Synthesis, Biological Evaluation, and Molecular Docking of Arylpyridines as Antiproliferative Agent Targeting Tubulin

Article abstract of DOI:10.1021/acsmedchemlett.0c00278

Mimicking different pharmacophoric units into one scaffold is a promising structural modification tool to design new drugs with enhanced biological properties. To continue our research on the tubulin inhibitors, the synthesis and biological evaluation of arylpyridine derivatives (9-29) are described herein. Among these compounds, 6-arylpyridines (13-23) bearing benzo[d]imidazole side chains at the 2-position of pyridine ring displayed selective antiproliferative activities against HT-29 cells. More interestingly, 2-trimethoxyphenylpyridines 25, 27, and 29 bearing benzo[d]imidazole and benzo[d]oxazole side chains displayed more broad-spectrum antitumor activities against all tested cancer cell lines. 29 bearing a 6-methoxybenzo[d]oxazole group exhibited comparable activities against A549 and U251 cells to combretastatin A-4 (CA-4) and lower cytotoxicities than CA-4 and 5-Fu. Further investigations revealed 29 displays strong tubulin polymerization inhibitory activity (IC50 = 2.1 μM) and effectively binds at the colchicine binding site and arrests the cell cycle of A549 in the G2/M phase by disrupting the microtubules network.

Full text of DOI:10.1021/acsmedchemlett.0c00278

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Letter
Synthesis, Biological Evaluation and Molecular Docking of
Arylpyridines as Anti-proliferative Agent Targeting Tubulin
JiaPeng He, Mao Zhang, Lv Tang, Jie liu, JiaHong Zhong, Wenya Wang,
Jiang-Ping Xu, Hai-Tao Wang, Xiao-Fang Li, and Zhong-Zhen Zhou
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00278 • Publication Date (Web): 15 Jul 2020
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ACS Medicinal Chemistry Letters
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Synthesis, Biological Evaluation and Molecular Docking of
Arylpyridines as Anti-proliferative Agent Targeting Tubulin
JiaPeng He,†, # Mao Zhang,†, # Lv Tang,†, # Jie liu,† JiaHong Zhong,† Wenya Wang,† Jiang-Ping Xu,†, ‡
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Hai-Tao Wang,† Xiao-Fang Li § and Zhong-Zhen Zhou*, †
†Innovation Program of Drug Research on Neurological and Metabolic Diseases, Guangdong Provincial Key Laboratory of
New Drug Screening, School of Pharmaceutical Sciences, Southern Medical University, Guangzhou, 510515, China
‡ Key Laboratory of Mental Health of the Ministry Education, Southern Medical University, Guangzhou 510515, China
§ Department of Hematology, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China.
ABSTRACT: Mimicking different pharmacophoric units into one
scaffold is a promising structural modification tool to design new drugs
with enhanced biological properties. To continue our research on the
tubulin inhibitors, the synthesis and biological evaluation of
arylpyridine derivatives (9-29) are described herein. Among these
compounds, 6-arylpyridines (13-23) bearing benzo[d]imidazole side
chains at 2-position of pyridine ring displayed selective antiproliferative
activities
against
HT-29
cells.
More
interestingly,
2-
trimethoxyphenylpyridines 25, 27, and 29 bearing benzo[d]imidazole
and benzo[d]oxazole side chains displayed more broad-spectrum
antitumor activities against all tested cancer cell lines. 29 bearing 6-
methoxybenzo[d]oxazole group exhibited comparable activities against
A549 and U251 cells to combretastatin A-4 (CA-4), and lower
cytotoxicities than CA-4 and 5-Fu. Further investigations revealed 29
display strong tubulin polymerization inhibitory activity (IC50 = 2.1
μM), and effectively bind at the colchicine binding site, and arrest the
cell cycle of A549 in the G2/M phase by disrupting microtubules network.
