Discovery of indoline derivatives as anticancer agents via inhibition of tubulin polymerization

Article abstract of DOI:10.1016/j.bmcl.2021.128131

Human esophageal squamous cell carcinoma (ESCC) is one of the most lethal cancers in human digestive system. It is necessary to discover novel antitumor agents for the treatment of esophageal cancers because of its poor prognosis. Indoline has been reported as an efficient anticancer fragment to design novel anticancer agents. In this work, indoline derivatives were designed, synthesized and explored their anticancer activity. Compound 9d, which exhibited potent antiproliferative activity with IC₅₀ values of 1.84 μM (MGC-803 cells), 6.82 μM (A549 cells), 1.61 μM (Kyse30 cells), 1.49 μM (Kyse450 cells), 2.08 μM (Kyse510 cells) and 2.24 μM (EC-109 cells), respectively. The most active compound 9d was identified as a tubulin inhibitor targeting colchicine binding site with an IC₅₀ value of 3.4 μM. Compound 9d could strongly suppress the tubulin polymerization in Kyse450 cells. The results of molecular docking also suggested compound 9d could tightly bind into the colchicine binding site of β-tubulin. Besides, compound 9d inhibited the growth of KYSE450 cells in time and dose-dependent manners. All the results suggest that the indoline derivatives might be a class of novel tubulin inhibitors with potential anticancer activity and is worthy of further study.

Full text of DOI:10.1016/j.bmcl.2021.128131

Journal Pre-proofs
Discovery of indoline derivatives as anticancer agents via inhibition of tubulin
polymerization
Shu-Yu Wang, Xu Liu, Ling-Wei Meng, Miao-Miao Li, Yin-Ru Li, Guang-Xi
Yu, Jian Song, Hong-Yu Zhang, Ping Chen, Sai-Yang Zhang, Tao Hu
PII:
S0960-894X(21)00357-7
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2021.128131
BMCL 128131
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
13 February 2021
28 April 2021
17 May 2021
Please cite this article as: Wang, S-Y., Liu, X., Meng, L-W., Li, M-M., Li, Y-R., Yu, G-X., Song, J., Zhang, H-
Y., Chen, P., Zhang, S-Y., Hu, T., Discovery of indoline derivatives as anticancer agents via inhibition of tubulin
polymerization, Bioorganic & Medicinal Chemistry Letters (2021), doi: https://doi.org/10.1016/j.bmcl.
2
021.128131
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Discovery of indoline derivatives as anticancer agents via
inhibition of tubulin polymerization
Shu-Yu Wang , Xu Liua, b, 1, Ling-Wei Menga, b , Miao-Miao Li , Yin-Ru Li , Guang-
a, 1
a
a
a
a, b
a
a
a, b, c,
Xi Yu , Jian Song , Hong-Yu Zhang , Ping Chen , Sai-Yang Zhang
*, Tao Hu
a,
*
a. School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, 450001, China
b. School of Pharmaceutical Sciences, Institute of Drug Discovery & Development, Key
Laboratory of Advanced Drug Preparation Technologies (Ministry of Education), Zhengzhou
University, Zhengzhou, 450001, China
c. Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
*
Corresponding authors: saiyangz@zzu.edu.cn; hnhutao@zzu.edu.cn
These authors contributed equally to this work
1
1

Abstract
Human esophageal squamous cell carcinoma (ESCC) is one of the most lethal
cancers in human digestive system. It is necessary to discover novel antitumor agents
for the treatment of esophageal cancers because of its poor prognosis. Indoline has been
reported as an efficient anticancer fragment to design novel anticancer agents. In this
work, indoline derivatives were designed, synthesized and explored their anticancer
activity. Compound 9d, which exhibited potent antiproliferative activity with IC50
values of 1.84 μM (MGC-803 cells), 6.82 μM (A549 cells), 1.61 μM (Kyse30 cells),
1
.49 μM (Kyse450 cells), 2.08 μM (Kyse510 cells) and 2.24 μM (EC-109 cells),
respectively. The most active compound 9d was identified as a tubulin inhibitor
targeting colchicine binding site with an IC value of 3.4 µM. Compound 9d could
5
0
2

strongly suppress the tubulin polymerization in Kyse450 cells. The results of molecular
docking also suggested compound 9d could tightly bind into the colchicine binding site
of β-tubulin. Besides, compound 9d inhibited the growth of KYSE450 cells in time and
dose-dependent manners. All the results suggest that the indoline derivatives might be
a class of novel tubulin inhibitors with potential anticancer activity and is worthy of
further study.
Keywords: Esophageal squamous cell carcinoma; Indoline; Anticancer activity;
Tubulin; Colchicine binding site
Human esophageal squamous cell carcinoma (ESCC) is one of the most fatal
malignant tumors in human digestive system. The great endeavors recently on its
1
treatment seem to have little effect and still carries a poor prognosis . There are more
than 500,000 newly diagnosed cases in the world each year, and about 400,000 cases
2
die of ESCC . Although, there are some small molecule drugs under clinical trials for
the treatment of ESCC 3-7, no significant improvements have been observed in the
8
overall survival rates of ESCC patients . Thus, there is an urgent need to develop
effective therapeutic agents targeting key targets or pathways for the treatment of
esophageal cancers.
Microtubules play an important role in the process of mitosis, cell division, signal
transduction and material transportation, which has been consider as an attractive and
successful drug target in cancer therapy 9-11. There are several tubulin inhibitors for the
clinical treatment, including paclitaxel/taxol, vincristine and vinblastine. However, no
colchicine site tubulin inhibitors are approved for clinical use, therefore, there is an
urgent need to develop new colchicine site tubulin inhibitors with high antitumor
activity and low side effects.
As a simple heterocyclic fusion scaffold, indoline has been widely designed as the
core scaffold of bioactive compounds with strong pharmacological significance,
especially the anticancer ability12-15. Indoline derivatives has also been used to design
novel colchicine binding site tubulin inhibitors with potent anticancer acctivity 9, 12, 14,
16-18.
1-Arylsulfonyl indoline 1 is a potent tubulin and HDAC dual inhibitor with
3

