Design, synthesis and biological evaluation of novel 4-phenoxypyridine based 3-oxo-3,4-dihydroquinoxaline-2-carboxamide derivatives as potential c-Met kinase inhibitors

Article abstract of DOI:10.1016/j.bioorg.2020.104371

Blocking c-Met kinase activity by small-molecule inhibitors has been identified as a promising approach for the treatment of cancers. Herein, we described the design, synthesis, and biological evaluation of a series of 4-phenoxypyridine-based 3-oxo-3,4-dihydroquinoxaline derivatives as c-Met kinase inhibitors. Inhibitory activitives against c-Met kinase evaluation indicated that most of compounds showed excellent c-Met kinase activity in vitro, and IC₅₀ values of ten compounds (23a, 23e, 23f, 23l, 23r, 23s, 23v, 23w, 23x and 23y) were less than 10.00 nM. Notably, three of them (23v, 23w and 23y) showed remarkable potency with IC₅₀ values of 2.31 nM, 1.91 nM and 2.44 nM, respectively, and thus they were more potent than positive control drug foretinib (c-Met, IC₅₀ = 2.53 nM). Cytotoxic evaluation indicated the most promising compound 23w showed remarkable cytotoxicity against A549, H460 and HT-29 cell lines with IC₅₀ values of 1.57 μM, 0.94 μM and 0.65 μM, respectively. Furthermore, the acridine orange/ethidium bromide (AO/EB) staining, cell apoptosis assays by flow cytometry, wound-healing assays and transwell migration assays on HT-29 and/or A549 cells of 23w were performed. Especially compound 23w, which displayed potent antitumor, apoptosis induction and antimetastatic activity, could be used as a promising lead for further development. Meanwhile, their preliminary structure-activity relationships (SARs) were also discussed.

Full text of DOI:10.1016/j.bioorg.2020.104371

Journal Pre-proofs
Design, synthesis and biological evaluation of novel 4-phenoxypyridine based
3-oxo-3,4-dihydroquinoxaline-2-carboxamide derivatives as potential c-Met
kinase inhibitors
Zhen Wang, Jiantao Shi, Xianglong Zhu, Wenwen Zhao, Yilin Gong,
Xuechen Hao, Yunlei Hou, Yajing Liu, Shi Ding, Ju Liu, Ye Chen
PII:
S0045-2068(20)31669-2
DOI:
https://doi.org/10.1016/j.bioorg.2020.104371
YBIOO 104371
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
2 August 2020
9 September 2020
8 October 2020
Please cite this article as: Z. Wang, J. Shi, X. Zhu, W. Zhao, Y. Gong, X. Hao, Y. Hou, Y. Liu, S. Ding, J. Liu, Y.
Chen, Design, synthesis and biological evaluation of novel 4-phenoxypyridine based 3-oxo-3,4-
dihydroquinoxaline-2-carboxamide derivatives as potential c-Met kinase inhibitors, Bioorganic Chemistry
(2020), doi: https://doi.org/10.1016/j.bioorg.2020.104371
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Design, synthesis and biological evaluation of novel 4-phenoxypyridine based 3-oxo-3,4-
dihydroquinoxaline-2-carboxamide derivatives as potential c-Met kinase inhibitors
Zhen Wanga, Jiantao Shia, Xianglong Zhua, Wenwen Zhaoa, Yilin Gonga, Xuechen Haoa,
Yunlei Houb, Yajing Liub, Shi Dinga, Ju Liua*, Ye Chena,**
a College of Pharmacy of Liaoning University, API Engineering Technology Research Center of Liaoning Province, 66 Chongshan Road,
Huanggu District, Shenyang 10036, P. R. China.
b Key Laboratory of Structure-Based Drug Design and Discovery (Shenyang Pharmaceutical University), Ministry of Education, 103
Wenhua Road, Shenhe District, Shenyang 110016, PR China
∗*Corresponding author: Ye Chen. Tel./Fax: 86-24-6220 2469. E-mail: sy-chenye@163.com.
∗Corresponding author: Ju Liu. Tel./Fax: 86-24-6220 2469. E-mail: liuju1216@126.com.
Abstract
Blocking c-Met kinase activity by small-molecule inhibitors has been identified as a promising approach for the
treatment of cancers. Herein, we described the design, synthesis, and biological evaluation of a series of 4-
phenoxypyridine-based 3-oxo-3,4-dihydroquinoxaline derivatives as c-Met kinase inhibitors. Inhibitory activitives
against c-Met kinase evaluation indicated that most of compounds showed excellent c-Met kinase activity in vitro, and
IC50 values of ten compounds (23a, 23e, 23f, 23l, 23r, 23s, 23v, 23w, 23x and 23y)were less than 10.00 nM. Notably,
three of them (23v, 23w and 23y) showed remarkable potency with IC50 values of 2.31 nM, 1.91 nM and 2.44 nM,
respectively, and thus they were more potent than positive control drug foretinib (c-Met, IC50 = 2.53 nM). Cytotoxic
evaluation indicated the most promising compound 23w showed remarkable cytotoxicity against A549, H460 and HT-
29 cell lines with IC50 values of 1.57 μM, 0.94 μM and 0.65 μM, respectively. Furthermore, the acridine
orange/ethidium bromide (AO/EB) staining, cell apoptosis assays by flow cytometry, wound-healing assays and
transwell migration assays on HT-29 and/or A549 cells of 23w were performed. Especially compound 23w, which
displayed potent antitumor, apoptosis induction and antimetastatic activity, could be used as a promising lead for
further development. Meanwhile, their preliminary structure-activity relationships (SARs) were also discussed.
Keywords: 4-Phenoxypyridine derivatives, c-Met inhibitors, Antitumor activity, Apoptosis, Migration.
1. Introduction
The regulation of intracellular signal transduction pathways is a key function of receptor tyrosine kinases (RTKs),
and thus, inappropriate activation of RTKs can lead to deleterious consequences with regard to downstream signaling
[1]. c-Met is a prototype member of a subfamily of heterodimeric receptor tyrosine kinases (RTKs) and is the receptor
for hepatocyte growth factor (HGF) [2]. Binding of HGF to its receptor c-Met, induces several complex signaling
pathways that result in cell proliferation, survival, motility, induction of cell polarity, scattering, angiogenesis and
invasion [3,4]. The physiological functions of the HGF/c-Met signaling pathway are restricted to embryonic

development, wound healing and tissue regeneration processes [5,6]. However, deregulation of the c-Met /HGF
pathway can lead to tumorigenesis and metastasis [7]. Amplification of the c-Met gene, overexpression of c-Met and/or
HGF and constitutive activation conferred by sequence mutations are some of the mechanisms of deregulation found
in many human cancers [8]. Consequently, pharmacological inhibition of c-Met activity has been considered to be an
emerging strategy for cancer therapies. [9].
At present, small molecule c-Met kinase inhibitors were mainly divided into two groups based on the structural
features and their different binding mode with the active site: type I inhibitors and type II inhibitors [10–13]. As
reported recently, certain mutations near the active site of c-Met may cause resistance of type I inhibitors. Type I
inhibitors function as an analog of ATP to competitively bind to the highly conservative hinge region of c-Met kinase
[14–17]. In contrast, type II inhibitors not only bind to the same area occupied by the type I inhibitors but also exploit
hydrogen bonding and hydrophobic interactions with the allosteric site, which beyond the entrance of c-Met’s active
site[18-20]. Consequently, type II inhibitors are postulated to be more effective than type I inhibitors against these
mutations, and study on type II inhibitors to develop novel chemical entities is much more rational and of great
significance. Recently, a series of small molecules with different scaffolds have been reported as type II c-Met
inhibitors and some of them are launched or in clinical trials, such as Cabozantinib [21], Foretinib [22], BMS-794833
[23], Altiratinib [24], BMS-777607 [25], NPS-1034[26] and MGCD-516[27] (Fig. 1).
H
H
N
H
N
F
F
H
N
H
N
N
H
N
X
F
O
O
O
O
O
N
O
F
F
O
O
O
F
5-atom linker
O
O
5-atom linker
5-atom linker
O
Cl
R
N
N
H
H2N
N
4 (Altiratinib )
1 (Cabozantinib)
3 (BMS-794833)
X = H, R = OCH3
c-Met ( IC50 = 2.7 nM )
c-Met ( IC50 = 1.3 nM )
2 (Foretinib
c-Met ( IC50 = 1.7 nM )
N
)
X = F, R =
O
c-Met ( IC50 = 0.4 nM )
N
H
H
H
N
N
O
H
F
N
F
F
N
O
F
N
N
O
O
O
O
O
O
F
O
O
5-atom linker
O
F
HN
S
5-atom linker
5-atom linker
Cl
H2N
N
N
N
H
N
N
7 (NPS-1034)
c-Met ( IC50 = 48 nM )
5 (BMS-777607 )
6 (MGCD-516)
c-Met ( IC50 = 20 nM )
c-Met ( IC50 = 3.9 nM )
Fig. 1. The representative c-Met kinase inhibitors.
As displayed in Fig 2, most of the type II c-Met inhibitors may be disconnected into three units according to their
structures and subunit functions. Reported SARs [28-31] of type II c-Met inhibitors suggested that moiety A (4-
phenoxypyridine core: usually substituted 4-phenoxypyridine, 4-phenoxypyrrolopyridine, 4-phenoxythienopyridine
and 4-phenoxyquinoline) and moiety B (usually substituted phenyl group) are crucial for kinase activity. The 4-

phenoxypyridine core was essential for furnishing hydrogen bonds with the backbone of c-Met kinase and responsible
for π-π stacked interactions with amino acid residue of the DFG motif. In addition, hydrophobic interaction was
generated by terminal aryl ring (moiety B) reaching into the hydrophobic pocket formed by amino acid residues. In
contrast, various linear chains and heterocyclic rings can be introduced to the main chain of moiety C (linker bridge).
Importantly, there should be two structural characteristics on the linker bridge (moiety C) between moiety A and B,
one is 5-atoms regulation (i.e., six chemical bonds distance between moiety A and B) and the other is possessing
hydrogen-bond donors or acceptors simultaneously [32–34].
To our knowledge, compounds bearing quinoxalinone fragment have been reported to use as a building block in
the design of anticancer agents. For example, compounds 8-11 (Fig. 2) displayed a multitude of biological activities
[35-38]. 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 3-oxo-3,4-
dihydroquinoxaline-2-carboxamide fragment was introduced into the moiety C via cyclization strategy based on the
‘5 atoms regulation’. Meanwhile, 4-phenoxypyridine was used as the moiety A. Substituted phenyl ring was reserved
as the moiety B. Small substituent X, R1 and R2 were introduced to investigate their effects on activity of the target
compounds. Accordingly, a series of 4-phenoxypyridine derivatives were designed and synthesized to study the
structure-activity relationships (SARs) and find promising antitumor agents (Fig. 2).
N
H
N
N
Br
N
N
CH3
O
N
N
O
O
N
H
HN
N
N
O
O
NH
O
H2NHN
O
HN
H
O
N
O
O
8
Cl
9
11
10
N
H
N
N
O
O
X = H or F
H
N
N
O
H
X
R
X
N
N
O
N
O
H
R2
moiety B
O
O
R'
H
5-atom linker
moiety C
O
N
O
R''
N
O
moiety A
different substituted phenyl
R1
N
N
H
General Structure
target compounds.
Fig. 2. Design strategy and structures of the target compounds.
In the current study, all target compounds were synthesized and evaluated for their in vitro inhibitory activitives
against c-Met kinase and some compounds were further evaluated for their cytotoxic activitives against human lung

