Design, synthesis and biological evaluation of 4-anilinoquinoline derivatives as novel potent tubulin depolymerization agents

Article abstract of DOI:10.1016/j.ejmech.2017.07.040

A series of novel 4-anilinoquinoline derivatives were synthesized and evaluated for their antiproliferative activities. Among them, 14h exhibited the most potent cytotoxic activity with IC₅₀ values ranging from 1.5 to 3.9 nM against all tested cancer cell lines, and showed promising efficacy in multidrug resistant cancer cells. Flow cytometry assay, immune-fluorescence staining, microtubule dynamics assays and competition assays with EBI identified that 14h was a novel tubulin depolymerization agent by binding to the colchicine site. Importantly, in vivo efficacy evaluation of HCT116 xenograft model, 14h showed efficient antitumor activity without significant loss in body weight. All the results indicated that 14h could be a promising candidate for the treatment of cancer.

Full text of DOI:10.1016/j.ejmech.2017.07.040

Accepted Manuscript
Design, synthesis and biological evaluation of 4-anilinoquinoline derivatives as novel
potent tubulin depolymerization agents
Yuanyuan Zhou, Wei Yan, Dong Cao, Mingfeng Shao, Dan Li, Fang Wang, Zhuang
Yang, Yong Chen, Linhong He, Taijin Wang, Mingsheng Shen, Lijuan Chen
PII:
S0223-5234(17)30562-7
10.1016/j.ejmech.2017.07.040
EJMECH 9603
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 23 March 2017
Revised Date: 19 May 2017
Accepted Date: 20 July 2017
Please cite this article as: Y. Zhou, W. Yan, D. Cao, M. Shao, D. Li, F. Wang, Z. Yang, Y. Chen, L. He,
T. Wang, M. Shen, L. Chen, Design, synthesis and biological evaluation of 4-anilinoquinoline derivatives
as novel potent tubulin depolymerization agents, European Journal of Medicinal Chemistry (2017), doi:
10.1016/j.ejmech.2017.07.040.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Graphical abstract
Design, synthesis and biological evaluation of 4-anilinoquinoline derivatives
as novel potent tubulin depolymerization agents

ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of 4-anilinoquinoline derivatives
as novel potent tubulin depolymerization agents
Yuanyuan Zhoua,1, Wei Yana,1, Dong Caob, Mingfeng Shaoa, Dan Lia, Fang Wanga,
Zhuang Yanga, Yong Chena, Linhong Hea, Taijin Wanga, Mingsheng Shena, Lijuan
Chena,c,*
a.State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center, Chengdu, 610041, China
b.Chengdu Institute of Chinese Herbal Medicine, Chengdu, 610043, China
c.School of Chemical Engineering, Sichuan University, Chengdu, China

ACCEPTED MANUSCRIPT
ABSTRACT:
A series of novel 4-anilinoquinoline derivatives were synthesized and evaluated
for their antiproliferative activities. Among them, 14h exhibited the most potent
cytotoxic activity with IC50 values ranging from 1.5 to 3.9 nM against all tested
cancer cell lines, and showed promising efficacy in multidrug resistant cancer cells.
Flow cytometry assay, immune-fluorescence staining, microtubule dynamics assays
and competition assays with EBI identified that 14h was a novel tubulin
depolymerization agent by binding to the colchicine site. Importantly, in vivo efficacy
evaluation of HCT116 xenograft model, 14h showed efficient antitumor activity
without significant loss in body weight. All the results indicated that 14h could be a
promising candidate for the treatment of cancer.
Keywords: quinoline, antiproliferative, microtubules, depolymerization, antimitotic.

ACCEPTED MANUSCRIPT
1. Introduction
Microtubules, mainly composed of α/β-tubulin heterodimers [1], are the basic
constituents of eukaryotic cell and play a crucial role in many fundamental cellular
processes such as cell division, formation, regulation of motility, cell signaling,
secretion, maintenance of cell shape, and intracellular transport [2-4]. Consequently,
the microtubule has become an attractive target for the design and development of
novel antimitotic agents for cancer therapy [5-7]. Antimitotic agents are generally
classified into two major categories: microtubule stabilizing agents (e.g., paclitaxel)
and polymerization inhibitors (e.g., colchicine) [8, 9]. Antitumor drugs targeting
microtubules can directly affect cell mitosis, and lead to cell division stagnated in
G2/M phase [6, 7].
These antimitotic agents were found targeting several different binding sites,
including the taxane site, the vinca site, the colchicine site, and so on [10-12]. In
contrast to develop the taxane or vinca alkaloid binding sites, more attention has been
drawn to the inhibitors which target to colchicine sites, due to its potential of
overcoming the ABC-transporter-mediated drug resistance [13-15]. Currently, several
colchicine site inhibitors have been reported, such as colchicine (1), combretastatin
A-4 (CA-4, 2), etc. CA-4 was first isolated from the African bush willow Combretum
caffrum, and exhibited potent antitumor activity against several cancer cells [16, 17].
CA4P (3), a water-soluble phosphate prodrug of CA-4, has been approved by both the
FDA and EMA for using in anaplastic thyroid cancer [17, 18]. Previously, a
4-anilinocoumarin derivative (4) was synthesized by our group and showed potent

ACCEPTED MANUSCRIPT
antiproliferative activity against all tested cancer cell lines. It had IC50 values ranging
from 3.3 to 23.4 nM. Compound 4 was also reported as microtubule agents by binding
to the colchicine site [19]. In the present study, a series of new quinoline derivatives
have been designed and synthesized as tubulin inhibitors, as an attempt to investigate
whether the coumarin ring could be replaced with the quinoline one.
Quinoline-based derivatives, an important class of heterocyclic compounds,
exhibit anticancer activity with different mechanisms including alkylating agents [20],
tyrosine kinase inhibitors [21, 22] and tubulin inhibitors [23, 24]. Molecular docking
analysis [25] (Figures S1 in Supporting Information) proved that it was feasible to
replace the coumarin ring of 4 with the quinoline ring, and compound 13b was
designed. According to the docking results, and it can be speculated that 13b is a
novel tubulin agent targeting colchicine sites. Based on the structural modification
and optimization of compound 13b, diverse derivatives (14a-m and 15a-e) were
synthesized and evaluated for their antiproliferative abilities. (Figure 1)