KEYWORDS: arylpyridines, antiproliferative activities, tubulin polymerization inhibitor, cell cycle arrest, molecular docking
It is widely known that malignant tumors always compromise
human health 1. However, recovery rates of cancer patients have
improved in recent years with the emergence of new antitumor
drugs. It is commonly known that microtubules, an effective
target for cancer chemotherapy, play important roles in various
cellular functions, and participate in the formation of mitotic
tubulin14. The cis-configuration of the double bond connecting
two ring moieties present in the structures is very important for
the high cytotoxic and anti-tubulin activities of combretastatin
derivatives. To avoid the cis-trans isomerization, many efforts
have focused on the replacement of the double bond with a ring
or other functionalities to increase the biological efficiency and
minimize possible metabolism14-15. Interestingly, numerous
novel microtubule-destabilizing agents with six-membered
heterocyclic bridging groups displayed potential activities.
2-3
bipolar spindles . By altering microtubule dynamics at the
mitotic stage, microtubule-targeting agents (MTAs), also called
antimitotic drugs, directly interrupt spindle microtubules and
arrest cells in the G2/M phase. Microtubules contain six unique
binding sites (taxanes, vinca alkaloids, epothilone, laulimalide,
pironetin, and colchicine domains)4-5. And the epothilone
binding site is an undistinct site, which has been proposed in the
taxane pocket of β-tubulin6. Currently, several MTAs are
approved for the treatment of tumors, and these include
paclitaxel 7-8, vinca alkaloids9, eribulin (vinca binding site
inhibitors)10, and ixabepilone (taxanes binding site inhibitor) 11.
Among them, sulfonamide tubulin polymerization inhibitor
16
(ABT-751, Figure 1)
with a pyridine bridging group is
reported to cause a significant reduction in rat tumor blood flow
and was well tolerated in the clinical trial 17-18. Pyridine-bridged
analogs (3) with a 3-atom distance between two phenyl rings
exhibited comparable antitumor activity to CA-4 and blocked
angiogenesis in vivo19.
Our recent research were mainly focused on potential
heterocyclic anticancer agents based on different kinds of
heterocyclic scaffolds.20-22 Our reported compounds 4 and 5
with purine bridging group displayed promising antitumor
activities and lower cytotoxicity in normal cells, but weak
tubulin polymerization inhibition activity 20. Nevertheless, the
potent biological profile encouraged us to continue our search
However, none of the approved tubulin inhibitors target the
colchicine domain for treating the tumor. Despite all this,
extensive efforts have been made to promote the discovery of
colchicine site inhibitors12-13. The cis-stilbene combretastatin A-
4 (CA-4, Fig. 1) is one of the most anti-tubulin active
compounds by strongly binding to the colchicine site in β-
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for high-potency antitumor drugs. Herein, different skeleton arylpyridines (9-29, Figure 1) with benzo[d]imidazole,
1
2
3
4
5
6
7
8
modifications on the CA-4 scaffold were described, especially
the replacement of the olefinic bond with pyridine ring and the
incorporation of five-membered heterocyclic rings (such as
benzo[d]imidazoles, benzo[d]thiazoles and benzo[d]oxazoles).
benzo[d]thiazole and benzo[d]oxazole side chains were
designed and synthesized in the current study. The
antiproliferative activities, arresting the cell cycle abilities, and
tubulin polymerization inhibitory activities of these obtained
derivatives were subsequently evaluated. To better understand
structure-activity relationships, molecular docking was also
performed.
Many tubulin polymerization inhibitors with these five-
23-27
membered heterocyclic rings displayed strong activities
,
such as compounds 6 27, 7 26, and 825. Consequently,
N
N
Me
9
N
N
NH
N
MeO
OMe
N
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
N
MeO
OMe
OH
OMe
MeO
MeO
HN
S
HO
O
OMe
CA-4
MeO
OH
OMe
R
1
2
3
4
5
ABT-751
R = H
R = MeO
OMe
NH
O
H
N
N
N
H
N
MeO
S
MeO
MeO
N
N
N
N
N
O
MeO
MeO
MeO
OMe
OMe
6
7
8
OMe
Me
Designed compounds
N
R3
R1
R3
R1
X
X
R4
N
X
R4
N
N
N
R2
N
R2
R2
R1
R3
24-29
R4
X = NH, O, S
9-23
Figure 1. Examples of some reported tubulin polymerization inhibitors and the proposed design of novel arylpyridine conjugates (9-29)
containing benzo[d]imidazole, benzo[d]thiazole and benzo[d]oxazole side chains.
Scheme 1. Synthetic routes for arylpyridines 9-29.
