Design, synthesis, and evaluation of new 2-oxoquinoline arylaminothiazole derivatives as potential anticancer agents

Article abstract of DOI:10.1016/j.bioorg.2020.104469

A series of novel 2-oxoquinoline derivatives containing arylaminothiazole were designed and synthesized as potential antitumor agents. The synthesized compounds were evaluated for their in vitro cytotoxicity activity against HeLa, NCI-H460, T24 and SKOV3

Full text of DOI:10.1016/j.bioorg.2020.104469

Journal Pre-proofs
Design, synthesis, and evaluation of new 2-oxoquinoline arylaminothiazole
derivatives as potential anticancer agents
Yilin Fang, Zhilin Wu, Mengwu Xiao, Li Wei, Kangming Li, Yuting Tang,
Jiao Ye, Jiannan Xiang, Aixi Hu
PII:
S0045-2068(20)31767-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.104469
YBIOO 104469
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Bioorganic Chemistry
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Accepted Date:
7 August 2020
14 October 2020
8 November 2020
Please cite this article as: Y. Fang, Z. Wu, M. Xiao, L. Wei, K. Li, Y. Tang, J. Ye, J. Xiang, A. Hu, Design,
synthesis, and evaluation of new 2-oxoquinoline arylaminothiazole derivatives as potential anticancer agents,
Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.104469
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Design, synthesis, and evaluation of new 2-oxoquinoline
arylaminothiazole derivatives as potential anticancer agents
Yilin Fang1,2, Zhilin Wu1, Mengwu Xiao1, Li Wei1, Kangming Li1, Yuting Tang1,
Jiao Ye1, Jiannan Xiang1 and Aixi Hu1,*
1College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, China
2State Key Laboratory for Chemistry and Molecular Engineering of Medicinal Resources,
Collaborative Innovation Center for Guangxi Ethnic Medicine, School of Chemistry and
Pharmaceutical Sciences, Guangxi Normal University
*Corresponding author: Ai-Xi Hu, axhu@hnu.edu.cn
Abstract
A series of novel 2-oxoquinoline derivatives containing arylaminothiazole were designed and
synthesized as potential antitumor agents. The synthesized compounds were evaluated for their in
vitro cytotoxicity activity against HeLa, NCI-H460, T24 and SKOV3 cancer cell lines using MTT
assay. Among them, compound A7 exhibited the most potent activity against the test cancer cell
lines, with the IC50 values ranged from 4.4 to 8.7 µM. The results of tubulin polymerization assay
showed that compound A7 could inhibit tubulin polymerization in vitro. Meanwhile, molecular
docking study revealed that A7 can bind to the colchicine site of tubulin and formed hydrogen
bonds with key amino acid residues in the active site. Further mechanism study demonstrated that
compound A7 blocked cell cycle arrest at G2/M phase, induced cell apoptosis and depolarized
mitochondria of HeLa cells. Collectively, our findings suggest that A7 could serve as a promising
lead for the development of more efficient microtubule polymerization inhibitors for cancer
therapy.
Keywords: 2-Oxoquinoline; Arylaminothiazole; Tubulin inhibitors; Anti-cancer activity;
Apoptosis

1. Introduction
Microtubules, as key components of the cytoskeleton, are cytoskeletal filaments comprised of
α- and β-tubulin heterodimers and play an essential role in a series of cellular processes such as
regulation of motility, cell signaling, formation and maintenance of cell shape, secretion, and
intracellular transport [1-4]. Because of the important roles of microtubules for the rapid dividing
of cancer cells, the discovery and development of small molecules that target tubulin to interfere
with tubulin dynamics becomes a well-verified strategy for cancer therapy [5-7]. Currently, a
number of small molecules as anti-tubulin agents derived from natural sources or obtained by
chemical synthesis have been reported and achieved notable success in cancer chemotherapy
[8-10]. Nevertheless, the emergence of high toxicity, multidrug resistance, side effects, poor
solubility, low oral bioavailability and complex synthesis limits their efficiency and long-term use
[11-13]. Therefore, the discovery and development of novel effective antimicrotubule agents for
overcoming the above limitations is urgently needed.
Quinolines and their hetero fused scaffolds are frequently found in natural alkaloids that exhibit
extensively biological and pharmacological activities and prevalent molecular structures in
chemistry, biology and medicine [14-16]. Based on the literature evidence the quinoline analogs
also exhibit anticancer activities with different mechanisms, including alkylating agents, tyrosine
kinase inhibitors, and tubulin inhibitors [17, 18]. Among the various types of active skeletons of
quinolines, 2-oxoquinolines, a class of heterocyclic molecular scaffold, received considerable
interest and attention due to their diverse pharmacological activities, such as antimicrobial [19],
antioxidant [20], anti-platelet [21], antimalarial [22], anti-inflammatory [23], especially good
anticancer effects [24-26]. In our previous study, we also found that 2-oxoquinoline phosphonate
derivatives exhibited potential cytotoxicity against cancer cells [27]. Therefore, 2-oxoquinoline
skeleton was chosen as an active pharmacal core to screen for new potential antitumor compounds
by the introduction of various functional groups. Meanwhile, previous studies have revealed that
aryl thiazole compounds that can disrupt tubulin polymerization, thereby inhibiting the production
of functional microtubules and cell mitosis [28, 29]. The polarity property of thiazole can be used
as a means to reduce the lipophilic character, improving water solubility, oral absorption and
pharmacokinetic parameters of the drugs itself [30]. In addition, we have been involved in the area
of thiazole derivatives for the past few years and various arylaminothiazole derivatives with
antitumor activities were synthesized successfully in our previous work [31-34]. Based on the
aforementioned results, we proposed in this work that the introduction of arylaminothiazole
moiety into 2-oxoquinoline may lead to good antitumor activity.
In continuation of our ongoing research program for the discovery of potential thiazole
derivatives, for present study, we aim to design a series of 2-oxoquinoline arylaminothiazoles
derivatives and evaluated them as tubulin-binding agents (Fig. 1). The representative compound
A7 was further docked with tubulin protein to explore the possible binding interactions and

investigated in vitro cytotoxicity of the target compounds. The preliminary mechanism of the
inhibitory potency was also investigated.
2. Results and discussion
2.1 Chemistry
Compounds A1-A28 was accomplished using the general method outlined in Scheme 1.
Starting materials, 2-chloro-3-formylquinoline (1) and 2-oxo-3-formyl-quinoline (2) were
prepared according to the reported procedures [35]. Compounds 3, 4 and 5 were synthesized
according to the reported procedures of our previous work [33]. Compound 3 was synthesized by
the aldol condensation reaction of pinacolone and 2-oxo-3-formylquinoline (2) under basic
conditions, and then compound 4 was prepared by the reduction of compound 3 with H2 in EtOH.
Compound 5 was synthesized by the reaction of compound 4 with CuBr2 in CHCl3 and EtOAc.
Target compounds A1-A28 were obtained by the compound 5 and different aryl thiourea in EtOH.
The structures of compounds A1-A28 were confirmed by 1H NMR and 13C NMR.
2.2. Biological Activity
2.2.1 Cytotoxicity Test
The in vitro cytotoxic potency of compounds A1-A28 were tested by MTT assay against
HeLa (Human cervical cancer cells), NCI-H460 (Human large cell lung cancer cell), T24 (Human
bladder cancer cells) and SKOV3 (Human ovarian cancer cells) tumor cell lines, and the
anticancer drugs 5-fluorouracil (5-FU) and CA-4 were chosen as a positive control. The IC50
values of the tested compounds for each cell line were summarized in Table 1.
As shown in Table 1, most of synthesized compounds (A1-A28) displayed moderate to
strong inhibitory activity against HeLa, NCI-H460, T24 and SKOV3 cell lines, with IC50 values
mostly in the micromole level. The anticancer effects of various arylaminothiazole substituent's
attached to 2-oxoquinoline was evaluated. In particular, the compounds A1, A7, A13, A14 and
A19 showed promising anti-proliferative activity with IC50 values ranging from 4.4-12.9 μM
respectively, against the four human cancer cell lines. Among them, compound A7, featuring 4-
fluorinephenyl moiety, displayed the most anti-proliferative activity against all the four human
cancer cell lines. The results revealed that the analogs A2-A5, obtained by inserting a methoxy or
methyl moieties in the benzene group, led to a significant decrease in potency compared with
other analogues substituted by halogen. A beneficial effect was observed with the modifications of
the C-4 position. The derivative A7 has approximately 4-fold more potent than A6 against HeLa.
Moreover, the modifications of 4-position in the phenyl ring with a chlorine or a bromine moiety
caused a decrease in potency compared with the modification of a fluorine. For example,
compound A7 exhibited better cytotoxicity activities than A10 and A12 against the test human
cancer cell lines.
As is shown in the table, a nitro substituent in the benzene ring has a weakened potency
comparing with halogen substituent analogues, which indicates that the nitro substituent might be

