Discovery of novel quinazolines as potential anti-tubulin agents occupying three zones of colchicine domain

Article abstract of DOI:10.1016/j.bioorg.2018.10.027

A series of novel quinazolines as tubulin inhibitors occupying three zones of colchicine domain have been designed and synthesized inspired by the recently disclosed crystal structure of verubulin analogue 6 with tubulin. Among the newly synthesized compounds, 19c showed noteworthy potency against K562, HepG2, KB, HCT-8 and MDB-MB-231 cancer cells. In vitro microtubule polymerization assays identified 19c as a potent tubulin assembly inhibitor, the binding mode of which with tubulin was confirmed by molecular modeling studies to occupy three zones of tubulin domain. Furthermore, 19c disrupted the intracellular microtubule network, caused G2/M phase arrest, induced cell apoptosis and depolarized mitochondria of K562 cells. 19c also reduced the cell migration and disrupted the capillary-like tube formation of human umbilical vein endothelial cells (HUVECs). Importantly, 19c significantly and dose dependently inhibited tumor growth in H22 liver cancer xenograft mouse model. All these results suggested that 19c deserves further research as a novel and potential anti-tubulin agent for the treatment of cancers.

Full text of DOI:10.1016/j.bioorg.2018.10.027

Accepted Manuscript
Discovery of Novel Quinazolines as Potential Anti-tubulin Agents Occupying
Three Zones of Colchicine domain
Wenlong Li, Ying Yin, Wen Shuai, Feijie Xu, Hong Yao, Jie Liu, Keguang
Cheng, Jinyi Xu, Zheying Zhu, Shengtao Xu
PII:
S0045-2068(18)30618-7
DOI:
https://doi.org/10.1016/j.bioorg.2018.10.027
YBIOO 2561
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
23 June 2018
10 October 2018
15 October 2018
Please cite this article as: W. Li, Y. Yin, W. Shuai, F. Xu, H. Yao, J. Liu, K. Cheng, J. Xu, Z. Zhu, S. Xu, Discovery
of Novel Quinazolines as Potential Anti-tubulin Agents Occupying Three Zones of Colchicine domain, Bioorganic
Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.2018.10.027
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Discovery of Novel Quinazolines as Potential Anti-tubulin
Agents Occupying Three Zones of Colchicine domain
a
a
a
a
a
b, c
Wenlong Li , Ying Yin , Wen Shuai , Feijie Xu , Hong Yao , Jie Liu , Keguang
c
a, *
d, *
a, *
Cheng , Jinyi Xu , Zheying Zhu , Shengtao Xu
aState Key Laboratory of Natural Medicines and Department of Medicinal Chemistry, China
Pharmaceutical University, 24 Tong Jia Xiang, Nanjing 210009, P. R. China
bDepartment of Organic Chemistry, China Pharmaceutical University, 24 Tong Jia Xiang, Nanjing
2
10009, P. R. China
cState Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, and
School of Chemistry and Pharmacy, Guangxi Normal University, Guilin 541004, China
dDivision of Molecular Therapeutics & Formulation, School of Pharmacy, The University of
Nottingham, University Park Campus, Nottingham NG7 2RD, UK
*Corresponding authors: jinyixu@china.com (J. Xu); Zheying.Zhu@nottingham.ac.uk (Z. Zhu);
cpuxst@163.com (S. Xu).
Abstract
A series of novel quinazolines as tubulin inhibitors occupying three zones of
colchicine domain have been designed and synthesized inspired by the recently
disclosed crystal structure of verubulin analogue 6 with tubulin. Among the newly
synthesized compounds, 19c showed noteworthy potency against K562, HepG2, KB,
HCT-8 and MDB-MB-231 cancer cells. In vitro microtubule polymerization assays
identified 19c as a potent tubulin assembly inhibitor, the binding mode of which with
tubulin was confirmed by molecular modelling studies to occupy three zones of tubulin
domain. Furthermore, 19c disrupted the intracellular microtubule network, caused
G2/M phase arrest, induced cell apoptosis and depolarized mitochondria of K562 cells.
1
9c also reduced the cell migration and disrupted the capillary-like tube formation of
human umbilical vein endothelial cells (HUVECs). Importantly, 19c significantly and
dose dependently inhibited tumor growth in H22 liver cancer xenograft mouse model.
All these results suggested that 19c deserves further research as a novel and potential
anti-tubulin agent for the treatment of cancers.
Key words: microtubule; tubulin inhibitor; colchicine domain; anti-vascular; anti-

tumor
1
. Introduction
Microtubules are ubiquitous cellular polymers that play diverse roles within the cell,
such as maintenance of cell structure, protein trafficking, chromosomal segregation,
and mitosis [1]. Microtubules are long and hollow structures with 5 nm walls
surrounding a cavity, and are made up of two globular protein subunits, α- and β-tubulin
[2-4].
Microtubule-targeting agents (MTAs) that stabilize or destabilize microtubule can
interfere with microtubule dynamics, which leads to mitotic block and cell apoptosis.
MTAs are divided into two classes including microtubule destabilizing agents and
[5]
microtubule stabilizing agents . At least three binding sites in tubulin including taxane,
vinca alkaloid and colchicine binding sites, have been identified, which can be
interfered by MTAs. The compounds targeting taxane binding site stabilize microtubule,
while those targeting vinca alkaloid and colchicine binding sites destabilize
microtubules [6]. Inhibitors that target colchicine binding site are characterized with
many advantages compared with those binding another two sites, such as simpler
structures, improved aqueous solubility, reduced toxicity and multidrug resistance
(MDR) effects. Notably, the colchicine-site binders also exhibit antiangiogenic and
vascular disrupting activities which are not found in other site binders [7]. Extensive
efforts have been made to develop colchicine binding site inhibitors (CBSIs) either
from natural products or synthetic origins, and several candidates are under clinical
investigations as potential novel anti-cancer agents [8, 9]
.
Colchicine-binding domain is a deep pocket located at the α-β interface of tubulin
heterodimers, which comprises a main site (colchicine binding site) and an additional
neighboring pocket [10]. The colchicine domain can be divided into three zones
including zones 1, 2 and 3 [11, 12]. Classical CBSIs are featured with an “aromatic ring -
bridge - aromatic ring” structure character exemplified by colchicine (1) and
combretastatin A-4 (2, CA-4) (Figure 1), and they mostly have globular or butterfly-
like shape forming a specific spatial conformation in which ring A and ring B occupy
zone 2 and zone 1 of the colchicine domain, respectively. CBSIs that simultaneously
occupy three zones in the cavity have been rarely reported.
ABT-751 (3), also named E7010, was a potent anti-proliferative agent firstly found
in 1992 [13] and was subsequently identified to target the colchicine site of tubulin [14]
.
In 2009, the crystal structure of ABT-751 with tubulin was obtained and it showed that
ABT-751 was buried more deeply than colchicine and occupied three zones of
colchicine domain at the same time. The 4-methoylphenyl of ABT-751 is
accommodated into zone 1 with pyridine ring in zone 2, and the phenol extends into the
deeper site of β-tubulin, namely zone 3, forming hydrogen bond with Tyrβ202 residue
[10].
Another reported compound that occupied all three zones of colchicine domain was

4
whose m-ethoxyaniline group was extended into zone 3 of colchicine domain and
formed hydrogen bond with Tyrβ202 [15]. Several residues in zone 2 and zone 3 differ
among the β tubulin isotypes, which may provide opportunities to improve the
selectivity of CBSIs to the isotype α β III tubulin isoform that is commonly
overexpressed in cancer cells [16,17]. Thus, the above-mentioned CBSIs with unique
binding poses may be conferred with specific targeting to tubulin isotypes, thereby
lowering general toxicity.
Verubulin (5) was a potent tubulin polymerization inhibitor targeting the colchicine
binding site, and demonstrated low nanomolar potency against diverse cancer cell lines
[18, 19].
Verubulin had entered into clinical trials for patients with advanced cancer [20]
,
but had been discontinued due to cardiovascular toxicity revealed in phase I and phase
II studies [21]. The binding mode of verubulin with tubulin was yet unknown until the
crystal structure of tubulin in complex with verubulin analogue 6 was determined
recently [22]. Surprisingly, the binding pose of 6 is flipped over 180° compared to the
published molecular modeling [23, 24]. The rings A and B of compound 6 occupied zone
2
, and the rings C and D extended into zone 1 (Figure 2). The N atom at the 1-position
of ring B interacted with the residue Cys241 via a water-mediated hydrogen bond. The
chlorine atom at 2-position of ring B pointed to the deep pocket of β-tubulin, namely
zone 3, reminding us that the position 2 of quinazoline may tolerate derivatization to
extend into zone 3, thus the novel CBSIs occupy all the three zones of colchicine
domain could be deigned. Inspired by the binding mode of ABT-751 with tubulin,
various substituted phenyl moieties that might form hydrogen bond with Tyrβ202
residue were introduced to the C2-position of quinazoline skeleton of verubulin (Figure
2
). Herein, we wish to report the synthesis and biological studies of these novel
quinazoline derivatives as tubulin inhibitors occupying three zones of colchicine
domain.
N
O
NHCOCH3
O
O
O
S
A
A
HO
NH HN
O
O
B
B
O
O
O
OH
O
O
OCH3
Colchicine (1)
Combretastatin A-4 (2, CA-4)
OCH3
ABT-751 (3)
OCH3
O
N
N
N
N
N
O
H N
2
N
N
N
O
H
O
N
N
Cl
4
Verubulin (5)
6