ACCEPTED MANUSCRIPT
Figure 1. Some reported tubulin depolymerization agents. 4-anilinoquinoline general
structure 13b is derived from a combination of 4-anilinocoumarin (4).
2. Results and discussion
2.1. Chemistry
4-Substituted quinoline derivatives (9a-d) were first prepared by the synthesis
method outlined in Scheme 1. 5a-c in phosphorus oxychloride refluxed for 12 h to
give 6a-c. 6a-c and 7a in isopropanol with catalytic amount of HCl (conc.) refluxed
for 2 h to give 8a-c. 6b reacted with 7b to give 8d by following the same route.
Compounds 8a-d reacted with sodium hydride and iodomethane to obtain 9a-d,
respectively.
Scheme 1. Reagents and conditions: (a) POCl3, 80 °C, 4 h. (b) HCl (conc.),
(Me)2CHOH, 80 °C, 2 h. (c) NaH, CH3I, DMF, 0 °C, 1h, rt, 1 h.
Compounds 13a-d and 14a-m were synthesized by the synthesis route shown in
Scheme 2. 6a-c and 10a with catalytic amount of HCl (conc.) refluxed in isopropanol
for 2 h to give 11a-c. 6b reacted with 10b to give 11d following the similar approach.
11a-d were treated with sodium hydride and iodomethane to give 12a-d, respectively.
Compounds 12a-d in ethanol and water were added iron powder and ammonium

ACCEPTED MANUSCRIPT
chloride to obtain 13a-d, respectively. Then we used the appropriate various acyl
chlorides to react with 13b to obtain 14a-m, respectively. As shown in Scheme 3,
compounds 15a-e were easily prepared from 14f react with different amines.
X
X
Cl
X
a
b
HN
N
NO2
N
N
NO2
H2N
NO2
N
R1
R1
R1
6a, R1=Cl
6b, R1=Me
6c, R1=H
11a, R1=Cl, X=O
11b, R1=Me, X=O
11c, R1=H, X=O
11d, R1=Me, X=S
12a, R1=Cl, X=O
12b, R1=Me, X=O
12c, R1=H, X=O
12d, R1=Me, X=S
10a, X=O
10b, X=S
X
X
R3
c
d
N
N
N
N
N
NH2
H
R1
R1
13a, R1=Cl, X=O
13b, R1=Me, X=O
13c, R1=H, X=O
13d, R1=Me, X=S
14a-m, R1=Me, X=O,
R3=Carbonyl derivatives
Scheme 2. Reagents and conditions: (a) HCl (conc.), (Me)2CHOH, 80 °C, 2 h. (b)
NaH, CH3I, DMF, 0 °C, 1 h, rt 1 h. (c) Fe powder, NH4Cl, CH3CH2OH/H2O (3:1, v/v),
80 °C. (d) RCO2Cl, Et3N, CH2Cl2, rt, 0.5 h.
Scheme 3. Reagents and conditions: (a) R’NH R, K2CO3, CH3CN, 80 °C, 2 h.
2.2. Biological evaluation
2.2.1. In vitro antiproliferative activities.

ACCEPTED MANUSCRIPT
The synthesized derivatives 9a-d and 13a-d were evaluated for antiproliferative
activities against MDA-MB-231 cells (human breast cancer cells) using MTT assay
and colchicine was included as the reference compound [26]. The results were
summarized in Table 1. Compound 9b (containing methyl in the 2-position of the
A-ring) displayed potent inhibitory activity with an IC50 value of 6.6 nM. In contrast,
both the 2-Cl analog (9a) and 2-H analog (9c) showed poor inhibitory activities (IC50 >
500 nM). The similar outcomes can be found in the comparison of 13a and 13C with
13b. It can be concluded that the methyl in the 2-position of the A-ring is necessary to
maintain the inhibitory activity of the series of compounds. It has been reported that
the in vitro phase I biotransformation of CA-4 in hepatic microsomal involves
O-demethylation [27]. The compounds of the 4-methylthio substituent group in the
B-ring were synthesized to block O-demethylation metabolic pathway. Compounds
9d and 13d (IC50, 15.5 and 9.5 nM, respectively) presented a slightly decreased
inhibition activities compared to 9b and 13b (IC50, 6.6 and 3.2 nM, respectively)
when methylthio was introduced to the para-position of benzene. It should be noted
that both 13b and 13d with amino group exhibited stronger inhibitory activities
compared to the corresponding compounds 9b and 9d. It can be concluded that the
amino was an important group to maintain the antiproliferative activity. Among the
above compounds, 13b (containing methyl in the 2-position of the A-ring, amino in
the 3-position and methoxy group in the 4-position of the B-ring) exhibited the
optimal antiproliferative activity.

ACCEPTED MANUSCRIPT
Table 1. The in vitro antiproliferative activities of 9a-d and 13a-d against
MDA-MB-231 cells
a
Compd
9a
X
O
O
O
S
R1
Cl
R2
H
IC50 , nM
>500
Me
H
H
6.6 ± 0.7
>500
9b
H
9c
Me
Cl
H
15.5 ± 0.2
43.1 ± 0.6
3.2 ± 0.8
>500
9d
O
O
O
S
NH2
NH2
NH2
NH2
13a
Me
H
13b
13c
Me
9.5 ± 0.4
38.2 ± 1.8
13d
colchicine
a 50% inhibitory concentration after drug treatment. Data are expressed as the mean ±
SD from the dose-response curves of at least three independent experiments.
Although 13b showed the best antiproliferative activity against MDA-MB-231
cells, the compound still suffered from poor water solubility. In order to improve the
solubility of 13b, we subsequently modified the 3-amino group on B-ring with
aliphatic chain to obtain diverse derivatives 14a-m and 15a-e. As expected in Table 2,
compounds containing different aliphatic chain alkanes, 14a-d, caused gradient
decrease of inhibitory activities with the increase of the length of the alkyl chain, with
IC50 values ranging from 9.1 to 82.4 nM. There was a slight decrease in inhibitory
activity compared to 13b when propylene bond was introduced to the amide to obtain