1
2
potential anticancer activity . Compound 1 exhibited striking tubulin inhibition
activity and HDAC1 inhibitory potency with IC50 values of 1.1 and 0.221 μM,
respectively. Besides, compound 1 displayed signifitant antiproliferative activity
against KB cells (IC50 = 79ꢀnM). 7-Aroyl-aminoindoline-1-sulfonamide 2 potently
inhibited the tubulin polymerization (IC = 1.1 μM) by binding to colchicine binding
5
0
site. Compound 2 effectiveily suppressed the growth of KB, MKN45, H460, HT29, and
1
6
TSGH cells with IC values ranging from 8.6 nM to 10.8 nM . Indoline 3 as a
5
0
microtubule-targeting agent showed significant antiproliferative activity against HCT-
16, PC3, HepG2 and SK-OV-3 cells with IC values ranging from 0.039 μM to 0.112
1
5
0
1
8
μM . Therefore, indoline might be a potential sacfflold for the design of tubulin
inhibitors with potential acticancer activity. In addition, trimethoxyphenyl-based
derivatives exhibited potent anticaner activity against cancer cells and
trimethoxyphenyl group is also an important pharmacophoric point for tubulin
binding10, 19-27. Compound 4 19 showed potent antiproliferative activity against SGC-
7
901 cells (IC50 = 0.054 μM) and tubulin inhibitory activity (IC50 = 9.17 μM).
Compound 5 20 potently inhibited the tubulin polymerization (IC = 1.9 μM) and the
5
0
2
7
growth of MGC-803 cells (IC = 0.035 μM). Compound 6 with a trimethoxyphenyl
5
0
group displayed low nanomolar antiproliferative efficacy on HeLa cells and potent
inhibitory activity of tubulin polymerization (IC = 1.2 μM) (Fig. 1).
5
0
O
O
N
N
S
S
O
O
N
N
S
O HN
H
O
O
O
H2N
O HN
N
N
O
O
3
1
2
O
N
O
HO
O
S
N
N
N
N
N
O
N
S
HO
N
O
O
O
O
O
O
O
O
O
4
5
6
Fig. 1. Structures of indoline-based and trimethoxyphenyl-based derivatives as tubulin inhibitors
4

Molecular hybridization strategy is a common method to design new compounds
by the combination of different biologically active parts to produce novel hybrids with
higher affinity and effectivenes. As the continuation of our group work on colchicine
binding site tubulin inhibitors and anticancer agents, we designed and synthesized a
series of indoline derivatives by the combination of anticancer scaffold indoline and
anti-tubulin pharmacodynamic trimethoxyphenyl group into one hybrid (Fig. 2).
Among
of
these
compounds
,
(6-bromoindolin-1-yl)(3,4,5-
trimethoxyphenyl)methanone (9d) was identified as a tubulin inhibitor. Compound 9d
exhibited potent antiproliferative activity against six tested human cancer cells. In the
further studies suggested compound 9d inhibited tubulin polymerization via binding to
the colchicine site and inhibited Kyse450 cells in time and dose-dependent manners.
O
N
NH O
S
O
N
R1
n1
N
Molecular hybridization
O
2
n2
O
N
R2
O
N
9a - 9p
O
O
4
Fig. 2. Design of indoline derivatives in this work.
The synthetic route of indoline derivatives is shown in Scheme 1. Commercially
available indolines (7) as the starting materials reacted with substituted acyl chlorides
o
(
8a-8l) presence of K CO in acetone at 60 C to give target compounds 9a-9p. Finally,
2
3
all the target compounds were fully characterized by NMR.
Cl
O
R1
n2
n1
R1
n1
N
a
N
O
H
n2
R2
R2
9
a-9p
7
8a-8l
o
Reagents and conditions: a) K CO , acetone, 60 C, 6 h
2
3
Scheme 1. Synthetic route of indoline derivatives
5