Figure 1. Representative tubulin inhibitors targeting colchicine binding site. Pink represents
moieties occupying zone 1, red represents moieties occupying zone 2 and blue represents moieties
extending into zone 3.
Zone 1
Zone 1
Zone 1
O
OCH3
OCH3
O
S
OCH3
C
N
D
NH
N
4
3
N
Zone 2
N
2
Zone 2
N
NH
OH
Zone 2
A
N
B
N
1
N
X
Cl
R
Zone 3
Zone 3
Zone 3
ABT-751 (3)
Quinazolines
6
Figure 2. The rational design of quinazolines as tubulin inhibitors occupying three zones of
colchicine domain.
2
Results and discussion
2
.1 Chemistry
Key intermediate 4-oxo-3,4-dihydroquinazoline-2-carboxylic acid ethyl ester (9)
was synthesized via a two-step acylation/cyclization sequence using anthranilamide (7)
as the starting material according to the literature report [25] (Scheme 1). Following
chlorination of 9 using POCl afforded 10 which underwent a nucleophilic addition
3
with N-methyl-4-methoxyaniline to give intermediate 11. Quinazoline-2-carboxylic
acid (12) was obtained by esterolysis of 11, following condensation reactions with
various anilines produced compounds 13a-c, and further deprotection of 13b gave
compound 13d.
Scheme 2 outlines the synthesis route of compounds 19a-t. The key intermediate 2,4-
dichloroquinazoline (17) was synthesized according to the literature report [26]. The
starting material 2-amino-1-methyl benzoate (14) was coupled with KCNO in acetic
acid at ambient temperature. Cyclization of methyl 2-ureidobenzoate (15) was
processed in KOH methanol solution (pH = 10) at reflux temperature to produce 16.
2
,4-Dichloroquinazoline (17) was then achieved by refluxing 16 with POCl in the
3
presence of DIPEA in toluene. Then, N-methyl-4-methoxyaniline was attached to the
-position of 2,4-dichloroquinazoline (17) through nucleophilic addition under basic
4
condition in THF at room temperature to form intermediate 18. Another nucleophilic
addition with various anilines or benzylamines under EtOH in sealed tube at 150 ℃
produced compounds 19a-t, and 19e was further deprotected to produce compound 19f.
Scheme 1. Synthesis of compounds 13a-da.

O
COOC H
2 5
OH
NH2
O
NH
a
COOC H5
b
N
c
2
NH2
+
NH2
COOC H5
2
N
COOC H
2 5
O
7
8
9
OCH3
OCH3
Cl
N
N
N
N
N
d
e
N
N
N
COOC H5
2
COOC H
2
5
COOH
10
11
12
OCH3
1
3a: R =
3b: R =
N
N
N
f
1
OTBS
OCH3
OH
N
H
N
g
R
13c: R =
3d: R =
O
13a-d
1
a
Reagents and conditions: (a) Et N, THF, 0 °C to rt; (b) NaOCH , EtOH, 0 °C to rt; (c) POCl ,
3
3
3
DIPEA, toluene, reflux; (d) N-methyl-4-methoxyaniline, concentrated HCl, isopropanol, rt; (e) 10%
NaOH aqueous, CH OH, reflux; (f) various anilines, Et N, HATU, CH CN, rt; (g) TBAF, THF, rt.
3
3
3
Scheme 2. Synthesis of compounds 19a-ta.
O
NH2
H
N
Cl
N
O
NH2
NH
a
b
c
N
NH
COOCH3
COOCH3
Cl
O
14
15
16
17
OCH3
OCH3
1
9a: n = 0, R =
19b: n = 1, R =
9c: n = 1, R =
19d: n = 1, R =
N
N
d
e
N
N
N
N
N
N
1
N
N
R
Cl
H
n
OCH3
18
19a-d
N
e
e
OCH3
OCH3
19e: n = 0, R , R3
= H, R2 = OTBS;
1
f
1
9f: n = 0, R , R3 = H, R2 = OH;
1
N
19g: n = 0, R , R3 = H, R2 = OCH3;
N
1
1
9h: n = 1, R , R3 = H, R2 = OCH3;
1
N
R2
N
19i: n = 1, R , R3 = H, R2 = F;
1
1
1
9j: n = 1, R , R3 = H, R2 = OCF3;
R3
N
N
N
N
19k: n = 1, R , R3 = H, R2 = OC H ;
19l: n = 1, R , R3 = H, R2 = OC H ;
19m: n = 1, R , R3
19n: n = 1, R , R3 = H, R1 = OCH3
n
1 2 5
H
R1
1
3
7
OCH3
R2
= H, R2 = CN;
19r-t
1
1
9e-q
R1
2
1
1
1
9r: R1 = CH , R = H;
19o: n = 1, R1 = H, R , R3 = OCH3;
3
2
2
9s: R1 = H, R2 = CH3;
9t: R , R = -CH CH CH CH -
19p: n = 1, R , R , R = OCH3;
1
2
3
1
2
2
2
2
2
19q; n = 1, R1 = H, R , R3 = -OCH CH -
2
2
2
a
Reagents and conditions: (a) KNCO, AcOH, H O, rt; (b) KOH, CH OH, H O, reflux; (c) POCl ,
2
3
2
3
DIPEA, toluene, reflux; (d) N-methyl-4-methoxyaniline, Et N, THF, rt; (e) various anilines or
3

benzylamines, EtOH, 150 ℃, sealed tube; (f) TBAF, THF, rt.
2
.2 In vitro anti-proliferative and anti-tubulin activity
The effect of linkers attached to the 2-position of quinazoline on anti-proliferative
activity was firstly investigated. Eight compounds 13a, 13c, 13d, 19a, 19b and 19f-h
bearing three kinds of linkers including amide bond, amino and aminomethyl were
screened for their anti-proliferative activities in human leukemia cell lines (K562) by
MTT assays. The results showed that compounds bearing amide bond (13a, 13c, 13d)
and amino (19a, 19g) exhibited no obvious cytotoxicity at both 1 μM and 0.1 μM
concentrations except 19f which had an inhibitive rate of 87.6% at 1 μM. However,
compounds with aminomethyl as the linker (19b, 19h) exhibited significant
cytotoxicity with the inhibitive rates of 95.3% and 80.5%, respectively at 1 μM
concentration (see supporting information S1). Thus, aminomethyl was identified as
the best linker that could orient additional phenyl moieties to zone 3 and was used for
the next derivatization.
Seventeen compounds 19b-d, 19f and 19h-t were further evaluated for their anti-
proliferative activities by MTT assays using the typical tubulin inhibitor ABT-751 that
occupies three zones of colchicine domain as the positive control. Various hydrogen
bond acceptors were introduced to the methylamino linker with the wish that they may
form hydrogen bonds with the Tyrβ202 residue in zone 3, resembling the interaction of
phenolic hydroxyl of ABT-751. As shown in Table 1, most of the compounds exhibited
potent anti-proliferative activities. Substitutions at the 3-position of the phenyl moiety
(19c, 19n) seem to be favorable for the improvement of activity, compound 19c bearing
3
-pyridine (IC = 0.10 μM) and compound 19n bearing 3-methoylphenyl (IC = 0.13
5
0
50
μM) were about 25 folds more potent than ABT-751 (IC = 2.82 μM). The effect of
5
0
substitutions at the linker on activity was also investigated, either the N-CH linker (19r)
3
or tetrahydroisoquinoline linker (19t) led to the dramatically loss of activity while the
methyl substitution at the methylene position (19s) maintained the activity.
Four representative compounds 19b, 19c, 19h and 19n were chosen to evaluate their
anti-proliferative activities against another four cancer cell lines including HepG2, KB,
HCT-8 and MDA-MB-231 (Table 2). Interestingly, 19n was less sensitive against these
four cancer cell lines than K562 cells with the IC values ranging from 0.74 to 1.17
5
0
μM, while 19c displayed no obvious selectivity among these four cancer cell lines with
the IC values ranging from 0.11 to 0.13 μM, which was about 50 folds more potent
5
0
than ABT-751.
In vitro tubulin polymerization inhibition assays further demonstrated that 19b, 19c,
1
9h and 19n were potent tubulin polymerization inhibitors. Among them, 19c (IC =
50
2
.45 μM) and 19h (IC = 2.67 μM) were comparable with ABT-751 (IC = 2.24 μM)
50 50

