Identification of novel N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas showing potent multi-tyrosine kinase inhibitory activities

Article abstract of DOI:10.1016/j.ejmech.2018.01.061

A total of 29 novel compounds bearing N¹-(2-aryl-1, 3-thiazolidin-4-one)-N³-aryl ureas were designed, synthesized and evaluated for their biological activities. The structure-activity relationships (SARs) and binding modes of this series of compounds were clarified together. Compound 29b was identified possessing high potency against multi-tyrosine kinases including Ron, c-Met, c-Kit, KDR, Src and IGF-1R, etc. In vitro antiproliferation and cytotoxicity of compound 29b against A549 cancer cell line were confirmed by IncuCyte live-cell imaging.

Full text of DOI:10.1016/j.ejmech.2018.01.061

Accepted Manuscript
1
3
Identification of novel N -(2-aryl-1, 3-thiazolidin-4-one)-N -aryl ureas showing potent
multi-tyrosine kinase inhibitory activities
Baohui Qi, Ying Yang, Huan He, Xupeng Yue, Yuting Zhou, Xing Zhou, Yuying Chen,
Min Liu, Anmian Zhang, Fachang Wei
PII:
S0223-5234(18)30074-6
10.1016/j.ejmech.2018.01.061
EJMECH 10138
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 September 2017
Revised Date: 18 January 2018
Accepted Date: 18 January 2018
Please cite this article as: B. Qi, Y. Yang, H. He, X. Yue, Y. Zhou, X. Zhou, Y. Chen, M. Liu, A. Zhang,
1
3
F. Wei, Identification of novel N -(2-aryl-1, 3-thiazolidin-4-one)-N -aryl ureas showing potent multi-
tyrosine kinase inhibitory activities, European Journal of Medicinal Chemistry (2018), doi: 10.1016/
j.ejmech.2018.01.061.
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ACCEPTED MANUSCRIPT
Identification of novel N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas showing
potent multi-tyrosine kinase inhibitory activities
Baohui Qi, Ying Yang, Huan He, Xupeng Yue, Yuting Zhou, Xing Zhou, Yuying Chen, Min Liu,
Anmian Zhang, Fachang Wei
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, Guangdong
Province, China
Graphical Abstract

ACCEPTED MANUSCRIPT
Identification of novel N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas showing
potent multi-tyrosine kinase inhibitory activities
Baohui Qi, Ying Yang, Huan He, Xupeng Yue, Yuting Zhou, Xing Zhou, Yuying Chen, Min Liu,
Anmian Zhang, Fachang Wei
Department of Bioengineering, Zhuhai Campus of Zunyi Medical University, Zhuhai 519041, Guangdong
Province, China
Abstract
A total of 29 novel compounds bearing N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl ureas were designed,
synthesized and evaluated for their biological activities. The structure-activity relationships (SARs) and
binding modes of this series of compounds were clarified together. Compound 29b was identified
possessing high potency against multi-tyrosine kinases including Ron, c-Met, c-Kit, KDR, Src and IGF-1R,
etc. In vitro antiproliferation and cytotoxicity of compound 29b against A549 cancer cell line were
confirmed by IncuCyte live-cell imaging.
Keywords: 1, 3-thiazolidin-4-one; urea; anticancer; tyrosine kinase; inhibitors
1
Introduction
C-Met, also referred to as hepatocyte growth factor receptor (HGFR), is a heterodimeric transmemeric
receptor tyrosine kinase composed of an extracellular α-chain and a membrane spanning β-chain that are
connected through a disulfide bond [1]. Aberrant c-Met signaling, resulting from MET genomic
amplification, c-Met overexpression, or c-Met mutations, could be found in a variety of human cancers and
often correlated with poor clinical outcomes [2]. Some literature disclosed that constitutive c-Met
activation due to MET amplification was found to be a driver of proliferation and survival of several gastric
and lung cancer cell lines, and has been linked to acquired resistance of lung cancers to epidermal growth
factor receptor (EGFR) inhibitors [3-5]. Thus, developing novel c-Met kinase inhibitors have been actively
pursued by researchers, especially small-molecule inhibitors targeting the catalytic domains of kinase [6,
7].
Cabozantinib, a typical multi-kinase inhibitor bearing quinoline pharmacophore, was approved for the
treatment of patients with progressive metastatic medullary thyroid cancer (MTC) [8]. In recent years, a
variety of small-molecule inhibitors bearing quinoline pharmacophores have emerged continually as shown
in Fig. 1 [9-12]. 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 [2].
(Fig. 1 should be listed here.)
As shown in Fig. 2, reported SARs [13-16] of quinoline-based inhibitors suggested that regions I and II,
which could form H-bonds with the backbone of c-Met kinase, are crucial for inhibitory activity. The
substituted phenyl ring (ring A) connecting region I and II was responsible for forming π-π stacking. In
addition, hydrophobic interaction was generated by terminal aryl ring reaching into the hydrophobic pocket
formed by amino acid residues.

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(Fig. 1 should be listed here.)
1, 3-Thiazolidin-4-one and its derivatives have attracted much attention over the years since they were
promising scaffolds that have occupied a prominent position in medicinal chemistry [17, 18]. The detailed
literature survey of 1, 3-thiazolidin-4-one and its derivatives demonstrated a wide broad-spectrum
pharmacological properties such as antioxidant, antimalarial, anti-HIV, anti-inflammatory, antibacterial and
anticancer, etc [19-22]. Published data indicated that anticancer activity of 1, 3-thiazolidin-4-ones may be
associated with their affinity to diverse targets including non-membrane protein tyrosine phosphatase
(SHP-2), JNK-stimulating phosphatase-1 (JSP-1), tumor necrosis factor TNF-α, anti-apoptotic biocomplex
Bcl-XL-BH3 and inregrin, etc [23].
Hence, based on the above SARs and the structural charactiristics of c-Met kinase inhibitors bearing
quinoline pharmacophores, we designed and synthesized hydrid compounds by linking the main structural
unit of 4-phenoxy-6, 7-disubstituted quinolines with 1, 3-thiazolidin-3-one scaffolds in attempt to maximize
their anticancer activity (Fig. 2). In this report, in vitro antiproliferation and cytotoxicity against A549
cancer cell line were further confirmed by Incucyte real-time imaging.
2 Results and discussion
2.1 Chemistry
(Scheme 1 should be listed here.)
Target compounds 11a-u were synthesized according to the routes outlined in Scheme 1 [24].
Commercially available 4-hydroxy-3-methoxyacetophenone was alkylated with 1-bromo-3-chloropropane
under basic condition to provide 1. Regioselective nitration and subsequent aminomethylenation with N,
N-dimethylformamide dimethylacetal (DMF-DMA) afforded intermediate 3. The intramolecular cyclization
in the presence of iron powder and acetic acid afforded 4, which underwent a nucleophilic substitution with
morpholine to give access to 5. The resultant hydroxyl moiety 5 was converted to 6 by nucleophilic
substitution with 4-fluoronitrobenzene under Cs2CO3 in MeCN. Intermediate 6 was reduced to provide
aniline 7 in excellent yields. By further treatment with 4-nitrophenyl carbonochloridate, the intermediate 7
was converted into amides 8 which was treated with hydrazine hydrate directly to furnish semicarbazide 9.
Intermediates 10a-u were available via the condensation of 9 with appropriate aldehydes. Finally, the target
compounds 11a-u were obtained via the cyclization with mercaptoacetic acid under SiCl4 [25].
(Scheme 2 should be listed here.)
As shown in Scheme 2, the resultant hydroxyl moiety 5 obtained previously was converted to the
corresponding chloride 12 on exposure to phosphorus oxychloride. Nucleophilic displacement of chlorine
atom in the 12 with 4-hydroxybenzaldehyde afforded 13 [26]. Finally, the intermediate 13 was condensed
with N-(2-fluorophenyl)hydrazinecarboxamide or N-(2, 4-difluorophenyl)hydrazine-carboxamide to afford
semicarbazones 14a and 14b, respectively. At last, the target compounds 15a-b were obtained according to
the same method (vide supra).

