Identification of novel quinoline analogues bearing thiazolidinones as potent kinase inhibitors for the treatment of colorectal cancer

Article abstract of DOI:10.1016/j.ejmech.2020.112643

In this investigation, a novel series of quinoline analogues bearing thiazolidinones were designed and synthesized based on our previous study. Among them, the most potent compound 11k, 4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phen

Full text of DOI:10.1016/j.ejmech.2020.112643

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Identification of Novel Quinoline Analogues Bearing Thiazolidinones as Potent Kinase
Inhibitors for the Treatment of Colorectal Cancer
Yuting Zhou, Xingwei Xu, Fei Wang, Huan He, Guowei Gong, Li Xiong, Baohui Qi
PII:
S0223-5234(20)30615-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112643
EJMECH 112643
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 May 2020
Revised Date: 30 June 2020
Accepted Date: 1 July 2020
Please cite this article as: Y. Zhou, X. Xu, F. Wang, H. He, G. Gong, L. Xiong, B. Qi, Identification
of Novel Quinoline Analogues Bearing Thiazolidinones as Potent Kinase Inhibitors for the
Treatment of Colorectal Cancer, European Journal of Medicinal Chemistry, https://doi.org/10.1016/
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Identification of Novel Quinoline Analogues Bearing Thiazolidinones as Potent
Kinase Inhibitors for the Treatment of Colorectal Cancer
1
Yuting Zhou, Xingwei Xu, Fei Wang, Huan He, Guowei Gong, Li Xiong, Baohui Qi*
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai, 519041, Guangdong
Province, China
Graphical Abstract
*Corresponding authors.
E-mail address: bhqi@zmu.gd.cn.

Identification of Novel Quinoline Analogues Bearing Thiazolidinones as Potent
Kinase Inhibitors for the Treatment of Colorectal Cancer
1
Yuting Zhou, Xingwei Xu, Fei Wang, Huan He, Guowei Gong, Li Xiong, Baohui Qi*
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai, 519041,
Guangdong Province, China
Abstract:
In this investigation, a novel series of quinoline analogues bearing thiazolidinones
were designed and synthesized based on our previous study. Among them, the most
potent
compound
11k,
4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxy
quinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide, possessed submicromolar
c-Met and Ron inhibitory activities. In addition, enzymatic assays against a
mini-panel of kinases (c-Kit, B-Raf, c-Src, IGF1R, PDGFRα and AXL) were
performed, the results showed that compound 11k exhibited moderate inhibitory
activity against PDGFRα, c-Src and AXL. MTT assay revealed in vitro antitumor
activities against HT-29 cells of compound 11k with an IC50 value of 0.31 μM which
was 9.3- and 34.2-fold more potent than that of Regorafenib (IC50=2.87 μM) and
Cabozantinib (IC50=10.6 μM). Preliminary antitumor mechanisms were also
investigated by cellular assays. Considerable cytotoxicity, antiproliferation and
induction of apoptosis of compound 11k in a dose- and time-dependent manner were
confirmed by IncuCyte live-cell imaging assays. Treatment with compound 11k
caused slight G2- or M-phase arrest in HT-29 cells. Further cell selectivity of
compound 11k showed that it was not active against human normal colorectal mucosa
epithelial cell FHC at 10.0 μg/mL. The above results support further structural
modification of compound 11k to improve its inhibitory activity, which will lead to
more potent anticancer agents.
Keywords:
Anticancer;
Tyrosine
Kinase;
Inhibitors;
Quinoline;
1,
3-Thiazolidin-4-one
1. Introduction
Numerous study indicated that receptor and non-receptor protein kinases played an
important role in malignant tumor development and progression [1, 2]. Thus, the
discovery of drugs targeting receptor or non-receptor protein kinases has comprised a
substantial proportion of pharmaceutical research over the past two decades in
worldwide. Among the 58 receptor tyrosine kinases, c-Met (Mesenchymal-epithelial
transition factor) and Ron (Récepteur d′Origine Nantais) has drawn significant
attention for their ability of the oncogenic forms to confer growth advantage,
*Corresponding authors.
E-mail address: bhqi@zmu.gd.cn.

protection from apoptosis, invasive properties, and resistance to chemotherapies [3-6].
c-Met, also known as hepatocyte growth factor receptor (HGFR), is a critical
member of receptor tyrosine kinase. The c-Met/HGF recruits numerous transduction
proteins and triggers its downstream signaling pathways, including phosphoinositide
3-kinase/threonine-protein kinase (PI3K/AKT) axis, mitogen-activated protein kinase
(MAPK) cascades, Rac1-Cdc42 and STAT pathways, etc [6, 7]. Overexpression and
mutation of c-Met is implicated in multiple tumor oncogenic processes, such as
tumorigenesis, metastasis, poor prognosis, angiogenesis, invasive growth, and drug
resistance [8-10].
Ron, also known as human macrophage stimulating 1 receptor (MST1R), is the
only other member of c-Met family. The structures of the two kinases are very similar,
and they share 34% overall homology and the tyrosine kinase region of the receptors
share 80% homology [11, 12]. RON gene is rarely or not expressed in normal
epithelial tissue, while in bladder, lung, thyroid, colon, skin, breast and pancreas
tumors it is highly expressed and is usually accompanied by the generation of variants
[13]. The phosphorylated Ron can activate a great number of transduction proteins
and trigger its downstream signaling cascades, including c-Src, β-catenin/TCF-4,
ERK1/2, MAPK, SMAD/TGF-β, JNK/STAT, and PI3K/AKT, etc [14-16]. The
dysregulation of Ron has shown to be associated extensively with the process of the
induction and development of tumorigenesis, such as cell proliferation, migration,
invasion, survival, angiogenesis, epithelial-to-mesenchymal transition (EMT) and
drug resistance [17, 18].
Over expression and/or aberrant activation of c-Met and Ron in different kinds of
cancers suggested that they were important targets for cancer therapy. In the past
decades, there has been a flourish of activity to develop small molecule c-Met and/or
Ron kinase inhibitors, and a number of structurally diverse inhibitors have been
reported, such as BMS777607, Crizotinib, Cabozantinib, MK-8033, Capmatinib and
Merestinib (Fig. 1) [19-25]. However, the majority of studies have been limited to in
vitro experiments due to unfavorable pharmacologic and/or pharmacokinetic
properties, as well as intolerable toxicity. Based on our previous study, we report the
identification of novel quinoline analogues bearing thiazolidinones as potent
inhibitors of c-Met and Ron kinases, as well as their antitumor activity and antitumor
mechanism. In addition, the structure-activity relationship (SAR) and docking studies
are also presented in the present work.

Fig. 1 The representative c-Met and/or Ron inhibitors.
2. The design of novel compounds
We previously disclosed
a
multi-tyrosine
kinase
inhibitor,
N1-(4-((7-(3-(4-ethylpiperazin-1-yl)propoxy)-6-methoxyquinolin-4-yl)oxy)-3,5-difluo
rophenyl)-N3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)urea, which showed IC50
values of 15.0 nM and 2.9 nM against c-Met and Ron, respectively (Fig. 2) [26]. The
results of SARs indicated: (1) the terminal heterocycles of part I reached out into the
solvent, and it was a potential position to improve the anticancer activity and the
aqueous solubility (Fig. 2 and Fig. 3C); (2) a phenyl ring substituted by fluorine atom
in Part II was well tolerated; (3) the nitrogen atom of quinoline form a strong H-bond
with Met1160 (Fig. 3B) [26].
Fig. 2 Our previous work and the design of novel compounds in the present work.
According to the structure of Vandetanib, we first modified the structure of the
compound obtained in our previous work by replacing of the
(4-ethylpiperazin-1-yl)propoxy
group
(part
I)
with
substituted
piperidine-1-carboxamide, a fragment bearing hydrophilic groups (Fig. 2). The best
position and the number of fluorine atoms were further investigated in the following
modification of part II. Subsequently, some compounds were synthesized by changing
the linker atom (O or S) between phenyl ring A and quinoline and the position (3- or
4-) of thiazolidinone urea attached to the phenyl ring A.

Fig. 3 The binding mode of compound obtained in our previous work with c-Met kinase (PDB ID:
3LQ8). The H-bonds were represented by green dotted lines.
Docking study showed that the distance between the oxygen in carbonyl group of
Pro1158 and the carbon at the 2-position of quinoline ring was 3.33 Å (Fig. 3A). Thus,
an additional H-bond might be formed by introducing a H-bond donor at the
2-position of quinoline. Hence, a 2-aminoquinoline derivate was designed and
synthesized in the present work.
Based on the above assumption, totally nineteen compounds were designed,
prepared, and evaluated for their biological activity in the present work.
3. Results and Discussion
3.1. Chemistry
All newly compounds were prepared by synthetic routes outlined in Schemes 1-5.
Scheme 1 showed the synthetic routes for target compounds 11a-o. Starting from
commercially available 7-(benzyloxy)-4-chloro-6-methoxyquinoline, substitution of
chloride with 4-nitrophenol or 4-nitrothiophenol gave nitro 2a-b, which were
converted to phenols 3a-b in the presence of HBr [27]. Reaction of 3a-b with
1-Boc-4-methanesulfonyloxypiperidine
or
1-Boc-4-(((methylsulfonyl)oxy)methyl)piperidine in DMF containing Cs2CO3
provided ethers 4a-c [28]. Piperidines 5a-c were obtained by clean deprotection of the
N-Boc group using CF3COOH in CH2Cl2. Treatment of 5a-c with different
isocyanates gave intermediates 6a-g, which were reduced by powdered iron in 90%
EtOH to afford amines 7a-g. To obtain semicarbazides 9a-g, the amines 7a-g were
reacted with phenyl chloroformate in the presence of pyridine to afford 8a-g, followed
by hydrazinolysis reaction with 80% hydrazine hydrate in xylene with vigorous