not a suitable match for this position. Additionally, different double substituted derivatives
A17-A25 were synthesized and evaluated for their antiproliferative activity. However, most of this
series displayed lower activities (IC50 > 10 μM) than the single substituted counterparts, except
compound A19, of which the 2-F-4-NO2 were substituted on the phenyl ring. Replacement of the
phenyl group by pyridine or naphthalen skeleton resulted in decreased activity. Interestingly, all
tested compounds exhibited lower cytotoxicity against human normal cell line than selected
cancer cell lines, suggesting that these compounds have a selective toxicity for the cancer cells.
Because compound A7 exerted the strongest cytotoxic effect against HeLa cell line, then this cell
line were selected for further mechanism study to address the cell death caused by compound A7,
and a concentration slightly higher than IC50 was used.
2.2.2. In vitro tubulin polymerization inhibition activity
It is well-known that microtubule-targeting agents can be divided into two types: microtubule
stabilizing agents (e.g., paclitaxel) and microtubule destabilizing agents (e.g., colchicine and CA-4)
[4]. In order to further confirm which type of microtubule-targeting agents that compound A7 is,
the in vitro tubulin polymerization inhibition activity of compound A7 was investigated.
Compound A7 were employed at respective 5 and 10 μM for microtubule dynamics assays with
paclitaxel and CA-4 as positive controls, respectively. As illustrated in Fig.2, paclitaxel (10 µM)
was found to stimulate tubulin polymerization, while CA-4 (10 µM) almost completely inhibited
tubulin polymerization as expected. For compound A7, an obvious concentration dependent
inhibition of polymerization was observed. These data indicated that A7 can effectively inhibit
tubulin polymerization and act as potential microtubule destabilizing agents for cancer therapy.
2.2.3. Molecular docking
To investigate the potential binding site of A7 with the tubulin−microtubule system,
molecular modeling studies were performed. The crystal structure of tubulin complexed with
colchicine (PDB: 3E22) was chosen as the docking protein [36]. The binding modes of A7 and
colchicine in the colchicine binding site of tubulin are depicted in Fig. 3. As shown in Fig. 3A, the
docking model proposed that A7 is extended tightly into the cavity of the tubulin active site,
adopting an energetically stable conformation. The oxygen atom on the 2-oxoquinoline moiety of
A7 formed two hydrogen bonds with the critical residue ASN249 and ALA250, which was
consistent with the observation that colchicine stabilizes the tubulin heterodimer (Fig. 3B) and
confirmed that this moiety was crucial for binding [18]. The arylaminothiazoles moiety pointed
toward the deep pocket of the colchicine site, and the fluorine atom of the terminal aromatic group
of A7 as an acceptor established a hydrogen bond with the amino hydrogen of LEU242. The
docking results demonstrated that A7 binds to the colchicine site of tubulin resembling the binding
pose of colchicine with tubulin (Fig. 3B).
2.2.4 Cell cycle distribution effect of compound A7

As most anti-mitotic agents display the ability to disrupt the regulated cell cycle distribution,
the effect of compounds A7 on cell cycle progression of HeLa cell lines was investigated by flow
cytometry at 5 μM and 10 μM for 24 h and shown in Fig. 4. Cell cycle analysis demonstrated that
compound A7 treatment concentration-dependently increased the population of cells in the G2/M
phases. These results accompanied by an increase in the population of cells in G2/M phase
compared with control. As shown in Fig. 4, the population of HeLa cells in the G0/G1 phase
decreased from 62.24% (control) to 46.81% and in the G2/M phase increased from 5.92% to
22.86% after treatment with 10 μM compound A7 for 24 h. These results indicated that cell cycle
mainly arrest in the G2/M stage contributed to the anticancer effects of compound A7 in HeLa
cells.
2.2.5 Compound A7 induced apoptosis in HeLa cells
The apoptosis ratios induced by compound A7 in HeLa tumor cells were quantitatively
evaluated by flow cytometry. Four quadrant images were observed by flow cytometric analysis:
the damaged cells appearing in the process of cell collection were showed in Q1 area, the early
apoptotic cells were displayed in Q3 region, the later period apoptotic cells were represented in Q2
area, and the normal cells were located in the Q4 area. The results were given in Fig. 5.
As shown in Fig. 5, the apoptosis ratios (late plus early apoptotic cells, Q2 + Q3 quadrants)
for compound A7 were significantly increased within the initial 24 h of treatment at the
concentration of 5 μM and 10 μM. The apoptosis of HeLa cells treated with compound A7
increased gradually in a concentration manner. The apoptosis ratios of compound A7 measured at
different concentration points were found to 8.82% (5 μM) and 22.96% (10 μM), respectively,
while that of control was only 2.33%. The results evidently manifested that compound A7
effectively induced apoptosis in HeLa cells in comparison with the control.
2.2.6 Apoptosis assessment by hoechst 33342 staining
Anticancer drugs-induced tumor cell apoptosis is usually accompanied by changes in cell
morphology. Hoechst 33342, which is a membrane permeable dye, can stain the cell nucleus with
blue fluorescence. Normal cells with equably light blue nuclei were evidently detected by
fluorescence microscope after dealing with Hoechst 33342, while apoptotic cells nuclei were
stained as bright blue because of karyopyknosis and chromatin condensation. However, the dead
cells nuclei could not be stained. HeLa cells treated with compound A7 at 5 μM and 10 μM for 24
h were stained with Hoechst 33342. HeLa cells not treated with the A7 used as control at for 24 h.
The results were given in Fig. 6. As shown in Fig. 6, cells not treated with compound A7 were
normally blue (in the web version). It should be noticed that, for A7 treatment, the cells exhibited
strong blue fluorescence and demonstrated typical apoptotic morphology after 24 h and
simultaneous changes in fluorescence due to the change of concentration.
2.2.7 Apoptosis assessment by acridine orange/ethidium bromide (AO/EB) staining
To further characterize the cell apoptosis induced by compound A7, AO/EB staining was
performed to evaluate the accompanying changes in morphology. Besides, AO can pass through