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As illustrated in Scheme 3 and Scheme 4, the synthesis of compounds 22a-d and 29a-b were done in
accordance with previously described procedures prepared for 11a-u.
(Scheme 3 should be listed here.)
(Scheme 4 should be listed here.)
2.2 Enzymatic Assay and SARs
Taking Cabozantinib and Staurosporine as references, all the newly synthesized compounds (11a-u,
15a-b, 22a-d and 29a-b) were evaluated for their ability to inhibit c-Met kinase using enzyme-linked
immunosorbent assays (ELISAs). The results are summarized in Table 1, 2, 3 and 4.
A series of compounds (11a-u) with different R1 were synthesized and evaluated for their inhibition of
c-Met kinase (Table 1). All compounds exhibited moderate to potent inhibitory activities against c-Met
kinase with IC50 values ranging from 0.05 to 0.688 µmol·L-1.
Firstly, a phenyl ring (ring B) was introduced at the C-2 of 1, 3-thiazolidin-4-one and compound 11a was
obtained. Satisfyingly, it displayed promising inhibition of c-Met kinase (IC50 = 0.097 µmol·L-1).
Replacement of the phenyl ring by a cyclohexyl ring led to an approximate 5-fold drop in potency (11u vs
11a). The introduction of pyridinyl ring 11p into R1 was detrimental to enzymatic potency (3.3-fold loss in
potency). In order to know if the “five-atom-rule” [16] was applicable for the designed compounds, benzyl
11s and phenethyl 11t were synthesized. In addition, loss of c-Met inhibition was observed compared 11s
(IC50 = 0.205 µmol·L-1) and 11t (IC50 = 0.331 µmol·L-1) with 11a (IC50 = 0.097 µmol·L-1). It suggested that
the presence of a phenyl ring directly attached to the C-2 of 1, 3-thiazolidin-4-one was of significance to
inhibitory potency. The bulky effect of the substituents likely weakened inhibitory potency, since the more
steric analogues 4-dimethylaminophenyl 11q and naphthyl 11r showed 4.4- and 6.7-fold lower potency
than phenyl 11a. Subsequently, a series of compounds (11b-o) with diversified small substituents at the
phenyl ring were explored. IC50 values indicated that it was well tolerated when the substituents were at the
ortho-position of phenyl ring, with following rank order: -CF3 < -Cl < -F. From the results above, we could
confirm that minor changes at R1 could influence c-Met kinase inhibitory activity in different extent.
(Table 1 should be listed here.)
In the following work, the 1, 3-thiazolidin-4-one moiety was moved to 4-position of phenyl ring A.
Unfortunately, the obtained compounds 15a (IC50 = 4.10 µmol·L-1) and 15b (IC50 = 3.35 µmol·L-1) were
significantly less potent compared to 11b (IC50 = 0.061 µmol·L-1) and 11e (IC50 = 0.141 µmol·L-1)
accordingly (Table 2).
(Table 2 should be listed here.)
Having identified well tolerated group on R1 (2, 6-F-Ph-) and the position of 1, 3-thiazolidin-4-one
moiety, our attention was turned to the investigation of R3 and four compounds (22a-d) were prepared
(Table 3). To our delight, the pyrrolyl derivative 22d exhibited optimal c-Met inhibition (IC50 = 0.037

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µmol·L-1).
(Table 3 should be listed here.)
Encouraged by the prominent activity of compound 22d, it was selected for further investigation focused
on the phenyl ring A. The IC50 values showed that the substitution by fluorine atom on the phenyl ring A
resulted a slight boost in c-Met inhibitory activity. The 3-fluoro analogue 29b displayed a better enzymatic
potency than the 2-fluoro analogue 29a, suggesting that 3-fluoro substitution is superior to 2-fluoro
substitution (Table 4).
(Table 4 should be listed here.)
2.3 Molecular docking study
In order to elucidate the binding mode of this series of compounds with the active site of c-Met kinase,
docking simulation studies of selected compound 29b were carried out by Molecular Operating
Environment (MOE, Chemical Computing Group Inc., Canada). As depicted in Fig.3A, compound 29b
adopted an extend conformation as type II kinase inhibitors [27] was buried into the binding pocket of
c-Met kinase completely. It occupied the ATP-binding site and formed two strong H-bonds (the quinoline-N
and Met1160, the urea-O and Lys1110) and one weak H-bond (the thiazolidinone-S and Glu1127). The
terminal 2, 6-difluorophenyl ring (ring B) inserted into a hydrophobic pocket formed by Phe1134, Phe1200
and Ala1221, etc, and notable arene-H interaction with Phe1134 was observed. In addition, π-π stacking
was formed between the central fluorophenyl ring (ring A) and the phenyl ring of Phe1223 (Fig. 3B and
3C).
(Fig. 3 should be listed here.)
2.4 IncuCyte live-cell imaging
In vitro antitumor activity was evaluated by IncuCyte live-cell imaging preliminarily. Taking
Cabozantinib as positive control, antiproliferation and cytotoxicity of compound 29b against non-small cell
lung cancer A549 were confirmed.
Time-lapse imaging of the negative control showed a standard growth curve up to approximate 55% cell
confluence level by day 3 (green curve, Fig.4A), while compound 29b treatment efficiently suppressed cell
proliferation depending on both concentration and time. As shown in Fig. 4A (the curves of phase object
confluence) and 4B (phase contrast images), compound 29b exhibited significant antiproliferation against
A549 cells after 72 h at the concentration of 0.041 µM (confluence: from 16% to 35%), which was superior
to that of Cabozantinib at the concentration of 3.33 µM (confluence: from 18% to 48%).
(Fig. 4 should be listed here.)
In addition, real-time cytotoxicity study of compound 29 against A549 cells was carried on meantime. To
our delight, compound 29b performed excellent cytotoxicity at the concentration of 10µM compared with