adenocarcinoma cell line (A549), human lung cancer cell lines (H460) and human colon cancer cell line (HT-29)
cancer cell lines. Their structure-activity relationships (SARs) were further explored. Furthermore, acridine
orange/ethidium bromide (AO/EB) staining, apoptosis, wound-healing assays and transwell migration assays of 23w
were performed on HT-29 and/or A549 cell lines. Additionally, a docking analysis was also performed to elucidate
the binding mode of the target compound 23w with c-Met kinase.
2. Results and discussion
2.1. Chemistry
The key intermediate 17a-17e were synthesized according to the routes outlined in Scheme 1. Commercially
available 4-chloropyridin-2-amine was condensed with cyclopropanecarbonyl chloride or acetyl chloride in the
presence of Et3N to provide 13a and 13b as a white solid. Meanwhile, the starting material picolinic acid 14 was
chlorinated with thionyl chloride, and then treated with cyclopropanamine in an ice-bath to give 4-chloro-N-
cyclopropylpicolinamide 15 as a light-yellow solid. Subsequently, a nucleophilic substitution of 13a, 13b or 15 with
2-fluoro-4-nitrophenol or 4-nitrophenol in refluxing chlorobenzene provided 16a-16e in a moderate yield. Reduction
of the nitro group of 16a-16e with iron powder and acetic acid in ethyl acetate /water (10:1 v/v) provided aniline
compound 17a-17e.
Cl
Cl
a
O
R'
N
N
H2N
N
H
X
NH2
X
NO2
12
13a, 13b
e
O
13a: R' = cyclopropyl
13b: R' = methyl
O
d
R1
N
R1
N
Cl
17a -17e
16a-16e
b, c
H
N
N
HO
N
O
O
O
16c, 17c: R1 =
16d, 17d: R1 =
X = F
X = H
N
H
15
14
O
O
O
X = F
X = H
N
16a, 17a: R1 =
16b, 17b: R1 =
H
N
H
H
N
16e, 17e: R1 =
X = F
N
H
O
Scheme 1. Reagents and conditions: (a) cyclopropanecarbonyl chloride or acetyl chloride, Et3N, CH2Cl2, rt, 12 h; (b) SOCl2, NaBr,
PhCl, 55 °C, 1 h, reflux, 20 h; (c) cyclopropanamine, Et3N, THF, 0 °C, 3 h; (d) 2-fluoro-4-nitrophenol or 4-nitrophenol,
chlorobenzene, reflux, 40 h; (e) Fe (powder), HOAc, ethyl acetate /water (10:1 v/v), reflux, 2 h.
The synthetic route of the target compounds was shown in Scheme 2. The key intermediates 3-oxo-3,4-
dihydroquinoxaline-2-carboxylic acid derivatives 22a-22o were synthesized by a convenient four-step route starting

from 1-fluoro-2-nitrobenzene and different substituted amines 18a-18o, which was described in detail in our previous
study[34, 39]. Commercially available 1-fluoro-2-nitrobenzene was condensed with different substituted amines 18a-
18o in the presence of NaH in DMF at RT for 16 h to obtain intermediates diphenyl amines 19a-19o as yellow solids,
followed by reduction with iron powder and acetic acid in CH3COOEt/H2O at reflux condition for 16 h to provide
compounds 20a-20o. Further, intermediate 20a-20o were treated with diethyl ketomalonate to generate 3-oxo-3,4-
dihydroquinoxaline-2-carboxylic acid esters 21a-21o, which were then converted to acid analogues 22a-22o by
hydrolysis in aqueous lithium hydroxide at RT for 2 h. Finally, acids 22a-22o were condensed with intermediate 17a-
17e in the presence of HATU and Et3N in DMF at room temperature to afford the target compounds 23a-23y.
O
O
NO2
NH2
N
N
a
b
c
d
OEt
OH
NH2
R
NH
R
NH
R
O
O
N
R
N
R
20a-20o
18a-18o
19a-19o
22a-22o
21a-21o
O
18a-23a, 23p, 23r, 23v, 23x: R = Ph;
18b-23b: R = cyclohexyl;
18c-23c, 23c: R = benzyl;
18d-23d, : R = phenethyl;
18e-23e, 20q, 20s, 20w, 20y: R = 4-FPh;
18f-23f: R = 4-ClPh;
18g-23g, R = 3-FPh;
18h-23h: R = 3-ClPh;
18i-23i: R = 2-FPh;
18j-23j, 20t: R = 2-ClPh;
18k-23k: R = 4-BrPh;
23a-23o, 23v, 23w:
23r-23u, 23x, 23y:
R1 =
N
H
O
N
H
R1 =
N
H
X
N
N
e
R
H
N
O
O
O
N
23p, 23q: R1 =
O
23a-23u: X = F
23v-23y: X = H
R1
18l-23l: R = 4-CH3Ph;
23a-23y
18m-23m, 20u: R = 4-OCH3Ph;
18n-23n: R = 2,4-diClPh;
18o-23o: R = 2,4-diOCH3Ph;
Scheme 2. 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, 2 h, then 6 N HCl; (e) 17a-17e, HATU, Et3N, DMF, 25 C,12 h.
2.2 Biological evaluation
2.2.1 In vitro c-Met kinase assays and analysis of the structure-activity relationships
All the newly synthesized 4-phenoxypyridine derivatives (23a-23y) were evaluated for their in vitro inhibitory
activity toward c-Met enzyme using mobility shift assay. Foretinib was used as a positive control, with the results
expressed as half-maximal inhibitory concentration (IC50) values presented in Table 1. The IC50 values are the average
of at least three independent experiments.
As illustrated in Table 1, all the tested compounds (except for 23p and 23q) showed excellent c-Met enzymatic
activity with IC50 values ranging from 1.91 to 202.72 nM, which indicated that introduction of the 3-oxo-3,4-
dihydroquinoxaline-2-carboxamide framework to “5-atom linker” moiety of pyridine-based compounds maintained
the c-Met inhibitory efficacy. Notably, ten of them (23a, 23e, 23f, 23l, 23r, 23s, 23v, 23w, 23x and 23y) exhibited

promising activity against c-Met kinase with IC50 values less than 10.00 nM, and compound 23w demonstrated the
best activity with an IC50 value of 1.91 nM.
Firstly, a phenyl ring (moiety B) was introduced at the C-1 of quinoxalin-2-one and compound 23a was obtained.
As expected, compound 23a showed promising inhibition of c-Met kinase with an IC50 value of 5.71 nM. Replacement
of the phenyl ring by a cyclohexyl ring led to an approximate 10-fold drop in potency (23a vs 23b). In order to know
if the “5-atoms regulation” was applicable for the designed compounds, benzyl and phenethyl were also introduced at
the C-1 of quinoxalin-2-one. Compared with 23a (R = Ph, IC50 = 5.71 nM), 23c (R = CH2Ph, IC50 = 23.27 nM) and
23d (R = CH2CH2Ph, IC50 = 52.33 nM) decreased potency as the distance between terminal phenyl and quinoxaline
increasing, indicated that a phenyl substituent at this position was detrimental to the potency. This characteristic was
consistent with our previous reported regulation of five atoms in which moiety A and moiety B are usually connected
by six chemical bonds.
Encouraged by the observations described above, further investigations were performed to study the inhibitory
potency effect of different substituents on the phenyl ring. The SARs based on IC50 values revealed that the inhibition
of c-Met kinase of compounds with substituent at 4-position of phenyl ring was higher than those with substituents at
2- or 3-position. For example, compound with 4-fluorophenyl group (23e, IC50 = 5.02 nM) displayed more potency
than those with 3-fluorophenyl (23g, IC50 = 10.23 nM) or 2-fluorophenyl group (23i, IC50 = 25.13 nM), and the same
trend was observed for the series of chlorophenyl substituted compounds (23f, 23h and 23j). IC50 values indicated that
halogen and methyl at the para position of the phenyl ring made a good contribution to enzyme activity, with following
rank order: -Br < -CH3 < -Cl < -F. However, the introduction of 4-methoxyphenyl group (23m, IC50 = 145.96 nM), 2,
4-dichlorophenyl group (23n, IC50 = 65.68 nM) and 2, 4-dimethoxyphenyl group (23o, IC50 = 202.72 nM) exhibited
an obvious negative effect on the enzyme activity respectively, compared to 23a, which had no substituent on the
phenyl ring.
In the following work, our modification focused on the R1 group. Firstly, the orientation of amide at the C-2
position of pyridine was inverted and two picolinamides were prepared. Unfortunately, the obtained compounds 23p
(IC50 = 4110 nM) and 23q (IC50 = 3442 nM) were significantly less potent compared to 23a (IC50 = 5.71 nM) and 23e
(IC50 = 5.02 nM). From the results, we could deduced that 2-pyridinylamino amide moiety was the essential group for
c-Met kinase inhibitory activity. Meanwhile, replacing the cyclopropyl group with methyl group was very well
tolerated (23r–23u) and maintained the biological activity, especially IC50 values against c-Met kinase of 23r (IC50
8.22 nM) and 23s (IC50 = 6.79 nM) were less than 10.00 nM.
=
Having identified well tolerated group on R and R1 moiety, our attention was turned to the investigation of