ACCEPTED MANUSCRIPT
14e, with an IC50 value of 43.4 nM. The results indicated that introduction of the
unsaturated bond decreased the antiproliferative activity. According to our previous
experience [19], chlorinated alkanes with different chain length were also introduced
to furnish derivatives 14f-i. As expected, the antiproliferative activities of these
compounds decreased with the increase of the length of the alkyl chain. Surprisingly,
14h with three carbon chlorinated alkane did not fit into the pattern. 14h showed
potent antiproliferative activity with an IC50 value of 3.9 nM against MDA-MB-231
cell. The specific mechanism is still unclear. The chlorine of 14f was replaced with
bromine to obtain compound 14j (IC50, 87.2 nM) resulting in decreased activity. The
results indicated that the chlorine product was more potent than bromine derivative.
Introduction of ester bonds to obtain 14k-m caused a great decrease of
antiproliferative activities compared to 13b. Then the chlorine of 14f was replaced
with a series of different amines to explore more effective compounds (15a-e), but the
tactics resulted in decreased activity (Table 3). The results suggested that chlorine
product was more potent than ester bonds or amines derivatives.
Table 2. The in vitro antiproliferative activities of 14a-m against MDA-MB-231 cells
a
a
Compd
14a
R3
IC50 , nM
Compd
14h
R3
IC50 , nM
9.1 ± 0.2
3.9 ± 0.1

ACCEPTED MANUSCRIPT
18.5 ± 1.5
58.4 ± 1.7
82.4 ± 6.3
43.4 ± 9.9
6.6 ± 3.9
363.3 ± 67.8
87.2 ± 14.3
>500
14b
14c
14d
14e
14f
14i
14j
14k
442.6 ± 32.8
185.5 ± 69.8
38.2 ± 1.8
14l
14m
96.6 ± 0.7
14g
colchicine
a 50% inhibitory concentration after drug treatment. Data are expressed as the mean ±
SD from the dose-response curves of at least three independent experiments.
Table 3. The in vitro antiproliferative activities of 15a-e against MDA-MB-231 cells
a
Compd
15a
R4
IC50 , nM
>500
394.0 ± 46.2
256.0 ± 7.5
>500
15b
15c
15d
83.6 ± 4.5
38.2 ± 1.8
15e
colchicine

ACCEPTED MANUSCRIPT
a 50% inhibitory concentration after drug treatment. Data are expressed as the mean ±
SD from the dose-response curves of at least three independent experiments.
Although several compounds displayed potent antitumor activity with low
nanmolar IC50 values, these compounds were also subject to the challenge of poor
water solubility. It is a major obstacle for further testing in vivo. According to the
solubility detecting results (Table S1 in Supporting Information), compound 14h
was identified for further biological activity evaluation.
The antiproliferative activity of 14h on normal human cell lines and various
human tumor cell lines was investigated, with colchicine as the positive control.
These tumor cells were derived from human colon, lung, ovarian and breast cancers
(Table 4). 14h possessed potent inhibitory activity with IC50 values ranging from 1.5
to 75.6 nM. It is worth noting that 14h is more potent than colchicine against all the
tested tumor cell lines.
Table 4. Activity of compound 14h and colchicine against various human tumor
cell lines and non-tumoral cell lines
a
IC50 , nM
tumor type
colon
cell line
14h
colchicine
HCT-8
HCT116
3.4 ± 0.2
2.7 ± 0.2
13.4 ± 0.1
13.9 ± 1.5
A549
3.2 ± 0.3
2.2 ± 0.2
32.7 ± 0.1
12.3 ± 0.6
lung
A2780S
ovarian
MCF-7
MDA-MB-231
1.5 ± 0.1
3.9 ± 0.1
13.4 ± 0.8
38.2 ± 1.8
breast
LO2
HEK-293
75.6 ± 0.9
45.3 ± 0.8
26.5 ± 0.7
10.2 ± 0.9
normal

ACCEPTED MANUSCRIPT
a 50% inhibitory concentration after drug treatment. Data are expressed as the mean ±
SD from the dose-response curves of at least three independent experiments.
2.2.2. Effect on multidrug resistant cells
Drug resistance is a major obstacle for the first-line chemotherapy [13, 28]. The
common mechanism of resistance identified in preclinical or clinical study involves
two main categories: the overexpression of a cellular membrane protein called
P-glycoprotein (P-gp) and changing in the levels of expression of different β-tubulin
isotypes (β-III gene) [29-33]. As it has been known, paclitaxel resistant HCT-8/T
(human colon cancer cell line), vinblastine resistant HCT-8/V (human colon cancer
lines), and adriamycin resistant MCF-7/ADR (human breast cancer cell line) are all
P-gp overexpressed, and paclitaxel resistant A2780/T (human ovarian cancer cell lines)
are β-tubulin III overexpressed [34-37]. Hence, the activity of 14h in these resistant
and their related sensitive cancer cells was compared, with paclitaxel, vinblastine and
adriamycin as reference compounds. As shown in Table 5, 14h exhibited potent
cytotoxic activity against A2780/T (IC50 of 3.7 nM), HCT-8/T (IC50 of 3.5 nM),
HCT-8/V (IC50 of 3.5 nM), and MCF-7/ADR (IC50 of 3.3 nM). Compared with
paclitaxel, colchicine, vinblastine, and adriamycin, 14h had much lower drug
resistance indexes (1.0 for HCT-8/T, 1.0 for HCT-8/V, 2.3 for MCF-7/ADR, 1.7 for
A2780/T) than paclitaxel (567.4 for HCT-8/T and 2042.3 for A2780/T), vinblastine
(101.8 for HCT-8/V) and adriamycin (1222.6 for MCF-7/ADR). These results