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Cabozantinib at the same concentration after 72 h (Fig. 5A and 5B). The cytotoxicity at lower concentration
was evaluated (data not all showed), and the results indicated that the cytotoxicity of compound 29b
(1.11µM) was almost equipotent to Cabozantinib (10µM).
(Fig. 5 should be listed here.)
2.5 Kinase profile of compound 29b
Compared with the c-Met IC50 values of compound 29b (0.021 µmol·L-1) and Cabozantinib (0.019
µmol·L-1), similar in vitro anticancer activities should be diaplayed. However, the results were not the case
(vide supra). It suggested that compound 29b might act through other mechanisms rather than only by
inhibiting c-Met kinase. In order to know the details, compound 29b was tested on a panel of eight other
kinases including c-Kit, Ron, KDR, RET, EGFR, Src, IGF-1R and AXL. As can be seen in Table 5,
compound 29b showed potent inhibitory activities against c-Kit, Ron, EGFR and Src with 1.8-, 22.2-, 3.8-
and 2.9-fold more potent than Cabozantinib, respectively. In addition, compound 29b demonstrated
moderate inhibition of IGF-1R, with at least 2 times more effective than Cabozantinib. Further studies on
the mechanism of these compounds are in progress.
(Table 5 should be listed here.)
3 Conclusion
In summary, we described the pharmacophore hybrid design, synthesis and examination of a series of
N1-(2-aryl-1, 3-thiazolidin-4-one)-N3-aryl urea derivatives in an attempt to develop effective anticancer
agents. Compound 29b was found to have promising multi-tyrosine kinase inhibitory activities with IC50
values ranging from 3.1 nM to 4.67 µM, including c-Met, c-Kit, Ron, EGFR, Src and IGF-1R. The results
of IncuCyte live-cell imaging study showed that compound 29b performed excellent antiproliferation and
cytotoxicity against non-small cell lung cancer A549 cells in comparison to Cabozantinib. Further studies
comprising improvement of activity, mechanism and in vivo anticancer activity are currently ongoing in our
group, and the results will be reported in a due course.
4 Experimental
4.1 Chemistry
Unless otherwise noted, all materials were obtained from commercial suppliers and used without further
purification. All melting points were obtained on a Büchi Melting Point B-540 apparatus (Büchi
1
Labortechnik, Flawil, Switzerland) and were uncorrected. H NMR and 13C NMR spectra were generated
on a 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). Mass spectra (MS) were taken in ESI mode on Agilent 1100 LC-MS (Agilent,
Palo Alto, CA, USA).
4.1.1 4-(3-Chloropropoxy)-3-methoxyacetophenone (1)
Anhydrous K2CO3 (165.6 g, 1.2 mol) and 1-bromo-3-chloropropane (105.4 g, 0.66 mol) was added to a
solution of 4-hydroxy-3-methoxy-acetophenone (100.0 g, 0.60 mol) in DMF (500 mL). The reaction
mixture was then stirred at rt for 6 h. The mixture was then poured into cold water (1500 mL) with

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vigorously agitating for 15 min, and the resulting precipitate was filtered off, washed with water, and dried
under vacuum to afford 1 as white solid, yield: 91.9% (133.8 g). MS (ESI) m/z 242.9 [M+H]+.
4.1.2 4-(3-Chloropropoxy)-5-methoxy-2-nitroacetophenone (2)
A stirred solution of 1 (113.8 g, 0.46 mol) in CH2Cl2 (675 mL) was cooled to -20 °C, and fuming nitric
acid (56.5 mL) was added at a rate such that the temperature maintained at -15 to -10 °C. The reaction
mixture was allowed to stir at about -10 °C for 5 h, then poured into cold water (250 mL). The organic
layer was separated and washed with saturated aqueous sodium bicarbonate solution (2 × 200 mL) and
water (200 mL) orderly, then concentrated under reduced pressure to afford 2 as light yellow solid, yield:
88.3% (117.5 g). 1H NMR (400 MHz, CDCl3) δ 7.64 (s, 1H), 6.76 (s, 1H), 4.26 (t, J = 8.0 Hz, 2H), 3.96 (s,
3H), 3.77 (t, J = 8.8 Hz, 2H), 2.50 (s, 3H), 2.29-2.37 (m, 2H). MS (ESI) m/z 288.1 [M+H]+.
4.1.3 1-(4-(3-Chloropropoxy)-5-methoxy-2-nitrophenyl)-3-(dimethylamino)prop-2-en-1-one (3)
DMF-DMA (217.5 mL, 1.61 mol) was added to a solution of 2 (116.3 g, 0.40 mol) in xylene (560 mL)
and the reaction was refluxed for 14 h. The solvent was evaporated under reduced pressure, and another
150 mL xylene was added. After stirred for 30 min at rt, the product was collected by filtration and then
dried under vacuum to yield 3 as yellow solid, yield: 79.6 % (109.1 g). MS (ESI) m/z 343.0 [M+H]+.
4.1.4 6-Methyloxy-7-(4-(3-chloropropoxy))-4-4-hydroxyquioline (4)
Fe (88.9 g, 1.56 mmol) was added to a solution of 12 (108.8 g, 0.31 mol) in acetic acid (545 mL) at
60 °C in batches, then the mixture was stirred at 100 °C with vigorous agitation for 3 h. The hot solution
was filtered through celite and washed with hot acetic acid (100 mL). The combined filtrate was cooled to
rt, and the resultant solid was collected by filtration which was recrystallized from acetic acid (3000 mL) to
afford 4 as light yellow solid, yield: 67.5% (56.5 g). MS (ESI) m/z 268.1 [M+H]+.
4.1.5 General procedure for preparation of 4-hydroxyquinoline derivates (5 and 16a-d)
To a stirring solution of 4 in acetonitrile (V: m = 8:1) was added different amines (5 eqiv.), and the
resulting reaction mixture was refluxed for 8 h. The solvent was removed under reduced pressure to get a
crude product which was purified by column chromatography on silica gel using DCM: MeOH 15:1 to give
the corresponding 4-hydroxyquinolines 5 and 16a-d.
4.1.5.1 6-Methyloxy-7-(4-(3-morpholinopropoxy))-4-quiolin-ol (5)
Light yellow solid, yield: 95.1% (21.2 g). MS (ESI) m/z 318.1 [M+H]+.
4.1.5.2 6-Methoxy-7-(3-thiomorpholinopropoxy)quinolin-4-ol (16a)
Light yellow solid, yield: 92.4% (3.5 g). MS (ESI) m/z 335.1 [M+H]+.
4.1.5.3 6-Methoxy-7-(3-(2-methylpiperidin-1-yl)propoxy)quinolin-4-ol (16b)
White solid, yield: 90.8% (3.0 g). MS (ESI) m/z 331.3 [M+H]+.
4.1.5.4 7-(3-(3, 5-dimethylpiperidin-1-yl)propoxy)-6-methoxyquinolin-4-ol (16c)
White solid, yield: 89.9% (3.4 g). MS (ESI) m/z 345.1 [M+H]+.
4.1.5.5 6-Methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quinolin-4-ol (16d)
Yellow solid, yield: 88.6% (7.8 g). MS (ESI) m/z 303. 3[M+H]+.
4.1.6 4-(4-nitrophenoxy)-6-methoxy-7-(3-morpholinopropoxy)quinoline (6)
To a round bottom flask was added 5 (21g, 66.0 mmol), acetonitrile (200 mL) and cesium carbonate
(43.0 g, 0.13 mol). The mixture was stirred at rt for 30 min at which time 4-fluoronitrobenzene (11.2g, 79.2
mmol) was added over a 10 min period. Refluxed for 2 h, the reaction was complete at which time about 75%
of the MeCN was removed under reduced pressure and the resulting solution was poured into ice water