The in vitro antiproliferative activity of new target compounds 9a-9p were
evaluated against three human cell lines (MGC-803, Kyse30, and A549 cells) using
CCK8 assays and the famous tubulin inhibitor Colchicine as a positive drug. The
following Table 1, Table 2 and Table 3 depicted the results of in vitro antiproliferative
activity.
To explore the relationships between chemical groups and antiproliferative
activity, compound 9a-9e were designed and synthesized. Representative chemical
features of indoline derivatives 9a-9e are a 3,4,5-trimethoxypheny moiety linked to a
substituted indoline ring with different substituents such as bromine atom, nitro, or H.
Most of indoline derivatives exhibited certain antiproliferative potency against three
human cancer cell lines. Particularly, compound 9d exhibited the better growth
inhibition of the tested three human cancer cell lines (MGC-803, Kyse30, and A549)
with IC values of 1.84 µM, 6.82 μM and 1.61 μM, respectively.Campared compounds
5
0
9
a with 9b and 9c, compounds with a indoline ring exhibited better antiproliferative
activity than the 1,2,3,4-tetrahydroquinoline ring (9b) and 2,3,4,5-tetrahydro-1H-
benzo[b]azepine ring (9c). Moreover, the substituent groups of indoline ring was
important for antiproliferative activity ( 5-Br > 5-NO > H).
2
Table 1. IC values of compounds 9a-9e against three human cancer cell lines.
5
0
R1
n1
N
O
O
O
O
9
a-9e
a
IC (μmol/L)
5
0
Compounds.
R1
n1
MGC-803
7.57 ± 0.06
4.41 ± 0.08
4.3 ± 0.14
1.84 ± 0.03
8.32 ± 0.14
A549
Kyse30
9
a
H
H
1
2
3
1
1
22.0 ± 0.05
23.7 ± 0.11
21.7 ± 0.07
6.82 ± 0.05
17.7 ± 0.17
7.72 ± 0.19
6.72 ± 0.17
16.55 ± 0.09
1.61 ± 0.06
6.55 ± 0.09
9
b
9
c
H
9
d
5-Br
5-NO2
9
e
6

colchicine
-
-
1.07 ± 0.11
0.19 ± 0.04
0.28 ± 0.04
aAntiproliferative activity was assayed by exposure for 48 hours.
In addition, the importance of 3,4,5-trimethoxyphenyl unit and carbon chain
length on antiproliferative activity was further explored. As shown in Table 2, the
inhibitory potency of compounds 9f-9j was decreased when the 3,4,5-trimethoxyphenyl
unit was replaced by phenyl groups with different substituents such as F, Br, CH3,
OCH or H groups, indicating that the 3,4,5-trimethoxyphenyl unit played an important
3
role in the inhibitory potency for the indoline derivatives. In addition, we also explored
the the importance of the carbon chain length on antiproliferative activity. As shown in
Table 2, when the carbon chain length is 1, compound (9k- 9o) including compound
9
p with a 3,4,5-trimethoxyphenyl unit showed weak inhibitory activity against three
cancer cells with IC values > 20ꢀμM. These results indicated that the carbon chain
5
0
length is also important for the antiproliferative activity.
Table 2. IC values of compounds 9f-9p against three human cancer cell lines.
5
0
Br
N
O
n2
R2
9
f - 9p
a
IC (μmol/L)
5
0
Compounds.
R2
n2
MGC-803
28.5 ± 1.17
24.1 ± 1.01
33.6 ± 1.24
A549
Kyse30
9
f
H
0
0
0
＞40
20.3 ± 1.18
22.1 ± 1.04
26.8 ± 1.22
9
g
4-F
4-Br
18.9 ± 1.23
9
h
＞40
9
i
4-CH3
4-OCH3
H
0
0
1
＞40
38.9 ± 1.23
21. 1 ± 1.67
28.5 ± 2.64
＞40
18.9 ± 1.21
＞40
9
j
18.8 ± 1.18
32.8 ± 2.12
9
k
7

9
l
4-F
4-Br
1
1
1
1
＞40
＞40
＞40
＞40
20.7 ± 1.28
33.2 ± 1.06
＞40
9
m
35.8 ± 1.17
38.2 ± 1.02
28.7 ± 1.08
9
n
4-CH3
4-OCH3
＞40
9
o
24.8 ± 2.13
9
p
3,4,5-OCH3
-
1
-
22.4 ± 1.26
1.07 ± 0.11
24.8 ± 2.13
0.19 ± 0.04
20.2 ± 1.08
0.28 ± 0.04
colchicine
aAntiproliferative activity was assayed by exposure for 48 hours.
Compound 9d exhibited the best antiproliferative potency against there huan
cancer cells than other compounds. In addition, Kyse30 cells were more sensitive to the
most of compounds including compound 9d than MGC-803 cells and A549 cells.
Therefore, compound 9d was also chosen to test the antiproliferative activity of another
three ESCC cell lines (Kyse450, Kyse510, and EC-109 cells) in vitro. As showen in
Table 3, compound 9d also displayed potent antiproliferative potency against Kyse450,
Kyse510, and EC-109 cells with IC values of 1.49 µM, 2.08 µM and 2.24 µM,
5
0
respectively. These results also suggested that Kyse450 cells were more sensitive to the
compound 9d than other cancer cells. Therefore, compound 9d and Kyse450 cells were
used for further detection.
Table 3. IC values of compound 9d against four human cancer cell lines.
5
0
Tumor type
Esophagus
Esophagus
Esophagus
Esophagus
Cell line
Kyse30
Kyse450
Kyse510
EC-109
IC (μM)
50
1.61 ± 0.06
1.49 ± 0.04
2.08 ± 0.05
2.24 ± 0.04
As its potential target protein, activity on tubulin polymerization of compound 9d
was first to be detected. To investigat the effects on microtubules, compound 9d was
selected to do immunofluorescence assay by staining tubulin. The immunofluorescence
results in Fig. 3A showed compound 9d inhibited tubulin polymerization starting from
the concentration of 1.25 μM in Kyse450 cells. Cell displayed disorganized spindle
which was consistent with the effect of another two famous tubulin inhibitors CA-4P,
8