HS
HO
R3
R1
S
H2N
H2N
N
R3
R1
O
44
45
N
N
K2CO3, C8H15BrN2 (10 mol%)
Toluene, 120oC
N
K2CO3, C8H15BrN2 (10 mol%)
Toluene, 120oC
R2
9-10
11-12
R2
H2N
R1
R3
R2
H
N
R3
R3
R1
Br
N
33
CHO
CHO
N
N
H2N
40-43
DMF, 120 o
R1
B(OH)2
30-32
N
R4
R1
Pb(PPh3)4, K2CO3
DME/EtOH/H2O, 80 o
C
R2
35-37
R2
13-23
C
N
N
CHO
H2N
H2N
NH
N
34
Br
N
MeO
MeO
40
R2
CHO
DMF, 120 o
C
OMe
24-25
Pb(PPh3)4, K2CO3
DME/EtOH/H2O, 80 o
R4
38-39
R1
R4
C
R3
HO
N
H2N
45-46
O
N+
Br-
N
C8H15BrN2
=
N
MeO
MeO
K2CO3, C8H15BrN2 (10 mol%)
Toluene, 120 o
C
OMe
R4
26-29
The synthetic routes of the arylpyridines (9-29) are illustrated
in Scheme 1. Overall, the arylpyridylaldehyde (35-39)
intermediates were prepared by the Suzuki reaction of
bromopicolinaldehydes (33-34) with the substituted
phenylboronic acid (30-32). Arylpyridines bearing
benzo[d]thiazole side chains (9-10) and benzo[d]oxazole side
chains (11-12 and 26-29) were prepared by one-pot aerobic
oxidative condensation of arylpyridylaldehydes (35-39) with
substituted
2-aminophenols
and
2-aminobenzenethiol
respectively, using 1-butyl-3-methylimidazolium bromide as
the catalyst. 6-arylpyridines (13-25) possessing the
benzo[d]imidazole side chains were synthesized by the
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heterocyclic condensation of arylpyridylaldehydes with
substituted o-phenylenediamines.
1
2
3
Table 1. Antiproliferative activity of arylpyridine derivatives 9-29
4
5
6
7
8
9
GI50 (μM) a
CC50 (μM) c
Polymerization
No.
9
Structure
[% (10 M), IC50 (μM)]b
HT-29
> 20
A549
U251
> 20
HT22
O
N
N
N
N
N
[d]
> 20
96 ± 1
89 ± 2
77 ± 2
81 ± 7
-
O
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
O
O
N
[d]
10
11
12
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
> 20
-
O
O
S
N
[d]
-
O
O
S
N
[d]
-
O
O
O
O
O
O
H
N
N
N
N
N
13
14
15
16
17
18
19
1.4 ± 0.3
5.1 ± 0.1
1.1 ± 0.1
5.4 ± 0.2
5.9 ± 0.2
4.6 ± 0.2
6.0 ± 1.2
> 10
> 10
> 10
> 10
> 10
> 10
> 10
> 10
78 ± 5
80 ± 8
90 ± 3
87 ± 4
74 ± 6
82 ± 5
96 ± 2
17.1 ± 1.2
O
H
N
N
[d]
> 10
-
O
H
N
N
> 10
16.0 ± 2.7
O
O
H
N
N
N
[d]
> 10
-
O
O
O
O
H
N
N
N
[d]
> 10
-
O
H
N
N
N
[d]
7.9 ± 1.5
-
O
O
H
Cl
N
N
N
[d]
> 10
-
O
O
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GI50 (μM) a
CC50 (μM) c
Polymerization
1
2
3
4
5
6
7
8
No.