phenoxy part (-X group) and four compounds (23v-23y) were prepared. To our delight, the IC50 values showed that
the fluorine atom was substituted with hydrogen atom resulted a slight boost in c-Met inhibitory activity. Compounds
23v, 23w and 23y showed the most potent activity with IC50 values of 2.31 nM, 1.91 nM and 2.44 nM, respectively,
which were better than that of positive control foretinib (IC50 = 2.53 nM).
Table 1
In vitro c-Met kinase inhibitory activities of target compounds 23a-23y.
X
R1
O
O
O
N
N
N
H
N
R
IC50 (nM) ± SD a
c-Met
Compd.
R
R1
X
O
O
O
O
O
O
O
23a
23b
23c
23d
23e
23f
23g
23h
F
F
F
F
F
F
F
F
5.71 ± 0.26
55.39 ± 3.57
23.27 ± 1.31
52.33 ± 2.96
5.02 ± 0.32
7.64 ± 0.98
10.23 ± 1.15
16.79 ± 1.22
N
H
N
H
N
H
N
H
F
N
H
Cl
N
H
N
H
F
O
O
N
H
Cl
F
23i
23j
F
F
25.13 ± 0.89
45.28 ± 4.18
N
H
Cl
O
N
H
O
O
O
Br
23k
23l
F
F
F
F
14.16 ± 1.01
8.55 ± 0.45
N
H
CH3
N
H
OCH3
23m
23n
145.96 ± 6.47
65.68 ± 5.03
N
H
Cl
O
O
N
H
Cl
H
3CO
23o
23p
23q
23r
23s
F
F
F
F
F
202.72 ± 12.65
4110 ± 93.27
3442 ± 78.53
8.22 ± 0.36
N
H
OCH3
H
N
O
O
H
N
F
O
O
N
H
F
6.79 ± 0.24
N
H
Cl
O
O
23t
F
62.47 ± 6.17
N
H
OCH3
23u
23v
23w
23x
F
H
H
H
184.36 ± 10.43
2.31 ± 0.10
1.91 ± 0.12
2.96 ± 0.14
N
H
O
N
H
O
F
N
H
O
O
N
H
F
23y
Foretinibb
H
--
2.44 ± 0.16
2.53 ± 0.10
N
H
--
--

a Data presented is the mean ± SD value of three independent determinations.
b Used as a positive control.
2.2.2 In vitro antiproliferative activity
Based on the in vitro c-Met kinase assay results, we selected four compounds which IC50 values against c-Met
kinase were less than 5.00 nM for their antiproliferative activitives against three c-Met overexpressed human cancer
cell lines, namely A549 (human lung adenocarcinoma cell line), H460 (human lung cancer cell line) and HT-29 (human
colon cancer cell line) together with foretinib as the positive control by a MTT assay. The results were outlined in
Table 2. As shown in Table 2, all the four compounds exhibited excellent antiproliferative activitives against the three
tested cell lines. Among them, compound 23v, 23w and 23x exhibited remarkable inhibitory activitives against HT-
29 cell lines with IC50 value of 0.83μM, 0.65 μM and 0.74 μM, respectively, which were more potent than that of the
positive control foretinib (IC50 = 0.98 μM). Meanwhile, Compound 23y also showed excellent antiproliferative against
H460 cell line with IC50 values of 0.79 μM, and it was slightly more potent than foretinib (IC50 = 0.83 μM).
Table 2
Antiproliferative activities of selected compounds 23v, 23w, 23x, 23y and Foretinib in vitro.
IC50 (μM) ±SDa
Compd.
A549
H460
HT-29
23v
23w
23x
2.17 ± 0.19
1.57 ± 0.05
4.84 ± 0.11
3. 35 ± 0.23
1.25 ± 0.14
0.87 ± 0.06
0.94 ± 0.03
1.82 ± 0.16
0.79 ± 0.06
0.83 ± 0.05
0.83 ± 0.07
0.65 ± 0.05
0.74 ± 0.02
1.01 ± 0.09
0.98 ± 0.06
23y
Foretinibb
a Data presented is the mean ± SD value of three independent determinations.
b Used as a positive control.
2.2.3. Apoptosis induction ability of 23w
According to the results of c-Met kinase assay and antiproliferative assay, acridine orange (AO)/ethidium bromide
(EB) assay were performed to observe the morphologic changes of apoptosis by fluorescene microscopy after the HT-
29 and A549 cells were treatment with compound 23w for 48 h [40, 41]. As shown in Fig. 3, almost all cells of
untreated control group were stained in green and showed normal morphology of regular roundness. Most HT-29 and
A549 cells treated with 1.0 μM and 10.0 μM of compound 23w displayed the morphological changes such as apoptotic
body formation, membrane blebbing, cell shrinkage and chromatin condensation, suggesting that compound 23w
induced cell death in HT-29 and A549 cancer cells via apoptosis in a dose dependent manner.

Fig. 3. AO/EB stained apoptosis of HT-29 and A549 cell lines with different concentrations of compound 23w.
In the next step, flowcytometry analysis with Annexin V/PI double staining was carried out to confirm apoptosis
induction by the compound 23w according to known procedures [42]. HT-29 and A549 cells were seeded per well in
six-well plates and were treated with compound 23w of different concentrations (0, 1.0 and 10.0 μM) for 48 h,
respectively. As depicted in Fig. 4, incubation of HT-29 cells with compound 23w led to a dose dependent apoptosis
increase, with the percentage of total apoptotic cells (early and late apoptotic cells) of 17.1 % and 44.2 % at 1.0 μM
and 10.0 μM, respectively, compared to the control (13.7%). Meanwhile, we found that treatment of A549 cells with
compound 23w also induced apoptosis in a dose dependent manner as the same trend observed in HT-29 cells.
According to these results, we conclude that compound 23w could effectively induce apoptosis in HT-29 and A549
cells.

Fig. 4. Compound 23w induces cell apoptosis of HT-29 and A549 cell line in vitro.
2.2.4. Cell migration inhibition ability of 23w
As migration is an important characteristic for metastatic cancers [43], we investigated the antimigratory effect
of compound 23w on A549 cells in vitro using wound-healing assay. As illustrated in the photomicrographs shown in
Fig. 5, untreated A549 cells (control group) almost filled up the wounded area 72 h after scratching the cell monolayer,
whereas treatment with 1.0 μM of compound 23w significantly suppressed wound healing in a time dependent manner.
These results indicate that compound 23w possesses a significant ability to inhibit the metastasis of A549 cells.
Fig. 5. Anti-migration activity of compound 23w against A549 cells in wound-healing migration model.
To further determine anti-migration ability of compound 23w, transwell migration assay was also performed [44].

The results revealed that compound 23w inhibited the migration of A549 cancer cells obviously at two concentrations
(1.0 and 10.0 μM), compared with the control group. Interestingly, the migration of A549 cells was significantly
inhibited by compound 23w and almost completely suppressed by this compound at the concentration of 10.0 μM (Fig.
6).
Fig. 6. Anti-migration activity of compound 23w against A549 cells in transwell migration model.
2.2.5. Binding model analysis
In order to better understand the binding mode, molecular docking of compound 23w was performed based on
the cocrystal structure of foretinib (GSK1363089) with c-Met (PDB code: 3LQ8). As shown in Fig. 7, binding model
of compound 23w with c-Met (PDB code: 3LQ8) were well consistent with type II c-Met inhibitors binding
conformation and bonding model. In the binding model, the 4-phenoxypyridine part (moiety A) was found that the N
atom of pyridine and the O atom of cyclopropanecarboxamide interacted with Met1160 via two hydrogen bonds.
Meanwhile, the pyridinyl ring and the phenyl ring at 4-position of pyridine formed two π–π interactions with Tyr1159
and Phe1223, respectively. In addition, the following interactions were formed in moiety A: (1): five π-Alkyl
interaction with TYR1159, Met1160, Ala1108, Ile1084 and Leu1157, respectively; (2): one π-lone pair interaction
with 1159; (3): one carbon hydrogen interaction with Pro1158; (4): one π-sigma interaction with Met1121. The 4-
fluorophenyl ring (moiety B) fitted into the hydrophobic pocket very well, and formed one π–alkyl interaction with
Met1131. The 3-oxo-3,4-dihydroquinoxaline-2-carboxamide moiety (moiety C) formed two hydrogen bonds with
Lys1110, one π–anion interaction with Asp1222, one carbonhydrogen bond interaction with Asp1222 and one π–alkyl
interaction with Met1131. All these interactions contribute to the tight binding and greatly enhance the inhibitory
potency of 23w.

Fig. 7. The mode of compound 23w bound to c-Met (PDB code: 3LQ8). The amino acid residues were displayed by red sticks and
compound was displayed by bright blue sticks. Green dotted lines represent H-bonds. (A) The overview of compound 23w docked into c-
Met. (B) The c-Met active site in complex with compound 23w. (C) 2D interactions of the docking model of compound 23w to c-Met.
3. Conclusion
In summary, a series of novel 4-phenoxypyridine derivatives containing 3-oxo-3,4-dihydroquinoxaline moiety as
c-Met inhibitors were designed, synthesized and evaluated for their biological activitives. The screening of enzyme
activities and cytotoxicity led to the identification of a most promising compound 23w (c-Met IC50 = 1.91 nM) with
IC50 values of 1.57 μM, 0.94 μM and 0.65 μM against A549, H460, and HT-29 cells, representing a promising lead
for further optimization. The The initial structure-activity relationships (SARs) analyses indicated that introduction of
the 3-oxo-3,4-dihydroquinoxaline-2-carboxamide framework to “5-atom linker” moiety of pyridine-based
compounds maintained the c-Met inhibitory efficacy and compounds without substituents or with a fluoro group at 4-
position on the terminal phenyl ring (moiety B) were more active than other substituents. Meanwhile, 2-pyridinylamino
amide (moiety A) maybe the essential group for c-Met kinase inhibitory activity. Furthermore, AO/EB assays, cell
apoptosis assays by flow cytometry, wound-healing assays and transwell migration assay on HT-29 and/or A549 cells
results indicated that compound 23w could induce HT-29 and A549 cells apoptosis, and inhibit A549 cells motility.
Further studies on structural optimization and biological activities about these derivatives are still underway in our

laboratory and will be reported in the future.
4. Experimental
4.1. Chemistry
Unless otherwise specified, all melting points were obtained on a Beijing Taike X-4 microscopy melting point
apparatus and were uncorrected. 1H NMR spectra were recorded on a Bruker Biospin 600 MHz or Bruker Biospin 400
MHz instrument using TMS as the internal standard. All chemical shifts were reported in ppm. IR spectra were
recorded as KBr pellets on a Perkin-Elmer Spcetrum one FT-IR spectrometer. MS spectra were obtained on an Agilent
6460 QQQ mass spectrometer (Agilent, USA) analysis system. 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.). 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 key intermediates 13a, 16a, 17a, 18a-18o, 19a-19o, 20a-20o, 21a-21o and 22a-22o were synthesized according
to our previous reported procedures [30, 34, 39].
4.1.1. General procedure for preparation of N-(4-chloropyridin-2-yl)alkylcarboxamide (13a, 13b)
Cyclopropanecarbonyl chloride or acetyl chloride (89.00 mmol) was dissolved in dried CH2Cl2 (30 mL) and drop-
wise added to a mixture of 4-chloropyridin-2-amine (8.80 g, 68.45 mmol), Et3N (20.78 g, 205.35 mmol) and CH2Cl2
(80 mL) in an ice bath, which was then removed to raise the temperature to room temperature and stirred for 12 h. The
resulting mixture was sequentially washed with 20% K2CO3 (50 mL × 3) and brine (50 mL × 3), and the organic phase
was separated, dried over anhydrous Na2SO4, filtered, and the filtrate was evaporated under reduced pressure. The
crude product obtained was purified by silica gel chromatography to give 13a or 13b as a white solid.
4.1.1.1. N-(4-chloropyridin-2-yl)cyclopropanecarboxamide (13a)
Yield: 72.7% %; IR (KBr) cm-1: 3435.3, 3241.8, 1706.5, 1670.1, 1588.4, 1573.5, 1537.4, 1403.5, 1257.0, 1212.6,
1189.5, 1149.6, 959.6, 872.9, 823.8, 710.1; 1H NMR (600 MHz, CDCl3) δ 8.79 (s, 1H), 8.31 (s, 1H), 8.16 (d, J = 5.4
Hz, 1H), 7.03 (dd, J = 5.4, 1.6 Hz, 1H), 1.60 – 1.49 (m, 1H), 1.17 – 1.09 (m, 2H), 0.97 – 0.87 (m, 2H); MS (ESI)
m/z(%): 197.1 [M+H]+.
4.1.1.2. N-(4-chloropyridin-2-yl)acetamide (13b)