and Colchicine. We next evaluated the effects of compound 9d on tubulin
polymerization in vitro with colchicine as a positive drug. As showen in Fig. 3B, when
tubulin was incubated with compound 9d (1 µM, 2 µM and 4 µM), the increased
tendency of the fluorescence intensity was obviously slowed down. Compound 9d
potently inhibited tubulin polymerization in a dose-dependent manner with an IC50
value of 3.4 µM. The inhibitory tubulin polymerization activity of compound 9 at
concentration of 4 µM was stronger than that of Colchicine at concentration of 3 µM,
while was weaker at 2 µM than that of Colchicine at 3 µM. The N, N’-ethylenebis
(iodoacetamide) (EBI) assay is usually used test the binding ability of small molecules
to β - tubulin at colchicine binding sites. In EBI assay, compound 9d preincubation
prevented the formation of EBI: β-tubulin adduct resulting in the decrease of the adduct
band comparing with EBI only (Fig.3C), indicating compound 9d could directly bind
to the colchicine binding site of β-tubulin. These results indicated that compound 9d
was a novel tubulin polymerization inhibitor tgargeting the cochicine binding site.
Fig. 3. Compound 9d inhibited tubulin polymerization. A. Immunofluorescence staining of tubulin
(green) and nucleus (blue), Kyse450 cells were treated with CA-4P (0.05µM), Colchicine (0.1µM),
Paclitaxel (0.05µM) and different concentration of compound 9d for 48 hours; B. Tubulin
polymerization inhibitory activity of compound 9d, concentration of Colchicine was 3 μmol/L; C.
EBI competition assay on Kyse450 cells;
9

Compound 9d displayed the potent tubulin polymerization inhibition activity, and
we then selected it as the optimized compound for the molecular docking studies by
MOE 2015.10. To investigate the binding site of compound 9d with the tubulin-
microtubule system, PDB code 1SA0 was selected. The docking results were listed in
Fig. 4. The docking result revealed that compound 9d could well bind into the
colchicine binding site of β-tubulin. The carbonyl group of compound 9d could form
an hydrogen bond with the main chain of Asp249, and the hydrogen bond distance was
2
.9 Å. The indoline group of compound 9d was located in a hydrophobic subpocket
consist of Ala180, Val181, Lys252, Thr310, Val313 and Lys350. In addition, the 3,4,5-
trimethoxyphenyl group occupied a hydrophobic cavity and involved in hydrophobic
interactions with Tyr200, Val236, Leu240, Leu250, Leu253 and Ile368. It was noticed
that hydrophobic interaction played a key role in the binding of compound 9d. These
interactions made compound 9d tightly bind into the colchicine binding site of β-tubulin
and exhibit strong inhibitory activity.
Fig. 4. The proposed binding mode of compound 9d in the colchicine binding site of tubulin(PDB
code: 1SA0). Compound 9d was shown in yellow stick. The hydrogen bond was shown in red dotted
lines.
10

Tubulin polymerization inhibition usually leads to cell inhibition, therefore, the
inhibition activity against Kyse450 cells of compound 9d was further detected. As
shown in Fig. 5A, with the increase of the concentrations of compound 9d, the cell
viability decreased evidently. In addition, the cell viability were also inhibited in a time-
dependent manner with the treatment of different concentrations of compound 9d (Fig.
5
B). Compound 9d also inhibited the proliferation of Kyse450 cells in a dose-dependent
manner (Fig. 5C). After treatment with a high concentration of compound 9d for 48
hours, the cell growth was less than 2 times, while the control group was more than 3.5
times (Fig. 5C). Some other obvious changes were also observed in Kyse450 cells after
the treatment with compound 9d for 48 hours. In morphology, Kyse450 cells became
rounder; the nucleus became smaller and concentrated; Kyse450 cells were induced to
death and the cell density decreased (Fig. 5D). The antiproliferation activity of
compound 9d was also evaluated by colony formation assay. As shown in Fig. 5E,
compound 9d inhibited the ability of Kyse450 cells after a 7 days treatment even with
a low concentration from 0.188 μM (Fig. 4E). These results suggested that the
compound 9d inhibited the proliferation of MGC-803 cells in dose-and time-dependent
manners.
11

Fig. 5. Compound 9d inhibited the activity of Kyse450 cells. A & B. Kyse450 cell viability. in (A)
cells were treated with indicated concentrations of compound 9d for 48 hours; C. Growth curve of
Kyse450 cells; D. Kyse450 cellular morphologies (upper panel), nucleus (mid panel) and cell death
by compound 9d induced (lower panel, dead cells were stained red). cells were treated with different
concentration of Avermectin for 48 hours, cells were stained with different dyes; E. Cell colony
formation assay. Kyse450 cells were treated with indicated concentrations of compound 9d for 7
days.
In this study, we reported indoline derivatives and their antiproliferative activity
against three human cancer cell lines (MGC-803, Kyse30 and A549). Compound 9d
showed potently inhibitiory activity against six human cancer cells, especially Kyse30
and Kyse450 cells with IC values of 1.61 and 1.49 µM, respectively. Compound 9d
5
0
exhibited broad-spectrum antiproliferative activity of ESCC cell lines especially
Kyse450 cells. According to the results of antiproliferative activity, compound 9d and
12