20
Structure
[% (10 M), IC50 (μM)]b
HT-29
A549
U251
> 10
HT22
H
Cl
N
N
N
1.2 ± 0.3
> 10
69 ± 4
19.0 ± 1.5
O
O
H
N
Cl
9
N
N
[d]
21
22
23
24
25
9.6 ± 0.6
0.96 ± 0.08
7.9 ± 0.1
12.5 ± 1.2
2.9 ± 0.2
8.8 ± 0.2
> 10
85 ± 1
-
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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58
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60
O
O
O
H
N
N
N
O
> 10
> 10
86 ± 3
20.5 ± 2.1
O
O
H
N
N
N
O
[d]
> 10
8.3 ± 0.9
85 ± 4
-
O
O
O
H
N
N
N
[d]
> 20
> 20
81 ± 3
-
O
O
O
H
N
N
N
2.8 ± 0.2
1.7 ± 0.2
44 ± 2 (11.7 ± 0.5)
2.7 ± 0.4
O
O
O
N
O
N
[d]
26
27
28
> 20
> 20
> 20
84 ± 2
58 ± 4
89 ± 3
-
O
O
O
N
N
5.2 ± 0.5
7.9 ± 1.1
4.6 ± 0.6
25.6 ± 0.6
O
O
O
O
O
N
N
[d]
> 20
> 20
> 20
-
O
O
O
O
N
N
29
2.1 ± 0.1
0.89 ± 0.1
0.27 ± 0.04
6 ± 2 (2.1 ± 0.2)
24.8 ± 0.7
O
O
O
DMSO
CA-4
5-Fu
\
\
\
100
\
OH
OMe
MeO
0.20 ± 0.06
0.82 ± 0.02
0.17 ± 0.02
5 ± 2 (1.6 ± 0.1)
13.9 ± 1.7
OMe
MeO
12.6 ± 1.6
10.7 ± 2.6
30.1 ± 2.9
\
8.1 ± 0.2
a GI50 is the dose for 50% cells growth inhibition after 48h of incubation. The GI50 values (means ± SDs) were averaged over at least two
independent experiments；b Tubulin polymerization assay conditions in vitro: tested compounds (10 M), and tubulin (4 mg/mL), 40 min,
37 oC; DMSO (0.1% v/v) was used as blank (100%: no inhibition). c CC50 values represent the 50% cytotoxic concentration after 48h. All
data were averaged over three separate experiments. All data were averaged over three separate experiments; d Not tested.
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Antiproliferative activities (GI50) of arylpyridines (9-29) were
position of side chains (benzo[d]imidazole, benzo[d]thiazole
and benzo[d]oxazole) in pyridine ring are essential for their
1
2
3
4
5
6
7
8
evaluated against three human cancer cell lines [HT-29 (Human
colon carcinoma), A549 (Human non-small cell lung cancer
cells), and U251 (glioma)], using fluorouracil (5-Fu) and CA-4
as the reference cytotoxic compounds. The growth inhibitory
concentration values (GI50) against tumor cells indicate 50%
growth inhibition of cell growth compared to untreated controls
after 48 h of incubation (Table 1).
selectivity
and
antiproliferative
activities.
Firstly,
dimethoxyphenylpyridines bearing benzo[d]imidazole side
chains showed selective antiproliferative activities against HT-
29 cells, such as compounds 13, 14, 16, 17, 19, 20, and 22.
Secondly, trimethoxyphenylpyridines bearing substituted
benzo[d]imidazole displayed more broad-spectrum antitumor
activities. For example, trimethoxyphenylpyridines 15 and 24
bearing non-substituted benzo[d]imidazole selectively
inhibited HT-29 cells. However, trimethoxyphenylpyridines 18,
21 and 25 bearing substituted benzo[d]imidazole displayed
good antitumor activity toward A549 cells or U251 cells.
As shown in Table 1, 6-arylpyridines bearing benzo[d]thiazole
side chains (9-10) and benzo[d]oxazole side chains (11-12) at
the 2-position of the pyridine ring displayed noninhibitory
activities toward tested cancer cell lines. However, 2-
arylpyridines bearing benzo[d]oxazole side chains (27 and 29)
at the 3-position of the pyridine ring showed satisfactory
activities toward all tested cancer cell lines and higher activities
than 5-Fu. Intriguingly, the activities of compound 29 against
the A549 and U251 cell lines reached sub-micromole values
(GI50 = 0.89 and 0.17 μM respectively), which were comparable
to that of the positive control CA-4. Besides, the toxicity
evaluation in mouse normal hippocampal neurons (HT22) cells
demonstrated that 27 and 29 exhibited lower cytotoxicities than
the positive control CA-4.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Trimethoxyphenylpyridines
25
bearing
5-methyl
benzo[d]imidazole side chains at 3-position of pyridine ring
displayed broad-spectrum antitumor activities toward tested
tumor cells.