Yield: 70.2%; 1H NMR (600 MHz, CDCl3) δ 8.66 (s, 1H), 8.31 (s, 1H), 8.15 (d, J = 5.4 Hz, 1H), 7.05 (dd, J =
5.4, 1.8 Hz, 1H), 2.21 (s, 3H); MS (ESI) m/z (%): 171.1 [M+H]+.
4.1.2. 4-Chloro-N-cyclopropylpicolinamide (15)
To a solution of picolinic acid (18.0 g, 146.21 mmol) and sodium bromide (3.01 g, 29.24 mol) in chlorobenzene
(50 mL), thionyl chloride (60.90 g, 511.80 mmol) was added slowly at room temperature. The reaction mixture was
stirred at 50 C for 30 min, and heated to 85 C and stirred for another 20 h. The completion of reaction was determined
by TLC, at which point the solvent was removed under reduced pressure to give a brown oil, which was immediately
dissolved in toluene (50 mL). Then cyclopropanamine (9.2 g, 160.90 mmol) in toluene (30 mL) was added dropwise
in an ice-bath. The mixture was stirred at 10 C for 3.0 h. The solvent was removed under reduced pressure. The
residue was dissolved in dried CH2Cl2 (100 mL) and the resulting mixture was sequentially washed with saturated
aqueous K2CO3 (50 mL × 3) and brine (50 mL × 3), and the organic phase was separated, dried, and evaporated. The
crude product obtained was purified by silica gel chromatography to afford 15 as a light-yellow solid (16.8 g, yield
58.4%). 1H NMR (600 MHz, DMSO-d6) δ 8.83 (d, J = 3.9 Hz, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.02 (d, J = 2.0 Hz, 1H),
7.75 (dd, J = 5.2, 2.1 Hz, 1H), 3.09 – 2.76 (m, 1H), 0.99 – 0.44 (m, 4H); MS (ESI) m/z (%): 197.1 [M+H]+.
4.1.3. General procedure for preparation intermediates of 4-(4-nitrophenoxy)pyridine (16a-16e)
A stirring mixture of compound 13a, 13b or 15 (40.68 mmol) and 2-fluoro-4-nitrophenol or 4-nitrophenol (101.71
mmol) in chlorobenzene (100 mL) was heated to 140 C for about 40 h. After cooling to room temperature, the reaction
mixture was concentrated under reduced pressure to yield a pale solid. The solid was dissolved in CH2Cl2 (150 mL),
and washed with saturated K2CO3 aqueous solution (80 mL × 4), then brine (60 mL × 4), and dried over anhydrous
Na2SO4, concentrated under reduced pressure to afford a brown solid, which was was purified by silica gel
chromatography to give 16a-16e as a light yellow solid.
4.1.3.1. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)cyclopropanecarboxamide (16a)
Yield: 55.3%; IR (KBr) cm-1: 3467.0, 3417.7, 1687.3, 1616.7, 1528.7, 1492.5, 1353.9, 1300.4, 1271.9, 1179.8,
1070.0, 875.2, 798.4; 1H NMR (600 MHz, DMSO-d6) δ 11.00 (s, 1H), 8.43 (dd, J = 10.3, 2.2 Hz, 1H), 8.30 (d, J = 5.7
Hz, 1H), 8.19 (dd, J = 9.0, 1.9 Hz, 1H), 7.76 (d, J = 2.2 Hz, 1H), 7.61 (t, J = 8.5 Hz, 1H), 6.86 (dd, J = 5.6, 2.2 Hz,
1H), 2.04 – 1.95 (m, 1H), 0.78 (t, J = 6.3 Hz, 4H); MS (ESI) m/z (%): 318.1 [M+H]+.
4.1.3.2. N-(4-(4-nitrophenoxy)pyridin-2-yl)cyclopropanecarboxamide (16b)
Yield: 58.8%; 1H NMR (600 MHz, CDCl3) δ 8.77 (s, 1H), 8.31 – 8.26 (m, 2H), 8.23 (d, J = 5.7 Hz, 1H), 7.92 (d,
J = 2.2 Hz, 1H), 7.24 – 7.13 (m, 2H), 6.72 (dd, J = 5.7, 2.3 Hz, 1H), 1.64 – 1.45 (m, 1H), 1.11 – 1.05 (m, 2H), 0.93 –
0.86 (m, 2H); MS (ESI) m/z(%): 300.1 M+H]+.

4.1.3.3. N-(4-(2-fluoro-4-nitrophenoxy)pyridin-2-yl)acetamide (16c)
Yield: 51.6%; 1H NMR (600 MHz, CDCl3) δ 8.58 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 8.17 – 8.09 (m, 2H), 7.89 (s,
1H), 7.37 – 7.29 (m, 1H), 6.71 (dd, J = 5.7, 2.3 Hz, 1H), 2.18 (s, 3H); MS (ESI) m/z(%): 292.1 [M+H]+.
4.1.3.4. N-(4-(4-nitrophenoxy)pyridin-2-yl)acetamide (16d)
Yield: 54.3%; 1H NMR (600 MHz, CDCl3) δ 8.36 – 8.27 (m, 3H), 8.22 (d, J = 5.7 Hz, 1H), 7.93 (s, 1H), 7.20 (d,
J = 9.1 Hz, 2H), 6.71 (dd, J = 5.7, 2.3 Hz, 1H), 2.19 (s, 3H); MS (ESI) m/z(%): 274.1 [M+H]+.
4.1.3.5. N-cyclopropyl-4-(4-nitrophenoxy)picolinamide (16e)
Yield: 62.7%; 1H NMR (600 MHz, CDCl3) δ 8.47 (d, J = 5.5 Hz, 1H), 8.21 – 8.09 (m, 2H), 8.02 (s, 1H), 7.71
(d, J = 2.5 Hz, 1H), 7.40 – 7.30 (m, 1H), 7.07 (dd, J = 5.5, 2.6 Hz, 1H), 3.24 – 2.64 (m, 1H), 0.90 – 0.85 (m, 2H), 0.68
– 0.63 (m, 2H); MS (ESI) m/z(%): 318.1 [M+H]+, 340.0 [M+Na]+.
4.1.4. General procedure for preparation intermediates of 4-(pyridin-4-yloxy)aniline (17a-17e)
A mixture of compound 16a-16e (18.00 mmol), iron powder (90.00 mmol), acetic acid (180.00 mmol), water (10
mL) and ethyl acetate (100 mL) was heated to reflux for 2 h. 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，filtered, and the filtrate was evaporated under reduced pressure when white solid appeared, which
was filtered to obtain 17a-17e as light yellow solid.
4.1.4.1. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)cyclopropanecarboxamide (17a)
Yield: 64.6%; IR (KBr) cm-1: 3411.2, 2025.9, 1736.6, 1617.8, 1510.4, 1426.8, 1207.1, 1163.3, 992.6, 955.9, 866.0,
821.9, 609.7, 468.1; 1H NMR (600 MHz, DMSO-d6) δ 10.79 (s, 1H), 8.15 (d, J = 5.7 Hz, 1H), 7.59 (s, 1H), 6.95 (t, J
= 9.0 Hz, 1H), 6.67 – 6.61 (m, 1H), 6.49 (dd, J = 13.1, 2.2 Hz, 1H), 6.40 (d, J = 8.7 Hz, 1H), 5.44 (s, 2H), 2.03 – 1.88
(m, 1H), 0.76 (br, 4H); MS (ESI) m/z(%): 288.1 [M+H]+, 310.1 [M+Na]+.
4.1.4.2. N-(4-(4-aminophenoxy)pyridin-2-yl)cyclopropanecarboxamide (17b)
Yield: 71.3%; 1H NMR (600 MHz, CDCl3) δ 9.15 (s, 1H), 8.06 (d, J = 5.8 Hz, 1H), 7.75 (s, 1H), 6.89 (d, J = 8.6
Hz, 2H), 6.69 (d, J = 8.6 Hz, 2H), 6.55 (dd, J = 5.7, 2.1 Hz, 1H), 3.61 (s, 2H), 1.54 (s, 1H), 1.18 – 0.91 (m, 2H), 0.92
– 0.58 (m, 2H); MS (ESI) m/z (%): 270.1 M+H]+, 292.1 [M+Na]+.
4.1.4.3. N-(4-(4-amino-2-fluorophenoxy)pyridin-2-yl)acetamide (17c)
Yield: 67.9%; 1H NMR (600 MHz, DMSO-d6) δ 10.49 (s, 1H), 8.14 (d, J = 5.7 Hz, 1H), 7.61 (s, 1H), 6.96 (t, J =
9.0 Hz, 1H), 6.61 (dd, J = 5.6, 2.1 Hz, 1H), 6.56 – 6.46 (m, 1H), 6.42 (d, J = 8.6 Hz, 1H), 5.45 (s, 2H), 2.04 (s, 3H);
MS (ESI) m/z(%): 262.1 [M+H]+, 284.1 [M+Na]+.
4.1.4.4. N-(4-(4-aminophenoxy)pyridin-2-yl)acetamide (17d)