Kyse450 cells were selected to do the further researches. Compound 9d was identified
as novel tubulin polymerization inhibitor with an IC50 value of 3.4 µM.
Immunofluorescence results indicated that compound 9d inhibited tubulin
polymerization starting from the concentration of 1.25 μM in Kyse450 cells. Compound
9
d produced a dose-dependent inhibitory effect of tubulin polymerization in vitro. EBI
assay results suggested compound 9d could directly bind to the colchicine binding site
of β-tubulin. The results of molecular docking also suggested compound 9d tightly bind
into the colchicine binding site of tubulin. Further results indicated compound 9d
inhibited Kyse450 cells in time and dose-denpendent manners. This work is a part of
indoline derivatives as anticancer anticancer agents, which suggests that the indoline
derivatives mighe be a class of potential anticancer compounds as novel tubulin
inhibitors.
Appendix A. supplementary data
The supporting information provided the date of NMR and HRMS spectra of 9d.
Acknowledgement
This work was supported by the National Natural Sciences Foundations of China
(No. 81703541, U2004123 for Sai-Yang Zhang and No. 81673322 for Yan-Bing Zhang)
and China Postdoctoral Science Foundation (No. 2018M632812 for Sai-Yang Zhang)
and the Henan Scientific Innovation Talent Team, Department for Education (No.
1
9ITSTHN001 for Wen Zhao, China). Henan Association of Science and Technology
(No. 2020HYTP056 for Sai-Yang Zhang, China) and Science and Technology
Department of Henan Province (No. 20202310144, for Sai-Yang Zhang, China).
Declaration of competing interest
The authors declared that there was no conflict of interest about this work.
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benzothiazole moiety as anti-gastric cancer agents in vitro via inhibiting tubulin
polymerization and activating the Hippo signaling pathway. Eur J Med Chem. 2020;203:
1
12618.
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1. Fu D-J, Li P, Wu B-W, Cui X-X, Zhao C-B, Zhang S-Y. Molecular diversity of
trimethoxyphenyl-1,2,3-triazole hybrids as novel colchicine site tubulin polymerization
inhibitors. Eur J Med Chem. 2019;165: 309-322.
2. Fu D-J, Yang J-J, Li P, et al. Bioactive heterocycles containing a 3,4,5-trimethoxyphenyl
fragment exerting potent antiproliferative activity through microtubule destabilization. Eur J
Med Chem. 2018;157: 50-61.
3. Jian S, Gao Q-L, Wu B-W, et al. Novel tertiary sulfonamide derivatives containing
benzimidazole moiety as potent anti-gastric cancer agents: Design, synthesis and SAR studies.
Eur J Med Chem. 2019;183: 111731.
15

2
2
2
2
4. Li Q, Jian X-E, Chen Z-R, et al. Synthesis and biological evaluation of benzofuran-based 3,4,5-
trimethoxybenzamide derivatives as novel tubulin polymerization inhibitors. Bioorg Chem.
2
020;102: 104076.
5. Yang F, Jian X-E, Diao P-C, Huo X-S, You W-W, Zhao P-L. Synthesis, and biological
evaluation of 3,6-diaryl-[1,2,4]triazolo[4,3-a]pyridine analogues as new potent tubulin
polymerization inhibitors. Eur J Med Chem. 2020;204: 112625.
6. Ma W, Chen P, Huo X, et al. Development of triazolothiadiazine derivatives as highly potent
tubulin polymerization inhibitors: Structure-activity relationship, in vitro and in vivo study.
Eur J Med Chem. 2020;208: 112847.
7. Huo X-S, Jian X-E, Ou-Yang J, et al. Discovery of highly potent tubulin polymerization
inhibitors: Design, synthesis, and structure-activity relationships of novel 2,7-diaryl-
[1,2,4]triazolo[1,5-a]pyrimidines. Eur J Med Chem. 2021: 113449.
Declaration of Interest Statement
The authors declare that they have no conflicts of interest.
Discovery of indoline derivatives as anticancer agents via
inhibition of tubulin polymerization
Shu-Yu Wang a, 1, Xu Liu a, b, 1, Ling-Wei Meng a, b, Miao-Miao Li , Sheng-Hui wang
a
a
a
a, b
a
a
a, b, c,
Yin-Ru Li , Jian Song , Hong-Yu Zhang , Ping Chen , Sai-Yang Zhang
*, Tao
a,
Hu *
d. School of Basic Medical Sciences, Zhengzhou University, Zhengzhou, 450001, China
e. School of Pharmaceutical Sciences, Institute of Drug Discovery & Development, Key
Laboratory of Advanced Drug Preparation Technologies (Ministry of Education), Zhengzhou
University, Zhengzhou, 450001, China
f. Henan Institute of Advanced Technology, Zhengzhou University, Zhengzhou 450001, China
*
Corresponding authors: saiyangz@zzu.edu.cn; hnhutao@zzu.edu.cn
These authors contributed equally to this work
1
16

1
1
. Experimental section
.1. General
All commercial materials were used without further purification. TLC analysis
was carried out on silica gel plates GF254 (Qingdao Haiyang Chemical, China). Silica
gel chromatography was carried out on 200-300 mesh gel. Anhydrous solvents and
reagents were dried by routine protocols. Melting points were determined on an X-5
1
13
micromelting apparatus and are uncorrected. H NMR and C NMR spectra were
recorded on a Bruker 400 MHz and 100 MHz spectrometer, respectively.
1
.2. Cell culture
MGC-803, A549, Kyse30, Kyse450, Kyse510, and Ec109 cell lines were cultured
in an atmosphere with 5% CO at 37 °C, using RPMI-1640 with 10% fetal bovine serum
2
(FBS), 100 U/ml penicillin and 0.1 mg/ml streptomycin as culture medium.
17