ACCEPTED MANUSCRIPT
indicated that 14h might be useful in the treatment of tumors with resistance to other
anti-tubulin drugs.
Table 5. Drug tolerances of 14h against different drug resistant cancer cells
a
IC50 , nM
compd
RRb
HCT-8
3.4 ± 0.2
17.4 ± 0.1
HCT-8/T
3.5 ± 0.3
1.0
14h
9885.0 ± 2.8
567.4
paclitaxel
IC50, nM
compd
RR
HCT-8
3.4 ± 0.2
20.2 ± 0.1
HCT-8/V
3.5 ± 0.7
1.0
14h
2059.0 ± 5.9
101.8
vinblastine
IC50, nM
compd
RR
MCF-7
1.5 ± 0.1
44.8 ± 0.1
MCF-7/ADR
3.3 ± 0.4
2.3
14h
54750.0 ± 6.6
1222.1
adriamycin
IC50, nM
compd
RR
A2780S
2.2 ± 0.2
12.3 ± 0.1
A2780/T
3.7 ± 0.5
1.7
14h
25120.0 ± 2.7
2042.3
paclitaxel
a 50% inhibitory concentration after drug treatment. Data are expressed as the mean ±
SD from the dose-response curves of at least three independent experiments.
b Resistance ratio: (IC50 of drug resistant cancer cell) / (IC50 of parental cancer cell).
2.2.3. Cell cycle effects.
To explore whether the cytotoxicity of 14h was due to the cell cycle arrest, the
effect on cell cycle progression was measured using propidium iodide (PI) staining by
flow cytometry in A2780S [38]. As depicted in Figure 2, 14h caused a remarkable

ACCEPTED MANUSCRIPT
G2/M arrest in a concentration-dependent manner. Only 15.34% of the A2780S cells
were arrested in G2/M phase in the control group after 24 h treatment, the percentage
of G2/M peak increased to 80.67% when cells were exposed to 30 nM of 14h. The
results suggest that 14h significantly induced cell cycle arrest in the G2/M phase in
A2780S cells.
Figure 2. Effects of 14h on cell cycle phase arrest in A2780S cells. Cells were treated
with 3, 10, and 30 nM of 14h for 24 h.
2.2.4. Immunofluorescence staining.
To investigate whether the antitumor activities of 14h was derived from an
interaction with tubulin, the ability of 14h to alter the microtubule network was
examined by treating with different concentrations of compound 14h for 24 h and
stained for DNA (blue) and α-tubulin (green). The confocal microscopy of A2780S
cells shown in Figure 3 exhibited a well-organized microtubule network in the
absence of drug treatment. Treatment with paclitaxel caused the microtubule fibers
near the nucleus assemble in clusters. In contrary, cellular microtubules in A2780S
cells almost completely depolymerized when exposed to colchicine at 100 nM. 14h
significantly disrupted microtubule formation and exhibited similar effects as

ACCEPTED MANUSCRIPT
colchicine. In comparison with colchicine, 14h obviously inhibited microtubules
polymerization at 5 nM; moreover 14h completely disrupted the microtubules
polymerization at a concentration of 50 nM. The results suggested that 14h is most
likely targeting tubulin and more active than colchicine as a novel polymerization
inhibitor.
Figure 3. Effects of 14h on the microtubule network of A2780S cells. A2780S cells
were untreated (control) and treated with 14h (5, 50 nM), paclitaxel (100 nM), or
colchicine (100 nM) at the appropriate concentration for 24 h. Microtubules and
unassembled tubulin are shown in green and the nuclei in blue.
2.2.5. Effects on microtubule dynamics.
It is well-known that the tubulin binding agents were divided into two types:
microtubule stabilizing agents (e.g., paclitaxel) and polymerization inhibitors (e.g.,
colchicine). 14h was employed at 0.4, 2, and 10 µM for microtubule dynamics assays

ACCEPTED MANUSCRIPT
to make clear which type of the compound 14h related to [39]. (Figure 4) Similar but
superior to colchicine, 14h can inhibit tubulin polymerization in a concentration-
dependent manner. These data indicated that the mechanism of 14h was in accordance
with polymerization inhibitors, suggesting that compound 14h might bind to the
colchicine site as a novel tubulin depolymerization agent.
Figure 4. Effect of 14h on tubulin polymerization assay. Colchicine (10 µM) was
used as a reference, while DMSO (0.1% v/v) was used as a vehicle control. Tubulin
had been preincubated for 5 min with 14h at 0.4, 2, and 10 µM at room temperature
before GTP was added to start the tubulin polymerization reactions. The reaction was
monitored at OD340 nm at 37 °C.
2.2.6. EBI competition assay.
N, N’-ethylenebis (iodoacetamide) (EBI), an alkylating agent, specifically
cross-link the Cys239 and the Cys354 residues of β-tubulin involved in the
colchicine-binding site [40]. An EBI competition assay was carried out in HepG2

ACCEPTED MANUSCRIPT
cells to confirm whether compound 14h directly binds to the colchicine binding site.
EBI could form a β-tubulin adduct which could be easily detected by Western blot
analysis. Antimitotic drugs occupied colchicine-binding site could prevent the
formation of the EBI: β-tubulin adduct [40]. Similar to colchicine, compound 14h (1,
5, and 25 µM) dose-dependently inhibited the formation of the β-tubulin adduct band,
resulting in the disappearance of the adduct band. Vincristine has a distinct binding
site from colchicine, was used as a negative control (Figure 5).
Figure 5. EBI assay: competition assay of 14h (1, 5, 25 µM), vincristine (5, 25 µM),
and colchicine (5 µM) with EBI (100 µM) on HepG2 cells.
2.3. Molecular modeling.
To rationalize our experimental findings, molecular docking simulations of
compound 14h on tubulin were performed using a procedure reported previously
[41-45]. The overview of the binding site of 14h is shown in Figure 6A. This binding
pocket is located at the interface between the α- and β-subunits of the tubulin dimer;
14h can enter the pocket like colchicine. The quinoline ring of 14h forms
hydrophobic interactions with the residues of Leu248β, Ala250β, Leu250β, and
Ala354β. The tail alkyl chain slightly extends into the α-subunit. (Figure 6B) The