(Table 2). Considering the good performance of 19c in both in vitro anti-proliferative
assays and in vitro tubulin polymerization inhibition assays, it was selected for further
biological studies.
Table 1. Anti-proliferative activities of compounds against K562 cell linea.
b
b
IC values (μM)
IC values (μM)
5
0
50
Compd.
Compd.
K562
K562
0.37±0.02
0.13±0.01
0.56±0.02
0.41±0.02
0.85±0.05
>5
1
9b
9c
9d
9f
9h
0.24±0.04
0.10±0.01
0.63±0.04
0.33±0.02
0.28±0.03
0.38±0.02
0.31±0.03
0.31±0.02
0.43±0.03
19m
19n
1
1
19o
1
19p
1
19q
1
9i
19r
1
9j
19s
0.65±0.03
>5
1
9k
9l
19t
1
ABT-751
2.82±0.10
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by the
MTT assay as described in the Experimental Section.
b
IC50 values are indicated as the mean ± SD (standard error) of at least three independent experiments.
a
Table 2. Anti-proliferative activities against four cancer cell lines , and ITP of compounds 19b,
1
9c, 19h, 19n and ABT-751.
b
IC values (μM)
5
0
Compd.
HepG2
KB
HCT-8
MDA-MB-231
ITPc
1
9b
9c
0.58±0.05
0.11±0.01
0.21±0.02
0.74±0.02
5.94±0.51
0.48±0.04
0.11±0.02
0.37±0.03
1.11±0.05
5.32±0.32
0.47±0.03
0.13±0.01
0.48±0.01
1.17±0.07
5.78±0.65
0.46±0.03
0.12±0.01
0.47±0.05
1.08±0.06
4.73±0.43
5.29±0.25
2.45±0.16
2.67±0.18
5.56±0.28
2.24±0.13
1
1
1
9h
9n
ABT-751
a
Cells were treated with different concentrations of the compounds for 72 h. Cell viability was measured by the
MTT assay as described in the Experimental Section.
b
IC50 values are indicated as the mean ± SD (standard error) of at least three independent experiments.
c
Inhibition of tubulin polymerization activity.
2
.3 Molecular modeling
To validate whether 19c would occupy three zones of colchicine domain as ABT-
7
51, molecular modeling studies were performed. ABT-751 was firstly redocked into
the colchicine domain resulting a similar conformation to that of ABT-751 in the crystal

structure with the root mean square deviation (RMSD) value of 0.93 (PDB code:
3
HKC), which indicated the feasibility of our docking method. The docking results of
1
9c displayed that it occupied three zones of colchicine domain in a similar positioning
of ABT-751 (Figure 3A). The 4-meothylphenyl moiety of 19c located in zone 1 that
was surrounded by residues Thr314, Lys352 and Asn258, and the quinazoline occupied
in zone 2 with the 1-N forming a critical hydrogen bond with residue Cys241. 3-
Pyridine moiety extended into the deep pocket and was fixed by the hydrogen bond
between N atom of pyridine and residue Tyr202. Verubulin was also docked into the
protein which indicated that it adopted the similar location with that of verubulin 6 in
the crystal structure (PDB code: 4I55), and 1-N formed a critical hydrogen bond with
residue Cys241 (Figure 3B). The quinazoline moiety of 19c was highly overlapped with
verubulin in the colchicine domain with the additional 3-pyridine moiety extended into
zone 3. All these docking results demonstrated that 19c was a colchicine binding site
inhibitor with a unique binding mode that it occupies three zones of colchicine domain.
Figure 3. Proposed binding models for representative compounds binding with tubulin (PDB code: 3HKC) (A)
ABT-751 (shown in blue) and 19c (shown in pink); (B) verubulin (shown in green) and 19c (shown in pink).
2
.4 Anti-microtubule effect of 19c in K562 cells
Immunofluorescent assay was performed to investigate the effect of compound 19c
on microtubule networks (Figure 4). Vehicle treated K562 cells exhibited normal
filamentous microtubules arrays. However, when treated with 19c at three different
concentrations (0.05 μM, 0.10 μM, and 0.20 μM) for 24 h, the microtubule networks in
cytosol were disrupted; these results indicated that 19c can induce a dose-dependent
collapse of the microtubule networks.

Figure 4. Effects of compound 19c on the cellular microtubule networks visualized by immunofluorescence. K562
cells were treated with vehicle control 0.1% DMSO, 19c (0.05 μM, 0.1 μM, and 0.2 μM). Then, the cells were fixed
and stained with anti-α-tubulin-FITC antibody (green), Alexa Fluor 488 dye and counterstained with DAPI (blue).
2
.5 Cell cycle analysis
Most microtubule polymerization inhibitors alter the tubulin-microtubule
equilibrium, causing mitotic arrest at G2/M phase and ultimately apoptotic cell death.
Thus, the most active compound 19c was examined for its effect on cell cycle
progression of K562 cells using propidiumiodide (PI) staining by flow cytometry
analysis. As depicted in Figure 5, treatment with compound 19c resulted in a gradual
accumulation of cells in the G2/M phase of cell cycle in a dose-dependent manner. The
percentages of cells arrested at the G2/M phase increased from 15.82% to 22.86%,
2
5.72%, and 27.67% at concentrations of 0, 0.05, 0.1, and 0.2 μM, respectively. These
results demonstrated that 19c can induce cell cycle arrest at G2/M phase.
2
.6 Cell apoptosis analysis
The effects of 19c on K562 cell apoptosis were further evaluated in Annexin V-FITC
and propidium iodide (PI) staining assays and analyzed by flow cytometry (Figure 6).

The percentage of apoptotic cells after 48 h treatment was only 5.74% in the control
+
−
+
+
group. However, the total numbers of early (Annexin -V /PI ) and late (Annexin-V /PI )
apoptotic cells increased to 19.8%, 37.4% and 65.0% after treatment with 19c at 0.05,
0
.1, and 0.2 μM for 48 h, respectively. These results confirmed that 19c effectively
induced cell apoptosis in K562 cells in a dose-dependent manner.
Figure 5． Compound 19c induced G2/M arrest in K562 cells. K562 cells were incubated with varying
concentrations of 19c (0, 0.05, 0.1, and 0.2 μM) for 48 h. Cells were harvested and stained with PI and then analyzed
by flow cytometry. The percentages of cells in different phases of cell cycle were analyzed by ModFit 4.1.

Figure 6. Compound 19c induced apoptosis in K562 cells. K562 cells were incubated with varying concentrations
of 19c (0, 0.05, 0.1, and 0.2 μM). After 48 h of incubation, cells were collected and stained with Annexin-V/PI,
followed by flow cytometric analysis. The percentages of cells in each stage of cell apoptosis were quantified by
flow cytometry: (upper left quadrant) necrosis cells; (upper right quadrant) late-apoptotic cells; (bottom left quadrant)
live cells; and (bottom right quadrant) early apoptotic cells.
2
.7 Mitochondrial membrane potential (MMP) analysis
In order to determine whether 19c-induced apoptosis was involved in a disruption of
mitochondrial membrane integrity, the fluorescent probe JC-1 was employed to
measure the MMP (Figure 7). When treated with 19c at concentrations of 0, 0.05, 0.10,
and 0.20 μM for 48 h, the number of K562 cells with collapsed MMP increased from
0
.63% to 18.69%, 31.52% and 62.27%, respectively, suggesting that 19c caused
mitochondrial depolarization of K562 cells in the process of apoptosis.