Yield: 70.7%; 1H NMR (600 MHz, CDCl3) δ 8.11 (s, 1H), 8.05 (d, J = 5.8 Hz, 1H), 7.76 (s, 1H), 6.90 (d, J = 8.7
Hz, 2H), 6.71 (d, J = 8.7 Hz, 2H), 6.55 (dd, J = 5.8, 2.3 Hz, 1H), 3.66 (s, 2H), 2.15 (s, 3H); MS (ESI) m/z(%):
244.1M+H]+, 266.1 [M+Na]+.
4.1.4.5. 4-(4-amino-2-fluorophenoxy)-N-cyclopropylpicolinamide (17e)
Yield: 76.8%; 1H NMR (600 MHz, CDCl3) δ 8.33 (d, J = 5.6 Hz, 1H), 8.02 (s, 1H), 7.66 (d, J = 2.5 Hz, 1H), 7.01
– 6.86 (m, 2H), 6.51 (dd, J = 11.9, 2.6 Hz, 1H), 6.45 (dd, J = 8.6, 1.7 Hz, 1H), 3.84 (s, 2H), 3.00 – 2.78 (m, 1H), 0.88
– 0.82 (m, 2H), 0.67 – 0.61 (m, 2H); MS (ESI) m/z (%): 288.1 M+H]+, 310.1[M+Na]+.
4.1.5. General procedure for Preparation of the target Compounds (23a-23y)
A mixture of the corresponding acids 22a‒22o (1.30mmol), 17a-17e (1.00 mmol), HATU (1.50 mmol), Et3N
(3.00 mmol), and DMF (8 ml) was stirred at room temperature for 12 hr. The residue was dissolved in CH2Cl2 (50
mL) and the resulting mixture was sequentially washed with 20% K2CO3 (30 mL × 3) and brine (30 mL × 3), and the
organic phase was separated, dried, and evaporated. The crude product obtained was purified by silica gel
chromatography to afford 23a-23y as light yellow solids.
4.1.5.1. N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-4-phenyl-3,4-dihydro
quinoxaline-2-carboxamide (23a).
Yield: 84.1%; M.p.: 254–256 C; IR (KBr, cm-1): 3395.3, 1693.2, 1646.0, 1596.3, 1578.4, 1535.0, 1512.6, 1420.0,
1306.0, 1201.7, 1171.0, 1092.3, 953.7, 829.4, 767.7, 701.3, 603.0; 1H NMR (600 MHz, DMSO-d6) δ 11.29 (s, 1H),
10.87 (s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.93 (dd, J = 12.6, 2.2 Hz, 1H), 7.70 (t, J = 7.6 Hz,
2H), 7.68 – 7.57 (m, 3H), 7.54 (d, J = 8.9 Hz, 1H), 7.48 (t, J = 8.1 Hz, 3H), 7.41 (t, J = 8.9 Hz, 1H), 6.74 (dd, J = 5.7,
2.4 Hz, 1H), 6.68 (d, J = 8.5 Hz, 1H), 2.04 – 1.90 (m, 1H), 0.89 – 0.65 (m, 4H); MS (ESI) m/z(%): 536.1 [M+H]+,
558.1 [M+Na]+; Anal. calcd. for C30H22FN5O4 (%): C, 67.28; H, 4.14; N, 13.08. Found (%): C, 67.39; H, 4.19; N,
13.14.
4.1.5.2. 4-cyclohexyl-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-3,4-dihydro
quinoxaline-2-carboxamide (23b).
Yield: 79.5%; M.p.: 267–270 C; IR (KBr, cm-1): 3430.5, 2933.3, 2851.6, 1687.3, 1593.8, 1425.2, 1307.1, 1206.6,
1176.1, 1097.4, 954.8, 864.2, 817.4, 758.8; 1H NMR (600 MHz, DMSO-d6) δ 11.13 (s, 1H), 10.88 (s, 1H), 8.22 (d, J
= 5.7 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.98 – 7.85 (m, 2H), 7.74 (t, J = 7.9 Hz, 1H), 7.66 (d, J = 2.2 Hz, 1H), 7.57 –
7.44 (m, 2H), 7.41 (t, J = 8.9 Hz, 1H), 6.75 (dd, J = 5.7, 2.3 Hz, 1H), 4.73 (s, 1H), 2.55 (s, 2H), 2.06 – 1.92 (m, 1H),
1.87 (d, J = 11.6 Hz, 2H), 1.80 – 1.64 (m, 3H), 1.61 – 1.41 (m, 2H), 1.42 – 1.13 (m, 1H), 0.92 – 0.64 (m, 4H); 13C
NMR (150 MHz, DMSO-d6) δ 173.28, 165.73, 162.90, 154.35, 153.88 (d, J = 246.2 Hz), 153.45, 149.96, 137.51 (d, J

= 9.7 Hz), 136.22 (d, J = 12.3 Hz), 132.45, 130.96, 124.67, 124.44, 116.69, 108.60 (d, J = 23.0 Hz), 107.55, 99.62,
28.07, 25.89, 25.23, 14.62, 8.10; MS (ESI) m/z (%): 542.2 [M+H]+, 564.2 [M+Na]+; Anal. calcd. for C30H28FN5O4
(%): C, 66.53; H, 5.21; N, 12.93. Found (%): C, 66.48; H, 5.26; N, 12.97.
4.1.5.3.
4-benzyl-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-3,4-dihydro
quinoxaline-2-carboxamide (23c).
Yield: 84.8%; M.p.: 253–255 C; IR (KBr, cm-1): 3432.6, 3060.4, 1690.4, 1595.4, 1536.4, 1506.4, 1467.8, 1431.7,
1388.2, 1300.6, 1206.3, 1101.8, 954.8, 874.0, 820.4, 764.0; 1H NMR (600 MHz, DMSO-d6) δ 11.25 (s, 1H), 10.88 (s,
1H), 8.22 (s, 1H), 8.07 – 7.96 (m, 1H), 7.93 (dd, J = 12.6, 2.2 Hz, 1H), 7.75 – 7.62 (m, 2H), 7.58 (d, J = 8.4 Hz, 1H),
7.54 (d, J = 8.8 Hz, 1H), 7.50 – 7.39 (m, 2H), 7.39 – 7.32 (m, 4H), 7.31 – 7.27 (m, 1H), 6.76 (d, J = 3.4 Hz, 1H), 5.59
(s, 2H), 2.10 – 1.80 (m, 1H), 0.96 – 0.44 (m, 4H); MS (ESI) m/z(%): 550.1 [M+H]+, 572.1 [M+Na]+; Anal. calcd. for
C31H24FN5O4 (%): C, 67.75; H, 4.40; N, 12.74. Found (%): C, 67.87; H, 4.44; N, 12.71.
4.1.5.4.
N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-4-phenethyl-3,4-dihydro
quinoxaline-2-carboxamide (23d).
Yield: 76.3%; M.p.: 241–243 C; IR (KBr, cm-1): 3454.5, 1693.5, 1593.2, 1535.3, 1423.5, 1311.6, 1182.4, 761.8;
1H NMR (600 MHz, DMSO-d6) δ 11.23 (s, 1H), 10.88 (s, 1H), 8.22 (d, J = 5.7 Hz, 1H), 8.01 – 7.96 (m, 1H), 7.93 (dd,
J = 12.6, 2.3 Hz, 1H), 7.82 – 7.74 (m, 2H), 7.67 (d, J = 2.2 Hz, 1H), 7.54 (dd, J = 8.8, 1.4 Hz, 1H), 7.51 – 7.47 (m,
1H), 7.42 (t, J = 8.9 Hz, 1H), 7.38 (d, J = 7.3 Hz, 2H), 7.34 (t, J = 7.6 Hz, 2H), 7.26 (t, J = 7.3 Hz, 1H), 6.75 (dd, J =
5.7, 2.4 Hz, 1H), 4.63 – 4.39 (m, 2H), 3.04 – 2.95 (m, 2H), 2.11 – 1.86 (m, 1H), 0.94 – 0.67 (m, 4H); 3C NMR (150
MHz, DMSO-d6) δ 173.29, 165.73, 162.46, 154.35, 153.89 (d, J = 246.2 Hz), 152.87, 150.92, 149.98, 138.23, 137.48
(d, J = 9.8 Hz), 136.28 (d, J = 12.2 Hz), 133.18, 132.88, 131.94, 130.77, 129.27, 128.91, 127.05, 124.68, 124.61,
116.76, 115.45, 108.69 (d, J = 23.0 Hz), 107.56, 99.64, 43.71, 33.06, 14.62, 8.11;MS (ESI) m/z(%): 586.1 [M+Na]+;
Anal. calcd. for C32H26FN5O4 (%): C, 68.20; H, 4.65; N, 12.43. Found (%): C, 68.18; H, 4.67; N, 12.48.
4.1.5.5.
N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide (23e).
Yield: 84.4%; M.p.: 262–264 C; IR (KBr, cm-1): 3421.4, 1693.6, 1648.8, 1597.0, 1507.8, 1427.3, 1309.3, 1259.3,
1207.2, 1176.4, 954.8, 765.5; 1H NMR (600 MHz, CDCl3) δ 11.91 (s, 1H), 8.32 (d, J = 7.0 Hz, 1H), 8.19 – 8.08 (m,
2H), 7.98 (dd, J = 12.1, 2.3 Hz, 1H), 7.79 (s, 1H), 7.63 – 7.56 (m, 1H), 7.55 – 7.49 (m, 1H), 7.45 – 7.33 (m, 5H), 7.15
(t, J = 8.6 Hz, 1H), 6.80 (d, J = 8.3 Hz, 1H), 6.60 (dd, J = 5.7, 2.2 Hz, 1H), 1.52 – 1.47 (m, 1H), 0.90 – 0.81 (m, 4H);
13C NMR (150 MHz, DMSO-d6) δ 173.28, 165.70, 162.68 (d, J = 246.6 Hz), 162.02, 154.33, 153.88 (d, J = 246.2 Hz),
153.24, 151.19, 149.98, 137.40 (d, J = 9.8 Hz), 136.33 (d, J = 12.2 Hz), 135.12, 132.74, 131.80, 131.14 (d, J = 8.9