Thirdly, 2-trimethoxyphenylpyridines (such as 27 and 29)
displayed higher activities than 2-dimethoxyphenylpyridines
(such as 26 and 28). As reported in the literature,
trimethoxyphenyl (TMP) moiety of combretastain A-4
analogues (tubulin colchicine binding site inhibitors) is an
important pharmacophore for interaction with tubulin15, 28. The
replacement of trimethoxyphenyl group with 3,4-
For arylpyridines (13-25) bearing benzo[d]imidazole side
chains, most of them exhibited very weak antiproliferative
activities against A549 and U251 cell lines, but highly
antiproliferative activities against HT-29 cells than 5-Fu (GI50
= 12.6 μM). Among these compounds, compounds 13, 15, 20,
and 22 displayed more potential activities (0.96 < GI50 < 1.4
μM) against HT-29 cells, and lower cytotoxicity in normal
HT22 cells than CA-4 and 5-Fu. Compound 25 bearing 5-
methyl benzo[d]imidazole side chains at 3-position of pyridine
ring showed good activities against HT29 (GI50 = 2.9 μM),
A549 (GI50 = 2.8 μM), and U251 (GI50 = 1.7 μM) cell lines, but
higher cytotoxicities in normal HT22 cells than the positive
control CA-4 and 5-Fu.
dimethoxyphenyl group can lead to a decrease in activity19, 24
.
The possible reason is that replacing trimethoxyphenyl group
with 3,4-dimethoxyphenyl group will cause the hydrogen bond
with tubulin to weaken. Furthermore, benzo[d]oxazole side
chains at 3-position of pyridine ring are favorable for
antiproliferative activities. 2-trimethoxyphenylpyridines 27 and
29 bearing 3-benzo[d]oxazole side chains displayed satisfactory
activities against all tested tumor cells, which were higher than
that of 5-Fu. Finally, introducing the methoxy group into
benzo[d]oxazole is conducive to the improvement of the
activity. The activities of compound 29 (0.89 < GI50 < 2.1 μM)
bearing 6-methoxybenzo[d]oxazole group was higher than that
of 27 (4.6 < GI50 < 7.9 μM) bearing benzo[d]oxazole group.
Further structure-activity relationship results indicated that the
number of methoxy groups on the benzene ring and suitable
Figure 2. A) Effect of arylpyridines 25, 29 and CA-4 on in vitro tubulin polymerization. DMSO (0.1% v/v) was used as vehicle control. B)
Effect of 25 on the cellular microtubule networks of A549 cells. C) Effect of 29 on the cellular microtubule networks of A549 cells.
Microtubules and DNA are stained in red and blue respectively. Untreated cells (DMSO, 0.1% v/v) were used as a negative control, and cells
treated with CA-4 (0.6 μM) were used as a positive control.
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A
CA4 (0.6 μM)
Control
1
2
3
4
G0/G1:7.11 %
G2/M:82.60 %
S-Phase:10.29 %
G0/G1:68.26 %
G2/M: 17.03 %
S-Phase:14.71 %
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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53
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58
59
60
25 (1 μM)
25 (2 μM)
β-Chain
α-Chain
G0G1: 65.15 %
G2M:19.70 %
S-Phase: 15.16 %
G0G1: 56.06 %
G2M: 33.44 %
S-Phase: 10.51 %
B
29 (2 μM)
29 (1 μM)
G0/G1: 3.88 %
β-Chain
G0/G1: 6.12 %
G2/M: 73.67 %
α-Chain
G2/M: 88.18
%
C
S-Phase:20.21
%
S-Phase:7.94
%
Figure 3. Analysis of the effects of 25 and 29 on the cell cycle
distribution in the A549 cells using the flow cytometry analysis.
DMSO (0.1% v/v) and CA-4 (0.6 μM) were utilized as a blank
control and a positive control respectively. DNA content was
determined by DNA intercalating dye and propidium iodide
staining.