Figure 7. Effect of 19c on the mitochondrial membrane potential of K562 cells. Incubation with different
concentrations (0, 0.05, 0.10, and 0.20 μM) of 19c in K562 cells for 48 h prior to staining with JC-1 dye, the number
of cells with collapsed mitochondrial membrane potentials was determined by flow cytometry analysis.
2
.8 In vitro evaluation of anti-vascular activity
To evaluate the anti-vascular activity of compound 19c, the HUVEC culture assay
was used to assess the ability to inhibit HUVEC migration, which is the key step to
generate new blood vessels. As shown in Figure 8A, the untreated cells migrated to fill
the area that was initially scraped after 24 h. In contrast, compound 19c significantly
inhibited the HUVEC migration in a dose-dependent manner. Then we further
evaluated the anti-vascular ability of compound 19c in a tube formation assay. After
being seeded on matrigel, HUVECs form the capillary-like tubules with multicentric
junctions. After exposure to 19c at doses of 0.05, 0.10, and 0.20 μM for 6 h, the
capillary-like tubes were interrupted in different levels (Figure 8B), indicating that 19c
effectively inhibited the tube formation of HUVECs. All these results demonstrated that
1
9c was a potent vascular disrupting agent.

Figure 8. Effects of 19c on the HUVECs migration and tube formation. (A) Scratches were created with sterile 200
μL pipette and images were captured using phase contrast microscopy at 0 h and 24 h after treatment with 0, 0.05,
0
.1, and 0.2 μM of 19c. (B) Images depicting the formation of HUVEC capillary-like tubular network by treatment
with 0, 0.05, 0.1, and 0.2 μM of 19c for 6h.
2
.9 In vivo anti-tumor activity
To evaluate the in vivo anti-tumor efficacy of 19c, human liver cancer xenograft was
established by subcutaneous inoculation of H22 cells into the right flank of mice. The
tumor size and the body weights of mice were monitored and recorded every 2 days.
Paclitaxel (PTX) was selected as the positive control. As shown in Figure 9A, the
reduction in tumor weight reached 86.6% at a dose of 8 mg/kg/2 day (i.v.) of PTX at
2
1 days after initiation of treatment as compared to vehicle. Compared with the vehicle
group, administration of 19c at 15 and 30 mg/kg by i.v. resulted in 50.9% and 62.6%
reduction of tumor growth, respectively (Figure 9A). Notably, no significant
differences in body weight or other adverse effects were observed upon treatment with
1
9c, while treatment with PTX led to a significant decrease of body weight (Figure 9B).
Thus, 19c was efficacious and safe in inhibiting tumor growth with a dose-dependent
manner in vivo and deserved further evaluation.

Figure 9. A) 19c treatment resulted in significantly lower tumor weight compared with controls. B) Body weight
changes of mice during treatment.
3
Conclusions
In summary, a series of novel quinazolines occupying three zones of colchicine
domain have been designed, synthesized inspired by the crystal structure of verubulin
analogue 6 and tubulin. Anti-proliferative screening of these newly synthesized
compounds validated the representative compound 19c as a high cytotoxic compound
with IC values ranging from 0.10 to 0.13 μM against a panel of cancer cell lines, which
5
0
was about 50 folds more potent than ABT-751. 19c also displayed comparable
inhibitory activity in tubulin assembly assays to ABT-751 with the IC value of 2.45
5
0
μM. The proposed binding mode of 19c with tubulin was confirmed by molecular
modeling studies which showed that it adopted a similar positioning of ABT-751 to
occupy three zones of colchicine domain. Further mechanism studies demonstrated that
1
9c caused cell cycle arrest in G2/M phase, induced cell apoptosis and depolarized
mitochondria of K562 cells. Moreover, 19c effectively and dose-dependently disrupted
microtubule networks of K562 cells in immunofluorescent assays. The wound healing
and tube formation assays also identified 19c as a potent vascular disrupting agent.
Finally, the in vivo anti-tumor activity of 19c was validated in H22 liver cancer
xenograft mouse model. Altogether, 19c may represent a novel class of anti-tubulin
agent with a unique binding mode with tubulin and deserves further investigation.
4
Experimental
4
4
.1 Chemistry
.1.1.General
Most chemicals and solvents were purchased from commercial sources. Further
1
purification and drying by standard methods were employed when necessary. H NMR
1
3
and C NMR spectra were recorded on Bruker-300 spectrometers in the indicated

solvents (TMS as internal standard). Data are reported as follows: chemical shift in ppm
d), multiplicity (s =singlet, d =doublet, t =triplet, q =quartet, brs = broad singlet, m
multiplet), coupling constant (Hz), and integration. High Resolution Mass
(
=
measurement was performed on Agilent QTOF 6520 mass spectrometer with electron
spray ionization (ESI) as the ion source. Purity of all tested compounds was ≥ 95%, as
estimated by HPLC analysis. Flash column chromatography was carried out using
commercially available silica gel (200-300 mesh) under pressure.
4
.1.2. Synthesis of intermediate 12.
Intermediate 10 was synthesized according the procedures reported in the previous
literature [26]. To a solution of 10 (3.62 g, 0.015 mol) and N-methyl-4-methoxyaniline
(2.31g, 0.017 mol) in 25 mL of anhydrous isopropanol (IPA) was added 0.6 mL of
concentrated HCl, and the mixture was stirred at room temperature overnight. The
mixture was extracted with CH Cl (3 × 50 mL). The combined organic layers were
2
2
then washed with brine, dried over anhydrous Na SO , and concentrated in vacuo to
2
4
afford crude product 11 (4.2 g, 81.6%) as yellow solid. To a solution of 11 (4.2 g, 0.012
mol) in 20 mL CH OH was added 20 mL 10% NaOH aqueous, and the mixture was
3
stirred at 80 °C for 2 h. The CH OH was then removed in vacuo and acidized with 10%
3
HCl aqueous to pH 3. The yellow precipitates were collected by filtration, washed with
1
water and dried to afford intermediate 12 (2.9 g, 76.3%) as yellow solid. H NMR
(
300MHz, DMSO-d ) δ7.91 - 7.83 (m, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.26 (d, J = 8.4
6
Hz, 2H), 7.20 (d, J = 7.6 Hz, 1H), 7.01 (d, J = 8.3 Hz, 2H), 6.96 (d, J = 8.2 Hz, 1H),
1
3
3
1
1
.78 (d, J = 3.2 Hz, 3H), 3.57 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 165.98, 161.84,
6
58.47, 153.54, 150.87, 140.55, 133.14, 128.80, 127.82, 126.75, 126.25, 115.84,
+
14.16, 55.85,43.07; ESI-MS m/z 309.1 [M+Na] 332.1.
4
.1.3. General procedures of compounds 13a-d.
To a solution of 12 (70 mg, 0.24 mmol) in 5 mL CH CN was added 3-
3
tetramethyluronium hexafluorophosphate (HATU) (94 mg, 0.27 mmol), Et N (63 μL,
3
0
.48 mmol), various anilines (0.28 mmol) were added. The mixture was stirred for 1 h,
and extracted with CH Cl (3 × 25 mL). The combined organic layers were then washed
2
2
with brine, dried over anhydrous Na SO , and concentrated in vacuo. The residue was
2
4
purified by flash column chromatography to afford the products 13a-c in 65-84% yields.
1
4
.1.3.1 Compound 13a. White solid, yield 65.1%. H NMR (300 MHz, DMSO-d ) δ
6
1
1.27 (s, 1H), 8.87 (s, 1H), 8.67 (d, J = 5.9 Hz, 2H), 8.18 (d, J = 5.8 Hz, 2H), 7.95 (d,
J = 8.3 Hz, 1H), 7.76 (t, J = 7.6 Hz, 1H), 7.29 (d, J = 8.2 Hz, 2H), 7.24 (s, 1H), 7.06 -
1
3
7
1
.02 (m, 2H), 3.82 (s, 3H), 3.68 (s, 3H); C NMR (75 MHz, DMSO) δ 163.21, 161.39,
58.12, 152.73, 150.62, 148.42, 146.85, 139.95, 132.76, 128.74, 127.33, 126.57,