agitation. Condensation of 9a-g with different benzaldehydes was carried out to yield
semicarbazones 10a-o in favor of catalytic HOAc in i-PrOH. Finally, target
compounds 11a-o were obtained via the cyclization with mercaptoacetic acid under
SiCl4 in dry CH2Cl2. Taking intermediate 3a as starting material, target compound 19
was prepared by similar synthetic routes outlined in Scheme 2.
NO2
NO2
NO2
NO2
Cl
X
X
X
O
O
X
O
O
O
O
v
iii
iv
i
ii
O
O
O
Ph
N
n
n
N
N
Ph
N
HO
N
N
HN
Boc
2a: X = O;
2b: X = S.
3a
: X = O;
3b: X = S.
4a
: X = O, n = 0;
4b: X = S, n = 0;
4c: X = O, n = 1.
5a: X = O, n = 0;
5b: X = S, n = 0;
5c: X = O, n = 1.
H
NH2
NO2
N
O
O
X
N
X
X
N
O
O
O
O
viii
vi
vii
n
n
O
N
n
O
H
H
N
H
N
N
N
N
N
R1
R1
R1
O
O
O
6a: X = O, n = 0, R1 = ethyl;
7a: X = O, n = 0, R1 = ethyl;
8a: X = O, n = 0, R1 = ethyl;
7b: X = O, n = 0, R1 = iospropyl;
7c: X = O, n = 0, R1 = n-butyl;
7d: X = O, n = 0, R1 = cyclohexyl;
7e: X = O, n = 0, R1 = benzyl;
7f: X = S, n = 0, R1 = iospropyl;
7g: X = O, n = 1, R1 = iospropyl.
8b: X = O, n = 0, R1 = iospropyl;
8c: X = O, n = 0, R1 = n-butyl;
8d: X = O, n = 0, R1 = cyclohexyl;
8e: X = O, n = 0, R1 = benzyl;
8f: X = S, n = 0, R1 = iospropyl;
8g: X = O, n = 1, R1 = iospropyl.
6b: X = O, n = 0, R1 = iospropyl;
6c: X = O, n = 0, R1 = n-butyl;
6d: X = O, n = 0, R1 = cyclohexyl;
6e: X = O, n = 0, R1 = benzyl;
6f: X = S, n = 0, R1 = iospropyl;
6g: X = O, n = 1, R1 = iospropyl.
R2
H
H
H
N
H
N
H
N
H
N
N
N
NH2
N
N
S
R2
O
O
O
X
N
X
N
X
O
ix
x
O
O
O
O
n O
n
n
O
N
H
N
H
H
N
N
N
N
N
R1
R1
R1
O
10a-o
11a-o
O
O
9a: X = O, n = 0, R1 = ethyl;
10a, 11a: n = 0, X = O, R1 = ethyl, R2 = 4-F; 10b, 11b: n = 0, X = O, R1 = isopropyl, R2 = 4-F;
9b: X = O, n = 0, R1 = iospropyl;
9c: X = O, n = 0, R1 = n-butyl;
9d: X = O, n = 0, R1 = cyclohexyl;
9e: X = O, n = 0, R1 = benzyl;
9f: X = S, n = 0, R1 = iospropyl;
9g: X = O, n = 1, R1 = iospropyl.
10c, 11c: n = 0, X = O, R1 = n-butyl; R2 = 4-F; 10d, 11d: n = 0, X = O, R1 = cyclohexyl, R2 = 4-F;
10e, 11e: n = 0, X = O, R1 = benzyl, R2 = 4-F; 10f, 11f: n = 0, X = O, R1 = isopropyl, R2 = H;
10g, 11g: n = 0, X = O, R1 = isopropyl, R2 = 3,4-F; 10h, 11h: n = 0, X = O, R1 = isopropyl, R2 = 2,3-F;
10i, 11i: n = 0, X = O, R1 = isopropyl, R2 = 2-F; 10j, 11j: n = 0, X = O, R1 = isopropyl, R2 = 2,4-F;
10k, 11k: n = 0, X = O, R1 = isopropyl, R2 = 2,6-F; 10l, 11l: n = 0, X = O, R1 = isopropyl, R2 = 3-F;
10m, 11m: n = 0, X = O, R1 = isopropyl, R2 =2,4,6-F; 10n,11n: n = 0, X = S, R1 = isopropyl, R2 = 2,6-F;
10o, 11o: n = 1, X = O, R1 = isopropyl, R2 = 2,6-F.
Scheme 1. Synthesis of target compounds 11a-o. Reagents and conditions: i) 4-nitrophenol (2a) or
4-nitrothiophenol (2b), PhCl, reflux, 16 h, 60.1-62.4%; ii) 33% HBr in HOAc, rt, 3 h, 64.2-66.4%; iii)
1-Boc-4-methanesulfonyloxypiperidine or 1-Boc-4-(((methylsulfonyl)oxy)methyl)piperidine, Cs2CO3,
DMF, 110 , 6 h, 46.7-55.3%; iv) CF3COOH, CH2Cl2, rt, 2 h, 74.8-79.6%; v) isocyanates, Et3N,
CH2Cl2, rt, 3-5 h, 76.4-84.2%; vi) Fe, 90% EtOH, HCl, reflux, 4-6 h, 77.4-85.8%; vii) phenyl
chloroformate, pyridine, CH2Cl2, rt, 2 h; viii) 80% hydrazine hydrate, xylene, 70 , 2 h, 31.9-41.5%; ix)
aldehydes, i-PrOH, HOAc (cat.), reflux, 2 h, 77.7-86.1%; x) mercaptoacetic acid, SiCl4, CH2Cl2, reflux,
6 h, 36.3-45.8%.

Scheme
2.
Synthesis
of target
compound
19. Reagents
and
conditions:
i)
1-Boc-3-methanesulfonyloxypiperidine, Cs2CO3, DMF, 110 , 6 h, 39.8%; ii) CF3COOH, CH2Cl2, rt,
2 h, 73.2%; iii) isopropyl isocyanate, Et3N, CH2Cl2, rt, 3 h, 79.3%; iv) Fe, 90% EtOH, HCl, reflux, 6 h,
81.6%; v) phenyl chloroformate, pyridine, CH2Cl2, rt, 2 h; vi) 80% hydrazine hydrate, xylene, 70 , 2
h, 34.2%; vii) 2,6-difluorobenzaldehyde, i-PrOH, HOAc (cat.), reflux, 2 h, 81.9%; viii) mercaptoacetic
acid, SiCl4, CH2Cl2, reflux, 6 h, 39.6%.
The synthesis of compound 31 was accomplished in a multiple process as shown in
Scheme 3. Intermediate 2a was cleanly oxidated by m-CPBA in CH2Cl2 to afford
quinoline N-oxide 20 [29]. Intermediate 24 was obtained via debenzylation,
nucleophilic substitution, deprotection of the N-Boc group and amination reaction by
similar synthetic methods preparing intermediate 6. Quinolines N-oxide 24 were
readily converted to 2-tert-butylaminoquinoline 25 by Ts2O and t-BuNH2 in CH2Cl2
[30]. In the following steps, intermediate 30 were synthesized using a method similar
to that for compounds 11a-o. Finally, the deprotection of t-butyl group was achieved
by stirring at 70 in the presence of CF3COOH in xylene.

NO2
NO2
NO2
NO2
O
O
O
O
O
O
iv
ii
Boc
O
O
i
iii
O
N
O
O
Ph
N
O
N
O
HO
N
O
Ph
N
22
20
21
2a
NO2
NO2
NO2
O
O
O
O
O
N
vii
v
vi
O
O
O
O
O
O
HN
N
N
N
N
H
H
N
O
N
O
N
H
25
23
24
H
N
NH2
H
N
NHNH2
OPh
O
O
N
O
O
O
O
O
O
viii
i
x
O
O
N
N
O
O
O
N
H
N
H
N
N
H
N
N
N
H
H
O
F
N
N
26
H
27
28
H
H
F
H
N
N
N
F
H
N
N
xii
O
N
S
x
xi
O
O
N
F
O
O
O
O
O
O
N
N
O
O
H
N
N
N
H
H
29
N
N
H
30
F
F
H
H
N
N
N
S
O
O
O
O
O
N
N
H
O
N
NH2
31
Scheme 3. Synthesis of compounds 31. Reagents and conditions: i) m-CPBA, CH2Cl2, rt, 67.7%; ii) 33%
HBr in HOAc, rt, 3 h, 65.1%; iii) 1-Boc-4-methanesulfonyloxypiperidine, Cs2CO3, DMF, 110 , 6 h,
49.3%; iv) CF3COOH, CH2Cl2, rt, 2 h, 78.5%; v) isopropyl isocyanate, Et3N, CH2Cl2, rt, 3 h, 74.3%; vi)
t-BuNH2, Ts2O, CH2Cl2, rt, 1 h, 62.1%; vii) Fe, 90% EtOH, HCl, reflux, 6 h, 76.2%; viii) phenyl
chloroformate, pyridine, CH2Cl2, rt, 2 h; ix) 80% hydrazine hydrate, xylene, 70 , 2 h, 38.6%; x)
2,6-difluorobenzaldehyde, i-PrOH, HOAc (cat.), reflux, 2 h, 78.4%; xi) mercaptoacetic acid, SiCl4,
CH2Cl2, reflux, 6 h, 41.0%; xii) CF3COOH, xylene, 70 , 5 h, 57.9%.
Scheme 4 presents the synthetic routes for target compound 46. Commercially
available 1-(3-hydroxy-4-methoxyphenyl)ethan-1-one was reacted with benzyl
bromide under basic condition in DMF to afford 32. Nitration of 32 with fuming nitric
acid in CH2Cl2 afforded nitro 33, which was then reduced by powdered iron to give
amine 33 in satisfactory yield [31]. Cyclization was accomplished in the presence of
MeONa and HCOOEt to yield quinoline 35 [32]. The resultant hydroxyl moiety 35
were converted to the corresponding chloride 36 on exposure to phosphorus
oxychloride in the presence of 4-dimethylaminopyridine. Subsequently, compound 46
was obtained using a method similar to that for compound 11o via SN2 nucleophilic
substitution, debenzylation, deprotection of the N-Boc group, amination, reduction,
acylation, hydrazinolysis, condensation and cyclization reaction.