1
.3. Cell viability
Growth inhibition of MGC-803, A549, Kyse30, Kyse 450, Kyse510, Ec109 cell
lines was examined by the CCK8 assays. Briefly, human cancer cells were seeded in
6 - well plates at a density of 3000–5000 cells/well and incubated 24 hours. The cells
9
were treated with compounds 9d or medium for different times and 5 μl CCK8 was
added to per well. After incubation for another 4 hours, the absorbance was measured
at 450 nM by a microplate reader (MK3, Thermo, Germany). Data are calculated by
SPSS version 10.0.
1
.4. Colony formation assay
Kyse450, cells were seeded in a 6-well plate at a density of 5 x 103 per well and
incubated at 37 °C in 5% CO for 24 h, then they were treated with different
2
concentrations of 9d. After 7 days, the culture medium was removed, and the cells were
washed with PBS and fix with 4% paraformaldehyde. Then stain the cells with 0.1%
crystal violet. The image was captured with camera, and the number of colonies were
quantified using Image J software (National Institutes)
1
.5. Immunostaining and microscopy
Cell slices were placed on the bottom of a 24-well plate. Kyse 450 cells were
seeded on the slices and incubated for 24 hours. After 48 hours, slices were fixed with
% paraformaldehyde for 20 minutes. 0.5 % Triton-X-100 was added and shake for 20
4
min. 0.1% BSA was used to block for 30 minutes and then removed. The slices were
co-incubated with anti-β-tubulin antibody (1:100) for 8 hours at 4 °C. Then slices were
washed by PBST 3 times, bind with secondary antibody with FITC signal (1:500) in a
dark. DAPI was used to stain for 5 min. After that, images were captured by Laser
scanning confocal microscope (Nikon, Japan).
1
.6. In vitro tubulin polymerization assay
An amount of 5.6 mg/ml tubulin was resuspended in PEM buffer (containing 80
mmol/L PIPES (Ph = 6.9)), 1 mmol/L EGTA, 0.5 mmol/L MgCl , 1 mmol/L ATP, 10.2%
2
18

(v/v) glycerol) and then was preincubated with certain concentrations of 9d, Colchicine
or vehicle DMSO at 0 ℃. The reaction was monitored by a spectrophotometer in
absorbance at 420 nm at 37 °C every minute (excitation wavelength is 340 nm).
1
.7. EBI competition assay
6
-well plates were seeded with Kyse 450 cells at 5 × 105 cells per well. Cells were
first incubated with certain concentrations of 9d, CA-4P, colchicine and paclitaxel or
DMSO for 2 hours and afterward treated with EBI (100 μmol/L) for 2 hours. Then the
cells were harvested and lysed. Cell extracts were used for Western blotting analysis.
1
.8. Western blot analysis
Treated and untreated cells were harvested and lysed. Cell extracts were denatured
by loading buffer and resolved by SDS-PAGE and transferred to nitrocellulose
membrane. The membranes were incubated with appropriate primary antibodies at 4 °C
for 10 hours after blocking with 5% skimmed milk. After conjugated with secondary
antibodies, the detection of proteins was carried out with an ECL kit.
1
.9. Molecular docking
The molecular docking study was performed using MOE 2015.10. The crystal
structure of tubulin (PDB ID: 1SA0) was retrieved from RCSB Protein Data Bank, and
then was prepared by adding hydrogen atoms, removing water molecules and repairing
the missing side chains. The protonation states of protein residues were calculated in
the pKa at 7. The ligand compound 9d was built in MOE 2015.10 and was prepared by
energy minimization and conformational search. The ligand was docked into the
colchicine binding site of tubulin and 20 poses were exported for the next analysis.
1
13
2
. H, and C-NMR of Compound 9a-9p
Indolin-1-yl(3,4,5-trimethoxyphenyl)methanone（9a）
o
1
White solid, m.p.: 105~106 C, yield:90%. H NMR (400 MHz, CDCl3) δ 7.27 (s,
H), 7.22 (d, J = 7.4 Hz, 1H), 7.12 (s, 1H), 7.02 (t, J = 7.1 Hz, 1H), 6.78 (s, 2H), 4.12
1
1
3
(
t, J = 8.0 Hz, 2H), 3.88 (d, J = 14.8 Hz, 9H), 3.13 (t, J = 8.3 Hz, 2H). C NMR (100
MHz, CDCl3) δ 168.58, 153.38, 142.51, 139.78, 132.42, 132.20, 127.24, 124.96,
23.95, 123.89, 104.55, 60.98, 56.29, 50.45, 28.06. HRMS (ESI): Calcd. C18H20NO4,
1
19