Hz), 130.50, 124.82, 124.67, 117.74, 117.59, 116.84, 115.95, 108.78 (d, J = 23.0 Hz), 107.56, 99.65, 14.60, 8.10; MS
(ESI) m/z(%): 554.1 [M+H]+, 576.1 [M+Na]+; Anal. calcd. for C30H21F2N5O4 (%): C, 65.10; H, 3.82; N, 12.65. Found
(%): C, 65.13; H, 3.87; N, 12.61.
4.1.5.6. 4-(4-chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide (23f).
Yield: 86.7%; M.p.: 251–253 C; IR (KBr, cm-1): 3434.0, 2923.1, 1693.3, 1597.8, 1577.5, 1532.5, 1506.2, 1427.6,
1308.2, 1262.0, 1205.2, 1089.3, 1017.9, 954.8, 828.5, 752.9; 1H NMR (600 MHz, CDCl3) δ 11.88 (s, 1H), 8.30 (t, J =
7.2 Hz, 1H), 8.25 (s, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.97 (dd, J = 12.1, 2.3 Hz, 1H), 7.79 (d, J = 1.6 Hz, 1H), 7.69 (d,
J = 8.6 Hz, 2H), 7.61 – 7.56 (m, 1H), 7.55 – 7.49 (m, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.32 (d, J = 8.6 Hz, 2H), 7.20 –
7.13 (m, 1H), 6.81 (d, J = 8.4 Hz, 1H), 6.60 (dd, J = 5.8, 2.3 Hz, 1H), 1.54 – 1.47 (m, 1H), 1.09 – 1.03 (m, 2H), 0.89
– 0.83 (m, 2H); MS (ESI) m/z(%): 569.3 [M]+, 592.1 [M+Na]+; Anal. calcd. for C30H21ClFN5O4 (%): C, 63.22; H,
3.71; N, 12.29. Found (%): C, 63.31; H, 3.77; N, 12.35.
4.1.5.7.
N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(3-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide (23g).
Yield: 75.2%; M.p.: 254-257 C; IR (KBr, cm-1): 3426.4, 3060.4, 1695.1, 1599.2, 1530.9, 1506.7, 1430.8, 1298.4,
1209.8, 1174.2, 1113.9, 872.5, 745.8; 1H NMR (600 MHz, CDCl3) δ 11.86 (s, 1H), 8.46 (s, 1H), 8.31 (d, J = 7.9 Hz,
1H), 8.11 (d, J = 5.7 Hz, 1H), 7.97 (dd, J = 12.1, 2.1 Hz, 1H), 7.80 (s, 1H), 7.74 – 7.67 (m, 1H), 7.59 (t, J = 7.4 Hz,
1H), 7.52 (t, J = 7.5 Hz, 1H), 7.43 – 7.35 (m, 2H), 7.23 – 7.08 (m, 3H), 6.80 (d, J = 8.4 Hz, 1H), 6.59 (dd, J = 5.7, 2.1
Hz, 1H), 1.57 – 1.47 (m, 1H), 1.09 – 1.02 (m, 2H), 0.89 – 0.81 (m, 2H); MS(ESI)m/z(%): 554.1 [M+H]+, 576.1
[M+Na]+; Anal. calcd. for C30H21F2N5O4 (%): C, 65.10; H, 3.82; N, 12.65. Found (%): C, 65.23; H, 3.83; N, 12.61.
4.1.5.8.
4-(3-chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide (23h).
Yield: 79.3%; M.p.: 235-238 C; IR (KBr, cm-1): 3421.9, 3060.4, 2923.1, 1694.0, 1580.4, 1507.1, 1428.3, 1319.6,
1
1208.5, 1174.5, 1097.4, 955.1, 871.1, 821.4, 751.8; H NMR (600 MHz, DMSO-d6) δ 11.26 (s, 1H), 10.88 (s, 1H),
8.21 (d, J = 5.7 Hz, 1H), 8.08 – 7.99 (m, 1H), 7.94 (dd, J = 12.6, 2.3 Hz, 1H), 7.77 – 7.70 (m, 2H), 7.69 (s, 1H), 7.66
(d, J = 2.2 Hz, 1H), 7.64 – 7.59 (m, 1H), 7.58 – 7.54 (m, 1H), 7.51 – 7.46 (m, 2H), 7.41 (t, J = 8.9 Hz, 1H), 6.97 – 6.49
(m, 2H), 2.09 – 1.79 (m, 1H), 0.98 – 0.52 (m, 4H); MS (ESI) m/z(%): 569.3 [M]+, 592.1 [M+Na]+; Anal. calcd. for
C30H21ClFN5O4 (%): C, 63.22; H, 3.71; N, 12.29. Found (%): C, 63.20; H, 3.74; N, 12.27.
4.1.5.9.
N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(2-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide. (23i).

Yield: 77.4%; M.p.: 257-259 C; IR (KBr, cm-1): 3423.6, 3071.4, 2923.1, 1693.7, 1597.6, 1503.3, 1425.6, 1306.4,
1207.0, 1182.5, 1105.7, 954.8, 753.7; 1H NMR (600 MHz, CDCl3) δ 11.84 (s, 1H), 8.40 – 8.26 (m, 2H), 8.11 (d, J =
5.8 Hz, 1H), 7.98 (dd, J = 12.1, 2.3 Hz, 1H), 7.80 (d, J = 1.7 Hz, 1H), 7.72 – 7.64 (m, 1H), 7.63 – 7.57 (m, 1H), 7.56
– 7.51 (m, 1H), 7.50 – 7.38 (m, 4H), 7.15 (t, J = 8.7 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.59 (dd, J = 5.8, 2.3 Hz, 1H),
1.55 – 1.46 (m, 1H), 1.09 – 1.04 (m, 2H), 0.89 – 0.82 (m, 2H); MS (ESI) m/z (%): 554.1 [M+H]+, 576.1 [M+Na]+;
Anal. calcd. for C30H21F2N5O4 (%): C, 65.10; H, 3.82; N, 12.65. Found (%): C, 65.16; H, 3.79; N, 12.69.
4.1.5.10. 4-(2-chlorophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide. (23j).
Yield: 80.4%; M.p.: 261-263 C; IR (KBr, cm-1): 3432.4, 2917.6, 1698.0, 1596.7, 1580.5, 1530.9, 1507.0, 1433.0,
1
1298.2, 1208.4, 1179.7, 1094.7, 1056.3, 954.8, 864.2, 749.2; H NMR (600 MHz, CDCl3) δ 11.87 (s, 1H), 8.63 (s,
1H), 8.32 (d, J = 8.0 Hz, 1H), 8.11 (d, J = 5.8 Hz, 1H), 7.98 (dd, J = 12.1, 2.1 Hz, 1H), 7.81 (s, 1H), 7.77 – 7.69 (m,
1H), 7.62 (s, 3H), 7.53 (t, J = 7.5 Hz, 1H), 7.42 (dd, J = 10.3, 5.0 Hz, 2H), 7.15 (t, J = 8.6 Hz, 1H), 6.68 (d, J = 8.3
Hz, 1H), 6.59 (dd, J = 5.7, 2.1 Hz, 1H), 1.57 – 1.47 (m, 1H), 1.07 (dd, J = 7.1, 4.0 Hz, 2H), 0.88 – 0.82 (m, 2H);
MS(ESI) m/z(%): 570.1 [M+H]+, 592.1 [M+Na]+; Anal. calcd. for C30H21ClFN5O4 (%): C, 63.22; H, 3.71; N, 12.29.
Found (%): C, 63.28; H, 3.76; N, 12.31.
4.1.5.11. 4-(4-bromophenyl)-N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide (23k).
Yield: 82.2%; M.p.: 269-271 C; IR (KBr, cm-1): 3433.9, 2923.1, 1692.9, 1596.7, 1533.1, 1506.2, 1462.3, 1427.9,
1305.9, 1205.9, 1179.7, 1012.4, 828.6; 1H NMR (600 MHz, DMSO-d6) δ 11.26 (s, 1H), 10.88 (s, 1H), 8.21 (d, J = 5.7
Hz, 1H), 8.03 (dd, J = 8.0, 1.2 Hz, 1H), 7.98 – 7.82 (m, 3H), 7.65 (d, J = 2.3 Hz, 1H), 7.63 – 7.57 (m, 1H), 7.55 (dd, J
= 8.9, 1.4 Hz, 1H), 7.51 – 7.44 (m, 3H), 7.41 (t, J = 8.9 Hz, 1H), 6.84 – 6.63 (m, 2H), 2.02 – 1.92 (m, 1H), 0.81 – 0.74
(m, 4H); MS (ESI) m/z(%): 636.0 [M+Na]+; Anal. calcd. for C30H21BrFN5O4 (%): C, 58.64; H, 3.45; N, 11.40. Found
(%): C, 58.60; H, 3.48; N, 11.39.
4.1.10.12. N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-4-(p-tolyl)-3,4-dihydro
quinoxaline-2-carboxamide (23l).
Yield: 85.8%; M.p.: 257-259 C; IR (KBr, cm-1): 3434.1, 2917.6, 1693.0, 1576.7, 1533.3, 1506,2, 1462.3, 1425.1,
1309.4, 1180.6, 1091.9, 828.6, 769.7; 1H NMR (600 MHz, DMSO-d6) δ 11.28 (s, 1H), 10.88 (s, 1H), 8.21 (d, J = 5.7
Hz, 1H), 8.02 (dd, J = 8.0, 1.2 Hz, 1H), 7.93 (dd, J = 12.6, 2.3 Hz, 1H), 7.66 (d, J = 2.3 Hz, 1H), 7.62 – 7.57 (m, 1H),
7.54 (dd, J = 8.8, 1.4 Hz, 1H), 7.52 – 7.45 (m, 3H), 7.41 (t, J = 8.9 Hz, 1H), 7.33 (d, J = 8.2 Hz, 2H), 6.85 – 6.59 (m,
2H), 2.45 (s, 3H), 2.04 – 1.88 (m, 1H), 0.82 – 0.72 (m, 4H); MS (ESI) m/z (%): 550.2 [M+H]+, 572.1 [M+Na]+; Anal.