ACCEPTED MANUSCRIPT
right length of the alkyl chain is very important, which may lead to the formation of
halogen bond, hydrophobic interactions or other interactions between the chlorine
atom of the alkyl chain and the amino acid residues of the α-subunit. This may explain
the fact that 14h is more potent compared to the other compounds.
Figure 6. The structures are docked into the colchicine-binding site (PDB code 5eyp)
(A) Superimposed molecular models of 14h (purple) and colchicine (green). (B)
Hydrophobic interactions between 14h and tubulin.
2.4. Animal tumor models and treatment.
HCT116 xenograft model was established in nude mice to determine whether the
antitumor ability of 14h could also be reflected in vivo. HCT116 cells (5×106) were
inoculated in each flank of null mice. Once the tumor volume reached a size of around
100 mm3, 14h was injected at doses of 2.5 mg/kg once every other day. 5-Fluorouracil
(5-Fu), as a positive control drug, was administrated every seven days with 100 mg/kg
iv for comparison [44]. As shown in Figure 7, 14h caused a significant suppression of
tumor growth and the average inhibitory rate reached 54.30% at day 18, while 5-Fu at
a dosage of 100 mg/kg caused 60.76% tumor reduction. Although 5-Fu at 100mg/kg

ACCEPTED MANUSCRIPT
showed better antitumor activity than 14h, the dose is much higher than 14h. The
results demonstrated that 14h could be used as a potential candidate for cancer
treatment. During the therapy, only slight weight loss (<10%) was observed in both of
the two drug treatment groups. Serious side-effects, such as feeding and abnormal
behavior, were not observed in 14h-treatment group.
Figure 7. Antitumor effect of 14h and 5-Fu on the HCT116 xenograft models
3. Conclusion
In our research, 26 novel 4-anilinoquinoline derivatives were synthesized and
evaluated for their antiproliferative activities. Among them, compound 14h exhibited
potent antitumor abilities in several human tumor cell lines including multidrug
resistant human tumor cell lines, and significantly induced cell cycle arrest in the
G2/M phase in A2780S cells. The immunofluorescence assays, microtubule dynamics
experiment, EBI assay and molecular modeling studies identified that compound 14h
is a novel depolymerization agent binding to the colchicine site. Then in vivo activity
was evaluated on HCT116 xenografts models where compound 14h showed a
favorable effect on inhibiting the tumor growth without significant body weight loss
or behavior disorders. Therefore, 14h as a novel class of tubulin depolymerization
agent may be a potent anticancer agent.

ACCEPTED MANUSCRIPT
4. Experimental
4.1. Chemistry.
All the chemical solvents and reagents used in this study were analytically pure
without further purification and commercially available. TLC was performed on 0.20
mm silica gel 60 F254 plates (Qingdao Ocean Chemical Factory, Shandong, China).
Visualization of spots on TLC plates was done by UV light and I2. NMR data were
1
measured for H at 400 MHz and for 13C at 101 MHz on a Bruker Avance 400
spectrometer (Bruker Company, Germany) using TMS as an internal standard. Mass
spectra (MS) were obtained by Q-TOF Priemier mass spectrometer (Micromass,
Manchester, UK).
4.1.1. General preparation of 9a-d (method A).
To a solution of 8a-d (1.0 equiv) in DMF cooled at 0°C was added sodium
hydride (60% oil suspension, 3.0 equiv), followed by methyl iodide (3.0 equiv). The
mixture was stirred at 0 °C for 1 h, then allowed to warm to room temperature and
stirred for 1 h. The reaction mixture was diluted with ethyl acetate, washed with
saturated NaHCO3 aq, brine, dried over Na2SO4, filtered and concentrated under
vacuum. The residue was purified by flash chromatography using ethyl acetate and
petroleum ether (v/v, 1:3) as eluent to give the title compounds 9a-d.
4.1.1.1. 2-chloro-N-(4-methoxyphenyl)-N-methylquinolin-4-amine (9a).
Compound 9a was prepared from 8a (0.33 g, 1.2 mmol), NaH (0.14 g, 3.5 mmol)
and CH3I (0.22 mL, 3.5 mmol) as described for method A as a white solid, yield 57%.

ACCEPTED MANUSCRIPT
1H NMR (400 MHz, CDCl3) δ 7.96 (dd, J = 8.4, 1.2 Hz, 1H), 7.81-7.74 (m, 1H),
7.62-7.56 (m, 1H), 7.29 (t, J = 7.6 Hz, 1H), 7.19 (d, J = 8.8 Hz, 2H), 6.93 (d, J = 9.2
Hz, 2H), 6.71 (s, 1H), 3.86 (s, 3H), 3.56 (s, 3H). 13C NMR (101 MHz, DMSO) δ
158.36, 156.90, 148.63, 141.43, 138.26, 131.33, 128.81, 127.10, 123.94, 123.63,
120.79, 115.86, 111.00, 55.81, 39.08. MS (ESI, m/z): 299.13 [M + H]+.
4.1.1.2. 2-methyl-N-(4-methoxyphenyl)-N-methylquinolin-4-amine (9b).
Compound 9b was prepared from 8b (0.13 g, 0.5 mmol), NaH (0.60 g, 1.5 mmol)
and CH3I (0.09 mL, 1.5 mmol) as described for method A as a white solid, yield 52%.
1H NMR (400 MHz, DMSO) δ 7.83 (d, J = 8.4 Hz, 1H), 7.56-7.46 (m, 2H), 7.21-7.14
(m, 1H), 7.06 (s, 1H), 6.90 (d, J = 9.0 Hz, 2H), 6.83 (d, J = 9.0 Hz, 2H), 3.70 (s, 3H),
13
3.36 (s, 3H), 2.62 (s, 3H). C NMR (101 MHz, DMSO) δ 159.62, 155.61, 153.43,
149.57, 144.34, 129.30, 128.96, 125.09, 124.41, 123.74, 121.71, 115.16, 111.95,
55.64, 43.48, 25.55. MS (ESI, m/z): 279.14 [M + H]+.
4.1.1.3. N-(4-methoxyphenyl)-N-methylquinolin-4-amine (9c).
Compound 9c was prepared from 8c (0.25 g, 1.0 mmol), NaH (0.12 g, 3.0 mmol)
and CH3I (0.187 mL, 3.0 mmol) as described for method A as a light yellow solid,
yield 56%. 1H NMR (400 MHz, CDCl3) δ 8.74 (d, J = 5.2 Hz, 1H), 8.02 (d, J = 8.4 Hz,
1H), 7.61 (d, J = 8.8 Hz, 1H), 7.57-7.51 (m, 1H), 7.24-7.16 (m, 1H), 6.98 (d, J = 5.1
Hz, 1H), 6.91 (d, J = 9.0 Hz, 2H), 6.79 (d, J = 9.0 Hz, 2H), 3.76 (s, 3H), 3.41 (s, 3H).
13C NMR (101 MHz, DMSO) δ 158.09, 150.86, 148.82, 144.01, 143.57, 140.37,
133.25, 131.27, 129.84, 128.66, 124.12, 122.10, 119.23, 116.58, 107.54, 57.57, 45.24.
MS (ESI, m/z): 265.13 [M + H]+.