[M+H]+m/z: 314.1392, found: 314.1398.
(3,4-Dihydroquinolin-1(2H)-yl)(3,4,5-trimethoxyphenyl)
methanone（9b）
o
1
White solid, m.p.: 102~103 C, yield:60%. H NMR (400 MHz, CDCl3) δ 7.06 (d,
J = 7.3 Hz, 1H), 6.91 (t, J = 7.3 Hz, 1H), 6.82 (t, J = 7.5 Hz, 1H), 6.66 (d, J = 7.8 Hz,
1
2
1
2
3
H), 6.50 (s, 2H), 3.82 (t, J = 6.5 Hz, 2H), 3.74 (s, 3H), 3.58 (s, 6H), 2.75 (t, J = 6.5 Hz,
1
3
H), 2.06 – 1.85 (m, 2H). C NMR (100 MHz, CDCl3) δ 168.60, 151.69, 138.80,
38.51, 130.65, 129.96, 127.16, 124.85, 124.34, 123.59, 105.48, 59.84, 55.01, 43.53,
5.94, 23.22. HRMS (ESI): Calcd. C19H22NO4, [M+H]+m/z: 328.1549, found:
28.1552.
(2,3,4,5-Tetrahydro-1H-benzo[b]azepin-1-yl)(3,4,5-
trimethoxyphenyl)methanone（9c）
o
1
White solid, m.p.: 144~145 C, yield:50%. H NMR (400 MHz, CDCl3) δ 7.16 (d,
J = 7.5 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 6.89 (t, J = 7.5 Hz, 1H), 6.60 (d, J = 7.7 Hz,
1
H), 6.38 (s, 2H), 4.95 (d, J = 13.2 Hz, 1H), 3.69 (s, 3H), 3.52 (s, 6H), 2.97 (t, J = 13.0
Hz, 1H), 2.82 (dd, J = 13.6, 4.4 Hz, 1H), 2.71 (t, J = 10.1 Hz, 1H), 2.02 (d, J = 11.5 Hz,
1
3
1
H), 1.89 (dd, J = 11.8, 7.5 Hz, 2H), 1.45 (d, J = 11.5 Hz, 1H). C NMR (100 MHz,
CDCl3) δ 167.16, 151.25, 143.46, 138.14, 138.01, 129.72, 128.78, 127.15, 126.31,
26.20, 105.23, 59.78, 54.85, 46.75, 33.85, 28.65, 25.27. HRMS (ESI): Calcd.
C20H24NO4, [M+H]+m/z: 342.1705, found: 342.1709.
1
(5-Bromoindolin-1-yl)(3,4,5-trimethoxyphenyl)methanone （ 9d）
o
1
White solid, m.p.: 126~127 C, yield:31%. H NMR (400 MHz, DMSO-d ) δ 7.89
6
(s, 1H), 7.48 (s, 1H), 7.37 (d, J = 7.9 Hz, 1H), 6.90 (s, 2H), 4.06 (t, J = 8.4 Hz, 2H),
1
3
3
.82 (d, J = 8.0 Hz, 6H), 3.73 (d, J = 4.9 Hz, 3H), 3.10 (t, J = 8.3 Hz, 2H). C NMR
(
100 MHz, DMSO-d ) δ 168.29, 153.28, 142.63, 139.31, 136.21, 132.46, 130.03,
6
1
28.30, 115.66, 107.00, 105.02, 60.57, 56.58. HRMS (ESI): Calcd. C H BrNO ,
18 19 4
[M+H]+m/z: 392.0497, found: 392.0499.
(5-bromoindolin-1-yl)(phenyl)methanone (9f)
o
1
White solid, m.p.: 131~132 C, yield:44%. H NMR (400 MHz, CDCl ) δ 7.66 –
3
7
.52 (m, 2H), 7.47 – 7.39 (m, 2H), 7.30 (d, J = 9.0 Hz, 4H), 3.69 (t, J = 14.6 Hz, 2H),
20

1
3
3
1
.06 (t, J = 14.6 Hz, 2H). C NMR (101 MHz, CDCl ) δ 168.98, 141.89, 136.57, 134.61,
3
30.52, 130.19, 128.65, 127.91, 127.06, 116.40, 50.80, 28.00.
(5-bromoindolin-1-yl)(4-fluorophenyl)methanone (9g)
o
1
White solid, m.p.: 128~129 C, yield: 40%. H NMR (400 MHz, CDCl ) δ 7.57
3
(dd, J = 8.5, 5.4 Hz, 2H), 7.33 (s, 1H), 7.26 (s, 1H), 7.14 (t, J = 8.6 Hz, 2H), 4.07 (d, J
=
7.3 Hz, 2H), 3.12 (t, J = 8.3 Hz, 2H). 13C NMR (101 MHz, CDCl ) δ 167.93, 165.14,
3
1
1
62.64, 141.80, 134.56, 132.59, 130.21, 129.60, 129.52, 127.98, 116.56, 115.89,
15.67, 50.85, 27.95.
(5-bromoindolin-1-yl)(4-bromophenyl)methanone (9h)
o
1
White solid, m.p.: 123~124 C, yield: 48%. H NMR (400 MHz, CDCl3) δ 7.60 (d,
J = 8.3 Hz, 2H), 7.43 (d, J = 8.3 Hz, 2H), 7.34 (s, 1H), 7.26 (s, 2H), 4.06 (s, 2H), 3.12
1
3
(
t, J = 8.3 Hz, 2H). C NMR (101 MHz, CDCl3) δ 167.84, 141.68, 131.92, 130.28,
1
28.84, 128.00, 124.99, 116.69, 50.85, 27.97.
(5-bromoindolin-1-yl)(p-tolyl)methanone (9i)
o
1
White solid, m.p.: 119~120 C, yield: 50%. H NMR (400 MHz, CDCl3) δ 7.45 (d,
J = 8.0 Hz, 2H), 7.32 (s, 1H), 7.25 (d, J = 10.2 Hz, 4H), 4.09 (t, J = 7.7 Hz, 2H), 3.10
1
3
(
t, J = 8.3 Hz, 2H), 2.41 (s, 3H). C NMR (101 MHz, CDCl3) δ 169.13, 142.05, 140.87,
1
0
34.65, 133.63, 130.13, 129.22, 127.87, 127.24, 118.41, 116.23, 50.76, 27.95, 21.51, -
.00.
(5-bromoindolin-1-yl)(4-methoxyphenyl)methanone (9j)
o
1
White solid, m.p.: 129~130 C, yield: 38%. H NMR (400 MHz, CDCl ) δ 7.54 (d,
3
J = 8.6 Hz, 2H), 7.32 (s, 1H), 7.26 (s, 2H), 6.94 (d, J = 8.7 Hz, 2H), 4.12 (t, J = 8.3 Hz,
1
3
2
1
2
H), 3.86 (s, 3H), 3.10 (t, J = 8.2 Hz, 2H). C NMR (100 MHz, CDCl ) δ 168.82,
3
61.47, 142.17, 134.67, 130.09, 129.36, 128.55, 127.89, 116.11, 113.82, 55.41, 50.93,
8.01.
1
-(5-bromoindolin-1-yl)-2-phenylethan-1-one (9k)
o
1
White solid, m.p.: 118~119 C, yield: 54%. H NMR (400 MHz, CDCl ) δ 8.13 (d,
3
J = 8.5 Hz, 1H), 7.37 – 7.32 (m, 2H), 7.31 – 7.28 (m, 3H), 7.25 (s, 2H), 4.06 (t, J = 8.5
1
3
Hz, 2H), 3.79 (s, 2H), 3.13 (t, J = 8.4 Hz, 2H). C NMR (101 MHz, CDCl ) δ 169.18,
3
1
4
42.22, 133.90, 133.38, 130.41, 129.06, 128.79, 127.53, 127.10, 118.44, 116.12, 48.26,
3.42, 27.77.
1
-(5-bromoindolin-1-yl)-2-(4-fluorophenyl) ethan-1-one (9l)
21