To validate the relationship between the antiproliferative
activities, in vitro microtubule dynamics of arylpyridine
derivatives were investigated using microtubule-destabilizing
agent CA-4 as controls. Tubulin polymerization assay results
indicated the tubulin polymerization inhibition ability of these
compounds was increased by introducing side chains into 3-
position of the pyridine ring. All arylpyridine derivatives 9-23
with side chains at 2-position of pyridine ring showed very
weak activities (74% to 96% polymerization grade compared
with the DMSO control). arylpyridine derivatives 24-29 with
side chains at 3-position of pyridine ring displayed strong to
weak polymerization inhibitory activities at a concentration of
10 m. Among these compounds, 25 and 27 with good activities
against all tested tumor cells produced good anti-tubulin
polymerization activities. As shown in Table 1 and Figure 2A,
29 bearing 6-methoxybenzo[d]oxazole group at the 3-position
of the pyridine ring showed best polymerization inhibitory
activity (IC50 = 2.1 μM), which was closed to that of CA-4 (IC50
Figure 4. A) The proposed binding models of 29 (shown in green
ball and stick model) with tubulin (PDB: 5LYJ). B) The native CA-
4 ligand is illustrated in a blue ball and stick model. The glide
docking scores of 29 and CA-4 are 8.349 and 8.864, respectively.
In the 2D representation (C) of the ligand−protein interactions, all
residues within 3 Å of the ligand (29) are shown. The
intermolecular hydrogen bond is colored in magenta dash line. The
π-cation interaction is colored in a red dash line (B) or a red solid
line (C).
Non-small cell lung cancer (NSCLC), the main subtype of
lung cancer, is the leading cause of cancer deaths worldwide.
And drugs for NSCLC has been a hot and difficult problem.
Thus, A549 cells were used in immunofluorescence staining
assay to examine the effects of the most potent compounds 25
and 29 on the cellular microtubule networks (Figure 2B). After
treatment with 25, 29, and CA4 for 18h, the microtubule
morphology (red) was visualized. As shown in Figure 2B, the
=
1.6 μM). These results demonstrated that their
antiproliferative activities may be caused by their anti-tubulin
polymerization activities.
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normal arrangement and
microtubule network was
a
4, forming π-cation interaction with β/Lys352 and hydrophobic
interactions with β/Asn258, β/Leu248, and α/Thr179. The 6-
methoxybenzo[d]oxazole scaffold occupied the position of the
3-hydroxy-4-methoxyphenyl moiety of CA-4. Compared with
CA-4, 6-methoxybenzo[d]oxazole group cannot form hydrogen
bonds with surrounding amino acid residues, but it formed more
hydrophobic interactions with the α/Val181, α/Val180,
β/Asn258, β/Met259, β/Val315, β/Ala317, β/Ile347, β/Asn350,
and β/Lys352 residues. To further confirm whether compound
29 could bind to the colchicine site on tubulin, the colchicine
competitive binding assay of compound 29 was performed
using CA-4 and paclitaxel as a positive control and a negative
control respectively. As shown in Figure 5, the fluorescence
(F) of the colchicine-tubulin complex was reduced by
compound 29 in a dose-dependent manner. Combined with the
results of the molecular modeling study, these indicated that the
binding site of 29 is located at the colchicine binding site of
tubulin.
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organization in untreated A549 cells. In contrast, the interphase
microtubule network around the cell nucleus was disrupted
dramatically by 25, 29, and CA4, resulting in disassembly and
fragmentation. These results confirm that 25 and 29 were like
CA-4 inducing tubulin depolymerization and disturbing
microtubule networks.
Tubulin depolymerization and disruption of microtubule
networks can induce the G2/M arrest. Thus, the arrest effects of
compound 25 and 29 on the cell cycle of A549 cells was
measured at different concentrations via flow cytometry after
48 h of treatment, and following propidium iodide (PI ) staining
of the cells (Figure 3). As illustrated in Figure 3, compounds
25, 29, and CA-4 showed the same behavior, arrested the cell
cycle of A549 cells at the G2/M phase. At the same
concentration, the percentage of cells in the G2/M phase
arrested by compound 29 was higher than the percentage of
cells in the G2/M phase arrested by compound 25. As the
concentration of 29 increased from 0 to 2 μM, the percentage of
cells in the G2/M phase was increased from 17.03% to 88.18%.
Thus, compound 29 caused a significant G2/M arrest in the
A549 cells in a concentration-dependent manner. The cell cycle
arrest effects of 29 correlated well with its strong anti-tubulin
and antiproliferative activities.