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(150 mL). The solid was filtered and dried to afford 6 as yellow solid, yield: 61.2% (17.8 g). MS (ESI) m/z
440.1 [M+H]+.
4.1.7 4-(6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yloxy)aniline (7)
A mixture of 6 (17.5 g, 39.8 mmol), Fe (11.1 g, 0.20 mol) and conc. HCl ( 1.0 mL) in ethanol-water (265
mL, 9:1 v/v) was refluxed with vigorous agitation for 3 h. The hot solution was filtered through celite and
the filter cake was washed with hot ethanol. The combined filtrate was concentrated under reduced pressure
to afford a yellow solid, which was recrystallized from ethanol (120 mL) to afford 7 as white solid, yield:
91.4% (14.9 g). MS (ESI) m/z 410.3 [M+H]+.
4.1.8 N1-(3-fluoro-4-(6-methoxy-7-(3-morpholino-propoxy)quinolin-4-yloxy)phenyl)semicarbazide (9)
To the mixture of 7 (14.5 g, 35.4 mmol) and dry pyridine (14.2 g, 0.18 mmol) in dry DCM (145 mL),
phenyl chloroformate (13.3 mL, 0.11 mol) was added dropwise at 0 °C. After the addition was completed,
the mixture was warmed to rt for 3 h. 50 mL saturated aqueous sodium bicarbonate solution was added and
the organic phase was washed with water (2 × 30 mL), dried over anhydrous Na2SO4, concentrated under
reduced pressure to afford corresponding phenylcarbamate 8, which were immediately used in the next step
without further purification.
A mixture of 8 and 80% hydrazine monohydrate (80 mL) in xylene (80 mL) was stirred at 70 °C for 3
h with vigorous agitation. After cooling to rt, the resulting precipitate was filtered off, washed with water,
and dried under vacuum to afford 9 as white solid, total yield: 53.9% (8.9 g). MS (ESI) m/z 468.2
[M+H]+.
4.1.9 General procedure for the synthesis of semicarbazones (10a-u)
To a solution of 9 (0.46 g, 1 mmol) in isopropanol (5 mL), 1.1 equiv of aldehydes and acetic acid (1 drop)
were added, and the mixture was refluxed for 5-6 h until TLC showed the completion of the reaction. After
cooling to room temperature, the resultant precipitate was filtered and dried under vacuum to afford 10a-u,
yield: 88.1% to 94.3%.
4.1.10 General procedure for the synthesis of target compounds (11a-u)
To a solution of 10a-u (0.8 mmol) in mercaptoacetic (2 mL), SiCl4 (3 drops) was added. The mixture was
stirred at 50 °C for approximate 6 h. CH2Cl2 (20 mL) and cold water (1 mL) was added to the reaction
mixture, and the solution was adjusted to pH 8 with the addition of 10% aq. NaOH. The organic phase was
separated and washed with water (2 × 10 mL), dried over anhydrous Na2SO4, concentrated under reduced
pressure to afford yellow oil which was further columned with a biotage system with 10:1 DCM/MeOH.
4.1.10.1
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-phenylthiazolidin-3-
yl)urea (11a)
1
White solid, yield: 49.8%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ9.03 (s, 1H), 8.67 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.58-7.67 (m, 1H), 7.56 (d, J = 9.2 Hz, 2H), 7.52 (s, 1H), 7.42-7.47 (m, 1H), 7.38
(s, 1H), 7.25-7.34 (m, 3H), 7.17 (d, J = 9.2 Hz, 2H), 6.40 (d, J = 5.2 Hz, 1H), 6.07 (s, 1H), 4.19 (t, J = 6.4
Hz, 2H), 3.94 (s, 3H), 3.78-3.90 (m, 2H), 3.58-3.60 (m, 4H), 2.43-2.49 (m, 2H), 2.35-2.44 (br, 4H),
1.94-2.00 (m, 2H). MS (ESI) m/z 629.2 [M+H]+.
4.1.10.2
1-(2-(2-fluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)
oxy)phenyl)urea (11b)

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1
White solid, yield: 49.0%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.99-9.07 (br, 1H), 8.68 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.58-7.68 (m, 1H), 7.55 (d, J = 9.2 Hz, 2H), 7.50 (s, 1H), 7.42-7.47 (m, 1H),
7.38 (s, 1H), 7.24-7.30 (m, 2H), 7.19 (d, J = 9.2 Hz, 2H), 6.41 (d, J = 5.2 Hz, 1H), 6.07 (s, 1H), 4.19 (t, J =
6.4 Hz, 2H), 3.93 (s, 3H), 3.78-3.90 (m, 2H), 3.58-3.60 (m, 4H), 2.45-2.49 (m, 2H), 2.34-2.43 (br, 4H),
1.94- 2.01 (m, 2H). 13C NMR (400 MHz, DMSO-d6) δ 169.90, 162.07, 160.55, 159.61, 154.25, 152.28,
149.83, 149.27, 148.89, 146.83, 137.19, 131.39, 131.30, 129.89, 125.34, 121.97, 120.55, 116.37, 116.16,
115.47, 108.95, 103.28, 99.63, 67.09, 66.68 (2C), 56.79, 56.17, 55.29, 53.84 (2C), 29.60, 26.15. MS (ESI)
m/z 648.1 [M+H]+.
4.1.10.3
1-(2-(3-fluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)
oxy)phenyl)urea (11c)
1
White solid, yield: 49.2%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.05-9.18 (br, 1H), 8.67 (s,
1H), 8.44 (d, J = 5.2 Hz, 1H), 7.53 (d, J = 9.2 Hz, 2H), 7.51 (s, 1H), 7.44-7.49 (m, 1H), 7.34-7.42 (m, 2H),
7.19 (d, J = 9.2 Hz, 2H), 6.41 (d, J = 5.2 Hz, 1H), 5.86 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.91-3.95 (m, 4H),
3.79 (d, J = 15.6 Hz, 1H), 3.58-3.60 (m, 4H), 2.45-2.49 (m, 2H), 2.35- 2.43 (br, 4H), 1.95-2.01 (m, 2H).
MS (ESI) m/z 648.1 [M+H]+.
4.1.10.4
1-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)
oxy)phenyl)urea (11d)
1
White solid, yield: 51.6%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.95-9.06 (br, 1H), 8.55 (s,
1H), 8.46 (d, J = 5.2 Hz, 1H), 7.51-7.58 (m, 5H), 7.39 (s, 1H), 7.17-7.27 (m, 4H), 6.42 (d, J = 5.2 Hz, 1H),
5.86 (s, 1H), 4.21 (t, J = 6.0 Hz, 2H), 3.94 (s, 3H), 3.76-3.91 (m, 2H), 3.57-3.67 (br, 4H), 2.59-2.64 (m, 2H),
2.32-2.50 (br, 4H), 1.96- 2.07 (m, 2H). MS (ESI) m/z 648.2 [M+H]+.
4.1.10.5
1-(2-(2,4-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4
-yl)oxy)phenyl)urea (11e)
1
White solid, yield: 52.9%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.02-9.09 (br, 1H), 8.68 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.67-7.76 (m, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.51 (s, 1H), 7.38 (s, 1H), 7.30-
7.36 (m, 1H), 7.14-7.22 (m, 3H), 6.42 (d, J = 5.2 Hz, 1H), 6.04 (s, 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.94 (s,
3H), 3.78-3.91 (m, 2H), 3.58-3.61 (m, 4H), 2.45-2.49 (m, 2H), 2.34-2.44 (br, 4H), 1.95-2.01 (m, 2H). MS
(ESI) m/z 648.1 [M+H]+.
4.1.10.6
1-(2-(3,4-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4
-yl)oxy)phenyl)urea (11f)
1
White solid, yield: 52.0%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.65 (s, 1H),
8.46 (d, J = 5.2 Hz, 1H), 7.67-7.74 (m, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.50 (s, 1H), 7.35-7.38 (m, 1H), 7.29-
7.34 (m, 1H), 7.14-7.21 (m, 3H), 6.43 (d, J = 5.2 Hz, 1H), 5.95 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s,
3H), 3.78-3.88 (m, 2H), 3.56-3.61 (m, 4H), 2.45-2.51 (m, 2H), 2.34- 2.42 (m, 4H), 1.94-2.01 (m, 2H). MS
(ESI) m/z 666.2 [M+H]+.
4.1.10.7
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4