o
1
White solid, m.p.: 126~127 C, yield: 49%. H NMR (400 MHz, CDCl ) δ 8.11 (d,
3
J = 8.4 Hz, 1H), 7.25 (dd, J = 8.7, 5.5 Hz, 4H), 7.03 (t, J = 8.7 Hz, 2H), 4.08 (t, J = 8.5
1
3
Hz, 2H), 3.74 (s, 2H), 3.16 (t, J = 8.4 Hz, 2H). C NMR (100 MHz, CDCl ) δ 168.98,
3
1
1
63.19, 160.75, 142.12, 133.35, 130.78, 130.70, 130.45, 129.62, 129.58, 127.58,
18.42, 116.24, 115.72, 115.50, 48.22, 42.32, 27.77.
1
-(5-bromoindolin-1-yl)-2-(4-bromophenyl) ethan-1-one (9m)
o
1
White solid, m.p.: 126~127 C, yield: 49%. H NMR (400 MHz, CDCl ) δ 8.10
3
(d, J = 8.4 Hz, 1H), 7.46 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 6.1 Hz, 2H), 7.17 (d, J = 8.2
1
3
Hz, 2H), 4.06 (t, J = 8.5 Hz, 2H), 3.72 (s, 2H), 3.15 (t, J = 8.5 Hz, 2H). C NMR (101
MHz, CDCl ) δ 168.53, 142.06, 133.33, 132.89, 131.83, 130.92, 130.46, 127.59, 121.15,
3
1
18.41, 116.28, 48.23, 42.57, 27.76.
1
-(5-bromoindolin-1-yl)-2-(p-tolyl) ethan-1-one (9n)
o
1
White solid, m.p.: 121~122 C, yield: 57%. H NMR (400 MHz, CDCl ) δ 8.13 (d,
3
J = 8.5 Hz, 1H), 7.26 (s, 2H), 7.16 (q, J = 8.0 Hz, 4H), 4.06 (t, J = 8.5 Hz, 2H), 3.75 (s,
1
3
2
1
4
H), 3.13 (t, J = 8.4 Hz, 2H), 2.33 (s, 3H). C NMR (101 MHz, CDCl ) δ 169.43,
3
42.27, 136.74, 133.36, 130.77, 130.40, 129.49, 128.91, 127.51, 118.44, 116.07, 48.24,
3.06, 27.78, 21.07.
1
-(5-bromoindolin-1-yl)-2-(4-methoxyphenyl) ethan-1-one (9o)
o
1
White solid, m.p.: 121~122 C, yield: 57%. H NMR (400 MHz, CDCl ) δ 8.13 (d,
3
J = 8.5 Hz, 1H), 7.26 (s, 2H), 7.21 (d, J = 8.4 Hz, 2H), 6.88 (d, J = 8.6 Hz, 2H), 4.07 (t,
1
3
J = 8.5 Hz, 2H), 3.79 (s, 3H), 3.73 (s, 2H), 3.14 (t, J = 8.4 Hz, 2H). C NMR (100
MHz, CDCl ) δ 169.56, 158.68, 142.27, 133.35, 130.42, 130.11, 127.52, 125.85, 118.44,
3
1
16.08, 114.22, 55.29, 48.21, 42.52, 27.80.
1
-(5-bromoindolin-1-yl)-2-(3,4,5-trimethoxyphenyl) ethan-1-one
(9p)
o
1
White solid, m.p.: 128~129 C, yield: 51%. H NMR (400 MHz, CDCl ) δ 8.13
3
(d, J = 8.3 Hz, 1H), 7.30 (d, J = 8.4 Hz, 2H), 6.51 (s, 2H), 4.10 (t, J = 8.5 Hz, 2H), 3.84
1
3
(
d, J = 6.1 Hz, 9H), 3.73 (s, 2H), 3.18 (t, J = 8.4 Hz, 2H). C NMR (101 MHz, CDCl )
3
δ 169.05, 153.42, 142.18, 137.06, 133.36, 130.48, 129.46, 127.59, 118.45, 116.24,
06.13, 60.85, 56.16, 48.29, 43.65, 27.83.
1
22

Graphical Abstract
23