Scheme 4. Synthesis of target compound 46. Reagents and conditions: i) benzyl bromide, K2CO3,
DMF, rt, 2 h, 89.8%; ii) fuming HNO3, -10 , 5 h, 73.6%; iii) Fe, 90% EtOH, HCl, reflux, 3 h, 78.5%;
iv) MeONa, HCOOEt, DME, rt, 4 h, 72.4%; v) 4-dimethylaminopyridine, POCl3, 110 , 6 h, 64.7%; vi)
4-nitrophenol, PhCl, reflux, 16 h, 58.6%; vii) 33% HBr in HOAc, rt, 3 h, 63.8%; viii)
1-Boc-4-(((methylsulfonyl)oxy)methyl)piperidine, Cs2CO3, DMF, 110 , 6 h, 47.3%; ix) CF3COOH,
CH2Cl2, rt, 2 h, 74.4%; x) isopropyl isocyanate, Et3N, CH2Cl2, rt, 3 h, 81.2%; xi) phenyl chloroformate,
pyridine, CH2Cl2, rt, 2 h; xii) 80% hydrazine hydrate, xylene, 70 , 2 h, 39.7%; xiii) aldehydes,
i-PrOH, HOAc (cat.), reflux, 2 h, 83.1%; xiv) mercaptoacetic acid, SiCl4, CH2Cl2, reflux, 6 h, 37.2%.
Synthetic routes for target compound 56 are displayed in Scheme 5. Commercially
available 7-(benzyloxy)-4-chloro-6-methoxyquinoline and 3-nitrophenol were
converted to target compound 56 via a series of similar reactions as described above.
Scheme 5. Synthesis of target compound 56. Reagents and conditions: i) 3-nitrophenol, PhCl, reflux,
16 h, 52.4%; ii) 33% HBr in HOAc, rt, 3 h, 62.0%; iii) 1-Boc-4-methanesulfonyloxypiperidine, Cs2CO3,
DMF, 110 , 6 h, 51.2%; iv) CF3COOH, CH2Cl2, rt, 2 h, 80.2%; v) isopropyl isocyanate, Et3N, CH2Cl2,
rt, 3 h, 76.5%; vi) Fe, 90% EtOH, HCl, reflux, 4 h, 81.2%; vii) phenyl chloroformate, pyridine, CH2Cl2,
rt, 2 h; viii) 80% hydrazine hydrate, xylene, 70 , 2 h, 40.3%; ix) 2,6-difluorobenzaldehyde, i-PrOH,
HOAc (cat.), reflux, 2 h, 76.9%; x) mercaptoacetic acid, SiCl4, CH2Cl2, reflux, 6 h, 35.2%.

3.2. Results and discussion
3.2.1. Structure-Activity Relationship
In our previous study, we found that the quinoline derivates bearing thiazolidinone
fragments showed potent in vitro kinase inhibitory activity against c-Met/Ron and
anticancer activity against various cancer cell lines, especially human colorectal
cancer cell line HT-29 [26]. Moreover, c-Met and Ron overexpression is correlated
with colorectal cancer (CRC) and CRC liver metastasis [33-35]. Thus, in vitro
anticancer activity of all newly synthesized compounds against HT-29 cells was
determined in the present study. Due to the weak antitumor activity of Cabozantinib
against HT-29 cells, Regorafenib treatment for metastatic colorectal cancer (mCRC)
in clinic was chosen as another positive control. The inhibitory potencies against
c-Met, Ron and HT-29 cells were shown in Table 1 and Table 2.
Table 1. In vitro kinase inhibitory activity and anticancer activity of compounds 11a-m.
Inhibitory activities
IC50 (μM)c
c-Met Ron
HT-29
IC50 (μM)d
Compd.
R1
R2
c-Meta
23.6
29.1
14.4
24.4
3.5
Ronb
25.9
33.7
19.9
26.2
4.1
11a
4-F
4-F
NDe
ND
ND
2.25 ± 0.25
0.86 ± 0.072
1.70 ± 0.18
3.42 ± 0.21
6.60 ± 0.46
1.26 ± 0.11
1.06 ± 0.085
0.79 ± 0.057
0.41 ± 0.034
0.95 ± 0.086
0.31 ± 0.022
0.67 ± 0.058
0.91 ± 0.079
2.87 ± 0.19
10.6 ± 1.12
11b
ND
11c
4-F
ND
ND
11d
4-F
ND
ND
11e
4-F
ND
ND
11f
H
31.2
16.1
19.5
41.1
19.8
65.0
22.5
31.2
ND
50.6
21.2
31.1
60.3
33.8
84.4
26.8
37.5
ND
1.68
ND
0.503
ND
11g
3,4-F
2,3-F
2-F
11h
ND
ND
11i
11j
0.531
ND
0.347
ND
2,4-F
2,6-F
3-F
11k
0.382
ND
0.122
ND
11l
11m
2,4,6-F
-
ND
ND
Regorafenib
-
-
ND
ND
Cabozantinib
-
98.6
75.2
ND
ND
a The kinase inhibition % at 1.0 μM are the average of two independent experiments.
b The kinase inhibition % at 0.5 μM are the average of two independent experiments.
c The IC50 values are the average of two independent experiments.
d The values were an average of three separate determinations and standard deviations were shown.
e ND: Not determined.
To examine the substitution effect of R1 in the piperidine urea moiety (Fig. 2),
various substituents were introduced, including benzyl group, isopropyl group and
ethyl group, etc (11a-e). According to the biological results, isopropyl group (11b)
was fixed as the optimal group. Comparing the inhibitory potencies of 11b to that of

11e, a preliminary conclusion might be made that the introduction of hydrophobic and
bulky group at R1 dramatically decreased the activity against all the two kinases and
HT-29 cells, such as benzyl group (11e).
In the following modification, our attention turned to the R2. Based on our previous
study, only well tolerated fluorine atoms were introduced. The potency contribution of
the ortho-fluoro substituent on the phenyl ring was demonstrated by 11i and 11k
compared to 11f. The addition of 2,6-difluoro (11k) imparted 4.4- and 4.1-fold
increase in enzymatic potency (c-Met IC50=0.382 μM and Ron IC50=0.122 μM) and a
4.1-fold increase of cellular potency (IC50=0.31 μM) relative to the 11f (c-Met
IC50=1.68 μM, Ron IC50=0.503 μM and HT-29 IC50=1.26 μM). As a general trend,
moving fluorine atom to meta- or para-position on the phenyl ring resulted in a loss of
activity against kinases and cancer cell.
Having identified well tolerated group on R1 (R1 = isopropyl group) and R2 (R2 = 2,
6-difluoro), we next further explored the SAR, including the linker atom between
quinoline and phenyl ring A (11n), 2-, 6- and 7-positions on quinoline (11o, 19, 31, 46)
and the position of thiazolidinone urea moiety linked on phenyl ring A (56). As
indicated in Table 2, the replacement of the oxygen (11k) with a sulfur linker (11n)
was tolerated except for the activity against HT-29. The introduction of an amino in
2-position of quinoline (31) imparted 1.8- and 4-fold decrease in c-Met and Ron
kinase inhibitory rate and 18-fold decrease of cell potency. Docking study showed
that compound 31 adopted a similar docking mode comparing to that of 11k, and an
additional H-bond was formed between the amino and the residue Pro1158 (Fig. 5B).
Unfortunately, the newly formed H-bond interaction was weak (data not shown). In
addition, the H-bond between nitrogen atom of quinoline and the residue Met1160
was weakened due to the changes of conformation resulted from the introduction of
NH2. Movement of the thiazolidinone urea moiety from 4- to 3-position on the phenyl
ring A led to a dramatically reduced enzymatic potency and cellular potency (56 vs
11k). The results of docking study indicated that the loss of a H-bond formed between
the oxygen atom in the urea moiety and Lys1110 might lead to the lower potency (Fig.
5D).
Finally,
the
effect
of
varying
the
methoxy
group
and
N-isopropylpiperidine-1-carboxamide on the quinoline was examined with the
ultimate goal to improve inhibitory activity and introduce water solubilizing groups.
Compared 11o with 11k, a slight drop in enzymatic potency and cellular potency was
observed by lengthening the distance between oxygen atom and piperidine ring.
Notably, compound 46 bearing the N-isopropylpiperidine-1-carboxamide on the
6-position of quinoline displayed over 25-fold lower potency against HT-29 cells
(IC50>10.0 μM) than compound 11o. The results indicated that a substituted
piperidine-1-carboxamide at the 7-position of quinoline was necessarily required for
kinases and cancer cell inhibition.

Table 2. In vitro kinase inhibitory activity and anticancer activity of compounds 11n-o, 19, 31, 46 and
56.
Inhibitory activities
IC50 (μM)c
HT-29
Compd.
IC50 (μM)d
c-Meta
Ronb
c-Met Ron
11n
11o
19
62.9
83.0
NDe
ND
ND
ND
ND
ND
ND
5.53
0.42 ± 0.031
0.39 ± 0.028
>10.0
58.4
31.7
34.7
86.1
39.7
21.1
31
5.60
46
56
20.7
16.0
25.3
29.3
ND
ND
ND
ND
>10.0
>10.0
ND
ND
ND
ND
Regorafenib
Cabozantinib
-
-
2.87 ± 0.18
10.6 ± 1.12
98.6
75.2
a The kinase inhibition % at 1.0 μM are the average of two independent experiments.
b The kinase inhibition % at 0.5 μM are the average of two independent experiments.
c The IC50 values are the average of two independent experiments.
d The values were an average of three separate determinations and standard deviations were shown.
e ND: Not determined.
3.2.2. Molecular docking study
In order to explore the binding mode of this series of compounds and explain the
SAR, docking simulation studies of representative compound 11k, 31 and 56 were
performed. As shown in Fig. 4B, 5B, 5D and 6, all compounds adopted an extend
conformation as type II kinase inhibitors. The most potent compound 11k formed
three canonical hydrogen bonds with the residues of Lys1110, Met1160 and Lys1161.
H-arene interactions were formed by quinoline and 2, 6-difluorophenyl fragment with

Val1092
and
Phe1134,
respectively.
In
addition,
the
N-isopropylpiperidine-1-carboxamide was protruded into solvent, and the terminal 2,
6-difluorophenyl ring inserted into a hydrophobic pocket formed by the residues of
Phe1134, Phe1200 and Ala1221, etc.
Fig. 4 The proposed binding mode of compound 11k with c-Met kinase (PBD ID: 3LQ8). A) The
structure of compound 11k. B) The interactions between compound 11k and c-Met. The compound was
shown by purple sticks, the H-bonds were represented by green dotted lines, and the arene-H
interaction was shown by blue dotted lines. C) 2D depiction of the ligand-protein interactions.
Fig. 5 The proposed binding mode of compounds 31 and 56 with c-Met kinase (PBD ID: 3LQ8). A)
The structure of compound 31. B) The interactions between compound 31 and c-Met. The compound

was shown by blue sticks, and the H-bonds were represented by green dotted lines. C) The structure of
compound 56. D) The interactions between compound 56 and c-Met. The compound was shown by
yellow sticks, and the H-bond was represented by green dotted lines.
As depicted in Fig. 6, compounds 11k, 31 and 56 were well embedded in the
binding pocket by similar alignment (Fig. 6). The conformations of three compounds
were slightly different which led to different enzymatic, and the details were
explained in the section of structure-activity relationship.
Fig. 6 The superposition of compounds 11k, 31 and 56. The compounds 11k, 31 and 56 were shown by
purple, blue and yellow sticks, respectively. The H-bonds were represented by green dotted lines.
3.2.3. IncuCyte live-cell imaging assays
Taking Regorafenib as positive control, antitumor mechanism and cytotoxicity
against normal cells of the most potent compound 11k were illustrated by IncuCyte
live-cell imaging assays in the following work.
3.2.3.1. Antiproliferation and cytotoxicity against HT-29
Real-time monitoring the confluence and the number of dead HT-29 cells was
performed to compare the effects of compound 11k and Regorafenib upon the kinetics
of antiproliferation and cytotoxicity. As shown in Fig. 7A and 8, a reduction in cell
proliferation induced by compound 11k was observed which was inferior to that of
Regorafenib at the same concentration. Exhilaratingly, 5.0 μg/mL of compound 11k
led to a significant cytotoxicity, and no cytotoxicity could be seen by treatment with
5.0 μg/mL of Regorafenib (Fig. 7B and 8). Lower concentration (1.67 μg/mL and
0.56 μg/mL) of compound 11k could also induced strong cytotoxicity, especially after
treatment for 36 h. The above results indicated that compound 11k could induce
antiproliferation and cytotoxicity in a dose- and time-dependent manner.