1
25.90, 115.76, 115.39, 114.56, 55.37, 45.82; HR-MS (ESI) m/z: calcd for C H N O
22 20 5 2
+
[
M+H] 386.1612, found 386.1606.
1
4
.1.3.2 Compound 13c. White solid, yield 72.0%. H NMR (300 MHz, CDCl ) δ 10.08
3
(s, 1H), 8.05 - 7.95 (m, 1H), 7.75 (d, J = 9.0 Hz, 2H), 7.64 - 7.59 (m, 1H), 7.15 (d, J =
8
.9 Hz, 2H), 7.11 - 7.06 (m, 1H), 7.06 - 7.03 (m, 1H), 6.95 (d, J = 2.5 Hz, 2H), 6.92
1
3
(
d, J = 2.6 Hz, 2H), 3.85 (s, 3H), 3.81 (s, 3H), 3.72 (s, 3H); C NMR (75 MHz, CDCl )
3
δ 161.28, 160.46, 158.01, 155.88, 152.37, 151.02, 140.08, 131.82, 130.74, 128.94,
26.93, 125.76, 125.66, 120.70, 115.77, 114.92, 113.75, 55.04, 54.99, 42.48; HR-MS
ESI) m/z: calcd for C H N O [M+H]+ 415.1765, found 415.1762.
1
(
2
4
23
4
3
4
.1.3.3 Compound 13d. 13b was obtained as colorless oil which was used for next step
directly. To a solution of 13b (50 mg, 0.10 mmol) in THF was added TBAF (30 mg,
.12 mmol), and the reaction was stirred at room temperature for 30 min. Then, the
mixture was extracted with CH Cl (3 × 25 mL). The combined organic layers were
0
2
2
washed with brine, dried over anhydrous Na SO , and concentrated in vacuo. The
2
4
residue was purified by flash column chromatography to afford the product 13d as
1
white solid in 62.3% yield over two steps. H NMR (300 MHz, DMSO-d ) δ 10.34 (s,
6
1
H), 9.33 (s, 1H), 7.90 (d, J = 8.3 Hz, 1H), 7.73 (d, J = 7.5 Hz, 1H), 7.67 (d, J = 8.8
Hz, 2H), 7.29 (d, J = 7.9 Hz, 2H), 7.21 (t, J = 7.8 Hz, 1H), 7.04 (s, 1H), 7.02 (d, J = 7.9
1
3
Hz, 2H), 6.81 (d, J = 8.8 Hz, 2H), 3.80 (s, 3H), 3.65 (s, 3H); C NMR (75 MHz, DMSO)
δ 161.31, 161.23, 157.96, 153.91, 153.88, 150.99, 140.26, 132.53, 130.06, 128.77,
1
27.34, 126.03, 125.80, 121.76, 115.59, 115.36, 115.13, 55.37, 42.67; HR-MS (ESI)
+
m/z: calcd for C H N O [M+H] 401.1608, found 401.1613.
2
3
21
4
3
4
.1.4. Synthesis of intermediate 18.
Intermediate 17 was synthesized according the procedures reported in the previous
literature [27]. To a solution of 17 (3.2 g, 0.016 mol) in 50 mL THF was added Et N (3.3
3
mL, 0.024 mol) and N-methyl-4-methoxyaniline (1.9 g, 0.014 mol), and the mixture
was stirred at room temperature overnight. The mixture was extracted with CH Cl (3
2
2
×
50 mL). The combined organic layers were then washed with brine, dried over
anhydrous Na SO , and concentrated in vacuo. The residue was then purified by flash
2
4
column chromatography using PE/EA 15:1 as the fluent to afford 18 (3.9 g, 81.3%) as
white powder. 1H NMR (300 MHz, CDCl ) δ 7.64 (d, J = 8.4 Hz, 1H), 7.47 (t, J = 7.8
3
1
3
Hz, 1H), 7.07 (d, J = 8.8 Hz, 2H), 6.89 (m, 4H), 3.78 (s, 3H), 3.52 (s, 3H); C NMR
(
75 MHz, CDCl ) δ 162.54, 158.68, 156.56, 152.98, 139.94, 132.57, 127.65, 127.49,
3
+
1
26.38, 124.93, 115.44, 114.78, 55.56, 43.26; ESI-MS m/z 299.1 [M+H] 300.1.
.1.5. General procedures of compounds 19a-t.
4
To a solution of 18 (75 mg, 0.25 mmol) in 5 mL EtOH in sealed tube was added
various anilines or benzylamines, the mixture was stirred at 150 °C. After completion,
the reaction was cooled to room temperature. In the cases of solid precipitation in the

reaction, the precipitates were collected by filtration, washed with cold EtOH and dried
to afford the final products. When no solid precipitated in the reaction, the solvent was
removed in vacuo and then purified by flash column chromatography to afford the final
products.
1
4
9
7
3
1
4
4
.1.5.1. Compound 19a. White solid, yield 76.7%. H NMR (300 MHz, DMSO-d ) δ
6
.36 (s, 1H), 9.33 (s, 1H), 9.31 (s, 1H), 7.77 - 7.67 (m, 2H), 7.35 (d, J = 8.9 Hz, 2H),
.16 (d, J = 7.8 Hz, 2H), 7.14 - 7.10 (m, 1H), 6.86 (d, J = 8.5 Hz, 1H), 3.83 (s, 3H),
1
3
.65 (s, 3H); C NMR (75 MHz, DMSO-d ) δ 161.83, 160.96, 158.52, 151.11, 150.14,
6
39.04, 137.76, 129.81, 127.73, 127.61, 126.15, 125.29, 115.52, 114.53, 109.23, 55.49,
+
3.25; HR-MS (ESI) m/z: calcd for C H N O [M+H] 358.1662, found 358.1659.
2
1
20
5
1
.1.5.2. Compound 19b. White solid, yield 66.9%. H NMR (300 MHz, CDCl ) δ 8.56
3
(d, J = 5.1 Hz, 2H), 7.57 (d, J = 8.3 Hz, 1H), 7.49 (t, J = 7.7 Hz, 1H), 7.36 (d, J = 5.1
Hz, 2H), 7.13 (d, J = 8.6 Hz, 2H), 6.97 (d, J = 8.7 Hz, 2H), 6.87 - 6.76 (m, 1H), 6.67
1
3
(
d, J = 8.6 Hz, 1H), 4.88 (s, 1H), 4.77 (s, 2H), 3.87 (s, 3H), 3.45 (s, 3H); C NMR (75
MHz, CDCl ) δ161.35, 159.42, 149.84, 147.90, 144.14, 138.26, 134.12, 134.10, 127.46,
3
1
4
4
27.22, 122.90, 122.06, 119.25, 115.70, 110.46, 77.48, 77.06, 76.64, 55.63, 44.13,
+
3.84; HR-MS (ESI) m/z: calcd for C H N O [M+H] 372.1819, found 372.1815.
2
2
22
5
2
1
.1.5.3. Compound 19c. White solid, yield 79.2%. H NMR (300 MHz, CDCl ) δ 8.58
3
(s, 1H), 8.46 - 8.30 (m, 1H), 7.72 (d, J = 7.9 Hz, 1H), 7.42 (d, J = 8.4 Hz, 1H), 7.36 –
7
6
.30 (m, 1H), 7.24 - 7.15 (m, 1H), 7.04 (d, J = 8.9 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H),
1
3
.65 (d, J = 6.9 Hz, 2H), 6.21 (s, 1H), 4.68 (s, 2H), 3.76 (s, 3H), 3.42 (s, 3H); C NMR
(
75 MHz, CDCl ) δ 179.38, 161.20, 158.41, 148.44, 147.96, 138.70, 138.68, 138.64,
3
1
4
4
34.82, 134.20, 132.87, 126.92, 126.53, 123.09, 121.51, 114.98, 110.50, 55.06, 43.07,
+
2.23; HR-MS (ESI) m/z: calcd for C H N O [M+H] 372.1819, found 372.1816.
2
2
22
5
1
.1.5.4. Compound 19d. White solid, yield 75.2%. H NMR (300 MHz, CDCl ) δ 8.21
3
(s, 1H), 7.68 (dd, J = 8.4, 2.5 Hz, 1H), 7.45 (d, J = 8.3 Hz, 1H), 7.40 - 7.34 (m, 1H),
7
6
.10 (d, J = 8.6 Hz, 2H), 6.94 - 6.87 (m, 2H), 6.83 (d, J = 8.4 Hz, 1H), 6.69 (m, 2H),
1
3
.10 (s, 1H), 4.67 (s, 2H), 3.92 (s, 3H), 3.82 (s, 3H), 3.48 (s, 3H); C NMR (75 MHz,
CDCl ) δ 162.99, 161.95, 157.60, 157.31, 151.59, 145.48, 140.46, 138.17, 131.73,
3
1
4
4
27.62, 126.87, 126.25, 123.87, 120.21, 114.67, 111.76, 110.25, 55.00, 52.91, 42.48,
+
2.01; HR-MS (ESI) m/z: calcd for C H N O [M+H] 402.1925, found 402.1918.
2
3
24
5
2
.1.5.5. Compound 19f. To a solution of 19e (50 mg, 0.10 mmol) in THF was added
TBAF (32 mg, 0.12 mmol), and the reaction was stirred at room temperature for 30
min. Then, the mixture was extracted with CH Cl (3 × 25 mL). The combined organic
2
2
layers were washed with brine, dried over anhydrous Na SO , and concentrated in
2
4
vacuo. The residue was purified by flash column chromatography to afford the product
1
1
9f as white solid in 46.8% yield over two steps. H NMR (300 MHz, DMSO-d ) δ
6
1
0.27 (s, 1H), 9.57 (s, 1H), 7.63 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 8.3 Hz, 1H), 7.42 (d, J
=
4.9 Hz, 2H), 7.39 (d, J = 4.9 Hz, 2H), 7.09 (d, J = 8.4 Hz, 2H), 6.97 (t, J = 7.9 Hz,