ACCEPTED MANUSCRIPT
4.1.1.4. 2-methyl-N-(4-(methylthio)phenyl) -N-methyl quinolin-4-amine (9d).
Compound 9d was prepared from 8d (0.14 g, 0.5 mmol), NaH (0.60 g, 1.5 mmol)
and CH3I (0.09 mL, 1.5 mmol) as described for method A as a light yellow solid, yield
49%. 1H NMR (400 MHz, CDCl3) δ 8.00 (d, J = 8.4 Hz, 1H), 7.66 (d, J = 8.0 Hz, 1H),
7.60 (t, J = 7.6 Hz, 1H), 7.32-7.27 (m, 1H), 7.18 (d, J = 8.8 Hz, 2H), 7.03 (s, 1H),
6.79 (d, J = 8.4 Hz, 2H), 3.43 (s, 3H), 2.71 (s, 3H), 2.44 (s, 3H). 13C NMR (101 MHz,
DMSO) δ 160.08, 152.99, 149.61, 147.96, 129.77, 129.45, 128.69, 125.22, 124.57,
122.58, 120.25, 115.34, 42.17, 25.47, 16.44. MS (ESI, m/z): 295.17 [M + H]+.
4.1.2. General preparation of 13a-d (method B).
To compound 13a-d (1.0 equiv) in a solution of ethanol and water (v/v 3:1) was
added iron powder (5.0 equiv) and ammonium chloride (5.0 equiv). The reaction was
stirred at 80 °C for 2 h, then cooled to room temperature, and filtered through Celite.
The filter cake was washed with dichloromethane, and the filtrate was concentrated
under pressure. The residue was dissolved in dichloromethane, washed with water,
dried over Na2SO4, filtered, and concentrated. The residue was purified by flash
chromatography using ethyl acetate and petroleum ether (v/v, 1:1) as eluent to give
the title compounds 13a-d.
4.1.2.1. N-(2-chloroquinolin-4-yl)-4-methoxy-N-methylbenzene-1, 3-diamine (13a)
Compound 13a was prepared from 12a (0.40 g, 1.2 mmol), Fe (0.32 g, 6.0 mmol)
and NH4Cl (0.33 g, 6.0 mmol) as described for method B as a white solid, yield 70%.
1H NMR (400 MHz, DMSO) δ 7.90 (d, J = 8.0 Hz, 1H), 7.73-7.59 (m, 2H), 7.36-7.31

ACCEPTED MANUSCRIPT
(m, 1H), 6.89 (d, J = 8.4 Hz, 1H), 6.70 (s, 1H), 6.59 (s, 1H), 6.50 (d, J = 8.4 Hz, 1H),
4.98 (s, 2H), 3.82 (s, 3H), 3.43 (s, 3H). 13C NMR (101 MHz, DMSO) δ 157.01,
148.69, 145.66, 141.15, 139.77, 138.64, 131.23, 127.02, 123.91, 123.42, 120.68,
114.44, 112.21, 111.89, 111.29, 55.92, 38.97. MS (ESI, m/z): 314.10 [M + H]+.
4.1.2.2. 4-methoxy-N-methyl-N-(2-methylquinolin-4-yl)benzene-1,3-diamine (13b)
Compound 13b was prepared from 12b (6.0 g, 18.5 mmol), Fe (5.2 g, 92.5 mmol)
and NH4Cl (5.0 g, 92.5 mmol) as described for method B as a light yellow solid, yield
78%. 1H NMR (400 MHz, CDCl3) δ 8.48 (d, J = 8.0 Hz, 1H), 7.58 (t, J = 7.6 Hz, 1H),
7.39 (d, J = 8.6 Hz, 1H), 7.14 (t, J = 7.6 Hz, 1H), 6.72 (d, J = 8.4 Hz, 1H), 6.67 (s,
1H), 6.55 (d, J = 2.0 Hz, 1H), 6.41 (dd, J = 8.4, 2.1 Hz, 1H), 4.04 (s, 2H), 3.87 (s, 3H),
13
3.53 (s, 3H), 2.89 (s, 3H). C NMR (101 MHz, DMSO) δ 155.35, 144.62, 143.40,
139.44, 132.00, 130.77, 129.14, 126.05, 124.88, 111.68, 109.66, 108.69, 55.95, 44.71,
22.94, 19.13, 14.02. MS (ESI, m/z): 294.15 [M + H]+.
4.1.2.3. 4-methoxy-N-methyl-N-(quinolin-4-yl)benzene-1,3-diamine (13c)
Compound 13c was prepared from 12c (0.50 g, 1.6 mmol), Fe (0.45 g, 8.0 mmol)
and NH4Cl (0.43 g, 8.0 mmol) as described for method B as a yellow solid, yield 73%.
1H NMR (400 MHz, DMSO) δ 8.91 (d, J = 8.4 Hz, 1H), 8.56 (d, J = 7.6 Hz, 1H), 8.12
(s, 2H), 7.85-7.81 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.75-6.65 (m, 2H), 6.58 (d, J =
8.0 Hz, 1H), 5.11 (s, 2H), 4.15 (s, 3H), 3.83 (s, 3H). 13C NMR (101 MHz, DMSO) δ
155.52, 147.85, 146.02, 139.59, 139.23, 134.65, 130.32, 127.29, 125.11, 118.96,
118.19, 113.16, 111.50, 110.91, 100.11, 56.04, 42.56. MS (ESI, m/z): 280.40 [M +
H]+.