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In summary, a series of arylpyridines 9-29 containing
benzo[d]imidazole, benzo[d]thiazole, and benzo[d]oxazole side
chains was successfully designed and synthesized. Among
these compounds, four 6-arylpyridines (13, 15, 20, and 22)
bearing benzo[d]imidazole side chains at 2-position of pyridine
ring showed selective antiproliferative activities against the
HT-29 colon carcinoma cell line in the range of 0.96 μM to 1.4
μM. 2-Trimethoxyphenylpyridines 25, 27, and 29 bearing
benzo[d]imidazole and benzo[d]oxazole side chains at 3-
position of pyridine ring displayed more broad-spectrum
antitumor activities against all tested cancer cell lines (HT29,
A549, and U251 cells). 2-Trimethoxyphenylpyridine 25
bearing 5-methyl benzo[d]imidazole side chains at 3-position of
pyridine ring showed good activities (1.7 < GI50 < 2.9 μM)
against all tested cancer cell lines (HT29, A549, and U251
cells), but higher cytotoxicities in normal HT22 cells than the
positive control CA-4 and 5-Fu. However, 2-
trimethoxyphenylpyridines 27 and 29 bearing the
benzo[d]oxazole side chains with satisfactory antiproliferative
activities against three different cancer cell lines displayed
lower cytotoxicities in normal HT22 cells than the positive
control CA-4 and 5-Fu. What's exciting is that compound 29
displayed comparable antiproliferative activities against A549
(GI50 = 0.82 μM) and U251 (GI50 = 0.17 μM) cell lines to CA-
4.
1.2
1.0
0.8
0.6
0.4
0.2
0.0
29
CA-4
Paclitaxel
0
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8
9 10 11 12 13 14 15 16
Concentration (M)
Figure 5. Based on fluorescence, colchicine competitive binding
assays of compound 29 were performed in the 5.0 μM colchicine-
tubulin complex at various concentrations, using CA-4 and
paclitaxel positive control and negative control respectively.
Percent inhibition was determined by the ratio of F/F0, whereas F0
is the fluorescence of samples without inhibitor, and F is the
fluorescence of samples with inhibitor at various concentrations.
Tubulin polymerization assay results indicated all 6-
arylpyridine derivatives 9-23 showed very weak activities,
whereas 2-arylpyridine derivatives 24-29 displayed weak to
strong polymerization inhibitory activities. Among these
In general, CA4-like derivatives with trimethoxyphenyl
groups and cis-conformation inhibits colchicine binding to
tubulin.15 To investigate the potential binding site of 29 in
tubulin (PDB: 5LYJ)28 at the colchicine site, molecular docking
simulations were performed in Maestro 11.1. After the native
ligand (CA-4) was re-docked into the active site cavity, the
Glide RMSD (resulting root mean square deviation) value was
given as 0.666 Å, which confirmed the reliability of our docking
method. As shown in Figure 4, 29 adopted a similar
conformation in the colchicine site to that of CA-4. The
trimethoxyphenyl moiety of 29 formed one hydrogen bond with
the β/Cys241 residue, and hydrophobic interactions with
β/Cys241, β/Leu242, β/Ala250, β/Lys352, and β/Ala354. The
intermolecular hydrogen bonding distance between 29 and
β/Cys241 residue is 2.50Å, which is shorter than that of CA-4
with β/Cys241 residue. Moreover, the pyridine bridging group
occupied the position of the double bond bridging group of CA-
compounds,
2-trimethoxyphenylpyridine
25
bearing
benzo[d]imidazole side chains showed moderate tubulin
polymerization inhibition activity (IC50 = 2.1 μM), whereas 2-
trimethoxyphenylpyridines 29 bearing the benzo[d]oxazole
side chains displayed strong tubulin polymerization inhibition
activity (IC50 = 2.1 μM). Further investigations revealed 29
effectively bind at the colchicine binding site, and arrest the cell
cycle of A549 in the G2/M phase by disrupting microtubules
network. These results provide further guidance for the
discovery of new CA-4 analogs with lower cytotoxicities.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/
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Page 8 of 16
10.
Aseyev, O.; Ribeiro, J. M.; Cardoso, F., Review on the
clinical use of eribulin mesylate for the treatment of breast cancer.
Expert Opin. Pharmacother. 2016, 17 (4), 589-600.