ACCEPTED MANUSCRIPT
-yl)oxy)phenyl)urea (11g)
1
White solid, yield: 50.5%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.03 (s, 1H), 8.83 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.47-7.55 (m, 4H), 7.38 (s, 1H), 7.16-7.21 (m, 4H), 6.42 (d, J = 5.2 Hz, 1H), 6.17
(s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.80-3.84 (br, 2H), 3.58-3.60 (m, 4H), 2.45- 2.49 (m, 2H),
2.33-2.44 (br, 4H), 1.95-2.01 (m, 2H). MS (ESI) m/z 666.2 [M+H]+.
4.1.10.8
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-(3,4,5-trifluoropheny
l)thiazolidin-3-yl)urea (11h)
White solid, yield: 56.0%, m.p. > 300 °C. 1H NMR (400 MHz, DMSO-d6) δ 9.57-9.76 (br, 1H),
9.02-9.19 (br, 1H), 8,44 (d, J = 5.2 Hz, 1H),7.52-7.62 (m, 4H), 7.51 (s, 1H), 7.38 (s, 1H), 7.19 (d, J = 8.8
Hz, 2H), 6.41 (d, J = 5.2Hz, 1H), 5.86 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.95-3.97 (m, 1H), 3.94 (s, 3H),
3.73-3.77 (m,1H), 3.54-3.64 (m, 4H), 2.47 (t, J = 7.2 Hz, 2H), 2.34-2.44 (br, 4H), 1.95-2.01 (m, 2H). MS
(ESI) m/z 684.1 [M+H]+.
4.1.10.9
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-(2,4,6-trifluoropheny
l)thiazolidin-3-yl)urea (11i)
1
White solid, yield: 47.3%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.04 (s, 1H), 8.79 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.55 (d, J = 8.8 Hz, 2H), 7.51 (s, 1H), 7.38 (s, 1H), 7.28-7.33 (m, 2H), 7.20 (d, J =
8.8 Hz, 2H), 6.42 (d, J = 5.2 Hz, 1H), 6.12 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.79-3.86 (m,
2H), 3.58-3.61 (m, 4H), 2.45- 2.49 (m, 2H), 2.35-2.43 (br, 4H), 1.95-2.01 (m, 2H). MS (ESI) m/z 684.1
[M+H]+.
4.1.10.10
1-(2-(4-chlorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)
oxy)phenyl)urea (11j)
1
White solid, yield: 56.9%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.94-9.11 (br, 1H), 8.60 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.47-7.54 (m, 7H), 7.38 (s, 1H), 7.19 (d, J = 8.8 Hz, 2H), 6.41 (d, J = 5.2 Hz,
1H), 5.85 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.76-3.92 (m, 2H), 3.58-3.60 (m, 4H), 2.45- 2.48
(m, 2H), 2.28-2.43 (br, 4H), 1.94-2.01 (m, 2H). MS (ESI) m/z 664.2 [M+H]+.
4.1.10.11
1-(2-(2-chlorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)
oxy)phenyl)urea (11k)
1
White solid, yield: 54.0%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.12 (s, 1H), 8.78 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.66-7.68 (m, 1H), 7.52-7.60 (m, 4H), 7.39-7.47 (m, 2H), 7.38 (s, 1H), 7.20 (d, J =
8.8 Hz, 2H), 6.42 (d, J = 5.2 Hz, 1H), 6.17 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.78-3.90 (m, 2H),
3.58-3.60 (m, 4H), 2.45- 2.49 (m, 2H), 2.33-2.44 (br, 4H), 1.94-2.01 (m, 2H). MS (ESI) m/z 664.2 [M+H]+.
4.1.10.12
1-(2-(3,5-dichlorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-
4-yl)oxy)phenyl)urea (11l)
1
White solid, yield: 51.7%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.97-9.05 (br, 1H), 8.64 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.74-7.80 (m, 4H), 7.54 (d, J = 8.8 Hz, 2H), 7.50 (s, 1H), 7.38 (s, 1H), 7.19
(d, J = 8.8 Hz, 2H), 6.41 (d, J = 5.2 Hz, 1H), 5.95 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.79-3.96

ACCEPTED MANUSCRIPT
(m, 2H), 3.58-3.61 (m, 4H), 2.46- 2.49 (m, 2H), 2.34-2.44 (br, 4H), 1.95-2.01 (m, 2H). MS (ESI) m/z 698.2
[M+H]+.
4.1.10.13
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-(4-(trifluoromethyl)
phenyl)thiazolidin-3-yl)urea (11m)
1
White solid, yield: 43.9%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.06-9.20 (br, 1H), 8.74 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.61-7.63 (m, 3H), 7.56 (d, J = 8.8 Hz, 2H), 7.38 (s, 1H), 7.20 (d, J = 8.8 Hz,
2H), 6.41 (d, J = 5.2 Hz, 1H), 5.87 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.75-3.99 (m, 2H),
3.58-3.60 (m, 4H), 2.43- 2.49 (m, 2H), 2.31-2.41 (br, 4H), 1.95-2.01 (m, 2H). 13C NMR (400 MHz,
DMSO-d6) δ 170.03, 162.11, 160.55, 159.63, 154.13, 152.28, 149.83, 149.27, 148.85, 146.83, 137.22,
131.44, 131.38, 129.04, 126.09, 123.20, 121.95, 120.52, 116.35, 116.18, 115.47, 108.95, 103.27, 99.62,
67.09, 66.68 (2C), 56.79, 56.17, 55.29, 53.84 (2C), 29.60, 26.15. MS (ESI) m/z 698.2 [M+H]+.
4.1.10.14
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-(2-(trifluoromethyl)
phenyl)thiazolidin-3-yl)urea (11n)
1
White solid, yield: 40.4%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.70 (s, 1H),
8.44 (d, J = 5.2 Hz, 1H), 7.92-7.94 (m, 1H), 7.80-7.84 (m, 1H), 7.74-7.76 (m, 1H), 7.58-7.62 (m, 1H), 7.53
(d, J = 8.8 Hz, 2H), 7.50 (s, 1H), 7.38 (s, 1H), 7.19 (d, J = 8.8 Hz, 2H), 6.42 (d, J = 5.2 Hz, 1H), 6.14 (s,
1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.82-3.98 (m, 2H), 3.58-3.60 (m, 4H), 2.45- 2.49 (m, 2H),
2.33-2.43 (br, 4H), 1.94-2.01 (m, 2H). MS (ESI) m/z 698.2 [M+H]+.
4.1.10.15
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-(3-(trifluoromethyl)
phenyl)thiazolidin-3-yl)urea (11o)
1
White solid, yield: 43.7%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.99-9.10 (br, 1H), 8.65 (s,
1H), 8.45 (d, J = 5.2 Hz, 1H), 7.90-7.95 (br, 1H), 7.82-7.84 (m, 1H), 7.73-7.75 (m, 1H), 7.58-7.62 (m, 1H),
7.63-7.67 (m, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.50 (s, 1H), 7.38 (s, 1H), 7.18 (d, J = 8.8 Hz, 2H), 6.40 (d, J =
5.2 Hz, 1H), 6.14 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.78-3.97 (m, 2H), 3.58-3.60 (m, 4H), 2.45-
2.49 (m, 2H), 2.32-2.43 (br, 4H), 1.94-2.01 (m, 2H). MS (ESI) m/z 698.2 [M+H]+.
4.1.10.16
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-(pyridin-4-yl)thiazol
idin-3-yl)urea (11p)
1
White solid, yield: 50.1%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.03-9.13 (br, 1H), 8.73 (s,
1H), 8.63 (d, J = 6.0 Hz, 2H), 8.45 (d, J = 5.2 Hz, 1H), 7.51-7.55 (m, 5H), 7.38 s, 1H), 7.19 (d, J = 8.8 Hz,
2H), 6.41 (d, J = 5.2 Hz, 1H), 5.86 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.76-3.96 (m, 5H), 3.58-3.60 (m, 4H),
2.45-2.49 (m, 2H), 2.27-2.42 (br, 4H), 1.91-2.01 (m, 2H). MS (ESI) m/z 631.2 [M+H]+.
4.1.10.17
1-(2-(4-(dimethylamino)phenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)qui
nolin-4-yl)oxy)phenyl)urea (11q)
1
White solid, yield: 40.6%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.83-8.98 (br, 1H), 8.45 (d,
J = 9.2 Hz, 1H), 8.38 (s, 1H), 7.53 (d, J = 9.2 Hz, 2H), 7.50 (s, 1H), 7.38 (s, 1H), 7.28-7.30 (m, 2H), 7.18
(d, J = 9.2 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H), 6.40 (d, J = 5.2 Hz, 1H), 5.72 (s, 1H), 4.19 (t, J = 6.4 Hz, 2H),