1
3
1
H), 6.86 (d, J = 8.3 Hz, 2H), 6.72 (d, J = 8.5 Hz, 1H), 3.83 (s, 3H), 3.57 (s, 3H); C
NMR (75 MHz, DMSO) δ 160.94, 158.90, 154.94, 138.08, 138.06, 134.25, 128.11,
1
27.64, 126.97, 124.07, 123.03, 118.43, 118.32, 115.54, 115.47, 110.66, 55.51, 43.67;
+
HR-MS (ESI) m/z: calcd for C H N O [M+H] 373.1659, found 373.1659.
2
2
21
4
2
1
4
.1.5.6. Compound 19g. White solid, yield 86.5%. H NMR (300 MHz, CDCl ) δ 10.48
3
(s, 1H), 7.56 (d, J = 8.8 Hz, 2H), 7.48 (t, J = 7.6 Hz, 1H), 7.31 (s, 1H), 7.20 (d, J = 8.4
Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.8 Hz, 2H), 6.82 (s, 1H), 6.67 (d, J =
1
3
8
1
1
.6 Hz, 1H), 3.88 (s, 3H), 3.80 (s, 3H), 3.62 (s, 3H); C NMR (75 MHz, CDCl ) δ
3
60.74, 159.22, 156.44, 150.40, 140.73, 137.21, 133.96, 129.21, 127.00, 126.85,
23.16, 122.90, 117.78, 115.35, 113.46, 109.75, 55.17, 54.95, 43.96; HR-MS (ESI) m/z:
+
calcd for C H N O [M+H] 387.1816, found 387.1814.
2
3
23
4
2
1
4
.1.5.7. Compound 19h. White solid, yield 73.2%. H NMR (300 MHz, CDCl ) δ 7.44
3
(d, J = 8.4 Hz, 1H), 7.37 (s, 1H), 7.34 (d, J = 7.8 Hz, 2H), 7.08 (d, J = 8.4 Hz, 2H), 6.88
-
6.85 (m, 5H), 6.66 (t, J = 7.7 Hz, 1H), 5.47 (s, 1H), 4.70 (s, 2H), 3.81 (s, 3H), 3.78 (s,
1
3
3
1
1
H), 3.46 (s, 3H); C NMR (75 MHz, CDCl ) δ 162.17, 158.26, 158.21, 157.30, 153.34,
3
41.10, 131.66, 131.33, 128.47, 126.82, 126.18, 124.96, 119.66, 114.56, 113.38,
12.17, 54.98, 54.78, 44.66, 42.25; HR-MS (ESI) m/z: calcd for
C H N O [M+H]+401.1972, found 401.1968.
2
4
25
4
2
1
4
1
7
1
1
1
.1.5.8. Compound 19i. White solid, yield 75.3%. H NMR (300 MHz, CDCl ) 8.64 (s,
3
H), 7.54 (d, J = 8.3 Hz, 1H), 7.47 (t, J = 7.6 Hz, 1H), 7.39 (dd, J = 8.4, 5.3 Hz, 2H),
.19 - 7.12 (m, 2H), 7.06 - 6.97 (m, 4H), 6.81 (t, J = 7.9 Hz, 1H), 6.67 (d, J = 8.5 Hz,
1
3
H), 4.73 (s, 2H), 3.87 (s, 3H), 3.57 (s, 3H); C NMR (75 MHz, CDCl ) δ 163.18,
3
60.76, 159.93, 158.93, 153.07, 133.59, 133.29, 128.56, 128.46, 126.96, 126.77,
22.41, 115.21, 115.06, 114.77, 109.82, 55.14, 43.90, 43.49; HR-MS (ESI) m/z: calcd
+
for C H FN O [M+H] 389.1772, found 389.1768 .
2
3
22
4
1
4
.1.5.9. Compound 19j. White solid, yield 83.3%. H NMR (300 MHz, CDCl ) δ 7.65
3
(s, 1H), 7.46 (dd, J = 8.3, 1.4 Hz, 1H), 7.41 (d, J = 1.3 Hz, 1H), 7.37 (d, J = 8.4 Hz,
2
6
H), 7.10 (d, J = 7.7 Hz, 2H), 7.07 (d, J = 7.0 Hz, 2H), 6.90 (d, J = 8.9 Hz, 2H), 6.75 -
1
3
.69 (m, 1H), 6.59 (d, J = 8.6 Hz, 1H), 4.69 (s, 2H), 3.79 (s, 3H), 3.46 (s, 3H); C NMR
(
75 MHz, CDCl ) δ 160.81, 158.90, 153.28, 147.85, 137.74, 136.58, 133.54, 128.20,
3
1
4
4
4
26.96, 126.92, 126.74, 122.32, 121.61, 120.59, 120.51, 118.18, 115.19, 109.89, 55.11,
+
3.83, 43.40; HR-MS (ESI) m/z: calcd for C H F N O [M+H] 455.1689, found
2
4
22
3
4
2
55.1687.
1
.1.5.10. Compound 19k. White solid, yield 82.1%. H NMR (300 MHz, CDCl ) δ 8.76
3
(
s, 1H), 7.50 (d, J = 8.3 Hz, 1H), 7.37 (t, J = 7.6 Hz, 1H), 7.26 (d, J = 8.2 Hz, 2H), 7.08
d, J = 8.3 Hz, 2H), 6.91 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.3 Hz, 2H), 6.70 (d, J = 7.3
(
Hz, 1H), 6.53 (d, J = 8.6 Hz, 1H), 4.61 (s, 2H), 3.92 (q, J = 6.9 Hz, 2H), 3.79 (s, 3H),
1
3
3
1
.52 (s, 3H), 1.31 (t, J = 6.9 Hz, 3H); C NMR (75 MHz, CDCl ) δ 160.57, 159.10,
3
57.77, 152.09, 140.61, 137.35, 133.81, 129.19, 128.25, 127.04, 126.77, 122.62,