Fig. 7 The curves of IncuCyte live-cell imaging assays in HT-29 cell lines. All error bars are expressed
as mean ± SD of three independent experiments. A) The curves of real-time cell confluence. The cell
population was monitored for 72 h using an IncuCyte ZOOM system in an incubator. B) The curves of
real-time cytotoxicity. The dead cell population was monitored for 72 h using an IncuCyte ZOOM
system in an incubator.
Fig. 8 The white light images (merged) of study on antiproliferation and cytotoxicity against HT-29 cell
lines after 48 h and 72 h of treatment with compound 11k, Regorafenib or 0.1% DMSO. Green
fluorescent cells were counted as dead cell. Nucleus were stained with the DNA fluorescent probe
YOYO-1 iodide.
3.2.3.2. Measurement of apoptosis
Inducing apoptosis is often considered as one of the major strategies for
antitumor-drug development. Thus, in order to characterize the mode of cell death
induced by compound 11k, apoptosis analysis was performed. 3.0 μg/mL of
compound 11k induced significant apoptosis on HT-29 cells after 36 h which was
much greater than that of Regorafenib (Fig. 9 and 10). Slight induction of apoptosis
was observed at lower concentration after treatment with compound 11k after 48 h.
Overall, the results showed that compound 11k could induce cell apoptosis in a time-
and dose-dependent manner.

Fig. 9 The apoptosis curves of IncuCyte live-cell imaging assays in HT-29 cells. All error bars are
expressed as mean ± SD of three independent experiments. The cell population was monitored for 72 h
using an IncuCyte ZOOM system in an incubator.
Fig. 10 The white light merged images of study on apoptosis on HT-29 cells after 48 h and 72 h of
treatment with compound 11k and 0.1% DMSO. Green fluorescent cells were counted as apoptotic
cells. Nucleus were stained with the CellEvent® Caspase 3/7 Green ReadyProbes® reagent.
3.2.3.3. Antiproliferation and cytotoxicity against FHC
Inspired by the potent antitumor activity of compound 11k, its kinetics of
antiproliferation and cytotoxicity on human normal colorectal mucosa epithelial cell
FHC was evaluated. As shown in Fig. 11A and Fig. 12, the confluence treated with
10.0 μg/mL of compound 11k reached to 26.7% after 72 h which was extremely
weaker than that of Regorafenib (13.5%). Lower concentration could not efficiently
inhibited cell proliferation (data not shown). A dose- and time-dependent increase of
dead cell numbers was exhibited by treatment with compound 11k (Fig. 11B and 12).
The kinetics of cytotoxicity of compound 11k (10.0 μg/mL) was almost equipotent to
that of Regorafenib (0.19 μg/mL) during the treatment time. The above results
indicated that compound 11k was much less toxic to normal colorectal mucosa

epithelial cells than that of Regorafenib.
Fig. 11 The curves of IncuCyte live-cell imaging assays in FHC cell lines. All error bars are expressed
as mean ± SD of three independent experiments. A) The curves of real-time cell confluence. The cell
population was monitored for 72 h using an IncuCyte ZOOM system in an incubator. B) The curves of
real-time cytotoxicity. The dead cell population was monitored for 72 h using an IncuCyte ZOOM
system in an incubator.
Fig. 12 The white light images (merged) of study on antiproliferation and cytotoxicity against FHC
cells after 24 h and 72 h of treatment with compound 11k, Regorafenib or 0.1% DMSO. Green
fluorescent cells were counted as dead cell. Nucleus were stained with the DNA fluorescent probe
YOYO-1 iodide.
3.2.4. Cell cycle analyses
Cell cycle analyses in HT-29 cells treated with compound 11k and Regorafenib was
performed. As shown in Fig. 13, no significant cell cycle arrest was found. Compound
11k slightly induced cell cycle arrest with a G2/M percentage of 4.4% at 3.0 μg/mL
compared to Regorafenib (2.0%) and 0.1% DMSO (2.4%). According to the results of
weak cell cycle arrest and strong induction of apoptosis of compound 11k, we could
deduce that other factors inducing of apoptosis might be the targets of compound 11k,
such as p53, JNK, Bcl-2 and Caspases, etc [36-38].

Fig. 13 Cell cycle progression analyses of HT-29 cells treated with compound 11k and Regorafenib for
48 h.
3.2.5. In vitro kinase profile
In order to investigate the potential targets preliminarily, the most potent compound
11k was screened against other six kinases, including c-Kit, B-Raf, c-Src, IGF1R,
PDGFRα and AXL. As shown in Table 3, compound 11k exhibited moderate
inhibitory activity against several kinases, such as c-Met, Ron and PDGFRα, etc. The
results indicated that compound 11k was not a highly selective c-Met/Ron dual
inhibitors. Thus, we deduced that the excellent anticancer activity might result from
multiple kinases inhibitory activity, and other targets will be investigated in our future
work.
Table 3 The kinase profile of compound 11k.
Kinase
c-Met
Ron
IC50 (μM)a
Kinase
PDGFRα
c-Src
IC50 (μM)
0.384
0.382
0.122
0.421
c-Kit
AXL
>10.0
IGF1R
B-Raf
>10.0
0.632
>10.0
a Values are expressed as the mean of two independent experiments.
4. Conclusions
In summary, a novel series of quinoline analogues bearing thiazolidinones were
designed, synthesized and evaluated for their biological activities in the present work.
Structure activity relationship analysis was performed based on biological evaluation
and
docking
study,
and
the
most
potent
4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxy
quinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11k) was identified as a
multi-kinase inhibitor. IncuCyte live-cell imaging assays indicated that compound 11k
performed excellent cytotoxicity, antiproliferation and induction of apoptosis on
HT-29 cells in a time- and dose-dependent manner with an efficacy that was

significantly greater than Regorafenib. In addition, compound 11k induced slight cell
cycle arrest in the G2/M stage in HT-29 cells. The results of induction of apoptosis
and cell cycle analysis indicated that other factors inducing of apoptosis might be the
targets of compound 11k. Further cell selectivity of compound 11k showed that it was
not active against human normal colorectal mucosa epithelial cell FHC at 10.0 μg/mL
or lower concentrations. The results of cell selectivity indicated that the toxicity to
normal cells of compound 11k was much lower than that of Regorafenib. The above
results support further structural modification of compound 11k to improve its
inhibitory activity, which will lead to more potent kinase inhibitors as anticancer
agents.
5. Experimental
5.1. Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and
1
13
used without further purification. H NMR and C NMR spectra were generated on
Bruker ARX-400 spectrometers (Bruker Bioscience, Billerica, MA, USA). Chemical
shifts are given in parts per million (ppm) relative to TMS as internal standards.
Column chromatography was carried out on silica gel (200-300 mesh). High
Resolution Mass spectra (HRMS) were taken in ESI mode on Agilent 6530 Q-TOF
(Agilent Technologies, CA, USA).
5.1.1. General procedure for the synthesis of 2a-b. To a mixture of
7-(benzyloxy)-4-chloro-6-methoxyquinoline (50 g, 167.2 mmol) in dry PhCl (300 mL)
was added 4-nitrophenol (418.1 mmol) for 2a or 4-nitrothiophenol for 2b (418.1
mmol). After refluxed for 16 h, the solvent was concentrated in vacuo, treated with
CH2Cl2 (500 mL), and washed with 10% NaOH aqueous solution (3×100 mL) and
water (100 mL). The organic layers were dried over MgSO4 and concentrated give the
crude products which were used for the next step without further purification.
5.1.1.1. 7-(benzyloxy)-6-methoxy-4-(4-nitrophenoxy)quinoline (2a). Tawny solid,
yield: 62.4%. HRMS (ESI) m/z 403.1345 [M+H]+, Calcd. for 403.1294.
5.1.1.2. 7-(benzyloxy)-6-methoxy-4-((4-nitrophenyl)thio)quinoline (2b). Brown
solid, yield: 60.1%. HRMS (ESI) m/z 419.1102 [M+H]+, Calcd. for 419.1066.
5.1.2. General procedure for the synthesis of 3a-b. Intermediates 2 (30.0 g) was
slowly added to 33% HBr in HOAc (150 mL) and the mixtures were stirred for 3 h at
room temperature during which time target products were precipitated. The mixture
was filtrated, and the solid was washed with isopropyl ether to give the products.
5.1.2.1. 6-methoxy-4-(4-nitrophenoxy)quinolin-7-ol (3a). Light solid, yield: 66.4%.
HRMS (ESI) m/z 313.0872 [M+H]+, Calcd. for 313.0824.
5.1.2.2. 6-methoxy-4-((4-nitrophenyl)thio)quinolin-7-ol (3b). Beige solid, yield:
64.2%. HRMS (ESI) m/z 329.0653 [M+H]+, Calcd. for 329.0596.
5.1.3. General procedure for the synthesis of 4a-c. To a mixture of intermediates
3a-b (0.1 mol) in DMF (100 mL) was added caesium carbonate (0.25 mol) and the
mixture was stirred vigorously at room temperature for 10 min. Different piperidines
(0.15 mol) were added and the mixture was heated to 110
for 6 h. The reaction
mixture was cooled to room temperature and poured into water (500 mL), filtered,
and washed with water to give crude 4a-c which were purified by silica gel column