ACCEPTED MANUSCRIPT
3.93 (s, 3H), 3.71-3.82 (m, 2H), 3.58-3.60 (m, 4H), 2.91 (s, 6H), 2.46-2.49 (m, 2H), 2.33-2.43 (br, 4H),
1.94-2.01 (m, 2H). MS (ESI) m/z 673.2 [M+H]+.
4.1.10.18
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(2-(naphthalen-2-yl)-4-oxothi
azolidin-3-yl)urea (11r)
1
White solid, yield: 50.6%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.99-9.09 (br, 1H), 8.69 (s,
1H), 8.43 (d, J = 5.2 Hz, 1H), 7.90-8.02 (m, 4H), 7.66- 7.71 (m, 1H), 7.55-7.58 (m, 2H), 7.53 (d, J = 8.8 Hz,
1H), 7.50 (s, 1H), 7.38 (s, 1H), 7.17 (d, J = 8.8 Hz, 2H), 6.40 (d, J = 5.2Hz, 1H), 6.00 (s, 1H), 4.19 (t, J =
6.4 Hz, 2H), 3.94-3.99 (m, 2H), 3.93 (s, 3H), 3.85 (d, J = 16.0 Hz, 1H), 3.56-3.63 (m, 4H), 2.45- 2.49 (m,
2H), 2.32-2.43 (br, 4H), 1.91-2.01 (m, 2H). MS (ESI) m/z 680.3 [M+H]+.
4.1.10.19
1-(2-benzyl-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phen
yl)urea (11s)
1
White solid, yield: 59.4%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.25 (s, 1H), 8.77 (s, 1H),
8.46 (d, J = 5.2 Hz, 1H), 7.63 (d, J = 9.2 Hz, 2H), 7.52 (s, 1H), 7.38 (s, 1H), 7.25-7.35 (m, 5H), 7.23 (d, J =
9.2 Hz, 2H), 6.44 (d, J = 5.2 Hz, 1H), 4.94-5.01 (m, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.58-3.60
(m, 4H), 3.35-3.50 (m, 2H), 3.25-3.29 (m, 1H), 2.90-2.95 (m, 1H), 2.45-2.52 (m, 2H), 2.32-2.44 (br, 4H),
1.95-2.01 (m, 2H). MS (ESI) m/z 644.2 [M+H]+.
4.1.10.20
1-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-3-(4-oxo-2-phenethylthiazolidin
-3-yl)urea (11t)
1
White solid, yield: 58.9%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.15-9.26 (br, 1H), 8.68 (s,
1H), 8.46 (d, J = 5.2 Hz, 1H), 7.60 (d, J = 8.8 Hz, 2H), 7.51 (s, 1H), 7.38 (s, 1H), 7.18-7.31 (m, 7H), 6.43
(d, J = 5.2 Hz, 1H), 7.75-7.77 (m, 1H), 4.19 (t, J = 6.4 Hz, 2H), 3.93 (s, 3H), 3.58- 3.69 (m, 6H), 2.66-2.75
(m, 2H), 2.45-2.49 (m, 2H), 2.38-2.43 (br, 4H), 2.22-2.31 (m, 1H), 1.94-2.03 (m, 3H). 13C NMR (400 MHz,
DMSO-d6) δ 169.69, 162.14, 160.56, 159.67, 154.51, 152.29, 149.84, 149.29, 148.86, 146.84, 141.38,
137.39, 128.90 (2C), 128.82 (2C), 126.47, 121.94, 120.72, 115.49, 108.96, 103.30, 99.64, 67.10, 66.69
(2C), 60.82, 56.18, 55.30, 53.85 (2C), 36.86, 30.43, 28.92, 26.16. MS (ESI) m/z 657.2 [M+H]+.
4.1.10.21
1-(2-cyclohexyl-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)p
henyl)urea (11u)
1
White solid, yield: 56.6%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.09 (s, 1H), 8.79 (s, 1H),
8.46 (d, J = 5.2 Hz, 1H), 7.61 (d, J = 9.2 Hz, 2H), 7.52 (s, 1H), 7.38 (s, 1H), 7.21 (d, J = 9.2 Hz, 2H), 6.43
(d, J = 5.2 Hz, 1H), 4.73-4.77 (br , 1H), 4.20 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.58-3.61 (m, 4H), 3.54 (s,
2H), 2.46-2.49 (m, 2H), 2.34-2.43 (br, 4H), 1.92-2.02 (m, 3H), 1.70-1.80 (m, 2H), 1.61-1.69 (m, 2H),
1.51-1.59 (m, 1H), 0.98-1.37 (m, 5H). 13C NMR (400 MHz, DMSO-d6) δ 169.91, 162.09, 160.57, 159.70,
154.41, 152.29, 149.84, 149.29, 148.82, 146.85, 137.42, 121.96, 120.61, 115.49, 108.97, 103.29, 99.65,
67.10, 66.70 (2C), 66.16, 56.19, 55.31, 53.86 (2C), 28.96, 28.67, 26.41, 26.18 (2C), 26.15, 25.47, 24.78.
MS (ESI) m/z 636.3 [M+H]+.
4.1.11 4-Chloro-6-methyloxy-7-(4-(3-morpholinopropoxy))quinolone (12)
Phosphorus oxychloride (24 mL) was added to 5 (3.0 g, 9.4 mmol) and the mixture heated to reflux for 3