1
2
3
4
Experimental
details
(synthetic
experimental
details,
pharmacological assays, molecular docking, and (1H and 13C)
spectral information) are found in the supporting information of
this article (PDF).
11.
Kaur, R.; Kaur, G.; Gill, R. K.; Soni, R.; Bariwal, J., Recent
developments in tubulin polymerization inhibitors: An overview. Eur.
J. Med. Chem. 2014, 87, 89-124.
12.
Xia, L. Y.; Zhang, Y. L.; Yang, R.; Wang, Z. C.; Lu, Y. D.;
5
6
7
8
AUTHOR INFORMATION
Wang, B. Z.; Zhu, H. L., Tubulin inhibitors binding to colchicine-site:
A Review from 2015 to 2019. Curr. Med. Chem. 2019, 26, 1-27.
13.
Corresponding Author
Naaz, F.; Haider, M. R.; Shafi, S.; Yar, M. S., Anti-tubulin
* (Zhong-Zhen Zhou) E-mail: zhouzz@smu.edu.cn
agents of natural origin: Targeting taxol, vinca, and colchicine binding
9
domains. Eur. J. Med. Chem. 2019, 171, 310-331.
Author Contributions
14.
Bukhari, S. N. A.; Kumar, G. B.; Revankar, H. M.; Qin, H.-
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#
These authors contributed equally to this work. In addition, all
L., Development of combretastatins as potent tubulin polymerization
inhibitors. Bioorg. Chem. 2017, 72, 130-147.
authors have given approval to the final version of the manuscript.
15.
Li, L.; Jiang, S.; Li, X.; Liu, Y.; Su, J.; Chen, J., Recent
ACKNOWLEDGMENT
advances in trimethoxyphenyl (TMP) based tubulin inhibitors targeting
the colchicine binding site. Eur. J. Med. Chem. 2018, 151, 482-494.
16.
Manero, G.; O'Brien, S. M.; Faderl, S.; Thomas, D.; Wierda, W.;
Kornblau, S.; Ferrajoli, A.; Albitar, M.; McKeegan, E.; Grimm, D. R.;
Mueller, T.; Holley-Shanks, R. R.; Sahelijo, L.; Gordon, G. B.;
Kantarjian, H. M.; Giles, F. J., Phase 1 study of ABT-751, a novel
microtubule inhibitor, in patients with refractory hematologic
malignancies. Clin. Cancer Res. 2005, 11 (18), 6615-6624.
This work was financially supported by Foundation for
Distinguished Young Teachers in Higher Education of Guangdong
Province (Yue Teacher (2014)145), Natural Science Foundation of
Guangdong Province (2018A030313046), and National Natural
Science Foundation of China (No. 81872735).
Yee, K. W.; Hagey, A.; Verstovsek, S.; Cortes, J.; Garcia-
ABBREVIATIONS
CA-4, Combretastatin-A4; NSCLC. Non-small cell lung cancer;
HT22, Hippocampal neuron (HT22); HT-29, Human colon
carcinoma); A549, Human non-small cell lung cancer cells; U251,
glioma); 5-Fu, fluorouracil;
17.
Rudin, C. M.; Mauer, A.; Smakal, M.; Juergens, R.; Spelda,
S.; Wertheim, M.; Coates, A.; McKeegan, E.; Ansell, P.; Zhou, X.;
Qian, J.; Pradhan, R.; Dowell, B.; Krivoshik, A.; Gordon, G., Phase I/II
study of pemetrexed with or without ABT-751 in advanced or
metastatic non-small-cell lung cancer. J. Clin. Oncol. 2011, 29 (8),
1075-1082.
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Control
CA4 (0.6 μM)
G0/G1:7.11 %
G2/M:82.60 %
S-Phase:10.29 %
G0/G1:68.26 %
G2/M: 17.03 %
S-Phase:14.71 %
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25 (1 μM)
25 (2 μM)
G0G1: 65.15 %
G2M:19.70 %
S-Phase: 15.16 %
G0G1: 56.06 %
G2M: 33.44 %
S-Phase: 10.51 %
29 (2 μM)
29 (1 μM)
G0/G1: 3.88 %
G2/M: 88.18 %
S-Phase:7.94 %
G0/G1: 6.12 %
G2/M: 73.67 %
S-Phase:20.21 %
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α-Chain
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