an intact cell membrane, then stain nuclear DNA, while EB only stains cells with damaged cell
membranes. Therefore, after simultaneously treating with AO and EB, normal cells will be stained
as green and early apoptotic cells will be stained as green yellow or display green yellow
fragments. And late apoptotic cells will be thickly stained as orange or show orange fragments and
non-apoptotic cells will be stained as orange-yellow. The apoptosis induction effect of compound
A7 against HeLa cells at the concentration of 5 μM and 10 μM for 24 h was tested by AO/EB
staining, and HeLa cells not treated with A7 were used as control for 24 h. The results are shown
in Fig. 7.
As shown in Fig. 7, the HeLa cells treated with A7 for 24 h, at the concentration of 5 μM and
10 μM, had evidently changed. The nuclei obviously stained evenly as yellow green or light
orange, and the cell morphology exhibited pycnosis, membrane blebbing and cell budding. These
phenomena were related to cell apoptosis. On the basis of the above observation, the cells
displayed an apoptotic morphology. These results demonstrated that compound A7 could cause the
decrease of apoptosis with low cytotoxicity.
2.2.8. Compound A7 triggers mitochondrial pathway dependent apoptosis
The loss of mitochondrial membrane potential (MMP) is regarded as an important factor in the
apoptotic pathway. [37] In order to further evaluate the apoptosis-inducing effect of compound A7,
MMP changes were detected using the fluorescent probe JC-1. HeLa cells were treated with
compound A7 (5, 10 μM) for 24 h using untreated cells as control. As shown in Fig. 8, our results
indicated that treatment with A7 led to the loss of MMP in HeLa cells. When HeLa cells were
exposed to A7, we found a dose-dependent enhancement in the green (monomeric) fluorescence
versus that of control, suggesting that compound A7 treatment induced mitochondrial membrane
permeability in HeLa cells. Taken together, these results clearly demonstrated that mitochondrial
function is extremely impaired in compound A7 caused apoptosis in HeLa cells.
3. Conclusion
In summary, a class of novel 2-oxoquinoline arylaminothiazole derivatives were designed
and synthesized, and evaluated as tubulin polymerization inhibitors. Anti-proliferative screening
of these compounds validated the representative compound A7 as a potent tubulin inhibitor with
IC50 values ranging from 4.4 to 8.7 μM against a panel of cancer cell lines. In addition, the
microtubule polymerization inhibitory action of A7 was investigated using an in vitro tubulin
polymerization assay and a virtual molecular docking study. These results confirmed that A7
effectively inhibited tubulin polymerization, and it bound to the colchicine site of tubulin. Further
mechanistic studies of A7 in HeLa cells were investigated by Hoechst 33258 staining, JC-1
mitochondrial membrane potential analysis and apoptosis detection. The results indicated that A7
induced cell cycle arrest in the G2/M phase and induced cell apoptosis in HeLa cells. Taken
together, these results suggested that compound A7 is a promising antitumor agent to inhibit

tubulin polymerization.
4. Experimental
4.1. Chemistry
All chemicals and solvents were analytical reagents and used directly without further
depuration. Melting points were determined using an X-4 electrothermal digital melting point
apparatus and are uncorrected. Nuclear magnetic resonance (NMR) was recorded on a Varian
INOVA-400 spectrometer in DMSO-d6 or CDCl3, whereas tetramethylsilane (TMS) (δ 0 ppm)
was used as an internal standard for spectra recorded.
4.1.1. General procedure for the synthesis of compounds 1 and 2
Compounds 1 and 2 were synthesized according to the literature [35].
4.1.2. General procedure for the synthesis of compounds 3-5
The synthesis of the key intermediate (3-5) reference the previous report [33].
4.1.3. General procedure for the synthesis of target compounds (A1-A28)
Compound 5 (1 mmol) was added into a refluxing solution of an appropriate aryl thiourea (1.2
mmol) in EtOH (15 mL) and refluxed for 4 h. After completion of the reaction as indicated by
TLC, the mixture was cooled to room temperature. The reaction mixture made alkaline with NH3
(aq) to pH = 8-9 and stirred for 30 min, then the reaction mixture was cooled and filtered to get the
crude products. The crude products were recrystallized from 95% alcohol to offer compounds
A1-A28.
3-((4-(tert-Butyl)-2-(phenylamino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A1).
Yield:
72.0%, m.p. 229~232°C. 1H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH), 9.92 (s, 1H, NH),
7.66 (d, J = 7.8 Hz, 1H), 7.60 (d, J = 8.3 Hz, 2H), 7.56 (s, 1H), 7.46 (t, J = 7.6 Hz, 1H), 7.34~7.25
(m, 3H), 7.14 (t, J = 7.5 Hz, 1H), 6.89 (t, J = 7.3 Hz, 1H), 4.00 (s, 2H, CH2), 1.35 (s, 9H, 3×CH3).
13C NMR (100 MHz, DMSO-d6) δ: 162.04, 159.05, 155.10, 141.84, 138.06, 136.69, 133.55,
130.29, 129.43, 128.05, 122.52, 121.25, 119.65, 116.94, 115.44, 115.31, 36.35, 31.11, 27.22.
3-((4-(tert-Butyl)-2-(o-tolylamino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A2).
Yield:
1
70.0%, m.p.161~165°C. H NMR (400 MHz, CDCl3) δ: 12.22 (s, 1H, NH), 7.69 (d, J = 8.2 Hz,
1H), 7.54 (s, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 7.6 Hz, 1H), 7.40 (d, J = 8.1 Hz, 1H),
7.23~7.14 (m, 3H), 6.96 (t, J = 7.4 Hz, 1H), 4.21 (s, 2H, CH2), 2.31 (s, 3H, CH3), 1.42 (s, 9H,
3×CH3). 13C NMR (100 MHz, CDCl3) δ: 158.86, 156.31, 134.16, 132.51, 127.84, 125.94, 124.97,
122.58, 122.19, 118.36, 117.71, 116.59, 115.19, 113.67, 112.53, 110.79, 110.39, 31.17, 26.00,
22.61, 12.94.
3-((4-(tert-Butyl)-2-(m-tolylamino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A3).
Yield:
71.8%, m.p. 210~212°C. 1H NMR (400 MHz, CDCl3) δ: 12.23 (s, 1H, NH), 7.54 (s, 1H), 7.51 (d,
J = 7.7 Hz, 1H), 7.47 (d, J = 7.3 Hz, 1H), 7.41 (d, J = 8.1 Hz, 1H), 7.22~7.17 (m, 2H), 7.13 (s,
2H), 6.85 (d, J = 7.2 Hz, 1H), 4.21 (s, 2H, CH2), 2.31 (s, 3H, CH3), 1.44 (s, 9H, 3×CH3). 13C NMR