ACCEPTED MANUSCRIPT
h. The contents were concentrated under reduced pressure and the pale residue was poured into ice-water
(50 mL) with vigorous agitation. The solution was treated with solid sodium bicarbonate to achieve pH to 8,
and the mixture was extracted with CH2Cl2 (3 × 20 mL). The combined organic layer was washed with
brine, then water, and dried over anhydrous Na2SO4, concentrated under reduced pressure to give 12 as
offwhite solid, yield: 94.4% (3.0 g). MS (ESI) m/z 337.1 [M+H]+.
4.1.12 4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)benzaldehyde (13)
A solution of 12 (3.0 g, 8.9 mmol) and 4-hydroxybenzaldehyde (1.3 g, 10.7 mmol) in chlorobenzene (30
mL) was refluxed for 13 h. After cooling to room temperature, the reaction mixture was concentrated under
reduced pressure to yield dark brown solid which was purified by silica gel column chromatography (eluent,
DCM:MeOH:Et3N = 100:2:1 to 100:5:1) to afford 13 as white solid, yield: 64.3% (2.4 g). MS (ESI) m/z
423.2[M+H]+.
4.1.13
1-(4-fluorophenyl)-3-(2-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-4-oxothia
zolidin-3-yl)urea (15a)
Compound 15a was prepared with similar procedure as compound 11a. White solid, yield: 60.1%,
1
m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ13C NMR (400 MHz, DMSO-d6) δ 8.99-9.05 (br, 1H),
8.68 (s, 1H), 8.46 (d, J = 5.2 Hz, 1H), 7.58-7.66 (m, 1H), 7.57 (d, J = 9.2 Hz, 2H), 7.52 (s, 1H), 7.42-7.48
(m, 1H), 7.36 (s, 1H), 7.24-7.31 (m, 2H), 7.19 (d, J = 9.2 Hz, 2H), 6.43 (d, J = 5.2 Hz, 1H), 6.01 (s, 1H),
4.19 (t, J = 6.4 Hz, 2H), 3.95 (s, 3H), 3.78-3.90 (m, 2H), 3.58-3.62 (m, 4H), 2.45-2.52 (m, 2H), 2.34-2.42
(br, 4H), 1.94- 2.01 (m, 2H). MS (ESI) m/z 648.2 [M+H]+.
4.1.14
1-(2,4-difluorophenyl)-3-(2-(4-((6-methoxy-7-(3-morpholinopropoxy)quinolin-4-yl)oxy)phenyl)-4-oxot
hiazolidin-3-yl)urea (15b)
Compound 15b was prepared with similar procedure as compound 11a. White solid, yield: 58.7%, m.p. >
1
300 °C. H NMR (400 MHz, DMSO-d6) δ13C NMR (400 MHz, DMSO-d6) δ 9.00-9.06 (br, 1H), 8.67 (s,
1H), 8.44 (d, J = 5.2 Hz, 1H), 7.67-7.75 (m, 1H), 7.54 (d, J = 8.8 Hz, 2H), 7.50 (s, 1H), 7.36 (s, 1H), 7.31-
7.36 (m, 1H), 7.14-7.21 (m, 3H), 6.43 (d, J = 5.2 Hz, 1H), 5.97 (s, 1H), 4.21 (t, J = 6.4 Hz, 2H), 3.96 (s,
3H), 3.79-3.93 (m, 2H), 3.58-3.62 (m, 4H), 2.43-2.49 (m, 2H), 2.34-2.43 (br, 4H), 1.95-2.00 (m, 2H). MS
(ESI) m/z 666.2 [M+H]+.
4.1.15 General procedure for the synthesis of target compounds (22a-d)
Compound 22a-d was prepared with similar procedure as compound 11a.
4.1.15.1
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-thiomorpholinopropoxy)quinol
in-4-yl)oxy)phenyl)urea (22a)
1
White solid, yield: 50.9%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.06 (s, 1H), 8.83 (s, 1H),
8.45 (d, J = 5.2Hz, 1H), 7.47-7.55 (m, 4H), 7.38 (s, 1H), 7.16-7.23 (m, 4H), 6.42 (d, J = 5.2 Hz, 1H), 6.17
(s, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.84 (s, 2H), 2.57-2.73 (m, 8H), 2.45-2.49 (m, 2H), 1.93-1.99
(m, 2H). MS (ESI) m/z 682.1 [M+H]+.
4.1.15.2
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-(2-methylpiperidin-1-yl)propo
xy)quinolin-4-yl)oxy)phenyl)urea (22b)

ACCEPTED MANUSCRIPT
1
White solid, yield: 49.0%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.14 (s, 1H), 8.87 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.47-7.55 (m, 4H), 7.38 (s, 1H), 7.16- 7.21 (m, 4H), 6.42 (d, J = 5.2 Hz, 1H), 6.17
(s, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.93 (s, 2H), 2.83-2.96 (m, 2H), 2.32-2.47 (m, 2H), 2.17-2.28 (br, 1H),
1.88-1.99 (m, 2H), 1.52-1.65 (m, 3H), 1.38-1.51 (m, 1H), 1.19-1.31 (m, 2H), 1.05 (d, J = 6.4 Hz, 3H). MS
(ESI) m/z 677.3 [M+H]+.
4.1.15.3
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((7-(3-(3,5-dimethylpiperidin-1-yl)propoxy)-6-m
ethoxyquinolin-4-yl)oxy)phenyl)urea (22c)
1
White solid, yield: 51.5%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.02 (s, 1H), 8.79 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.47- 7.55 (m, 4H), 7.37 (s, 1H), 7.16- 7.21 (m, 4H), 6.42 (d, J = 5.2 Hz, 1H), 6.17
(s, 1H), 4.18 (t, J = 6.4 Hz, 2H), 3.94 (s, 3H), 3.84 (s, 2H), 2.84-2.87 (m, 2H), 2.41-2.48 (m, 2H), 1.93-2.03
(m, 2H), 1.54-1.70 (m, 3H), 1.37-1.49 (m, 2H), 0.82-0.83 (m, 6H), 0.45-0.54 (m, 1H). MS (ESI) m/z 692.3
[M+H]+.
4.1.15.4
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propoxy)quino
lin-4-yl)oxy)phenyl)urea (22d)
1
White solid, yield: 47.3%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.01 (s, 1H), 8.80 (s, 1H),
8.45 (d, J = 5.2 Hz, 1H), 7.47-7.55 (m, 4H), 7.38 (s, 1H), 7.16-7.21 (m, 4H), 6.42 (d, J = 5.2 Hz, 1H), 6.17
(s, 1H), 4.20 (t, J = 4.8 Hz, 2H), 3.95 (s, 3H), 3.84 (s, 2H), 2.62-2.73 (m, 2H), 2.56-2.63 (m, 4H), 1.97-2.05
(m, 2H), 1.68-1.76 (br, 4H). MS (ESI) m/z 650.2 [M+H]+.
4.1.16 General procedure for the synthesis of target compounds (29a-b)
Compound 29a-b was prepared with similar procedure as compound 11a.
4.1.16.1
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(3-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propo
xy)quinolin-4-yl)oxy)phenyl)urea (29a)
1
White solid, yield: 46.6%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 8.94 (s, 1H), 8.74 (s, 1H),
8.47 (d, J = 5.2 Hz, 1H), 8.01-8.05 (m, 1H), 7.49-7.56 (m, 1H), 7.48 (s, 1H), 7.39 (s, 1H), 7.31-7.34 (m,
1H), 7.19-7.23 (m, 2H), 7.06-7.09 (m, 1H), 6.52 (d, J = 5.2 Hz, 1H), 6.19 (s, 1H), 4.20 (t, J = 4.8 Hz, 2H),
3.93 (s, 3H), 3.85 (s, 2H), 2.64-2.72 (m, 2H), 2.53-2.64 (br, 4H), 1.97-2.05 (m, 2H), 1.68-1.78 (br, 4H). MS
(ESI) m/z 668.2 [M+H]+.
4.1.16.2
1-(2-(2,6-difluorophenyl)-4-oxothiazolidin-3-yl)-3-(2-fluoro-4-((6-methoxy-7-(3-(pyrrolidin-1-yl)propo
xy)quinolin-4-yl)oxy)phenyl)urea (29b)
1
White solid, yield: 45.9%, m.p. > 300 °C. H NMR (400 MHz, DMSO-d6) δ 9.29 (s, 1H), 9.02 (s, 1H),
8.46 (d, J = 5.2 Hz, 1H), 7..63-7.67 (m, 1H), 7.53 (s, 1H), 7.47-7.54 (m, 1H), 7.39 (s, 1H), 7.35-7.37 (m,
1H), 7.26-7.29 (m, 2H), 7.16-7.20 (m, 2H), 6.42 (d, J = 5.2 Hz, 1H), 6.15 (s, 1H), 4.20 (t, J = 4.8 Hz, 2H),
3.95 (s, 3H), 3.84 (s, 2H), 2.62-2.71 (m, 2H), 2.56-2.62 (m, 4H), 1.98-2.05 (m, 2H), 1.68-1.77 (br, 4H). 13
C
NMR (400 MHz, DMSO-d6) δ 168.96, 162.49, 160.00, 159.87, 155.24, 154.24, 152.32, 149.97, 148.91,
146.80, 138.85, 138.75, 135.10, 134.93, 132.18, 124.58, 121.95, 115.63, 115.25, 114.90, 108.97, 102.35,
99.50, 67.09, 66.68 (2C), 56.79, 56.16, 55.29, 53.84 (2C), 29.60, 26.15. MS (ESI) m/z 668.2 [M+H]+.
4.2 ELISA Kinase Assay