1
17.58, 115.27, 114.04, 109.48, 62.93, 55.17, 44.07, 43.70, 29.18; HR-MS (ESI) m/z:
+
calcd for C H N O [M+H] 415.2129, found 415.2120.
2
5
27
4
2
1
4
.1.5.11. Compound 19l. White solid, yield 76.5%. H NMR (300 MHz, CDCl ) δ 7.45
3
(d, J = 8.5 Hz, 1H), 7.37 (d, J = 6.9 Hz, 1H), 7.35 - 7.30 (m, 2H), 7.09 (d, J = 8.4 Hz,
2
H), 6.89 (s, 1H), 6.89 - 6.86 (m, 2H), 6.83 (s, 2H), 6.66 (t, J = 7.7 Hz, 1H), 5.45 (s,
H), 4.68 (s, 2H), 4.58 - 4.48 (m, 1H), 3.81 (s, 3H), 3.46 (s, 3H), 1.32 (d, J = 6.1 Hz,
1
1
3
6
H); C NMR (75 MHz, CDCl ) δ 162.14, 158.14, 157.33, 156.51, 153.07, 141.02,
3
1
31.39, 128.49, 126.83, 126.20, 124.81, 124.81, 119.72, 115.43, 114.57, 112.11, 69.43,
+
5
4.99, 44.67, 42.29, 21.59; HR-MS (ESI) m/z: calcd for C H N O [M+H] 429.2285,
2
6
29
4
2
found 429.2275.
1
4
9
7
5
1
1
.1.5.12. Compound 19m. White solid, yield 78.9%. H NMR (300 MHz, CDCl ) δ
3
.19 (s, 1H), 7.64 (d, J = 8.0 Hz, 2H), 7.57 - 7.49 (m, 4H), 7.17 (d, J = 8.7 Hz, 2H),
.01 (d, J = 8.3 Hz, 2H), 6.86 (t, J = 7.1 Hz, 1H), 6.65 (d, J = 8.6 Hz, 1H), 4.84 (d, J =
1
3
.9 Hz, 2H), 3.89 (s, 3H), 3.53 (s, 3H); C NMR (75 MHz, CDCl ) δ 160.51, 159.28,
3
52.35, 142.84, 142.79, 140.24, 136.95, 134.13, 131.94, 127.29, 126.93, 123.10,
18.15, 117.51, 115.36, 110.71, 109.53, 55.18, 44.13, 43.70; HR-MS (ESI) m/z: calcd
+
for C H N O [M+H] 396.1819, found 396.1814.
2
4
22
5
1
4
.1.5.13. Compound 19n. White solid, yield 88.1%. H NMR (300 MHz, CDCl ) δ 7.46
3
(s, 1H), 7.35 (t, J = 7.6 Hz, 1H), 7.16 (t, J = 7.9 Hz, 1H), 7.05 (d, J = 8.4 Hz, 2H), 6.93
(d, J = 6.8 Hz, 2H), 6.88 (d, J = 8.7 Hz, 2H), 6.74 - 6.60 (m, 3H), 5.87 (s, 1H), 4.66 (s,
1
3
2
1
1
H), 3.77 (s, 3H), 3.72 (s, 3H), 3.45 (s, 3H); C NMR (75 MHz, CDCl ) δ 161.05,
3
59.32, 158.56, 158.55, 139.64, 139.62, 138.43, 138.41, 133.06, 129.04, 126.97,
26.97, 126.59, 121.75, 119.22, 115.04, 112.42, 112.34, 55.10, 54.81, 44.75, 43.23;
+
HR-MS (ESI) m/z: calcd for C H N O [M+H] 401.1972, found 401.1969.
2
4
25
4
2
1
4
.1.5.14. Compound 19o. White solid, yield 79.2%. H NMR (300 MHz, CDCl ) δ 7.44
3
(d, J = 8.4 Hz, 1H), 7.39 - 7.33 (m, 1H), 7.09 (d, J = 8.8 Hz, 2H), 7.01 - 6.94 (m, 2H),
6
2
.92 – 6.84 (m, 3H), 6.82 (d, J = 8.1 Hz, 1H), 6.69 - 6.64 (m, 1H), 5.50 (s, 1H), 4.69 (s,
1
3
H), 3.86 (s, 3H), 3.85 (s, 3H), 3.81 (s, 3H), 3.47 (s, 3H); C NMR (75 MHz, CDCl )
3
δ 162.17, 158.27, 157.32, 153.32, 148.49, 147.59, 141.04, 132.13, 131.36, 126.81,
1
4
4
4
26.19, 124.93, 119.70, 119.39, 114.56, 112.15, 110.65, 110.61, 55.44, 55.36, 54.98,
+
5.09, 42.26; HR-MS (ESI) m/z: calcd for C H N O [M+H] 431.2078, found
2
5
27
4
3
31.2074.
.1.5.15. Compound 19p. White solid, yield 68.3%. H NMR (300 MHz, CDCl ) δ 8.96
1
3
(s, 1H), 7.58 - 7.46 (m, 2H), 7.19 (d, J = 8.8 Hz, 2H), 7.01 (d, J = 8.9 Hz, 2H), 6.88 -
6
3
1
1
.82 (m, 1H), 6.69 (s, 2H), 6.65 (d, J = 8.8 Hz, 1H), 4.72 (s, 2H), 3.89 (s, 9H), 3.83 (s,
1
3
H), 3.64 (s, 3H); C NMR (75 MHz, CDCl ) δ 160.57, 159.19, 152.88, 152.23, 140.36,
3
37.15, 136.69, 133.96, 132.66, 126.98, 126.89, 122.83, 117.43, 115.32, 109.48,
03.84, 60.31, 55.79, 55.16, 44.79, 43.69; HR-MS (ESI) m/z: calcd for C H N O
2
6
29
4
4
+
[
M+H] 461.2183, found 461.2181.

1
4
.1.5.16. Compound 19q. White solid, yield 81.4%. H NMR (300 MHz, CDCl ) δ 7.45
3
(
d, J = 8.3 Hz, 1H), 7.41 - 7.30 (m, 1H), 7.26 (s, 1H), 7.15 (d, J = 7.9 Hz, 1H), 7.09 (d,
J = 8.6 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 6.84 (s, 1H), 6.79 – 6.62 (m, 2H), 5.63 (s,
H), 4.67 (s, 2H), 4.54 (t, J = 8.7 Hz, 2H), 3.81 (s, 3H), 3.48 (s, 3H), 3.16 (t, J = 8.7
1
1
3
Hz, 2H); C NMR (75 MHz, CDCl ) δ 162.07, 158.78, 157.78, 157.47, 140.84, 131.50,
3
1
1
31.37, 127.11, 126.82, 126.75, 126.21, 124.43, 124.06, 124.06, 119.86, 114.63,
12.00, 108.50, 70.76, 54.99, 44.89, 42.33, 29.24; HR-MS (ESI) m/z: calcd for
+
C H N O [M+H] 413.1972, found 413.1966.
2
5
25
4
2
1
4
.1.5.17. Compound 19r. White solid, yield 76.3%. H NMR (300 MHz, CDCl ) δ 8.24
3
(s, 1H), 7.48 - 7.42 (m, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.14 (d, J = 8.4 Hz, 2H), 6.95 (d,
J = 8.4 Hz, 2H), 6.87 (d, J = 8.2 Hz, 2H), 6.76 (d, J = 4.0 Hz, 2H), 5.01 (s, 2H), 3.85
1
3
(
s, 3H), 3.79 (s, 3H), 3.54 (s, 3H), 3.42 (s, 3H). C NMR (75 MHz, CDCl ) δ 160.69,
3
1
1
58.52, 158.27, 141.56, 139.08, 132.73, 128.75, 128.37, 126.95, 126.10, 121.73,
21.62, 121.49, 114.92, 113.56, 110.25, 57.71, 55.08, 54.79, 52.31, 43.02; HR-MS (ESI)
+
m/z: calcd for C H N O [M+H] 415.2129, found 415.2126.
2
5
27
4
2
1
4
.1.5.18. Compound 19s. White solid, yield 81.2%. H NMR (300 MHz, CDCl ) δ 8.95
3
(
s, 1H), 7.51 (d, J = 8.3 Hz, 1H), 7.46 (d, J = 7.1 Hz, 1H), 7.39 (d, J = 8.3 Hz, 2H), 7.14
d, J = 8.7 Hz, 2H), 6.99 (d, J = 8.9 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 6.83 – 6.74 (m,
(
1
3
1
1
H), 6.59 (d, J = 8.6 Hz, 1H), 5.29 - 5.11 (m, 1H), 3.88 (s, 3H), 3.78 (s, 3H), 3.54 (s,
1
3
H), 1.66 (d, J = 6.9 Hz, 3H); C NMR (75 MHz, CDCl ) δ 160.37, 159.10, 158.21,
3
51.36, 140.40, 137.25, 135.31, 133.77, 127.00, 126.80, 126.61, 122.59, 117.37,
15.26, 113.47, 109.36, 55.15, 54.76, 50.89, 43.66, 22.89; HR-MS (ESI) m/z: calcd for
+
C H N O [M+H] 415.2129, found 415.2122.
2
5
27
4
2
1
4
.1.5.19. Compound 19t. White solid, yield 80.1%. H NMR (300 MHz, CDCl ) δ 7.52
3
(d, J = 8.4 Hz, 1H), 7.37 - 7.32 (m, 1H), 7.16 (d, J = 8.3 Hz, 1H), 7.09 (d, J = 8.4 Hz,
2
1
3
1
1
2
H), 6.98 - 6.89 (m, 1H), 6.87 (d, J = 7.9 Hz, 2H), 6.77 (d, J = 8.3 Hz, 1H), 6.71 (s,
H), 6.68 - 6.56 (m, 1H), 5.01 (s, 2H), 4.19 (t, J = 5.9 Hz, 2H), 3.80 (s, 3H), 3.79 (s,
1
3
H), 3.54 (s, 3H), 2.96 (t, J = 5.9 Hz, 2H); C NMR (75 MHz, CDCl ) δ 161.75, 157.57,
3
57.46, 157.30, 153.47, 141.24, 136.16, 131.23, 127.02, 126.85, 126.58, 126.07,
25.19, 119.53, 114.56, 112.89, 111.82, 111.44, 54.98, 54.81, 45.51, 42.30, 41.10,
+
9.03; HR-MS (ESI) m/z: calcd for C H N O [M+H] 427.2129, found 427.2199.
2
6
27
4
2
4
4
.2. Pharmacology
.2.1. In vitro anti-proliferative assay
K562, HepG2, KB, HCT-8 and MDB-MD-231 cells were purchased from Nanjing
KeyGen Biotech Co. Ltd. (Nanjing, China). The cytotoxicity of the test compounds was
determined using the MTT assay. Briefly, the cell lines were incubated at 37 °C in a
humidified 5% CO incubator for 24 h in 96-microwell plates. After medium removal,
2
1
00 mL of culture medium with 0.1% DMSO containing the test compounds at different