(100 MHz, CDCl3) δ: 158.76, 156.30, 134.94, 134.58, 132.67, 132.57, 127.28, 125.16, 124.37,
122.65, 119.21, 117.90, 115.10, 114.03, 110.78, 110.21, 109.86, 31.06, 25.88, 22.53, 16.61.
3-((4-(tert-Butyl)-2-(p-tolylamino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A4).
Yield:
1
73.8%, m.p. 224~227°C. H NMR (400 MHz, CDCl3) δ: 12.03 (s, 1H ， NH), 7.53 (s, 1H),
7.50~7.45 (m, 2H), 7.40 (d, J = 8.1 Hz, 1H), 7.22~7.17 (m, 3H), 7.09 (d, J = 8.4 Hz, 2H), 4.21 (s,
2H, CH2), 2.28 (s, 3H, CH3), 1.41 (s, 9H, 3×CH3) ； 13C NMR (100 MHz, CDCl3) δ: 163.63,
160.89, 155.73, 138.11, 137.35, 132.76, 132.27, 129.85, 127.49, 122.59, 120.08, 118.13, 115.59,
114.73, 36.03, 30.85, 27.40, 20.71.
3-((4-(tert-Butyl)-2-((4-methoxyphenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one (A5).
Yield: 73.8%, m.p. 224~227°C. 1H NMR (400 MHz, DMSO-d6) δ: 11.89 (s, 1H, NH), 9.67 (s, 1H,
NH), 7.65 (d, J = 7.7 Hz, 1H), 7.55 (s, 2H), 7.51 (d, J = 9.0 Hz, 2H), 7.46 (t, J = 7.7 Hz, 1H), 7.31
(d, J = 8.2 Hz, 1H), 6.88 (d, J = 9.1 Hz, 2H), 3.97 (s, 2H, CH2), 3.70 (s, 3H, OCH3), 1.34 (s, 9H,
3×CH3). 13C NMR (100 MHz, DMSO-d6) δ：162.06, 159.92, 154.99, 154.21, 138.26, 136.47,
135.58, 133.67, 130.15, 128.05, 122.34, 119.69, 118.72, 115.32, 114.66, 114.50, 55.64, 36.31,
31.16, 27.26.
3-((4-(tert-Butyl)-2-((2-fluorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A6).
Yield: 84.5%, m.p. 226~230°C. 1H NMR (400 MHz, CDCl3) δ: 11.77 (s, 1H, NH), 8.11 (t, J = 8.1
Hz, 1H), 7.55~7.44 (m, 3H), 7.38 (d, J = 8.1 Hz, 1H), 7.29 (d, J = 8.4 Hz, 1H), 7.20 (t, J = 7.5 Hz,
1H), 7.10 (dd, J = 18.3, 10.0 Hz, 2H), 6.93 (dd, J = 11.9, 6.2 Hz, 1H), 4.23 (s, 2H), 1.43 (s, 9H,
3×CH3). 13C NMR (100 MHz, CDCl3) δ: 163.42, 137.41, 137.30, 136.13, 132.75, 129.96, 129.73,
127.55, 124.64, 122.70, 121.89, 120.03, 118.03, 117.23, 116.18, 115.49, 114.96, 114.77, 36.21,
30.85, 27.35.
3-((4-(tert-Butyl)-2-((4-fluorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A7).
1
Yield 45.0%, m.p. 170~173°C. H NMR (400 MHz, CDCl3) δ: 12.07 (s, 1H, NH), 7.63 (s, 1H),
7.53~7.46 (m, 3H), 7.35~7.32 (m, 2H), 7.19 (t, J = 7.5 Hz, 1H), 6.98 (t, J = 8.6 Hz, 3H), 4.19 (s,
2H, CH2), 1.41 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 166.43, 149.23, 144.04, 138.37,
137.56, 132.18, 131.17, 130.94, 130.02, 129.01, 127.94, 125.83, 123.26, 119.53, 116.80, 115.31,
35.34, 30.27, 27.70.
3-((4-(tert-Butyl)-2-((2-chlorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A8).
Yield: 64.3%, m.p. 223~225°C. 1H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH), 9.31 (s, 1H,
NH), 8.46 (d, J = 8.2 Hz, 1H), 7.65 (d, J = 7.8 Hz, 1H), 7.59 (s, 1H), 7.44 (dd, J = 12.6, 7.8 Hz,
2H), 7.34~7.27 (m, 2H), 7.15 (t, J = 7.5 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 4.02 (s, 2H, CH2), 1.35
(s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO) δ: 161.82, 158.89, 154.30, 138.13, 136.46, 133.40,
129.98, 129.73, 127.90, 127.83, 122.69, 122.13, 121.56, 120.21, 119.46, 117.24, 115.11, 113.79,
36.08, 30.92, 27.08.
3-((4-(tert-Butyl)-2-((3-chlorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A9).
Yield: 75.4%, m.p. 271~273°C. 1H NMR (400 MHz, DMSO) δ: 11.92 (s, 1H, NH), 10.08 (s, 1H,
NH), 7.65 (t, J = 7.9 Hz, 3H), 7.56 (s, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.34~7.31 (m, 3H), 7.14 (t, J =

7.5 Hz, 1H), 4.00 (s, 2H, CH2), 1.35 (s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO) δ: 162.01,
158.77, 155.10, 140.85, 138.26, 136.51, 133.52, 130.13, 129.14, 128.01, 124.37, 122.29, 119.64,
118.40, 116.00, 115.29, 36.28, 31.10, 27.20.
3-((4-(tert-Butyl)-2-((4-chlorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one (A10).
1
Yield: 70.7%, m.p. 227–229°C. H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH), 10.08 (s,
1H, NH ), 7.65 (t, J = 7.9 Hz, 3H), 7.56 (s, 1H), 7.46 (t, J = 7.7 Hz, 1H), 7.32 (dd, J = 8.2, 4.8 Hz,
3H), 7.14 (t, J = 7.5 Hz, 1H), 4.00 (s, 2H, CH2), 1.35 (s, 9H, 3×CH3). 13C NMR (100 MHz,
DMSO-d6) δ：162.09, 158.77, 155.10, 140.85, 138.26, 136.51, 133.52, 130.13, 129.14, 128.01,
124.37, 122.29, 119.64, 118.40, 116.00, 115.29, 36.28, 31.10, 27.20.
3-((2-((2-Bromophenyl)amino)-4-(tert-butyl)thiazol-5-yl)methyl)quinolin-2(1H)-one (A11).
1
Yield: 70.4%, m.p. 253~257°C. H NMR (400 MHz, DMSO) δ 11.91 (s, 1H, NH), 9.12 (s, 1H,
NH), 8.29 (d, J = 8.3 Hz, 1H), 7.66 (d, J = 7.3 Hz, 1H), 7.61~7.57 (m, 2H), 7.46 (t, J = 7.1 Hz,
1H), 7.36~7.31 (m, 2H), 7.15 (t, J = 8.0 Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 4.00 (s, 2H, CH2), 1.33
(s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.99, 159.37, 154.51, 139.56, 138.16,
136.78, 133.52, 133.34, 130.26, 128.71, 128.04, 123.97, 122.45, 121.37, 119.64, 117.39, 115.29,
112.99, 36.28, 31.11, 27.32.
3-((2-((4-Bromophenyl)amino)-4-(tert-butyl)thiazol-5-yl)methyl)quinolin-2(1H)-one (A12).
1
Yield: 70.2%, m.p. 150~153°C. H NMR (400 MHz, CDCl3) δ: 12.16 (s, 1H, NH), 7.65 (s, 1H),
7.55~7.48 (m, 2H), 7.44 (d, J = 8.1 Hz, 1H), 7.40~7.37 (m, 3H), 7.28 (s, 2H), 4.19 (s, 2H, CH2),
1.41 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 163.38, 139.44, 137.48, 136.23, 133.85,
132.81, 132.19, 130.06, 129.73, 127.57, 122.78, 121.38, 119.14, 117.21, 115.49, 115.29, 36.13,
30.79, 27.44.
3-((4-(tert-Butyl)-2-((3-(trifluoromethyl)phenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-
one (A13). Yield: 43.7%, m.p. 225~228°C. 1H NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H, NH),
10.32 (s, 1H, NH), 8.49 (s, 1H), 7.66 (d, J = 7.7 Hz, 1H), 7.59~7.58 (m, 2H), 7.52~7.44 (m, 2H),
7.32 (d, J = 8.2 Hz, 1H), 7.22 (d, J = 7.6 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 4.02 (s, 2H, CH2), 1.36
(s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 162.00, 158.55, 155.09, 142.48, 138.27,
136.58, 133.46, 130.31, 130.17, 130.00, 128.03, 126.17, 122.31, 120.39, 119.63, 117.08, 116.73,
115.29, 112.99, 36.25, 30.99, 27.17.
3-((4-(tert-Butyl)-2-((4-(trifluoromethyl)phenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-
1
one (A14). Yield: 71.8%, m.p. 210~212°C. H NMR (400 MHz, CDCl3) δ: 11.73 (s, 1H, NH),
7.93 (s, 1H), 7.53–7.44 (m, 4H), 7.39 (t, J = 8.0 Hz, 2H), 7.25–7.17 (m, 2H), 4.22 (s, 2H, CH2),
1.43 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 158.56, 154.00, 136.11, 132.59, 132.40,
127.62, 125.11, 124.78, 122.66, 117.85, 115.13, 113.63, 111.33, 110.63, 109.10, 31.31, 25.91,
22.45.
3-((4-(tert-Butyl)-2-((2-nitrophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A15).
1
Yield: 75.9%, m.p. 265~269°C. H NMR (400 MHz, DMSO-d6) δ: 11.93 (s, 1H, NH), 10. 05 (s,
1H, NH), 8.83 (d, J = 9.4 Hz, 1H), 8.30 (d, J = 2.7 Hz, 1 H), 8.25~8.22 (m, 1H), 7.66 (d, J = 7.7