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Kinase activities were evaluated according to the reported protocol [14, 28]. Shortly, in an ELISAs, 20
µg/mL poly (Glu, Tyr) 4:1 was precoated as substrate in 384-well plates. 50 µL of 10 µmol·L-1 ATP solution
diluted in kinase reaction buffer (50 mmol·L-1 HEPES, pH 7.4, 50 mmol·L-1 MgCl2, 0.5 mmol·L-1 MnCl2,
0.2 mmol·L-1 Na3VO4, 1 mmol·L-1 DTT ) was added to each well. Compounds were tested from 1 µmol·L-1,
3-fold dilution, 10 points, in duplicate. The kinase reaction was initiated by addition of purified tyrosine
kinase protein diluted in 40 µL of kinase reaction buffer solution. After incubation for 1 h at 37 °C, the
plate was washed 3 times with phosphate buffered saline (PBS). 100 µL of antiphosphotyrosine (PY99)
antibody was added. After 0.5 h of incubation at 37 °C, the plate was washed 3 times with PBS. A solution
of 100 µL of horseradish peroxidase-conjugated goat anti-mouse IgG was added. The plate was reincubated
at 37 °C for 0.5 h and washed. 100 µL of a solution containing 0.03% H2O2 and 2 mg/mL
o-phenylenediamine in 0.1 mmol·L-1 citrate buffer, pH 5.5, was added and samples were incubated at rt
until color appeared. The reaction was terminated by addition of 2 mol·L-1 H2SO4 (50 µL), and the plate
was read using a multiwell spectrophotometer at 490 nm. IC50 values were calculated from the inhibition
curves.
4.3 IncuCyte live-cell imaging assays
A total of 5×103 A549 cells grown in 100µL Dulbecco’s Modified Eagle Media (DMEM) with serum
(10 % FBS) were seeded in 96-well plates respectively and incubated in a tissue culture incubator at 37 °C
and 5% CO2 in an IncuCyte (Essen BioScience). Assay was performed according to the manufacturer’s
protocol. All samples consisted of three replicates. Images were captured every 2 h over 72 h to monitor
proliferation and cytotoxicity.
Acknowlegements
This work was supported by the National Natural Science Foundation of China (21562053), Science and
Technology Foundation of Guizhou Province (Qian Ke He J Zi [2014]2181), Science and Technology
Cooperation Plan of Guizhou Province (Qian Ke He LH Zi[2015]7523)
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Table 1 c-Met kinase inhibitory activities of compounds 11a-u.
c-Met
IC50 (µmol·L-1)a
c-Met
IC50 (µmol·L-1)
0.495
Compd.
R1
Compd.
R1
11a
11b
11c
Ph-
0.097
11m
11n
11o
4-CF3-Ph-
2-CF3-Ph-
3-CF3-Ph-
2-F-Ph-
3-F-Ph-
0.061
0.358
0.308
0.415
11d
11e
11f
11g
11h
11i
4-F-Ph-
2, 4-F-Ph-
3, 4-F-Ph-
2, 6-F-Ph-
3, 4, 5-F-Ph-
2, 4, 6-F-Ph-
0.196
0.141
0.337
0.050
0.580
0.094
11p
11q
11r
11s
11t
0.320
0.427
0.659
0.205
0.331
0.688
11u
11j
11k
11l
4-Cl-Ph-
2-Cl-Ph-
0.348
0.169
0.658
Cabozantinib
Staurosporine
-
-
0.019
0.128
3, 5-Cl-Ph-
a) Values are expressed as the mean of two independent experiments.

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Table 2 c-Met kinase inhibitory activities of compounds 15a-b.
c-Met
IC50a (µmol·L-1)
Compd.
R2
15a
2-F-Ph-
4.10
15b
2, 4-F-Ph-
3.35
Cabozantinib
Staurosporine
-
0.019
-
0.128
a) Values are expressed as the mean of two independent experiments.

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Table 3 c-Met kinase inhibitory activities of compounds 22a-d.
c-Met
IC50a (µmol·L-1)
Compd.
22a
R3
0.154
22b
0.046
0.114
0.037
N
22c
22d
Cabozantinib
Staurosporine
-
-
0.019
0.128
a) Values are expressed as the mean of two independent experiments.

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Table 4 c-Met kinase inhibitory activities of compounds 29a-b.
c-Met
IC50a (µmol·L-1)
Compd.
R4
29a
2-F
3-F
-
0.030
29b
0.021
Cabozantinib
Staurosporine
0.019
-
0.128
a) Values are expressed as the mean of two independent experiments.

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Table 5 Kinase profile of compound 29b.
Kinase (IC50a, µmol·L-1)
Compd.
c-Kit
0.029
0.053
Ron
0.0031
0.069
KDR
0.021
RET
0.152
0.037
AXL
0.062
0.0095
EGFR IGF-1R
Src
29b
0.812
3.11
4.67
>10
0.061
0.178
Cabozantinib
0.00048
a) Values are expressed as the mean of two independent experiments.

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Fig. 2 The reported SARs of c-Met kinase inhibitors and the design of target compounds.

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Fig.3 The proposed binding mode of compound 29b with c-Met kinase (PBD ID: 3LQ8). A) The structure of compound 29b. B)
Binding pocket of c-Met. C) The interactions between compound 29b and c-Met. The compound was shown by green sticks, the
H-bonds were represented by red dotted lines, and the arene-H interaction was shown by green dotted lines. D) 2D depiction of
the ligand-protein interaction.

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Fig. 1 The representative c-Met kinase inhibitors bearing quinoline pharmacophores and drugs bearing thiazolidinone scaffolds
.

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Fig. 4 Real-time cell confluence study in non-small cell lung cancer A549. A) The cell population was monitored for 72 h using
an IncuCyte ZOOM system in an incubator (5% CO2 and 37 °C). A549 cells were incubated with 0.1% DMSO or exposed to
compound 29b or Cabozantinib at different concentration. Error bars: ± SD from n = 3 and all SD values are below 0.05. B)
Phase contrast images (white light) of A549 cells after 3 days of treatment with compound 29b, Cabozantinib or not.

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Fig. 5 Real-time cytotoxicity study in non-small cell lung cancer A549. A) The dead cell population was monitored for 72 h
using an IncuCyte ZOOM system in an incubator (5% CO2 and 37 °C). Error bars: ± SD from n = 3 and all SD values are below
0.05. B) Phase-contrast images (red light) of cells after 3 days of treatment with 10 µM of compound 29b, 10 µM Cabozatinib or
negative control. Red fluorescent cell was counted as dead cell.

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Scheme 1. Synthesis of compounds 11a-u. Reagents and conditions: i) 1-bromo-3-chloropropane, K2CO3, DMF, rt; ii) fuming
HNO3, CH2Cl2, -10°C; iii) DMF-DMA, xylene, reflux; iv) Fe, AcOH, 100 °C; v) morpholine, CH3CN, reflux; vi)
4-fluoronitrobenzene, Cs2CO3, CH3CN, reflux; vii) Fe, conc. HCl, 95% EtOH-H2O, reflux; viii) 4-nitrophenyl carbonochloridate,
CH2Cl2, pyridine, rt; ix) NH2NH2·H2O, xylene, 70°C; x) aldehyde, i-PrOH, HOAc, reflux; xi) SiCl4, mercaptoacetic acid, 50 °C.

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Scheme 2. Synthesis of compounds 15a-b. Reagents and conditions: i) POCl3, reflux; ii) 4-hydroxybenzaldehyde, PhCl, reflux;
iii) i-PrOH, HOAc, reflux; iv) SiCl4, mercaptoacetic acid, 50 °C.