calcd. for C31H24FN5O4 (%): C, 67.75; H, 4.40; N, 12.74. Found (%): C, 67.83; H, 4.49; N, 12.77.
4.1.5.13. N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-methoxyphenyl)-3-oxo-3,4-
dihydroquinoxaline-2-carboxamide (23m).
Yield: 88.9%; M.p.: 210-212 C; IR (KBr, cm-1): 3423.4, 2913.1, 1693.8, 1643.4, 1597.2, 1577.5, 1508.8, 1462.4,
1424.5, 1303.1, 1252.8, 1182.3, 1031.6, 954.8, 831.8, 767.2; 1H NMR (600 MHz, DMSO-d6) δ 11.29 (s, 1H), 10.87
(s, 1H), 8.21 (d, J = 5.7 Hz, 1H), 8.01 (dd, J = 8.0, 1.1 Hz, 1H), 7.93 (dd, J = 12.6, 2.3 Hz, 1H), 7.66 (d, J = 2.2 Hz,
1H), 7.63 – 7.57 (m, 1H), 7.54 (dd, J = 8.9, 1.4 Hz, 1H), 7.50 – 7.44 (m, 1H), 7.44 – 7.34 (m, 3H), 7.30 – 7.15 (m,
2H), 6.79 – 6.68 (m, 2H), 3.87 (d, J = 9.4 Hz, 3H), 2.14 – 1.71 (m, 1H), 0.98 – 0.47 (m, 4H); MS (ESI) m/z (%): 566.1
[M+H]+, 588.1 [M+Na]+; Anal. calcd. for C31H24FN5O5 (%): C, 65.84; H, 4.28; N, 12.38. Found (%): C, 65.86; H,
4.31; N, 12.34.
4.1.5.14.
N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(2,4-dichlorophenyl)-3- oxo-
3,4-dihydroquinoxaline-2-carboxamide (23n).
Yield: 89.6%; M.p.: 240-243 C; IR (KBr, cm-1): 3433.3, 3076.9, 2923.1, 1693.4, 1596.7, 1533.6, 1506.9, 1428.3,
1303.2, 1205.1, 1097.4, 872.5, 762.8; 1H NMR (600 MHz, DMSO-d6) δ 11.26 (s, 1H), 10.88 (s, 1H), 8.21 (d, J = 5.7
Hz, 1H), 8.15 – 8.03 (m, 2H), 7.93 (dd, J = 12.6, 2.3 Hz, 1H), 7.79 (dd, J = 8.5, 2.3 Hz, 1H), 7.76 – 7.69 (m, 1H), 7.70
– 7.59 (m, 2H), 7.61 – 7.54 (m, 1H), 7.52 (dd, J = 11.2, 4.1 Hz, 1H), 7.42 (t, J = 8.9 Hz, 1H), 6.79 – 6.68 (m, 2H), 2.01
– 1.93 (m, 1H), 0.82 – 0.72 (m, 4H); MS (ESI) m/z (%): 626.0 [M+Na]+; Anal. calcd. for C30H20 Cl2FN5O4 (%): C,
59.62; H, 3.34; N, 11.59. Found (%): C, 59.58; H, 3.37; N, 11.64.
4.1.5.15. N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(2,4-dimethoxyphenyl)-3- oxo-
3,4-dihydroquinoxaline-2-carboxamide (23o).
Yield: 86.6%; M.p.: 241-244 C; IR (KBr, cm-1): 3423.5, 3071.4, 2840.6, 1688.3, 1600.9, 1509.3, 1463.8, 1427.6,
1308.5, 1210.8, 1114.2, 1029.6, 954.8, 832.6, 765.5; 1H NMR (600 MHz, DMSO-d6) δ 11.28 (s, 1H), 10.87 (s, 1H),
8.21 (d, J = 5.7 Hz, 1H), 8.00 (d, J = 7.0 Hz, 1H), 7.92 (dd, J = 12.6, 2.3 Hz, 1H), 7.65 (d, J = 2.1 Hz, 1H), 7.62 – 7.58
(m, 1H), 7.55 (d, J = 10.1 Hz, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.41 (t, J = 8.9 Hz, 1H), 7.29 (d, J = 8.6 Hz, 1H), 6.89 (d,
J = 2.5 Hz, 1H), 6.80 – 6.71 (m, 3H), 3.89 (s, 3H), 3.72 (s, 3H), 2.02 – 1.88 (m, 1H), 0.84 – 0.70 (m, 4H); MS (ESI)
m/z (%): 596.2 [M+H]+, 618.1 [M+Na]+; Anal. calcd. for C32H26FN5O6 (%): C, 64.53; H, 4.40; N, 11.76. Found (%):
C, 64.48; H, 4.45; N, 11.74.
4.1.5.16.
N-(4-((2-(cyclopropylcarbamoyl)pyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-4-phenyl-3,4-dihydro
quinoxaline-2-carboxamide(23p).
Yield: 83.4%; M.p.: 269-271 C; IR (KBr, cm-1): 3400.5, 3072.6, 2924.1, 1687.7, 1595.1, 1535.3, 1298.1, 1213.2,

1085.9, 916.9, 846.8, 761.9; 1H NMR (600 MHz, DMSO-d6) δ 11.33 (s, 1H), 8.76 (d, J = 4.4 Hz, 1H), 8.52 (d, J = 5.5
Hz, 1H), 8.03 (d, J = 7.8 Hz, 1H), 7.98 (d, J = 12.1 Hz, 1H), 7.76 – 7.67 (m, 2H), 7.66 – 7.56 (m, 3H), 7.54 – 7.38 (m,
5H), 7.22 (d, J = 2.9 Hz, 1H), 6.68 (d, J = 8.4 Hz, 1H), 2.97 – 2.79 (m, 1H), 0.79 – 0.56 (m, 4H); MS (ESI) m/z (%):
536.1 [M+H]+, 558.1 [M+Na]+; Anal. calcd. for C30H22FN5O4 (%): C, 67.28; H, 4.14; N, 13.08. Found (%): C, 67.34;
H, 4.16; N, 13.15.
4.1.5.17. N-(4-((2-(cyclopropylcarbamoyl)pyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-fluorophenyl)-3-oxo-3,4-dihydro
quinoxaline-2-carboxamide (23q).
Yield: 80.5%; M.p.: 210-212 C; IR (KBr, cm-1): 3371.6, 3072.6, 1687.7, 1591.3, 1510.3, 1296.2, 1199.7, 952.8,
862.5, 761.9; 1H NMR (600 MHz, DMSO-d6) δ 11.32 (s, 1H), 8.76 (d, J = 5.0 Hz, 1H), 8.52 (d, J = 5.6 Hz, 1H), 8.10
– 7.91 (m, 2H), 7.68 – 7.37 (m, 9H), 7.22 (dd, J = 5.6, 2.6 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 3.00 – 2.77 (m, 1H), 0.75
– 0.61 (m, 4H); MS (ESI) m/z (%): 554.2 [M+H]+, 576.2 [M+Na]+; Anal. calcd. for C30H21F2N5O4 (%): C, 65.10; H,
3.82; N, 12.65. Found (%): C, 65.16; H, 3.80; N, 12.70.
4.1.5.18.
N-(4-((2-acetamidopyridin-4-yl)oxy)-3-fluorophenyl)-3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-
carboxamide (23r)
Yield: 79.9%; M.p.: 230-233 C; IR (KBr, cm-1): 3435.1, 2923.1, 1695.1, 1598.1, 1533.6, 1507.2, 1462.3, 1428.9,
1265.2, 1205.1, 1020.6, 971.2, 869.7; 1H NMR (600 MHz, DMSO-d6) δ 11.29 (s, 1H), 10.57 (s, 1H), 8.20 (d, J = 5.7
Hz, 1H), 8.09 – 7.99 (m, 1H), 7.94 (dd, J = 12.6, 2.3 Hz, 1H), 7.75 – 7.58 (m, 5H), 7.55 (dd, J = 8.8, 1.3 Hz, 1H), 7.52
– 7.45 (m, 3H), 7.42 (t, J = 8.9 Hz, 1H), 6.77 – 6.58 (m, 2H), 2.05 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 169.92,
165.66, 162.11, 154.35, 153.88 (d, J = 246.1 Hz), 153.12, 151.36, 149.99, 137.43 (d, J = 9.8 Hz), 136.38 (d, J = 12.4
Hz), 135.61, 135.04, 132.68, 131.78, 130.69, 130.48, 130.04, 128.75, 124.77, 124.62, 116.82, 115.94, 108.77 (d, J =
22.9 Hz), 107.39, 99.90, 24.28; MS (ESI) m/z(%):532.1 [M+Na]+; Anal. calcd. for C28H20FN5O4 (%): C, 66.01; H,
3.96; N, 13.75. Found (%): C, 66.12; H, 4.01; N, 13.80.
4.1.5.19. N-(4-((2-acetamidopyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-
2- carboxamide (23s).
Yield: 80.9%; M.p.: 254-256 C; IR (KBr, cm-1): 3429.0, 2917.6, 2851.6, 1694.3, 1596.7, 1583.0, 1533.6, 1507.8,
1426.6, 1265.6, 1204.4, 1152.3, 965.7, 760.0; 1H NMR (600 MHz, DMSO-d6) δ 11.28 (s, 1H), 10.57 (s, 1H), 8.20 (d,
J = 5.7 Hz, 1H), 8.07 – 8.00 (m, 1H), 7.94 (dd, J = 12.6, 2.3 Hz, 1H), 7.67 (s, 1H), 7.64 – 7.58 (m, 1H), 7.55 (dd, J =
8.8, 3.9 Hz, 5H), 7.48 (t, J = 7.6 Hz, 1H), 7.42 (t, J = 8.9 Hz, 1H), 6.77 – 6.67 (m, 2H), 2.04 (s, 3H); MS (ESI) m/z
(%): 550.1 [M+Na]+; Anal. calcd. for C28H19F2N5O4 (%): C, 63.76; H, 3.63; N, 13.28. Found (%): C, 63.88; H, 3.59;
N, 13.23.

4.1.5.20. N-(4-((2-acetamidopyridin-4-yl)oxy)-3-fluorophenyl)-4-(2-chlorophenyl)-3-oxo-3,4-dihydroquino xaline-
2-carboxamide (23t).
Yield: 85.6%; M.p.: 220-222 C; IR (KBr, cm-1): 3428.9, 2917.6, 2846.2, 1695.5, 1637.9, 1508.9, 1536.3, 1503.4,
1481.5, 1429.8, 1265.4, 1203.9, 1149.6, 1094.7, 749.8; 1H NMR (600 MHz, DMSO-d6) δ 11.28 (s, 1H), 10.58 (s, 1H),
8.20 (d, J = 5.7 Hz, 1H), 8.11 – 8.03 (m, 1H), 7.94 (dd, J = 12.6, 2.3 Hz, 1H), 7.89 – 7.83 (m, 1H), 7.74 – 7.61 (m,
5H), 7.60 – 7.55 (m, 1H), 7.51 (t, J = 7.6 Hz, 1H), 7.43 (t, J = 8.9 Hz, 1H), 6.71 (dd, J = 5.7, 2.4 Hz, 1H), 6.62 (d, J =
8.4 Hz, 1H), 2.05 (s, 3H); MS (ESI) m/z (%): 544.1 [M+H]+, 566.1 [M+Na]+; Anal. calcd. for C28H19ClFN5O4 (%): C,
61.83; H, 3.52; N, 12.88. Found (%): C, 61.76; H, 3.48; N, 12.90.
4.1.5.21.
N-(4-((2-acetamidopyridin-4-yl)oxy)-3-fluorophenyl)-4-(4-methoxyphenyl)-3-oxo-3,4-dihydroquinoxa
line-2-carboxamide (23u).
Yield: 82.4%; M.p.: 239-241 C; IR (KBr, cm-1): 3435.5, 3076.9, 2917.6, 1684.9, 1646.8, 1603.3, 1510.5, 1465.0,
1423.1, 1297.7, 1251.8, 1198.1, 1166.0, 1113.9, 1078.2, 758.3; 1H NMR (600 MHz, DMSO-d6) δ 11.29 (s, 1H), 10.57
(s, 1H), 8.20 (d, J = 5.7 Hz, 1H), 8.02 (dd, J = 8.0, 1.1 Hz, 1H), 7.94 (dd, J = 12.7, 2.3 Hz, 1H), 7.68 (s, 1H), 7.63 –
7.58 (m, 1H), 7.57 – 7.52 (m, 1H), 7.50 – 7.45 (m, 1H), 7.44 – 7.35 (m, 3H), 7.22 (d, J = 8.9 Hz, 2H), 6.78 – 6.65 (m,
2H), 3.88 (s, 3H), 2.05 (s, 3H); MS (ESI) m/z (%): 562.1 [M+Na]+; Anal. calcd. for C29H22FN5O5 (%): C, 64.56; H,
4.11; N, 12.98. Found (%): C, 64.64; H, 4.09; N, 13.06.
4.1.5.22. N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)phenyl)-3-oxo-4-phenyl-3,4-dihydroquinoxa line-2-
carboxamide (23v).
Yield: 82.9%; M.p.: 237-239 C; IR (KBr, cm-1): 3433.3, 1687.7, 1535.3, 1506.4, 1421.5, 1305.8, 1207.4, 758.0;
1H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.82 (s, 1H), 8.19 (d, J = 5.7 Hz, 1H), 8.02 (dd, J = 8.0, 1.2 Hz, 1H),
7.81 (d, J = 8.9 Hz, 2H), 7.70 (t, J = 7.6 Hz, 2H), 7.68 – 7.61 (m, 2H), 7.62 – 7.56 (m, 1H), 7.51 – 7.42 (m, 3H), 7.21
(d, J = 8.9 Hz, 2H), 6.76 – 6.61 (m, 2H), 2.02 – 1.90 (m, 1H), 0.86 – 0.68 (m, 4H); MS (ESI) m/z (%): 540.1 [M+Na]+;
Anal. calcd. for C30H23N5O4 (%): C, 69.62; H, 4.48; N, 13.53. Found (%): C, 69.74; H, 4.49; N, 13.46.
4.1.5.23.
N-(4-((2-(cyclopropanecarboxamido)pyridin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)-3-oxo-3,4-dihydro
quinoxaline-2-carboxamide (23w).
Yield: 81.6%; M.p.: 245-247 C; IR (KBr, cm-1): 3422.3, 3063.0, 2374.4, 1687.7, 1591.3, 1508.3, 1425.4, 1305.8,
1217.1, 1166.9, 952.8, 839.0, 763.8; 1H NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 10.82 (s, 1H), 8.19 (d, J = 5.7
Hz, 1H), 8.02 (dd, J = 8.0, 1.1 Hz, 1H), 7.81 (d, J = 8.9 Hz, 2H), 7.65 (d, J = 2.3 Hz, 1H), 7.64 – 7.57 (m, 1H), 7.58 –
7.50 (m, 4H), 7.48 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 8.9 Hz, 2H), 6.76 – 6.64 (m, 2H), 2.05 – 1.84 (m, 1H), 0.85 – 0.65
(m, 4H); 13C NMR (150 MHz, DMSO-d6) δ 173.15, 166.21, 162.67 (d, J = 246.5 Hz), 161.76, 154.26, 153.31, 151.62,