Hz, 1H), 7. 63 (s, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.15 (t, J = 7.2 Hz, 1H),
4.07 (s, 2H, CH2), 1.38 (s, 9H, 3×CH3) ；13C NMR (100 MHz, DMSO-d6) δ ：161.94, 157.44,
155.04, 138.36, 136.85, 136.71, 135.71, 133.32, 130.23, 128.01, 126.15, 124.51, 122.32, 121.52,
120.72, 120.16, 119.58, 115.30, 36.27, 30.98, 27.38。
3-((4-(tert-Butyl)-2-((4-nitrophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A16).
Yield: 69.0%, m.p. 285~288°C. 1H NMR (400 MHz, DMSO-d6) δ 12.05 (s, 1H, NH), 10.77 (d, J
= 8.1 Hz, 1H, NH), 8.21 (d, J = 9.0 Hz, 2H), 7.81 (d, J = 9.2 Hz, 2H), 7.60 (s, 1H), 7.47 (t, J = 7.6
Hz, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.27 (d, J = 8.7 Hz, 1H), 7.15 (t, J = 7.6 Hz, 1H), 4.06 (s, 2H,
CH2), 1.38 (s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.75, 161.56, 157.40, 147.44,
140.08, 137.20, 135.44, 134.57, 129.97, 127.89, 125.84, 122.02, 117.25, 116.10, 115.07, 113.77,
36.17, 30.93, 27.11.
3-((4-(tert-Butyl)-2-((2,6-dimethylphenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A17). Yield: 71.6%, m.p. 203~205°C. 1H NMR (400 MHz, DMSO-d6) δ: 11.85 (s, 1H, NH), 8.81
(s, 1H, NH), 7.57 (d, J = 7.6 Hz, 1H), 7.48 (s, 1H), 7.44 (d, J = 8.0 Hz, 1H), 7.30 (d, J = 8.2 Hz,
1H), 7.14 (t, J = 7.5 Hz, 1H), 7.10~7.04 (m, 3H), 3.91 (s, 2H, CH2), 2.22 (s, 6H, 2×CH3), 1.30 (s,
9H, 3×CH3). 13C NMR (101 MHz, DMSO-d6) δ: 164.09, 161.82, 154.77, 138.74, 138.05, 136.29,
136.04, 133.61, 129.91, 128.67, 127.68, 126.79, 122.13, 119.39, 115.13, 114.11, 36.01, 31.00,
27.36, 18.46.
3-((2-((3,5-Bis(trifluoromethyl)phenyl)amino)-4-(tert-butyl)thiazol-5-yl)methyl)quinolin-2-
1
(1H)-one (A18). Yield: 51.9%, m.p. 215~218°C. H NMR (400 MHz, CDCl3) δ: 11.15 (s,
1H, NH), 8.08 (s, 2H), 7.52 (s, 1H), 7.50~7.45 (m, 3H), 7.32 (d, J = 7.7 Hz, 1H), 7.20 (t, J = 7.2
Hz, 1H), 4.22 (s, 2H, CH2), 1.45 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 162.85, 150.29,
137.62, 137.20, 132.82, 132.50, 131.89, 130.25, 127.66, 124.44, 122.88, 121.73, 119.79, 117.41,
117.34, 117.06, 115.28, 36.02, 30.74, 27.37.
3-((4-(tert-Butyl)-2-((2-fluoro-4-nitrophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(A19). Yield: 64.4%, m.p. 270–275°C. H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH),
10.03 (s, 1H, NH), 8.83 (d, J = 9.2 Hz, 1H), 8.28 (d, J = 2.3 Hz, 1H), 8.23 (d, J = 9.3 Hz, 1H),
7.66~7.64 (m, 2H), 7.47 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 8.1 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 4.08
(s, 2H, CH2), 1.39 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 158.72, 157.79, 132.55,
132.45, 127.84, 127.16, 125.01, 122.63, 120.53, 117.69, 116.63, 116.53, 115.13, 114.06, 112.37,
110.67, 30.73, 25.63, 22.90.
3-((4-(tert-Butyl)-2-((2-chloro-4-nitrophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A20). Yield: 65.9%, m.p. 273~277°C.1H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH),
10.22 (s, 1H, NH), 8.36 (d, J = 8.2 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1 H), 7.70 (t, J = 7.8 Hz, 1H), 7.
65 (d, J = 7.8 Hz, 1H), 7.61(s, 1H ), 7.45 (t, J = 7.3 Hz, 1H), 7.32 (d, J = 8.2 Hz, 1H), 7.16 (t, J =
7.5 Hz, 1H), 4.05 (s, 2H, CH2), 1.33 (s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.96,
159.64, 154.73, 140.76, 140.26, 138.44, 136.93, 133.41, 130.27, 128.10, 125.61, 124.48, 122.32,
121.02, 119.97, 119.62, 117.64, 115.34, 36.26, 31.25, 27.18.