concentrations was added to each well and incubated at 37 °C for another 72 h. The
MTT (5 mg/mL in PBS) was added and incubated for another 4 h, the optical density
was detected with a microplate reader at 490 nm. The IC values were calculated
5
0
according to the dose-dependent curves. All the experiments were repeated in at least
three independent experiments.
4
.2.2. In vitro tubulin polymerization inhibitory assay
An amount of 2 mg/mL tubulin (Cytoskeleton) was resuspended in PEM buffer
containing 80 mM piperazine-N,N’-bis(2-ethanesulfonic acid) sequisodium salt PIPES
pH 6.9), 0.5 mM EGTA, 2 mM MgCl , and 15% glycerol. Then the mixture was
(
2
preincubated with compounds or vehicle DMSO on ice. PEG containing GTP was
added to the final concentration of 3 mg/mL before detecting the tubulin polymerization
reaction. After 30 min, the absorbance of different concentrations was detected by a
spectrophotometer at 340 nm at 37 °C. The area under the curve was used to determine
the concentration that inhibited tubulin polymerization by 50% (IC ), which was
5
0
calculated with GraphPad Prism Software version 5.02.
.2.3. Molecular modeling study
The X-ray structure of the ABT-751-α,β-tubulin complex was downloaded from the
4
Protein Data Bank (PDB code: 3HKC). The protein was prepared by removal of the
stathmin-like domain, subunits C and D, water molecules and ABT-751 using
Discovery Studio modules. The docking procedure was performed by employing
DOCK program in Discovery Studio 3.0 software, and the structural image was
obtained using PyMOL software.
4
.2.4. Immunofluorescence staining
K562 cells were seeded into 6-well plates and then treated with vehicle control 0.1%
DMSO, 19c (0.05 μM, 0.1 μM, 0.2 μM) for 24 h. The cells were fixed with 4%
paraformaldehyde and then penetrated with PBS for three times. After blocking for 20
min by adding 50-100 μL goat serum albumin at room temperature, cells were
incubated with a monoclonal antibody (anti-α-tubulin) at 37 °C for 2 h. Then the cells
were washed three times by PBS following staining by fluorescence antibody and
labeling of nuclei by 4,6-diamidino-2-phenylindole (DAPI). Cells were finally
visualized using a fluorescence microscope (OLYMPUS, Japan).
4
.2.5. Cell cycle analysis
K562 cells were seeded into 6-well plates and incubated at 37 °C in a humidified 5%
CO incubator for 24 h, and then with vehicle control 0.1% DMSO, 19c (0.05 μM, 0.1
2
μM, 0.2 μM) for 48 h. The collected cells were fixed by adding 70% ethanol at 4 °C for
1
2 h. Subsequently, the cells were resuspended in PBS containing 100 mL RNase A
and 400 mL of propidium iodide for 30 min. The DNA content of the cells was
measured using a FACS Calibur flow cytometer (BectoneDickinson, San Jose, CA,
USA).

4
.2.6. Cell apoptosis analysis
After treatment with vehicle control 0.1% DMSO, 19c (0.05 μM, 0.1 μM, 0.2 μM)
for 48 h, the cells were washed twice in PBS, centrifuged and resuspended in 500 mL
AnnexinV binding buffer. The cells were then harvested, washed and stained with 5
mL Annexin V-APC and 5 mL 7-AAD in the darkness for 15 min. Apoptosis was
analyzed using a FACS Calibur flow cytometer (BectoneDickinson, San Jose, CA,
USA).
4
.2.7. Mitochondrial membrane potential analysis
After treatment with vehicle control 0.1% DMSO, 19c (0.05 μM, 0.1 μM, 0.2 μM)
for 48 h, the cells were washed in PBS and resuspended in 500 mL JC-1 incubation
buffer at 37 °C for 15 min. Then, 19c was immediately assessed for a red fluorescence
using a microplate reader (ELx80, Bio-Tek, USA). The fluorescent signal of monomers
was measured with an excitation wavelength of 488 nm. The percentage of cells with
healthy or collapsed mitochondrial membrane potentials was monitored by flow
cytometry analysis (Bectone-Dickinson, San Jose, CA, USA).
4
.2.8. Wound healing assay
K562 cells were grown in 6-well plates for 24 h. Scratches were made in confluent
monolayers using 200 µL pipette tip. Then, wounds were washed twice with PBS to
remove non-adherent cell debris. The media containing different concentrations (0.05
μM, 0.1 μM, 0.2 μM) of the compound 19c were added to the petridishes. Cells which
migrated across the wound area were photographed using phase contrast microscopy at
0
h and 24 h.
4
.2.9. Tube formation assay
EC Matrigel matrix was thawed at 4 °C overnight, and HUVECs suspended in
DMEM were seeded in 96-well culture plates at a cell density of 50,000 cells/well after
polymerization of the Matrigel at 37 °C for 30 min. They were then treated with 20 μL
different concentrations (0.05 μM, 0.1 μM, 0.2 μM) of compound 19c or vehicle for 6
h at 37 °C. Then, the morphological changes of the cells and tubes formed were
observed and photographed under inverted microscope (OLYMPUS, Japan).
4
.2.10. In vivo anti-tumor evaluation
Five-week-old male Institute of Cancer Research (ICR) mice were purchased from
6
Shanghai SLAC Laboratory Animals Co. Ltd. A total of 1 × 10 H22 cells were
subcutaneously inoculated into the right flank of ICR mice according to protocols of
tumor transplant research, to initiate tumor growth. After incubation for one day, mice
were weighted and at random divided into four groups of eight animals. The groups
treated with 19c were administered 15, 30 mg/kg in a vehicle of 10% DMF/2% Tween
8
0/88% saline, respectively. The positive control group was treated with PTX (8 mg/kg)
every 2 days by intravenous injection. The negative control group received a vehicle of
0% DMF/2% Tween 80/88% saline through intravenous injection. Treatments of 19c
1

were done at a frequency of intravenous injection one dose per day for a total 21
consecutive days while the positive group was treated with PTX one dose per two days.
The mice were sacrificed after the treatments and the tumors were excised and weighed.
The inhibition rate was calculated as follows: Tumor inhibitory ratio (%) = (1-average
tumor weight of treated group/average tumor weight of control group) × 100%.
Acknowledgments
The authors acknowledge the National Natural Science Foundation of China (No.
8
1673306, 81703348), The Open Project of State Key Laboratory of Natural Medicines,
China Pharmaceutical University (No. SKLNMKF 201710), Key Laboratory for the
Chemistry and Molecular Engineering of Medicinal Resources (Guangxi Normal
University), Ministry of Education of China (No. CMEMR2013-B05), and China
Postdoctoral Science Foundation (No. 2017T100424) for financial support.
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Highlights:





A series of quinazolines have been designed to occupy three zones of colchicine domain.
19c showed the most potent in vitro anti-proliferative activity.
19c was identified as a tubulin polymerization inhibitor with potent vascular disrupting activity.
19c was validated to occupy three zones of colchicine domain on in silico studies.
19c exhibited potent tumor growth inhibition in H22 liver cancer xenograft mouse model.

Graphic abstract