ACCEPTED MANUSCRIPT
4.1.2.4. 4-methylthio-N-methyl-N-(2-methylquinolin-4-yl)benzene-1,3-diamine (13d)
Compound 13d was prepared from 12d (0.40 g, 1.2 mmol), Fe (0.33 g, 6.0 mmol)
and NH4Cl (0.32 g, 6.0 mmol) as described for method B as a light yellow solid, yield
77%. 1H NMR (400 MHz, CDCl3) δ 8.57 (d, J = 8.4 Hz, 1H), 7.68-7.57 (m, 1H), 7.41
(d, J = 8.8 Hz, 1H), 7.33 (d, J = 8.4 Hz, 1H), 7.22-7.15 (m, 1H), 6.76 (s, 1H), 6.57 (d,
J = 2.3 Hz, 1H), 6.43 (dd, J = 8.4, 2.4 Hz, 1H), 4.59 (s, 2H), 3.59 (s, 3H), 2.94 (s, 3H),
2.38 (s, 3H). 13C NMR (101 MHz, DMSO) δ 156.89, 155.23, 149.46, 148.25, 140.40,
133.00, 132.64, 126.47, 125.95, 121.22, 118.74, 118.01, 113.18, 109.98, 108.12,
44.81, 20.62, 16.78. MS (ESI, m/z): 310.20 [M + H]+.
4.1.3. General preparation of 14a-m (method C).
To a solution of 13b (0.9 g, 0.34 mmol，1.0 equiv) in dichloromethane (9.0 mL)
was added RCO2Cl (0.41 mmol，1.2 equiv) and triethylamine (0.095 mL, 0.68 mmol,
2.0 equiv), and the mixture stirred at room temperature for 30 min. When completed,
the solvent was removed under vacuum and the residue was purified by flash
chromatography using dichloromethane and methanol (v/v, 9:1) as eluent to give
compounds 14a-m.
4.1.3.1.
N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)acetamide
(14a)
Compound 14a was prepared from 13b and acetyl chloride as described for
1
method C as a light yellow solid, yield 41%. H NMR (400 MHz, CDCl3) δ 8.42 (s,
1H), 8.18 (d, J = 8.0 Hz, 1H), 7.83 (s, 1H), 7.60-7.48 (m, 2H), 7.19-7.11 (m, 1H), 6.83

ACCEPTED MANUSCRIPT
(s, 1H), 6.69 (d, J = 8.8 Hz, 1H), 6.46 (dd, J = 8.4, 2.4 Hz, 1H), 3.86 (s, 3H), 3.49 (s,
3H), 2.79 (s, 3H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO) δ 169.07, 158.67,
154.13, 147.72, 146.32, 143.40, 129.71, 128.82, 127.69, 125.30, 124.73, 121.13,
118.55, 116.82, 112.21, 111.09, 56.36, 43.91, 24.59, 24.37. MS (ESI, m/z): 336.20 [M
+ H]+.
4.1.3.2. N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)propionamide
(14b)
Compound 14b was prepared from 13b and propionyl chloride as described for
1
method C as a light yellow solid, yield 48%. H NMR (400 MHz, CDCl3) δ 8.44 (s,
1H), 8.07 (d, J = 8.4 Hz, 1H), 7.82 (s, 1H), 7.60-7.50 (m, 2H), 7.19-7.13 (m, 1H),
6.87 (s, 1H), 6.66 (d, J = 8.8 Hz, 1H), 6.45-6.37 (m, 1H), 3.86 (s, 3H), 3.46 (s, 3H),
2.75 (s, 3H), 2.45 (q, J =7.6 Hz, 2H), 1.28-1.24 (m, 3H). 13C NMR (101 MHz, DMSO)
δ 172.69, 158.75, 154.11, 146.23, 143.50, 129.67, 128.85, 125.29, 124.72, 121.19,
118.47, 116.68, 112.20, 111.14, 56.39, 43.94, 29.65, 24.69, 9.99. MS (ESI, m/z):
350.17 [M + H]+.
4.1.3.3.
N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)butyramide
(14c)
Compound 14c was prepared from 13b and butyryl chloride as described for
method C as a light yellow solid, yield 43%. 1H NMR (400 MHz, CDCl3) δ 8.41 (s,
1H), 7.95 (d, J = 8.0 Hz, 1H), 7.82 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 7.6
Hz, 1H), 7.16 (t, J = 7.6 Hz, 1H), 6.91 (s, 1H), 6.62 (d, J = 8.8 Hz, 1H), 6.35 (d, J =
7.2 Hz, 1H), 3.80 (s, 3H), 3.42 (s, 3H), 2.69 (s, 3H), 2.37 (t, J = 7.2 Hz, 2H),