chromatography (eluent, CH2Cl2:MeOH = 100:1 to 15:1) to afford 4a-c.
5.1.3.1.
tert-butyl
4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carboxylate
(4a).
Yellow solid, yield: 52.1%. HRMS (ESI) m/z 496.2166 [M+H]+, Calcd. for 496.2084.
5.1.3.2.
tert-butyl
4-((6-methoxy-4-((4-nitrophenyl)thio)quinolin-7-yl)oxy)piperidine-1-carboxylate (4b).
Yellow solid, yield: 46.7%. HRMS (ESI) m/z 512.1913 [M+H]+, Calcd. for 512.1855.
5.1.3.3.
tert-butyl
4-(((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)methyl)piperidine-1-carboxylate
(4c). Yellow solid, yield: 55.3%. HRMS (ESI) m/z 510.2302 [M+H]+, Calcd. for
510.2240.
5.1.4. General procedure for the synthesis of 5a-c. To a mixture of intermediates 4
(50.0 mmol) and CH2Cl2 (100 mL) was added CF3COOH (50 mL). The solution was
stirred for 2 h at room temperature and concentrated in vacuo. CH2Cl2 (200 mL) was
added to the residue and saturated NaHCO3 aqueous solution was added until the pH
was adjusted to 8. The organics were dried over Na2SO4, concentrated in vacuo, and
the residue was used for the next step without further purification.
5.1.4.1. 6-methoxy-4-(4-nitrophenoxy)-7-(piperidin-4-yloxy)quinoline (5a). Dark
yellow oil, yield: 79.6%. HRMS (ESI) m/z 396.1601 [M+H]+, Calcd. for 396.1559.
5.1.4.2. 6-methoxy-4-((4-nitrophenyl)thio)-7-(piperidin-4-yloxy)quinoline (5b).
Dark yellow oil, yield: 74.8%. HRMS (ESI) m/z 412.1403 [M+H]+, Calcd. for
412.1331.
5.1.4.3. 6-methoxy-4-(4-nitrophenoxy)-7-(piperidin-4-ylmethoxy)quinoline (5c).
Dark yellow oil, yield: 77.5%. HRMS (ESI) m/z 410.1764 [M+H]+, Calcd. for
410.1716.
5.1.5. General procedure for the synthesis of 6a-g. A solution of isocyanates (24.0
mmol) in dry CH2Cl2 (10mL)was added dropwise to a cooled solution of
intermediates 5a-c (20.0 mmol) and Et3N (30.0 mmol) in dry CH2Cl2 (100 mL). The
mixture was then stirred for 3-5 h at room temperature. After complete conversion of
the starting material, saturated NaHCO3 aqueous solution (20 mL) was added. The
organic phase was separated, dried over Na2SO4 overnight, and concentrated in vacuo
to afford 6a-g.
5.1.5.1.
N-ethyl-4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carboxamid
e (6a). Yellow solid, yield: 82.9%. 1H NMR (400 MHz, DMSO-d6) δ: 8.44 (d, J = 5.2
Hz, 1H), 7.92 (m, 2H), 7.49 (s, 1H), 7.15 (m, 3H), 6.43 (d, J = 5.2 Hz, 1H), 3.92 (s,
3H), 3.64-3.70 (m, 1H), 3.09-3.16 (m, 4H), 2.95-3.02 (m, 2H), 1.98-2.02 (m, 2H),
1.67-1.73 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H); HRMS (ESI) m/z 467.1983 [M+H]+,
Calcd. for 467.1931.
5.1.5.2.
N-isopropyl-4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carboxa
mide (6b). Yellow solid, yield: 84.2%. 1H NMR (400 MHz, DMSO-d6) δ: 8.46 (d, J =
5.2 Hz, 1H), 7.91 (m, 2H), 7.48 (s, 1H), 7.15 (m, 3H), 6.45 (d, J = 5.2 Hz, 1H), 3.93
(s, 3H), 3.66-3.72 (m, 1H), 3.10-3.16 (m, 3H), 2.95-3.02 (m, 2H), 1.98-2.01 (m, 2H),

1.52-1.61 (m, 2H), 1.07 (s, 3H), 1.06 (s, 3H); HRMS (ESI) m/z 481.2161 [M+H]+,
Calcd. for 481.2087.
5.1.5.3.
N-butyl-4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carboxamid
e (6c). Yellow solid, yield: 78.8%. HRMS (ESI) m/z 495.2268 [M+H]+, Calcd. for
495.2244.
5.1.5.4.
N-cyclohexyl-4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carbox
amide (6d). Yellow solid, yield: 76.4%. HRMS (ESI) m/z 537.2220 [M+H]+, Calcd.
for 537.2172.
5.1.5.5.
N-benzyl-4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carboxami
de (6e). Light yellow solid, yield: 83.1%. HRMS (ESI) m/z 545.1908 [M+H]+, Calcd.
for 545.1859.
5.1.5.6.
N-isopropyl-4-((6-methoxy-4-((4-nitrophenyl)thio)quinolin-7-yl)oxy)piperidine-1-carb
oxamide (6f). Yellow solid, yield: 78.7%. HRMS (ESI) m/z 497.1907 [M+H]+, Calcd.
for 497.1859.
5.1.5.7.
N-isopropyl-4-(((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)methyl)piperidine-1-
carboxamide (6g). Yellow solid, yield: 78.2%. HRMS (ESI) m/z 511.2069 [M+H]+,
Calcd. for 511.2015.
5.1.6. General procedure for the synthesis of 7a-g. Powered iron (0.06 mol) and
concentrated HCl (2 drops) was added to a suspension of intermediates 6a-g (0.02
mol) in 90% EtOH (100 mL). The mixture was refluxed with vigorous agitation for
4-6 h. The hot mixture was filtered through celites and the filtrate was evaporated in
vacuo to afford 7a-g.
5.1.6.1.
4-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)-N-ethylpiperidine-1-carboxami
de (7a). Yellow solid, yield: 83.4%. HRMS (ESI) m/z 437.2243 [M+H]+, Calcd. for
437.2189.
5.1.6.2.
4-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carbox
amide (7b). Yellow solid, yield: 81.0%. HRMS (ESI) m/z 451.2397 [M+H]+, Calcd.
for 451.2345.
5.1.6.3.
4-((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)-N-butylpiperidine-1-carboxami
de (7c). Yellow solid, yield: 85.8%. HRMS (ESI) m/z 465.2574 [M+H]+, Calcd. for
465.2502.
5.1.6.4.
N-cyclohexyl-4-((6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)piperidine-1-carbox
amide (7d). Yellow solid, yield: 78.9%. HRMS (ESI) m/z 491.2708 [M+H]+, Calcd.
for 491.2658.
5.1.6.5.

4-((4-((4-aminophenyl)thio)-6-methoxyquinolin-7-yl)oxy)-N-benzylpiperidine-1-carbo
xamide (7e). Yellow solid, yield: 81.7%. HRMS (ESI) m/z 499.2407 [M+H]+, Calcd.
for 499.2345.
5.1.6.6.
4-((4-((4-aminophenyl)thio)-6-methoxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-ca
rboxamide (7f). Yellow solid, yield: 80.4%. HRMS (ESI) m/z 467.2163 [M+H]+,
Calcd. for 467.2117.
5.1.6.7.
4-(((4-(4-aminophenoxy)-6-methoxyquinolin-7-yl)oxy)methyl)-N-isopropylpiperidine-
1-carboxamide (7g). Yellow solid, yield: 77.4%. HRMS (ESI) m/z 481.2338 [M+H]+,
Calcd. for 481.2273.
5.1.7. General procedure for the synthesis of 9a-g. To a solution of 7 (10.0 mmol)
and dry pyridine (42.0 mmol) in dry CH2Cl2 (50 mL), phenyl chloroformate (20.0
mmol) in dry CH2Cl2 (5 mL) was added dropwise at 0 °C. After the addition was
completed, the solution was stirred at room temperature for 2 h. Saturated NaHCO3
aqueous solution (2×20 mL) was added to the above solution and the organic phase
was separated, washed with 20 mL water, dried over anhydrous Na2SO4, concentrated
in vacuo to afford intermediates 8a-g, which were immediately used in the following
reaction without further purification.
80% Hydrazine monohydrate (15 mL) was added to a solution of 8a-g in xylene
(15 mL), and the mixture was stirred vigorously at 70 for 2 h. After cooling to room
temperature, the solvent was concentrated under reduced pressure. The residue was
purified by silica gel column chromatography (eluent, CH2Cl2: MeOH: Et3N =
100:2:1 to 100:10:1) to afford 9a-g.
5.1.7.1.
N-ethyl-4-((4-(4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)piperi
dine-1-carboxamide (9a). Light yellow solid, yield: 36.4% (two steps). HRMS (ESI)
m/z 495.2414 [M+H]+, Calcd. for 495.2356.
5.1.7.2.
4-((4-(4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)-N-isopropylp
iperidine-1-carboxamide (9b). Light yellow solid, yield: 39.8% (two steps). HRMS
(ESI) m/z 509.2584 [M+H]+, Calcd. for 509.2512.
5.1.7.3.
N-butyl-4-((4-(4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)piperi
dine-1-carboxamide (9c). Light yellow solid, yield: 35.3% (two steps). HRMS (ESI)
m/z 563.2734 [M+H]+, Calcd. for 563.2669.
5.1.7.4.
N-cyclohexyl-4-((4-(4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)
piperidine-1-carboxamide (9d). Light yellow solid, yield: 41.5% (two steps). HRMS
(ESI) m/z 549.2883 [M+H]+, Calcd. for 549.2825.
5.1.7.5.
N-benzyl-4-((4-(4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)pipe
ridine-1-carboxamide (9e). Light yellow solid, yield: 31.9% (two steps). HRMS (ESI)
m/z 557.2590 [M+H]+, Calcd. for 557.2512.