3-((4-(tert-Butyl)-2-((3-chloro-4-fluorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(A21). Yield: 63.6%, m.p. 196~199°C. H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH),
10.16 (s, 1H, NH), 8.14~8.12 (m, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.58 (s, 1H), 7.47 (t, J = 7.7 Hz,
1H), 7.44~7.39 (m, 1H), 7.36~7.30 (m, 2H), 7.15 (t, J = 7.5 Hz, 1H), 4.02 (s, 2H, CH2), 1.37 (s,
9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.82, 158.48, 154.81, 152.90, 150.52, 139.00,
138.10, 136.38, 133.29, 129.97, 127.84, 122.12, 119.46, 117.81, 117.36, 117.15, 116.79, 116.23,
115.11, 36.10, 30.88, 27.02.
3-((4-(tert-Butyl)-2-((2,3-dichlorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A22). Yield: 71.7%, m.p. 228~231°C. 1H NMR (400 MHz, CDCl3) δ: 12.13 (s, 1H, NH), 8.20 (d,
J = 8.0 Hz, 1H), 7.54 (s, 1H), 7.52–7.46 (m, 2H), 7.43 (t, J = 8.0 Hz, 1H), 7.22~7.16 (m, 2H), 7.07
(d, J = 8.0 Hz, 1H), 4.25 (s, 2H, CH2), 1.44 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ:
158.41, 152.83, 133.90, 132.57, 128.04, 127.90, 127.67, 125.10, 124.79, 122.90, 122.62, 117.81,
117.68, 115.12, 112.65, 110.77, 110.32, 31.41, 25.99, 22.54.
3-((4-(tert-Butyl)-2-((3,4-dichlorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(A23). Yield: 73.8%, m.p. 210~212°C. H NMR (400 MHz, DMSO-d6) δ: 11.92 (s, 1H, NH),
10.28 (s, 1H, NH), 8.22 (d, J = 2.5 Hz, 1H), 7.66 (d, J = 7.8 Hz, 1H), 7.58 (s, 1H), 7.51 (d, J = 8.8
Hz, 1H), 7.47 (t, J = 7.2 Hz, 1H), 7.41~7.38 (m, 1H), 7.33 (d, J = 8.2 Hz, 1H), 7.15 (t, J = 7.5 Hz,
1H), 4.03 (s, 2H, CH2), 1.37 (s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.81, 158.12,
154.94, 141.62, 138.10, 136.42, 133.25, 131.38, 130.87, 130.00, 127.86, 122.14, 121.75, 119.46,
117.88, 116.92, 116.80, 115.11, 36.11, 30.88, 27.02.
3-((4-(tert-Butyl)-2-((3,5-dichlorophenyl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A24). Yield: 82.9%, m.p. 223~226°C. 1H NMR (400 MHz, CDCl3) δ: 11.94 (s, 1H, NH), 7.50 (s,
1H), 7.48~7.41 (m, 4H), 7.38 (d, J = 8.2 Hz, 1H), 7.18 (t, J = 7.5 Hz, 1H), 6.92 (s, 1H), 4.22 (s,
2H, CH2), 1.41 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 158.71, 153.34, 151.27, 137.64,
132.75, 132.37, 130.41, 127.50, 125.14, 122.64, 117.92, 116.52, 115.16, 111.84, 110.74, 110.35,
31.36, 25.94, 22.48.
3-((4-(tert-Butyl)-2-((2,6-dibromo-4-(trifluoromethoxy)phenyl)amino)thiazol-5-yl)methyl)qui-
1
nolin-2(1H)-one (A25). Yield: 61.9%, m.p. 215~219°C. H NMR (400 MHz, CDCl3) δ: 12.37 (s,
1H, NH), 7.52 (s, 1H), 7.49~7.48 (m, 3H), 7.41 (t, J = 7.1 Hz, 1H), 7.21~7.12 (m, 2H), 4.08 (s,
2H, CH2), 1.41 (s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 158.72, 157.79, 140.91, 132.55,
132.45, 131.22, 127.84, 127.16, 125.01, 122.63, 120.53, 117.69, 116.63, 116.53, 115.13, 114.06,
112.32, 110.67, 30.73, 25.63, 22.90.
3-((4-(tert-Butyl)-2-(pyridin-2-ylamino)thiazol-5-yl)methyl)quinolin-2(1H)-one (A26). Yield:
43.6%, m.p. 268~271°C. 1H NMR (400 MHz, DMSO-d6) δ: 11.90 (s, 1H, NH), 11.03 (s, 1H, NH),
8.18 (d, J = 4.6 Hz, 1H), 7.67~7.60 (m, 2H), 7.46 (t, J = 7.8 Hz, 2H), 7.33 (d, J = 8.2 Hz, 1H),
7.12 (t, J = 7.5 Hz, 1H), 7.07 (d, J = 8.4 Hz, 1H), 6.84 (t, J = 8.0 Hz, 1H), 4.05 (s, 2H, CH2), 1.34
(s, 9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.87, 155.40, 153.29, 152.28, 146.71,
138.05, 137.92, 136.19, 133.84, 129.77, 127.93, 122.08, 119.48, 116.96, 115.79, 115.11, 110.87,

35.89, 30.97, 26.89.
3-((4-(tert-Butyl)-2-(naphthalen-1-ylamino)thiazol-5-yl)methyl)quinolin-2(1H)-one
(A27).
Yield: 56.8%, m.p. 165~168°C. 1H NMR (400 MHz, CDCl3) δ: 11.96 (s, 1H, NH), 8.08 (d, J = 8.9
Hz, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.75 (d, J = 7.4 Hz, 1H), 7.58 (d, J = 8.2 Hz, 1H), 7.52~7.47 (m,
4H) 7.42 (d, J = 9.4 Hz, 1H), 7.40~7.33 (m, 2H), 7.17 (t, J = 7.4 Hz, 1H), 4.19 (s, 2H, CH2), 1.43
(s, 9H, 3×CH3). 13C NMR (100 MHz, CDCl3) δ: 158.69, 157.38, 132.48, 131.59, 129.52, 127.83,
124.95, 123.75, 122.60, 121.61, 121.43, 121.24, 120.96, 119.30, 117.69, 116.03, 115.17, 111.02,
110.70, 110.24, 31.15, 26.01, 22.61.
3-((4-(tert-Butyl)-2-((2-methylquinolin-8-yl)amino)thiazol-5-yl)methyl)quinolin-2(1H)-one
1
(A28). Yield: 85.9%, m.p. 260~264°C. H NMR (400 MHz, DMSO-d6) δ: 11.88 (s, 1H, NH),
10.01 (s, 1H, NH), 8.57 (s, 1H), 8.22 (d, J = 8.4 Hz, 1H), 7.65~7.63 (m, 2H), 7.51~7.47 (m, 4H),
7.34 (d, J = 8.2 Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 4.07 (s, 2H, CH2), 2.72 (s, 3H, CH3), 1.42 (s,
9H, 3×CH3). 13C NMR (100 MHz, DMSO-d6) δ: 161.85, 158.23, 157.04, 138.16, 137.03, 136.77,
136.55, 136.42, 133.40, 129.99, 127.84, 126.55, 126.36, 123.00, 122.13, 119.49, 118.99, 117.10,
115.13, 112.71, 36.10, 31.00, 27.16, 25.03.
4.2. Cytotoxic activity
The MTT method was used to evaluate the cytotoxic activity of the synthesized
2-oxoquinoline arylaminothiazole derivatives A1-28. All tested compounds were dissolved in
DMSO and subsequently diluted in culture medium before treatment of cultured cells. The HeLa,
NCI-H460, T24 and SKOV3 cells lines used in this study were all obtained from the Institute of
Biochemistry and Cell Biology, China Academy of Sciences. All cells lines were seeded in
96-well plates at a density of 5×104 cells/mL with a volume of 200 μL DMEM and then cultivated
o
at 37 C for 24 h. In order to investigate the potential of compounds A1-28, 5-FU, a commercial
classical anti-cancer drug was used as a reference organic drug, were added to each well. After
co-incubation for 48 h, 60 μL of MTT solution in phosphate buffered saline (PBS, 2.5 mg/mL)
o
was added to each well, and the cells were incubated for another 4 h at 37 C in a humidified
atmosphere of 5% CO2/95% air. After drawing-off of the culture medium, 150 mL of DMSO was
added to dissolve formazan crystal; the percentage of cell viability was determined using a
microplate reader. The absorbance was read by enzyme labeling instrument with 570 nm wave
length measurement. The cytotoxicity was estimated based on the percentage cell survival in a
dose dependent manner relative to the negative control. The final IC50 values were calculated by
the Bliss method. All assays were conducted with four parallel samples.
4.3. Tubulin polymerization assay in vitro assays
Tubulin polymerization assay was monitored by the change in optical density at 340 nm
using a modification of methods described by Jordan et al. [38] Purified brain tubulin
polymerization kit was purchased from Cytoskeleton (BK006P, Denver, CO). The final buffer
concentrations
for
tubulin
polymerization
contained
80.0
mM
piperazine-N,N′-bis(2-ethanesulfonic acid)sequisodium salt (pH=6.9), 2.0 mM MgCl2, 0.5 mM