ACCEPTED MANUSCRIPT
13
1.85-1.66 (m, 2H), 1.01 (t, J = 7.6 Hz, 3H). C NMR (101 MHz, DMSO) δ 171.81,
159.56, 153.45, 149.48, 145.83, 144.01, 129.24, 129.00, 128.72, 125.03, 124.48,
121.84, 117.93, 116.35, 112.08, 56.35, 43.56, 38.44, 25.52, 18.96, 14.07. MS (ESI,
m/z): 364.21 [M + H]+.
4.1.3.4. N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)pentanamide
(14d)
Compound 14d was prepared from 13b and pentanoyl chloride as described for
method C as a light yellow solid, yield 46%. 1H NMR (400 MHz, CDCl3) δ 8.65 (d, J
= 8.4 Hz, 1H), 8.54 (s,1 H), 7.90 (s, 1H), 7.62 (t, J = 7.6 Hz, 1H), 7.32 (d, J = 8.4 Hz,
1H), 7.16 (t, J = 7.6 Hz, 1H), 6.82 (d, J = 8.4 Hz, 1H), 6.75-6.62 (m, 2H), 3.96 (s, 3H),
3.62 (s, 3H), 2.98 (s, 3H), 2.44 (t, J = 7.6 Hz, 2H), 1.76-1.66 (m, 2H), 1.47-1.36 (m,
2H), 0.95 (t, J = 7.2 Hz, 3H). 13C NMR (101 MHz, DMSO) δ 172.34, 157.22, 154.60,
148.62, 140.78, 139.69, 132.80, 129.41, 126.54, 125.84, 121.33, 120.53, 118.77,
117.97, 112.61, 106.78, 56.55, 45.67, 36.17, 27.60, 22.24, 20.19, 14.20. MS (ESI,
m/z): 378.25 [M + H]+.
4.1.3.5.
N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)acrylamide
(14e)
Compound 14e was prepared from 13b and acryloyl chloride as described for
method C as a light yellow solid, yield 46%. 1H NMR (400 MHz, DMSO) δ 9.35 (s,
1H), 7.87 (s, 1H), 7.82 (d, J = 8.8 Hz, 1H), 7.55-7.46 (m, 2H), 7.22-7.16 (m, 1H),
7.08 (s, 1H), 6.95 (d, J = 8.8 Hz, 1H), 6.73-6.60 (m, 2H), 6.15 (dd, J = 16.8, 2.0 Hz,
1H), 5.72-5.62 (m, 1H), 3.80 (s, 3H), 3.36 (s, 3H), 2.62 (s, 3H). 13C NMR (101 MHz,

ACCEPTED MANUSCRIPT
DMSO) δ 164.04, 157.23, 154.72, 148.86, 140.87, 139.79, 132.78, 132.17, 129.03,
127.69, 126.53, 125.88, 121.86, 120.62, 119.13, 118.07, 112.76, 106.97, 56.62, 20.23.
MS (ESI, m/z): 348.05 [M + H]+.
4.1.3.6.
2-chloro-N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)acetamide
(14f)
Compound 14f was prepared from 13b and 2-chloroacetyl chloride as described
for method C as a light yellow solid, yield 47%. 1H NMR (400 MHz, DMSO) δ 9.48
(s, 1H), 7.88-7.74 (m, 2H), 7.59-7.47 (m, 2H), 7.20 (t, J = 7.2 Hz, 1H), 7.09 (s, 1H),
6.97 (t, J = 8.4 Hz, 1H), 6.74-6.62 (m, 1H), 4.34 (s, 2H), 3.81 (s, 3H), 3.36 (s, 3H),
2.63 (s, 3H). 13C NMR (101 MHz, DMSO) δ 165.28, 158.94, 153.93, 148.12, 146.15,
143.64, 129.62, 127.99, 125.16, 124.80, 121.35, 118.99, 116.17, 112.46, 111.67, 56.53,
43.79, 24.81. MS (ESI, m/z): 370.20 [M + H]+.
4.1.3.7.
3-chloro-N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)propanamide
(14g)
Compound 14g was prepared from 13b and 3-chloropropanoyl chloride as
described for method C as a light yellow solid, yield 45%. 1H NMR (400 MHz, CDCl3)
δ 8.39 (s, 1H), 8.03 (d, J = 8.0 Hz, 1H), 7.92 (s, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.54 (t,
J = 7.6 Hz, 1H), 7.18 (t, J = 7.6 Hz, 1H), 6.89 (s, 1H), 6.67 (d, J = 8.8 Hz, 1H), 6.42
(d, J = 8.8 Hz, 1H), 3.89 (t, J = 6.4 Hz, 2H), 3.85 (s, 3H), 3.44 (s, 3H), 2.87 (t, J = 6.4
Hz, 2H), 2.73 (s, 3H). 13C NMR (101 MHz, DMSO) δ 164.02, 157.00, 155.09, 148.64,

ACCEPTED MANUSCRIPT
141.09, 132.57, 132.18, 128.99, 127.68, 126.45, 125.80, 121.59, 118.90, 118.34,
112.72, 107.35, 56.61, 45.45, 20.70. MS (ESI, m/z): 384.18 [M + H]+.
4.1.3.8.
4-chloro-N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)butanamide
(14h)
Compound 14h was prepared from 13b and 4-chlorobutanoyl chloride as
described for method C as a light yellow solid, yield 49%. 1H NMR (400 MHz, CDCl3)
δ 8.35 (d, J = 2.4 Hz, 1H), 7.96 (d, J = 8.4 Hz, 1H), 7.84 (s, 1H), 7.62 (dd, J = 8.4, 0.8
Hz, 1H), 7.55-7.50 (m, 1H), 7.20-7.15 (m, 1H), 6.92 (s, 1H), 6.65 (d, J = 8.8 Hz, 1H),
6.38 (dd, J = 8.8, 2.4 Hz, 1H), 3.83 (s, 3H), 3.67 (t, J = 6.4 Hz, 2H), 3.43 (s, 3H), 2.70
13
(s, 3H), 2.61 (t, J = 6.8 Hz, 2H), 2.26-2.15 (m, 2H). C NMR (101 MHz, CDCl3) δ
169.92, 159.15, 154.13, 148.99, 144.01, 143.70, 129.02, 128.47, 124.97, 124.35,
121.93, 116.90, 113.69, 112.14, 110.42, 55.97, 44.43, 42.96, 34.38, 27.85, 25.26. MS
(ESI, m/z): 398.34 [M + H]+.
4.1.3.9.
5-chloro-N-(2-methoxy-5-(methyl(2-methylquinolin-4-yl)amino)phenyl)pentanamide
(14i)
Compound 14i was prepared from 13b and 5-chloropentanoyl chloride as
1
described for method C as a light yellow solid, yield 47%. H NMR (400 MHz,
DMSO) δ 9.05 (s, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.19 (t, J =
7.6 Hz, 1H), 7.06 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.62 (dd, J = 8.6, 2.0 Hz, 1H),
3.80 (s, 3H), 3.63 (t, J = 9.0 Hz, 2H), 3.35 (s, 3H), 2.62 (s, 3H), 2.38 (t, J = 7.2 Hz,