5.1.7.6.
4-((4-((4-(hydrazinecarboxamido)phenyl)thio)-6-methoxyquinolin-7-yl)oxy)-N-isopro
pylpiperidine-1-carboxamide (9f). Light yellow solid, yield: 34.7% (two steps).
HRMS (ESI) m/z 525.2352 [M+H]+, Calcd. for 525.2284.
5.1.7.7.
4-(((4-(4-(hydrazinecarboxamido)phenoxy)-6-methoxyquinolin-7-yl)oxy)methyl)-N-is
opropylpiperidine-1-carboxamide (9g). Light yellow solid, yield: 34.7% (two steps).
HRMS (ESI) m/z 523.2761 [M+H]+, Calcd. for 523.2669.
5.1.8. General procedure for the synthesis of 10a-o. To a solution of semicarbazide
9a-g (2.0 mmol) and aldehyde (2.4 mmol) in dry i-PrOH (10 mL), HOAc (2 drops)
were added. After refluxing for 2 h, the mixture was cooled to 0 °C, and the resultant
precipitate was filtered, washed with cold i-PrOH and dried under vacuum to afford
10a-o. If no precipitate appeared, silica gel column chromatography (eluent, CH2Cl2:
MeOH: Et3N = 100:2:1 to 100:10:1) was used to isolated the target compounds.
5.1.8.1.
(E)-N-ethyl-4-((4-(4-(2-(4-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-m
ethoxyquinolin-7-yl)oxy)piperidine-1-carboxamide (10a). White solid, yield: 85.0%.
HRMS (ESI) m/z 601.2617 [M+H]+, Calcd. for 601.2575.
5.1.8.2.
(E)-4-((4-(4-(2-(4-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10b). White solid, yield:
81.6%. HRMS (ESI) m/z 615.2783 [M+H]+, Calcd. for 615.2731.
5.1.8.3.
(E)-N-butyl-4-((4-(4-(2-(4-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-m
ethoxyquinolin-7-yl)oxy)piperidine-1-carboxamide (10c). White solid, yield: 83.1%.
HRMS (ESI) m/z 629.2937 [M+H]+, Calcd. for 629.2888.
5.1.8.4.
(E)-N-cyclohexyl-4-((4-(4-(2-(4-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy
)-6-methoxyquinolin-7-yl)oxy)piperidine-1-carboxamide (10d). White solid, yield:
82.4%. HRMS (ESI) m/z 655.3102 [M+H]+, Calcd. for 655.3044.
5.1.8.5.
(E)-N-benzyl-4-((4-(4-(2-(4-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-
methoxyquinolin-7-yl)oxy)piperidine-1-carboxamide (10e). White solid, yield: 86.1%.
HRMS (ESI) m/z 663.2783 [M+H]+, Calcd. for 663.2731.
5.1.8.6.
(E)-4-((4-(4-(2-benzylidenehydrazine-1-carboxamido)phenoxy)-6-methoxyquinolin-7-
yl)oxy)-N-isopropylpiperidine-1-carboxamide (10f). White solid, yield: 78.7%.
HRMS (ESI) m/z 597.2877 [M+H]+, Calcd. for 597.2825.
5.1.8.7.
(E)-4-((4-(4-(2-(3,4-difluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-metho
xyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10g). White solid, yield:
84.7%. HRMS (ESI) m/z 633.2701 [M+H]+, Calcd. for 633.2637.
5.1.8.8.
(E)-4-((4-(4-(2-(2,3-difluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-metho

xyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10h). White solid, yield:
83.9%. HRMS (ESI) m/z 633.2703 [M+H]+, Calcd. for 633.2637.
5.1.8.9.
(E)-4-((4-(4-(2-(2-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10i). White solid, yield:
82.6%. HRMS (ESI) m/z 615.2803 [M+H]+, Calcd. for 615.2731.
5.1.8.10.
(E)-4-((4-(4-(2-(2,4-difluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-metho
xyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10j). White solid, yield:
82.0%. HRMS (ESI) m/z 633.2701 [M+H]+, Calcd. for 633.2637.
5.1.8.11.
(E)-4-((4-(4-(2-(2,6-difluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-metho
xyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10k). White solid, yield:
80.1%. HRMS (ESI) m/z 633.2705 [M+H]+, Calcd. for 633.2637.
5.1.8.12.
(E)-4-((4-(4-(2-(3-fluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10l). White solid, yield:
76.9%. HRMS (ESI) m/z 615.2801 [M+H]+, Calcd. for 615.2731.
5.1.8.13.
(E)-N-isopropyl-4-((6-methoxy-4-(4-(2-(2,4,6-trifluorobenzylidene)hydrazine-1-carbo
xamido)phenoxy)quinolin-7-yl)oxy)piperidine-1-carboxamide (10m). White solid,
yield: 80.7%. HRMS (ESI) m/z 651.2605 [M+H]+, Calcd. for 651.2543.
5.1.8.14.
(E)-4-((4-((4-(2-(2,6-difluorobenzylidene)hydrazine-1-carboxamido)phenyl)thio)-6-m
ethoxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (10n). White solid,
yield: 77.7%. HRMS (ESI) m/z 649.2473 [M+H]+, Calcd. for 649.2409.
5.1.8.15.
(E)-4-(((4-(4-(2-(2,6-difluorobenzylidene)hydrazine-1-carboxamido)phenoxy)-6-meth
oxyquinolin-7-yl)oxy)methyl)-N-isopropylpiperidine-1-carboxamide (10o). White
solid, yield: 83.5%. HRMS (ESI) m/z 647.2855 [M+H]+, Calcd. for 647.2793.
5.1.9. General procedure for the synthesis of target compounds 11a-o. To a solution
of semicarbazides 10a-o (0.5 mmol) and mercaptoacetic acid (0.5 mL) in dry CH2Cl2
(10 mL), SiCl4 (20 drops) were added at room temperature. After the resulting
mixture refluxed for 6 h, the reaction mixture was cooled to room temperature before
quenched by ice. The solution was adjusted to pH 9 with 10% NaOH aqueous
solution. The organic phase was separated and washed with water (2×5 mL),
concentrated in vacuo to afford yellow oil which was purified by silica gel column
chromatography (eluent, CH2Cl2: MeOH: Et3N = 100:5:1 to 100:10:1) to afford target
compounds.
5.1.9.1.
N-ethyl-4-((4-(4-(3-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-meth
1
oxyquinolin-7-yl)oxy)piperidine-1-carboxamide (11a). White solid, yield: 36.3%. H
NMR (400 MHz, DMSO-d6) δ: 9.45 (br, 1H), 9.06 (br, 1H), 8.45 (d, J = 5.2 Hz, 1H),
7.52-7.57 (m, 5H), 7.49 (s, 1H), 7.21-7.25 (m, 2H), 7.17 (m, 1H), 7.14 (m, 1H),

6.50-6.53 (m, 1H), 6.40 (d, J = 5.2 Hz, 1H), 5.85 (s, 1H), 4.76-4.82 (m, 1H), 3.93 (s,
3H), 3.85-3.89 (m, 1H), 3.72-3.77 (m, 3H), 3.09-3.17 (m, 2H), 3.20-3.07 (m, 2H),
1.98-2.02 (m, 2H), 1.53-1.60 (m, 2H), 1.02 (t, J = 7.2 Hz, 3H); 13C NMR (100 MHz,
DMSO-d6) δ: 169.4, 161.7, 160.6, 157.4 (2C), 154.2, 150.5, 149.3, 149.0, 146.7,
137.2, 132.3, 131.9, 121.9 (2C), 116.0, 115.9, 115.7, 115.2, 112.9, 112.7, 110.8, 103.3,
100.1, 73.9, 56.9, 56.2, 42.0, 41.5 (2C), 30.7 (2C), 29.6, 13.9. HRMS (ESI) m/z
697.2304 [M+Na]+, Calcd. for 697.2221.
5.1.9.2.
4-((4-(4-(3-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyquin
olin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11b). White solid, yield: 41.7%.
1H NMR (400 MHz, DMSO-d6) δ: 8.97 (br, 1H), 8.54 (s, 1H), 8.45 (d, J = 5.2 Hz,
1H), 7.52-7.58 (m, 5H), 7.49 (s, 1H), 7.22-7.26 (m, 2H), 7.18 (m, 1H), 7.15 (m, 1H),
6.41 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 5.85 (s, 1H), 4.77-4.81 (m, 1H), 3.93 (s, 3H),
3.86-3.90 (m, 1H), 3.73-3.81 (m, 4H), 3.09-3.16 (m, 2H), 1.98-2.01 (m, 2H),
1.52-1.61 (m, 2H), 1.07 (s, 3H), 1.06 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
169.7, 161.8, 160.6, 157.3 (2C), 154.2, 150.5, 149.3, 149.0, 146.7, 137.2, 132.1, 131.9,
121.9 (2C), 116.0, 115.9, 115.7, 115.2, 112.9, 112.7, 110.8, 103.3, 100.1, 73.9, 56.9,
56.2, 42.3, 41.5 (2C), 30.7 (2C), 29.6, 23.4 (2C). HRMS (ESI) m/z 711.2481
[M+Na]+, Calcd. for 711.2377.
5.1.9.3.
N-butyl-4-((4-(4-(3-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-meth
1
oxyquinolin-7-yl)oxy)piperidine-1-carboxamide (11c). White solid, yield: 40.4%. H
NMR (400 MHz, DMSO-d6) δ: 8.97 (br, 1H), 8.54 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H),
7.51-7.58 (m, 5H), 7.49 (s, 1H), 7.22-7.26 (m, 2H), 7.18 (m, 1H), 7.15 (m, 1H), 6.49
(m, 1H), 6.41 (d, J = 5.2 Hz, 1H), 5.85 (s, 1H), 4.77-4.81 (m, 1H), 3.92 (s, 3H),
3.86-3.90 (m, 1H), 3.73-3.79 (m, 3H), 3.10-3.17 (m, 2H), 3.00-3.05 (m, 2H),
1.98-2.01 (m, 2H), 1.52-1.60 (m, 2H), 1.36-1.43 (m, 2H), 1.23-1.32 (m, 2H), 0.88 (m,
3H). HRMS (ESI) m/z 725.2625 [M+Na]+, Calcd. for 725.2534.
5.1.9.4.
N-cyclohexyl-4-((4-(4-(3-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6
-methoxyquinolin-7-yl)oxy)piperidine-1-carboxamide (11d). White solid, yield: 40.0%.
1H NMR (400 MHz, DMSO-d6) δ: 8.94 (br, 1H), 8.51 (s, 1H), 8.45 (d, J = 5.2 Hz,
1H), 7.51-7.58 (m, 5H), 7.49 (s, 1H), 7.22-7.26 (m, 2H), 7.18 (m, 1H), 7.15 (m, 1H),
6.40 (d, J = 5.2 Hz, 1H), 6.21 (m, 1H), 5.84 (s, 1H), 4.76-4.80 (m, 1H), 3.92 (s, 3H),
3.86-3.91 (m, 1H), 3.72-3.79 (m, 3H), 3.09-3.16 (m, 2H), 1.97-2.01 (m, 2H),
1.74-1.77 (m, 2H), 1.67-1.70 (m, 2H), 1.52-1.59 (m, 3H), 1.18-1.28 (m, 4H),
1.05-1.15 (m, 2H);1.07 (s, 3H), 1.06 (s, 3H). HRMS (ESI) m/z 751.2791 [M+Na]+,
Calcd. for 751.2690.
5.1.9.5.
N-benzyl-4-((4-(4-(3-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-met
hoxyquinolin-7-yl)oxy)piperidine-1-carboxamide (11e). Light yellow solid, yield:
1
41.3%, purity: 98.24%. H NMR (400 MHz, DMSO-d6) δ: 9.06 (br, 1H), 8.64 (br,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.52-7.58 (m, 5H), 7.50 (s, 1H), 7.30-7.33 (m, 2H),
7.19-7.27 (m, 6H), 7.13-7.18 (m, 3H), 6.41 (d, J = 5.2 Hz, 1H), 5.83 (s, 1H),