ethylene glycol bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA), 1 mM GTP, and
10.2% glycerol. Test compounds were added in different concentrations, and then all components
except the purified tubulin were warmed to 37 °C. The reaction was initiated by the addition of
tubulin to a final concentration of 3.0 mg/mL. Paclitaxel was used as positive controls under the
same conditions. The optical density was measured for 40 min at 1 min intervals in BioTek’s
Synergy 4 multifunction microplate spectrophotometer with a temperature controlled cuvette
holder. Assays were performed according to the manufacturer’s instructions and under conditions
similar to those employed for the tubulin polymerization assays described above. [39]
4.4. Molecular docking
All the docking studies were carried out using Sybyl-X 2.0 on a windows workstation. The
crystal structure of the tubulin in complex with colchicine was retrieved from the RCSB Protein
Data Bank (PDB: 3E22.pdb). [36] The synthetic chalcone analogues were selected for the docking
studies. The 3D structures of these selected compounds were first built using Sybyl-X 2.0 sketch
followed by energy minimization using the MMFF94 force field and Gasteiger-Marsili charges.
We employed Powell’s method for optimizing the geometry with a distance dependent dielectric
constant and a termination energy gradient of 0.005 kcal/mol. All the selected compounds were
automatically docked into the colchicine binding pocket of tubulin by an empirical scoring
function and a patented search engine in the Surflex docking program. The polar hydrogen atoms
were added and also the automated docking manner was applied in the present work. Other
parameters were established by default to estimate the binding affinity characterized by the
Surflex-Dock scores in the software. Surflex-Dock total scores, which were expressed in -log10
(Kd) units to represent binding affinities, were applied to evaluate the ligand-receptor interactions
of newly designed molecules. A higher score represents stronger binding affinity. The optimal
binding pose of the docked compounds was selected based on the Surflex scores and visual
inspection of the docked complexes.
4.5. Cell cycle distribution analysis
Cell cycle distributions in cells were determined through PI staining, as previously described
[40]. In brief, after HeLa cells were treated with compound A7, the suspension and adherent
portions were collected into flow cytometry tubes and centrifuged at 1000 rpm for 5 min to obtain
cell pellets. The cells were washed with PBS and then fixed with 70% ethanol (–20°C) overnight.
Fixed cells were washed with PBS and sequentially incubated with RNase A (0.1 mg/mL) for 30
min and PI (50 mg/mL) for 15 min in the dark. The distribution of the cell cycle was determined
using flow cytometry. The data were analyzed using ModFit software (Becton Dickinson, San Jose,
CA, USA). All assays were conducted with three parallel samples, with 1×104 cells collected for
each sample.
4.6. Apoptosis detection via flow cytometry

The extent of apoptosis was quantitatively measured using Annexin V binding assay [40].
HeLa cells were cultured in Minimum Essential Medium (MEM) supplemented with 10%
heat-inactivated fetal bovine serum (FBS). Cells were plated in a six-well culture dish at a density
of 2.0 × 104 cells/well and cultured for 24 h at 37°C in 5%CO2. After the medium was removed, 1
mL of fresh MEM containing the indicated concentrations of compound A7 was added and the
cells were cultured at 37°C in 5%CO2. Then, the cells floating in the supernatant were combined
with the adherent fraction and washed with PBS thrice. The cells were incubated with Annexin
V-FITC and PI for 15 min at 37°C in the dark. The samples were immediately analyzed using a
Kaluza flow cytometry Software. A total of 2×104 cells were collected for each sample. All
experiments were performed in triplicate.
4.7. Hoechst 33342 staining
HeLa cells were seeded in six-well plates at a density of 1×104 cells/mL and then cultivated
o
at 37 C for 24 h. And the cells were treated with compound A7 (5 μM and 10 μM, respectively)
for 24 h. After being washed twice with PBS, the cells were stained with 1 mL of Hoechst 33342
(Dojindo Laboratories) for 15 minutes and then again washed twice with PBS. The stained nuclei
were observed under a Nikon Te2000 fluorescence microscope using 350 nm excitation and 460
nm emission.
4.8. AO/EB staining
HeLa cells were plated in a glass-bottom dish (glass diameter 10 mm) at 1.0 × 104 cells/dish
and cultured for 24 h at 37 °C in 5% humidified CO2. After the culture medium was removed, and
1 mL of fresh medium plus 10% fetal bovine serum and supplemented with compound A7 (5 μM
or 10 μM) was added to cells. After incubation for 24 h at 37°C in 5% CO2, then 1 mL of AO/EB
(Dojindo Laboratories) stain (20 μM) was added to and the cells were washed twice with PBS and
were observed by Nikon Te2000 fluorescence microscope. In the case of the compound
(A7)-AO-EB system, the cells were cultured for 20 minutes, and CLSFM observations were
carried out with excitation by an argon laser (488 nm for AO) and Helium-Neon laser (543 nm for
EB). The emitted fluorescence was detected through a 505–550 nm bandpass filter and a 610–690
nm long-pass filter, respectively.
4.9. Mitochondrial membrane potential analysis
Mitochondrial depolarization was assayed in HeLa cells using a JC-1 probe (Beyotime,
Haimen, China). Briefly, cells cultured in six-well plates after the indicated treatment were
incubated with an equal volume of JC-1 staining solution (5 μg/mL) at 37 °C for 20 min and
rinsed twice with PBS. Mitochondrial membrane potentials were monitored by determining the
relative amounts of dual emissions from mitochondrial JC-1 monomers or aggregates using a
Nikon ECLIPSETE2000-S fluorescent microscope (488 nm excitation and 525 nm emission
filters).
4.10 Statistics

The data were processed by the Student' st -test with the significance level P≤0.05 using SPSS.
Acknowledgments
The authors would like to thank Professor Kemin Wang (State Key Laboratory of
Chemo/Biosensing and Chemometrics, Hunan University) and Professor Jiannan Xiang (College
of Chemistry and Chemical Engineering, Hunan University) for their help in biological assays.
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Schemes
Scheme 1. Synthesis of compounds A1~A28. Reagents and conditions: (a) DMF, POCl3, 90oC, 16 h; (b) 70%
AcOH, 95oC, 6 h; (c) NaOH, EtOH, reflux, 2 h; (d) Raney Ni, EtOH, 90oC, 8 h; (e) CuBr2, CHCl3–EtOAc, 80oC, 4
h; (f) 1. EtOH; 2. NH3. H2O, reflux.

Figures
A1：Ar=C6H5；A2：Ar=2-CH3C6H4；A3：Ar=3-CH3C6H4；A4：Ar=4-CH3C6H4；A5：Ar=4-CH3OC6H4；
A6：Ar=2-FC6H4；A7：Ar=3-FC6H4；A8：Ar=2-BrC6H4；A9：Ar=4-BrC6H4；A10：Ar=2-ClC6H4；
A11：Ar=3-ClC6H4；A12：Ar=4-ClC6H4；A13：Ar=3-CF3C6H4；A14：Ar=4-CF3C6H4；A15：Ar=2-NO2C6H4
；A16：Ar=4-NO2C6H4；A17：Ar=2,6-diCH3C6H3；A18：Ar=3,5-diCF3C6H3；A19：Ar=2-F-3-NO2C6H3；
A20：Ar=3-Cl-4-F-C6H3；A21：Ar=2,3-diClC6H3；A22：Ar=3,4-diClC6H3；A23：Ar=3,5-diClC6H3；
A24：Ar=2-Cl-4-NO2C6H3；A25：Ar=2,6-diBr-4-CF3OC6H2；A26：Ar=2-C5H4N；A27：Ar=1-C10H9；
A28：Ar=2-CH3C9H5N-8-
Fig. 1. Design of novel 2-oxoquinoline arylaminothiazole derivatives as antitumor agents.
Fig. 2. Effects of compound A7 on microtubule dynamics. Polymerization of tubulin at 37 °C in
the presence of paclitaxel (10 µM), CA-4 (10 µM), compound A7 (5 and 10 µM) was monitored
continuously by recording the absorbance at 340 nm over 60 min. The reaction was initiated by
the addition of tubulin to a final concentration of 3.0 mg/mL.