149.93, 149.87, 136.03, 135.06, 132.58, 131.84, 131.15 (d, J = 8.9 Hz), 130.45, 124.77, 121.92, 121.67, 117.74, 117.59,
115.91, 108.34, 100.70, 14.58, 8.05; MS (ESI) m/z(%): 558.1 [M+Na]+; Anal. calcd. for C30H22FN5O4 (%): C, 67.28;
H, 4.14; N, 13.08. Found (%): C, 67.36; H, 4.10; N, 13.06.
4.1.5.24.
N-(4-((2-acetamidopyridin-4-yl)oxy)phenyl)-3-oxo-4-phenyl-3,4-dihydroquinoxaline-2-carboxamide
(23x).
Yield: 79.5%; M.p.: 243-245 C; IR (KBr, cm-1): 3267.4, 3061.0, 1693.5, 1591.3, 1537.3, 1502.6, 1425.4, 1301.9,
1211.3, 1159.2, 1091.7, 844.8;1H NMR (600 MHz, DMSO-d6) δ 11.10 (s, 1H), 10.52 (s, 1H), 8.18 (d, J = 5.7 Hz, 1H),
8.02 (d, J = 7.9 Hz, 1H), 7.82 (d, J = 8.9 Hz, 2H), 7.74 – 7.56 (m, 5H), 7.51 – 7.42 (m, 3H), 7.21 (d, J = 8.9 Hz, 2H),
6.71 – 6.61 (m, 2H), 2.04 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 169.81, 166.14, 161.85, 154.28, 153.20, 151.76,
150.00, 149.88, 136.04, 135.65, 134.98, 132.52, 131.82, 130.69, 130.43, 130.02, 128.76, 124.73, 121.84, 121.66,
115.91, 108.22, 100.96, 24.29; MS (ESI) m/z (%): 492.2 [M+H]+, 514.2 [M+Na]+; Anal. calcd. for C28H21N5O4 (%):
C, 68.42; H, 4.31; N, 14.25. Found (%): C, 68.38; H, 4.34; N, 14.19.
4.1.5.25.
N-(4-((2-acetamidopyridin-4-yl)oxy)phenyl)-4-(4-fluorophenyl)-3-oxo-3,4-dihydroquinoxaline-2-
carboxamide (23y).
Yield: 79.4%; M.p.: 253-256 C; IR (KBr, cm-1): 3064.9, 1695.4, 1587.4, 1367.5, 1220.9, 11161.2, 1020.3, 968.3,
846.8; 1H NMR (600 MHz, DMSO-d6) δ 11.09 (s, 1H), 10.52 (s, 1H), 8.18 (d, J = 5.7 Hz, 1H), 8.02 (dd, J = 8.0, 1.1
Hz, 1H), 7.82 (d, J = 8.9 Hz, 2H), 7.67 (s, 1H), 7.65 – 7.43 (m, 6H), 7.21 (d, J = 8.9 Hz, 2H), 6.71 (d, J = 8.5 Hz, 1H),
6.66 (dd, J = 5.7, 2.4 Hz, 1H), 2.04 (s, 3H); 13C NMR (150 MHz, DMSO-d6) δ 169.81, 166.13, 162.67 (d, J = 246.6
Hz), 161.75, 154.28, 153.33, 151.58, 150.01, 149.88, 136.02, 135.06, 132.58, 131.85, 131.15 (d, J = 9.1 Hz), 130.46,
124.77, 121.83, 121.68, 117.74, 117.59, 115.91, 108.22, 100.96, 24.28; MS (ESI) m/z (%): 510.2 [M+H]+, 532.2
[M+Na]+; Anal. calcd. for C28H20FN5O4 (%): C, 66.01; H, 3.96; N, 13.75. Found (%): C, 66.05; H, 4.00; N, 13.74.
4.2. Pharmacology
4.2.1. c-Met kinase assay
The in vitro enzymatic assays were performed by mobility shift assay. The solution of peptide substrates, ATP,
appropriate kinase (Carna), and various concentrations of tested compounds were mixed with the kinase reaction buffer
(50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, 2 mM DTT), with blank DMSO as the negative control.
The kinase reaction was initiated by the addition of tyrosine kinase proteins diluted in 39 µL of kinase reaction buffer
solution and incubated at 28 °C for 1 h. Then, 25 µL of stop buffer (100 mM HEPES, pH = 7.5, 0.015% Brij-35, 0.2%
Coating Reagent #3, 50 mM EDTA) was added to stop reaction. The data were cellected on Caliper at 320 nm and 615
nm and converted to inhibition values. IC50 was presented in MS Excel and the curves fitted by XLfit excel add-in

version
4.2.2. MTT assay in vitro
The anti-proliferative activities of tested compounds were evaluated against A549, H460 and HT-29 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.
4.2.3. AO/ EB assay
A549 or HT-29 cells were added to a final concentration of 1×106/mL in a 6-well plate, and the plate was
incubated for 24 h. Cells were treated with various concentrations of compound 23w. After being cultured for 48 h,
control cells and treated cells were washed with PBS which stored at 4 °C, and then dual fluorescent staining solution
(20 μL) containing 100 μg/mL AO and 100 μg/mL EB was added to each well for 10 min, and then covered with a
coverslip. The morphology of apoptotic cells was examined using fluorescent microscope.
4.2.4. Apoptosis assay
Apoptosis was measured by flow cytometry using Annexin V/propidium iodide (PI) double staining. HT-29 or
A549 cells were added to a final concentration of 1×106/mL in a 6-well plate, and the plate was incubated for 24 h.
Cells were treated with various concentrations of compound 23w. After being cultured for 48 h, control cells and
treated cells were harvested and washed with PBS, and then resuspended in 100μL 1×binding buffer incubated in the
mixture of 5μL Annexin V-FTIC and 5μL PI for 10 min at room temperature in dark place. The cells were resuspended
in 400μL 1×binding buffer just before flow cytometric analysis.
4.2.5. Wound-healing assay
A549 cells were added to a final concentration of 1×106 cells /mL in a 6-well plate, and the plate was incubated
for 24 h. Twenty-four hours later, when the cells reached confluency, scratches were created with sterile 1.0 mL pipette

tips and images were captured using phase contrast microscopy at 0 h, 12 h, 36 h and 72 h after treatment with 1.0 μM
of compound 23w.
4.2.6. Transwell migration assay
A549 cells were plated at a concentration of 1x105 cells / mL per well in 6-well plates for 8 h and then treated
with various concentrations of compound 23w for 24 h. Then cells were harvested and resuspended as single cells in
RPMI1640 with 1.0% FBS at the concentration of 1x105cells/mL. Abave cells solution 100 μL were added to the upper
chamber and the lower chamber was filled with 600 μL RPMI1640 supplemented with 10% FBS. Cells were allowed
to migrate for 24 h at 37 C in a 5% CO2 humidified incubator. The experiment was terminated by discarding the
medium and fixing the cells with MeOH for 30 min. Non-invading cells on the upper side of the insert were removed
by a cotton-tipped applicator. Staining of the cells on the bottom of the membrane was performed with crystal violet
(0.1%) for 10 min at room temperature and washed with PBS. Then lower surface was captures and counted under an
inverted microscope (Olympus).
4.2.7. Molecular docking study
The crystal structure of c-Met (PDB code: 3LQ8) in complex with foretinib (GSK1363089) was used for
molecular modeling. 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 protein coordinates (PDB code: 3LQ8) were downloaded
from the Protein Data Bank (http://www.rcsb.org/pdb/). For enzyme preparation, the hydrogen atoms were added. The
whole c-Met enzyme was defined as a receptor and the site sphere was selected on the basis of the ligand binding
location of foretinib. Compound foretinib was removed and compound 23w was placed.. Accelrys Discovery Studio
6.0 system was used for graphic display.
Acknowledgments
The authors thank the financial support of General Project of Education Department of Liaoning Province (project
no. LJC201907), Natural Science Foundation of Liaoning Provincial Department of Science and Technology (project
no. 2019-ZD-0191), Shenyang Science & Technology Project (project no. 18-013-0-03) and The National College
Students' innovation and entrepreneurship training program (project no. D201912311824318027)
Appendix A. Supplementary data
Supplementary data associated with this article can be found in the online version,

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