J = 6.0 Hz, 2H), 3.96 (s, 3H),
3.02 (m, 6H), 2.31 (m, 2H), 1.95 (m, 4H); MS (ESI) m/z(%): 694.4 [M+H]+; Anal. calcd. for C38H33ClFN5O5 (%):
C, 65.75; H, 4.79; N, 10.09; Found (%): C, 65.89; H, 4.88; N, 9.02.
6.7. Pharmacology
6.7.1.
MTT assay in vitro
The anti-proliferative activities of compounds 19–46 were evaluated against HT-29, H460, A549 and MKN-45
cell lines using the standard MTT assay in vitro, with Foretinib as the positive control. The cancer cell lines were
cultured in minimum essential medium (MEM) supplement 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 tested compounds 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 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 compounds were tested three times in each of the cell lines. 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.
6.7.2.
HTRF kinase assay
The c-Met kinase activity was evaluated using homogeneous time-resolved fluorescence (HTRF) assays as
previously reported protocol.28,29 In addition, the most promising compound 41 was further evaluated against other
five tyrosine kinase (c-Kit, PDGFRα, Ron, VEGFR-2, Flt-3, EGFR and ALK) using the same screening method.
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) 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 plate was read using Envision (Perkin Elmer) 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.
Acknowledgments
This work was supported by grant (No. 81573295) from the National Natural Science Foundation of China and
National Science Fund for fostering talents in basic science (J1103606).
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version,
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Legends

Figure 1. Structures of small-molecule c-Met inhibitors
Figure 2. Anticancer agents bearing quinoxalinone moiety
Figure 3. Design strategy and general structure of the target compounds
Scheme 1. Reagents and conditions: (a) amines, NaH, DMF, RT, 16 h; (b) Fe powder, HOAc, CH3COOEt/H2O,
reflux, 6 h; (c) diethyl ketomalonate, toluene, reflux, 12 h; (d) LiOH, THF/ H2O, RT, 1.5 h, then 6 N HCl; (e)
toluene, SOCl2, reflux, 5 h; Na2CO3, CH2Cl2, 25 °C, 3 h.
Table 1
Structures and cytotoxicity of compounds 19-46
Table 2
c-Met kinase activity of selected compounds 25, 26, 28, 29, 33, 37, 39, 41, 44 and Foretinib in vitro.
Table 3
Inhibition of tyrosine kinases by compound 41
Figure 4. A：The c-Met active site in complex with compound 41, the proteins were displayed by silver ribbon. Compound 41 was
shown in colored sticks (cyan:carbon atom, blue: nitrogen atom, red: oxygen atom, light cyan: fluorine atom). H-bonding interactions
between the 41 and c-Met were indicated with dotted lines in green, and Pi-Sigma interaction was shown in purple dotted lines. B：
Superposed docking poses of foretinib (purple) and compound 41 (blue) in c-Met.

►A series of 4-phenoxyquinoline derivatives bearing an 3-oxo-3,4-dihydroquinoxaline moiety were designed and
synthesized.
►The target compounds showed potent antitumor activity.
►Compound 41showed more potent against five cell lines than foretinib.
►Compound 41 showed an IC50 value of 0.90 nM against c-Met kinase.
►Compound 41 showed moderate-to-excellent selectivity against 5 other RTK kinases.

Graphical abstract
A series of novel 4-(2-fluorophenoxy)quinoline derivatives containing 3-oxo-3,4-dihydroquinoxaline moiety
were designed, synthesized and evaluated for their cytotoxicity, enzymatic assays and docking analysis.