Fig. 3. The proposed molecular mode and interaction of compound A7 and 3E22-colchicine in
complex with tubulin (PDB ID: 3E22). (A) Binding modes of compound A7 in the active site of
3E22. (B) Comparison of the binding modeling of colchicines (in green), and the energetically
most favorable pose of A7 (in gray) in complex with tubulin obtained by molecular docking
simulation. The compounds and important amino acids in the binding pockets are shown in stick
model, whereas tubulin is depicted in the ribbon model.
Fig. 4. Compound A7 induced cell cycle arrest in G2/M phase on HeLa cells. Effects of compound
A7 at various concentrations on the cell cycle of HeLa cells treated for 24 h. The results are
presented as the percentage distributions for specific phases. Results are representative of at least
three independent experiments and shown as the mean ± S.D. *P < 0.05, **P < 0.01 compared with a
control group.
Fig. 5. Compound A7 induced apoptosis on HeLa cells. Apoptosis of HeLa cells exposed to
different concentrations of compound A7 for 24 h. Apoptotic cells were determined by flow
cytometry using Annexin V/PI.

Fig. 6. Compound A7 induced morphological changes related to apoptosis in HeLa cells. HeLa
cells were treated with A7 (5 and 10 μM, for 24 h), incubated with Hoechst 33342. Images were
acquired using a Nikon Te2000 deconvolution microscope (magnification 200×).
Fig. 7. Compound A7 induced apoptotic in HeLa cells were determined by AO/EB staining and
were photographed via fluorescence microscopy. HeLa cells were treated with A7 (5 and 10 μM,
for 24 h), incubated with AO/EB. Images were acquired using a Nikon Te2000 deconvolution
microscope (magnification 200×).
Fig. 8. Compound A7 decreased the mitochondrial membrane potential (MMP) of HeLa cells. (A)
The HeLa cells were treated with A7 at the tested concentrations (5 and 10 μM) for 24 h, followed

by incubation with the fluorescence probe JC-1 for 20 min. Then, the cells were visualized by
fluorescence microscope. (B) The HeLa cells were treated with A7 at 5 and 10 μM for 24 h,
followed by incubation with the fluorescence probe JC-1 for 20 min, and quantitatively detected
by flow cytometric assay.

Table 1. IC50 values of compounds A1~A28 against four selected cell lines.
IC50 (μM)a
Compd.
Ar
HeLa
NCI-H460
8.7±0.6
T24
SKOV3
7.8±0.4
>100
LO2
88.4±2.9
>100
C6H5
6.4±0.5
>100
7.2±0.3
>100
A1
A2
2-CH3C6H4
3-CH3C6H4
4-CH3C6H4
4-CH3OC6H4
2-FC6H4
>100
73.2±2.1
49.8±2.4
37.5±1.2
15.7±1.1
4.4±0.7
19.6±0.9
13.4±0.5
7.3±0.6
27.1±0.7
12.3±0.8
8.9±0.5
4.6±0.4
28.7±1.5
24.5±1.2
>100
76.8±0.8
50.2±0.3
43.2±1.5
20.1±0.5
5.8±0.5
75.4±0.2
50.9±0.7
42.7±1.3
22.1±1.2
6.2±0.3
24.5±1.1
20.1±1.2
9.5±0.9
29.2±1.8
13.3±0.6
10.2±0.7
7.1±1.1
32.5±1.5
25.2±2.1
>100
82.1±0.4
54.5±1.7
46.6±0.9
23.4±1.4
8.7±0.6
26.9±1.4
21.4±1.1
>100
A3
>100
A4
>100
A5
>100
A6
4-FC6H4
97.8±2.2
>100
A7
2-ClC6H4
21.6±1.3
18.3±0.8
8.4±0.6
A8
3-ClC6H4
>100
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
A20
A21
A22
A23
A24
A25
4-ClC6H4
9.0±0.7 93.6±3.5
2-BrC6H4
27.8±2.4
13.9±0.8
9.5±0.3
29.8±2.1
15.7±0.9
>100
>100
4-BrC6H4
3-CF3C6H4
4-CF3C6H4
2-NO2C6H4
4-NO2C6H4
2,6-diCH3C6H3
3,5-diCF3C6H3
2-F-4-NO2C6H3
2-Cl-4-NO2C6H3
3-Cl-4-FC6H3
2,3-diClC6H3
3,4-diClC6H3
3,5-diClC6H3
11.7±0.5 95.9±1.7
5.4±0.2
6.3±0.6
34.1±0.7
30.9±2.3
>100
89.2±4.7
>100
31.4±2.3
28.5±1.4
>100
>100
>100
11.4±0.4
8.6±0.4
14.6±0.5
15.9±0.3
26.0±0.9
24.5±0.6
20.7±1.2
17.8±0.4
12.9±0.9
19.1±1.5
20.0±1.1
30.2±0.8
29.4±0.7
25.7±1.3
33.2±1.8
20.7±1.2
13.8±0.7
17.5±1.3
23.2±0.5
27.1±1.4
31.7±1.0
26.7±0.9
37.7±2.7
21. 8±0.9
>100
12.5±0.5 97.1±4.4
21.1±1.9
27.3±1.0
33.7±1.6
30.5±0.3
30.1±1.2
39.9±2.9
>100
>100
>100
>100
>100
>100
2,6-diBr-4-CF3OC6H2 36.3±1.1

pyridin-2-yl
41.4±0.7
>100
44.5±2.6
>100
49.3±1.4
>100
47.6±3.2
>100
>100
>100
A26
A27
naphthalen-1-yl
2-methylquinolin-8-yl 31.0±0.5
32.2±0.6
63.2±2.1
0.4±0.1
40.5±1.6
59.3±1.5
0.2±0.1
36.2±1.3
67.4±1.6
0.5±0.2
>100
A28
-
-
56.5±3.4
0.6±0.2
>100
5-FU
CA-4
0.7±0.3
a IC50 values are presented as the mean ± SD (standard error of the mean) from five independent experiments.

Design, synthesis, and evaluation of new 2-oxoquinoline arylaminothiazole derivatives as
potential anticancer agent
Yi-Lin Fang, Zhi-Lin Wu, Meng-Wu Xiao, Li Wei, Kang-Ming Li, Yu-Ting Tang, Jiao Ye,
Jian-Nan Xiang and Ai-Xi Hu*
A7: Ar=4-FC6H4, IC50 HeLa (4.4 μM)
The compound A7 exhibited potential cytotoxic activity with the IC50 values ranged from 4.4 to 8.7 µM.
The mechanism study demonstrated that compound A7 inhibited tubulin polymerization, blocked cell
cycle arrest at G2/M phase and induced cell apoptosis of HeLa cells.

Highlights

A series of novel 2-oxoquinoline arylaminothiazole derivatives were designed and
synthesized as potential antitumor agents.




Representative compound A7 displayed potent anti-proliferative activity in vitro.
A7 could inhibit tubulin polymerization in vitro.
Molecular modeling suggested that A7 can bind to the colchicine site of tubulin.
A7 effectively induced apoptosis and blocked cell cycle arrest at G2/M phase on HeLa cells.