4.79-4.84 (m, 1H), 4.27 (m, 2H), 3.93 (s, 3H), 3.74-3.90 (m, 5H), 3.17-3.23 (m, 2H),
2.01-2.04 (m, 2H), 1.55-1.64 (m, 2H). HRMS (ESI) m/z 759.2481 [M+Na]+, Calcd.
for 759.2377.
5.1.9.6.
N-isopropyl-4-((6-methoxy-4-(4-(3-(4-oxo-2-phenylthiazolidin-3-yl)ureido)phenoxy)q
uinolin-7-yl)oxy)piperidine-1-carboxamide (11f). White solid, yield: 44.6%. 1H NMR
(400 MHz, DMSO-d6) δ: 8.95 (br, 1H), 8.55 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H),
7.49-7.54 (m, 6H), 7.35-7.43 (m, 3H), 7.18 (m, 1H), 7.15 (m, 1H), 6.41 (d, J = 5.2 Hz,
1H), 6.22 (m, 1H), 5.83 (s, 1H), 4.76-4.82 (m, 1H), 3.93 (s, 3H), 3.83-3.89 (m, 1H),
3.73-3.79 (m, 4H), 3.10-3.16 (m, 2H), 1.97-2.01 (m, 2H), 1.52-1.61 (m, 2H), 1.07 (s,
3H), 1.06 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ: 169.7, 164.0, 160.5, 157.3 (2C),
154.2, 150.5, 149.3, 149.0, 146.8, 137.1, 132.2, 131.9, 121.9 (2C), 120.7, 115.7, 115.4,
115.2, 112.9, 112.7, 110.8, 103.4, 100.1, 73.8, 56.9, 56.2, 42.3, 41.5 (2C), 30.7 (2C),
29.6, 23.4 (2C). HRMS (ESI) m/z 693.2539 [M+Na]+, Calcd. for 693.2471.
5.1.9.7.
4-((4-(4-(3-(2-(3,4-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11g). White solid, yield:
1
44.7%. H NMR (400 MHz, DMSO-d6) δ: 9.01 (s, 1H), 8.57 (s, 1H), 8.45 (d, J = 5.2
Hz, 1H), 7.64-7.68 (m, 1H), 7.43-7.54 (m, 5H), 7.36-7.38 (m, 1H), 7.18 (m, 1H), 7,16
(m, 1H), 6.41 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 5.85 (s, 1H), 4.77-4.82 (m, 1H), 3.93
(s, 3H), 3.90-3.94 (m, 1H), 3.74-3.81 (m, 4H), 3.10-3.16 (m, 2H), 1.97-2.01 (m, 2H),
1.52-1.61 (m, 2H), 1.07 (s, 3H), 1.06 (s, 3H). HRMS (ESI) m/z 729.2386 [M+Na]+,
Calcd. for 729.2283.
5.1.9.8.
4-((4-(4-(3-(2-(2,3-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11h). White solid, yield:
1
40.1%. H NMR (400 MHz, DMSO-d6) δ: 9.02 (s, 1H), 8.72 (s, 1H), 8.45 (d, J = 5.2
Hz, 1H), 7.52-7.55 (m, 3H), 7.48 (s, 1H), 7.44-7.47 (m, 2H), 7.25-7.30 (m, 1H), 7.19
(m, 1H), 7,17 (m, 1H), 6.41 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 6.07 (s, 1H), 4.77-4.81
(m, 1H), 3.93 (s, 3H), 3.73-3.89 (m, 5H), 3.10-3.16 (m, 2H), 1.97-2.02 (m, 2H),
1.52-1.61 (m, 2H), 1.08 (s, 3H), 1.06 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ:
169.2, 164.0, 160.5, 157.3 (2C), 154.2, 150.5, 149.3, 149.0, 146.8, 137.1, 132.2, 131.4,
121.9 (2C), 120.7, 115.7, 115.4, 115.0, 112.9, 112.8, 110.8, 103.4, 100.1, 73.8, 56.9,
56.2, 42.3, 41.5 (2C), 30.8 (2C), 29.6, 23.3 (2C). HRMS (ESI) m/z 729.2386
[M+Na]+, Calcd. for 729.2283.
5.1.9.9.
4-((4-(4-(3-(2-(2-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyquin
olin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11i). White solid, yield: 45.8%.
1H NMR (400 MHz, DMSO-d6) δ: 8.99 (br, 1H), 8.61 (s, 1H), 8.45 (d, J = 5.2 Hz,
1H), 7.52-7.55 (m, 3H), 7.49 (s, 1H), 7.39-7.48 (m, 2H), 7.32-7.34 (m, 1H), 7.20-7.22
(m, 1H), 7.18 (m, 1H), 7,16 (m, 1H), 6.41 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 5.86 (s,
1H), 4.76-4.81 (m, 1H), 3.93 (s, 3H), 3.89 (m, 1H), 3.74-3.79 (m, 4H), 3.09-3.16 (m,
2H), 1.97-2.01 (m, 2H), 1.53-1.61 (m, 2H), 1.07 (s, 3H), 1.06 (s, 3H). HRMS (ESI)
m/z 711.2492 [M+Na]+, Calcd. for 711.2377.

5.1.9.10.
4-((4-(4-(3-(2-(2,4-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11j). White solid, yield:
38.6%. 1H NMR (400 MHz, DMSO-d6) δ: 9.07 (br, 1H), 8.70 (s, 1H), 8.45 (d, J = 5.2
Hz, 1H), 7.68-7.74 (m, 1H), 7.52-7.55 (m, 3H), 7.49 (s, 1H), 7.29-7.33 (m, 1H),
7.16-7.19 (m, 3H), 6.41 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 6.04 (s, 1H), 4.76-4.82 (m,
1H), 3.93 (s, 3H), 3.86-3.90 (m, 1H), 3.79-3.80 (m, 1H), 3.73-3.78 (m, 3H), 3.09-3.16
(m, 2H), 1.97-2.01 (m, 2H), 1.52-1.61 (m, 2H), 1.07 (s, 3H), 1.06 (s, 3H). HRMS
(ESI) m/z 729.2411 [M+Na]+, Calcd. for 729.2283.
5.1.9.11.
4-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11k). White solid, yield:
1
38.9%, purity: 99.65%. H NMR (400 MHz, DMSO-d6) δ: 9.04 (s, 1H, NH), 8.84 (s,
1H, NH), 8.45 (d, J = 5.2 Hz, 1H, quinoline-2H), 7.50-7.54 (m, 4H, Ar-H), 7.49 (s,
1H, Ar-H), 7.16-7.20 (m, 4H, Ar-H), 6.41 (d, J = 5.2 Hz, 1H, quinoline-3H), 6.22 (m,
1H, NH), 6.17 (s, 1H, thiazolidinone-2H), 4.76-4.82 (m, 1H, isopropyl-H), 3.93 (s, 3H,
CH3O-), 3.79-3.83 (s, 2H, thiazolidinone-5H), 3.73-3.80 (m, 3H, piperidinyl-H),
3.10-3.16 (m, 2H, piperidinyl-H), 1.97-2.01 (m, 2H, piperidinyl-H), 1.52-1.61 (m, 2H,
13
piperidinyl-H), 1.07 (s, 3H, isopropyl-H), 1.06 (s, 3H, isopropyl-H); C NMR (100
MHz, DMSO-d6) δ: 169.7, 164.0, 160.6, 157.3 (2C), 154.2, 150.5, 149.3, 149.0, 146.8,
137.1, 132.1, 131.9, 121.9 (2C), 120.7, 115.7, 115.4, 115.2, 112.9, 112.7, 110.8, 103.4,
100.1, 73.9, 56.9, 56.2, 42.3, 41.5 (2C), 30.7 (2C), 29.6, 23.4 (2C). HRMS (ESI) m/z
729.2411 [M+Na]+, Calcd. for 729.2283.
5.1.9.12.
4-((4-(4-(3-(2-(3-fluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyquin
olin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11l). White solid, yield: 38.9%.
1H NMR (400 MHz, DMSO-d6) δ: 9.01 (s, 1H), 8.68 (s, 1H), 8.45 (d, J = 5.2 Hz, 1H),
7.61-7.65 (m, 1H), 7.52-7.54 (m, 3H), 7.49 (s, 1H), 7.41-7.46 (m, 1H), 7.23-7.29 (m,
2H), 7.18 (m, 1H), 7,16 (m, 1H), 6.41 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 6.07 (s, 1H),
4.76-4.81 (m, 1H), 3.93 (s, 3H), 3.86-3.90 (m, 1H), 3.79-3.81 (m, 1H), 3.73-3.77 (m,
3H), 3.10-3.16 (m, 2H), 1.97-2.02 (m, 2H), 1.52-1.60 (m, 2H), 1.07 (s, 3H), 1.06 (s,
3H). HRMS (ESI) m/z 711.2452 [M+Na]+, Calcd. for 711.2377.
5.1.9.13.
N-isopropyl-4-((6-methoxy-4-(4-(3-(4-oxo-2-(2,4,6-trifluorophenyl)thiazolidin-3-yl)ur
eido)phenoxy)quinolin-7-yl)oxy)piperidine-1-carboxamide (11m). White solid, yield:
1
36.7%. H NMR (400 MHz, DMSO-d6) δ: 9.04 (s, 1H), 8.80 (s, 1H), 8.45 (d, J = 5.2
Hz, 1H), 7.52-7.54 (m, 3H), 7.49 (s, 1H), 7.28-7.32 (m, 2H), 7.19 (m, 1H), 7.17 (m,
1H), 6.42 (d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 6.11 (s, 1H), 4.77-4.80 (m, 1H), 3.93 (s,
3H), 3.82-3.87 (m, 2H), 3.74-3.79 (m, 3H), 3.10-3.15 (m, 2H), 1.97-2.01 (m, 2H),
1.53-1.60 (m, 2H), 1.08 (s, 3H), 1.06 (s, 3H). HRMS (ESI) m/z 747.2342 [M+Na]+,
Calcd. for 747.2189.
5.1.9.14.
4-((4-((4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenyl)thio)-6-meth
oxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (11n). White solid, yield:

1
39.5%. H NMR (400 MHz, DMSO-d6) δ: 9.14 (s, 1H), 8.86 (s, 1H), 8.40 (d, J = 5.2
Hz, 1H), 7.58-7.60 (m, 2H), 7.47-7.53 (m, 4H), 7.32 (s, 1H), 7.16-7.20 (m, 2H), 6.61
(d, J = 5.2 Hz, 1H), 6.22 (m, 1H), 6.16 (s, 1H), 4.76-4.82 (m, 1H), 3.93 (s, 3H), 3.84
(s, 2H), 3.73-3.79 (m, 3H), 3.08-3.15 (m, 2H), 1.97-2.01 (m, 2H), 1.51-1.60 (m, 2H),
13
1.07 (s, 3H), 1.06 (s, 3H); C NMR (100 MHz, DMSO-d6) δ: 169.7, 163.7, 160.5,
157.3 (2C), 150.5, 149.3, 149.0, 146.8, 137.1, 132.2, 131.9, 130.5, 121.9 (2C), 120.7,
115.7, 115.4, 115.2, 112.9, 112.7, 110.8, 103.4, 100.2, 73.8, 56.9, 56.2, 42.4, 41.5
(2C), 30.6 (2C), 29.6, 23.4 (2C). HRMS (ESI) m/z 745.2191 [M+Na]+, Calcd. for
745.2054.
5.1.9.15.
4-(((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxy
quinolin-7-yl)oxy)methyl)-N-isopropylpiperidine-1-carboxamide (11o). White solid,
yield: 40.8%, purity: 97.35%. 1H NMR (400 MHz, DMSO-d6) δ: 9.02 (s, 1H), 8.81 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.48-7.54 (m, 4H), 7.38 (s, 1H), 7.16-7.20 (m, 4H),
6.42 (d, J = 5.2 Hz, 1H), 6.17 (s, 1H), 6.09-6.11 (m, 1H), 4.76-4.82 (m, 1H),
4.00-4.03 (m, 4H), 3.93 (s, 3H), 3.83 (s, 2H), 3.73-3.77 (m, 1H), 2.64-2.70 (m, 2H),
2.01 (br, 1H), 1.76-1.78 (m, 2H), 1.16-1.25 (m, 2H), 1.06 (s, 3H), 1.05 (s, 3H).
HRMS (ESI) m/z 743.2507 [M+Na]+, Calcd. for 743.2439.
5.1.10.
3-((4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-methoxyq
uinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide (19). Taking intermediate 3a
and tert-butyl 3-((methylsulfonyl)oxy)piperidine-1-carboxylate as starting materials,
target compound was prepared following the synthetic procedure of 11k. White solid,
yield: 34.8%.1H NMR (400 MHz, DMSO-d6) δ: 9.03 (s, 1H), 8.84 (s, 1H), 8.44 (d, J
= 5.2 Hz, 1H), 7.49-7.54 (m, 4H), 7.47 (s, 1H), 7.16-7.21 (m, 4H), 6.42 (d, J = 5.2 Hz,
1H), 6.21 (m, 1H), 6.16 (s, 1H), 4.76-4.81 (m, 1H), 3.92 (s, 3H), 3.79-3.83 (m, 2H),
3.73-3.79 (m, 3H), 3.10-3.17 (m, 2H), 1.89-1.96 (m, 2H), 1.48-1.59 (m, 2H), 1.07 (s,
3H), 1.06 (s, 3H). HRMS (ESI) m/z 729.2411 [M+Na]+, Calcd. for 729.2283.
5.1.11. 7-(benzyloxy)-6-methoxy-4-(4-nitrophenoxy)quinoline 1-oxide (20).
Intermediate 2a (20.1 g, 50.0 mmol) in CH2Cl2 (200 mL) was treated with m-CPBA
(10.4 g, 60.0 mmol) at room temperature for 6 h. The reaction mixture was washed
with NaHCO3 aqueous solution (30 mL) and water (30 mL). The organic phase was
separated, dried over Na2SO4, and evaporated under vacuum to afford 20 as yellow
solid, yield: 67.7%. HRMS (ESI) m/z 419.1309 [M+H]+, Calcd. for 419.1243.
5.1.12.
7-((1-(isopropylcarbamoyl)piperidin-4-yl)oxy)-6-methoxy-4-(4-nitrophenoxy)quinolin
e 1-oxide (24). Prepared by the method described for the synthesis of intermediate 6b.
Yellow solid. HRMS (ESI) m/z 497.2064 [M+H]+, Calcd. for 497.2036.
5.1.13.
4-((2-(tert-butylamino)-6-methoxy-4-(4-nitrophenoxy)quinolin-7-yl)oxy)-N-isopropylp
iperidine-1-carboxamide (25). To a solution of quinoline N-oxide 24 (9.9 g, 20.0
mmol) and tert-butylamine (12.3 mL, 120.0 mmol) in CH2Cl2 at 0
was added
4-methylbenzenesulfonic anhydride (16.3 g, 50.0 mmol) in portions while
maintaining the reaction temperature at 0-5 °C. The mixture was warmed to room

temperature and stirred for 1 h. The reaction mixture was quenched with NaHCO3
aqueous solution (30 mL). The organic layer was separated, concentrated and the
crude product was purified by silica gel column chromatography to afford title
intermediate as light yellow solid, yield: 62.1%. HRMS (ESI) m/z 552.2870 [M+H]+,
Calcd. For 552.2822.
5.1.14.
4-((2-(tert-butylamino)-4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)p
henoxy)-6-methoxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide
(30).
Prepared following the synthetic procedure of 11k. White solid, yield: 36.1%. HRMS
(ESI) m/z 778.3244 [M+H]+, Calcd. For 778.3198.
5.1.15.
4-((2-amino-4-(4-(3-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)ureido)phenoxy)-6-
methoxyquinolin-7-yl)oxy)-N-isopropylpiperidine-1-carboxamide
(31).
To
a
suspension of 30 (0.39 g, 0.5 mmol) in xylene (3 mL), CF3COOH (3 mL) was added.
The solution was stirred for 5 h at 70 °C. The resulting mixture was concentrated, and
the residue was dissolved in CH2Cl2 (10 mL) which was basified by 10 % NaOH
aqueous solution. CH2Cl2 was separated, concentrated in vacuo and the oil was
purified by silica gel column chromatography (eluent, CH2Cl2: MeOH: Et3N =
100:5:1 to 100:20:1) to afford the title compound as a white solid, yield: 57.9%,
1
purity: 98.14%. H NMR (400 MHz, DMSO-d6) δ: 9.02 (s, 1H), 8.82 (s, 1H),
7.50-7.54 (m, 4H), 7.48 (s, 1H), 7.16-7.21 (m, 4H), 6.46 (s, 1H), 6.27 (br, 2H), 6.20
(m, 1H), 6.16 (s, 1H), 4.75-4.82 (m, 1H), 3.92 (s, 3H), 3.79-3.84 (m, 2H), 3.72-3.77
(m, 3H), 3.10-3.16 (m, 2H), 1.96-2.01 (m, 2H), 1.53-1.61 (m, 2H), 1.07 (s, 3H), 1.06
(s, 3H). HRMS (ESI) m/z 744.2534 [M+Na]+, Calcd. For. 744.2392.
5.1.16. 1-(3-(benzyloxy)-4-methoxyphenyl)ethan-1-one (32). To a mixture of
1-(3-hydroxy-4-methoxyphenyl)ethan-1-one (16.6 g, 0.1 mol) and K2CO3 (34.5 g,
0.25 mol) in DMF (80 mL), benzyl bromide ( 18.6 g, 0.11mol) was added dropwise at
room temperature. After stirring for 2 h, the reaction mixture was poured into cold
water (400 mL). The suspended solid was obtained by filtration and washed by water.
White solid, yield: 89.8%. HRMS (ESI) m/z 257.1212 [M+H]+, Calcd. For 257.1178.
5.1.17. 1-(5-(benzyloxy)-4-methoxy-2-nitrophenyl)ethan-1-one (33). Fuming HNO3
(16 mL) was added dropwise at -10 to a solution of 32 (25.7 g, 0.1 mol) in CH2Cl2
(200 mL). After stirring for 5 h at -10 , the solution was poured into ice water (100
mL). The organic layer was washed with saturated sodium bicarbonate (2×50 mL),
and brine (50 mL). The organic layers were dried over Na2SO4, filtered, and
concentrated under vacumm to provide 33 as a yellow solid, yield: 73.6%. HRMS
(ESI) m/z 302.1064 [M+H]+, Calcd. For 302.1028.
5.1.18. 1-(2-amino-5-(benzyloxy)-4-methoxyphenyl)ethan-1-one (34). Powered iron
(0.3 mol) and concentrated HCl (10 drops) was added to a suspension of intermediates
33 (0.1 mol) in 90% EtOH (300 mL). The mixture was refluxed with vigorous
agitation for 3 h. The hot mixture was filtered through celites and the filtrate was cool
to 0 . The precipitate was filtered and dried to afford 34 as a yellow solid, yield:
78.5%. HRMS (ESI) m/z 272.1329 [M+H]+, Calcd. For 272.1287.
5.1.19. 6-(benzyloxy)-7-methoxyquinolin-4-ol (35). Intermediate 34 (27.2 g, 0.1 mol)