N-Arylsulfonylsubstituted-1H indole derivatives as small molecule dual inhibitors of signal transducer and activator of transcription 3 (STAT3) and tubulin

Article abstract of DOI:10.1016/j.bmc.2017.11.023

Signal transducer and activator of transcription (STAT3) is a proposed therapeutic target for the development of anti-cancer agents. In this report, a series of N-arylsulfonylsubstituted-1H indole derivatives were designed and synthesized as STAT3 inhibitors, their anti-proliferative activities were evaluated against a number of tumor cells, some potent compounds exhibited IC₅₀ values less than 10 μM. The most potent compound 4a was further confirmed to inhibit STAT3 phosphorylation at Tyr705. It was further revealed that 4a arrested the cell cycle at the G2/M phase and inhibited tubulin polymerization. This study describes a series of N-arylsulfonylsubstituted-1H indole derivatives as potent anti-cancer agents targeting both STAT3 and tubulin.

Full text of DOI:10.1016/j.bmc.2017.11.023

Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
N-Arylsulfonylsubstituted-1H indole derivatives as small molecule dual
inhibitors of signal transducer and activator of transcription 3 (STAT3)
and tubulin
⇑
Qiang Zhou, Jinjin Zhu, Jinglei Chen, Peng Ji, Chunhua Qiao
Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, College of Pharmaceutical Sciences, Soochow University, 199 Ren Ai Road, Suzhou 215123,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Signal transducer and activator of transcription (STAT3) is a proposed therapeutic target for the develop-
ment of anti-cancer agents. In this report, a series of N-arylsulfonylsubstituted-1H indole derivatives
were designed and synthesized as STAT3 inhibitors, their anti-proliferative activities were evaluated
Received 5 September 2017
Revised 1 November 2017
Accepted 12 November 2017
Available online xxxx
against a number of tumor cells, some potent compounds exhibited IC50 values less than 10 lM. The most
potent compound 4a was further confirmed to inhibit STAT3 phosphorylation at Tyr705. It was further
revealed that 4a arrested the cell cycle at the G2/M phase and inhibited tubulin polymerization. This
study describes a series of N-arylsulfonylsubstituted-1H indole derivatives as potent anti-cancer agents
targeting both STAT3 and tubulin.
Keywords:
Signal transducer and activator of
transcription (STAT3) inhibitors
Tubulin polymerization inhibitors
Anti-cancer agents
Ó 2017 Published by Elsevier Ltd.
N-arylsulfonylsubstituted-1H indole
derivatives
1. Introduction
Most reported small molecule inhibitors of STAT3 were divided
into two categories: DNA-binding domain inhibitors and/or SH2-
The cytoplasmic signal transducer and activator of transcription
(STAT) family proteins regulate the gene expression related to the
cell proliferation, differentiation, apoptosis, immune-response,
and angiogenesis.1 There are seven different isoform members in
this family: STAT1, STAT2, STAT3, STAT4, STAT5a, STAT5b and
STAT6. Although structurally related,2 they participate in different
cellular processes. In particular, the STAT3 protein attracted a great
attention for its crucial function in cancer development. As part of
Janus kinase (Jak) signaling pathway, STAT3 is activated by both
receptor and non receptor tyrosine kinase via the phosphorylation
cascade.3 The SH2 domain Tyr705 phosphorylation would trans-
form inactive STAT3 into its active form, resulting in protein dimer-
ization. STAT3 dimer then dissociated from the receptor and
translocated from the cytosol into the nucleus to combine with
the target genes, and stimulate the expression of the target pro-
teins.4 The instantaneous activation of STAT3 in normal cell differs
from the continuous activation in the cancer cell. Due to its impor-
tant role in malignant transformation and tumorgenesis, STAT3
was considered as a promising anticancer drug target.5
binding domain inhibitors.5 As the first reported nonpeptide small
molecule STAT3 inhibitor, STA-216 (Fig. 1), was discovered through
virtual screening. On the basis of STA-21, more synthetic com-
pounds like LLL-3, LLL-127 (Fig. 1) were designed and revealed.
Stattic was one of the most potent STAT3 inhibitors.8 Derived from
Stattic, compound HJC01469 (Fig. 1) displayed even higher potency
and promoted cell apoptosis. In addition to these above mentioned
compounds, other structurally distinct molecules were
investigated, including: S3I-20110, platinum (IV) complexes11
,
InS3-5412, NSC-74338013 (Fig. 1). Despite the above mentioned
research effort, none of these molecules was advanced to a
successful clinical trial, probably due to the lack of ideal physical
and chemical properties. Therefore, novel STAT3 inhibitors with
distinctive scaffold need to be further explored.
Microtubule/tubulin is aggregated by the a- and b-tubulin het-
erodimer through so called ‘‘head to tail” manner to assemble into
a tubular structure.14 In the cell mitosis (M) phase, microtubules
presented as a spindle to guide the genetic material distribute
evenly into two daughter cells.15 The proper dynamic balance
between polymerization and depolymerization of microtubules is
critical for the maintenance of cell morphology, cell signal trans-
duction, and cell division.16 Either promotion or inhibition of tubu-
lin polymerization would disrupt mitosis and lead to cell death. The
⇑
Corresponding author.
E-mail address: qiaochunhua@suda.edu.cn (C. Qiao).
https://doi.org/10.1016/j.bmc.2017.11.023
0968-0896/Ó 2017 Published by Elsevier Ltd.
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Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 1. Representative inhibitors of STAT3 and microtubule.
Fig. 2. Design rationale of prepared compounds.
rapid growth of cancer cell makes this polymerization/depolymer-
ization process more active than that in the normal cell. Conse-
quently, microtubule has been considered a good target for
anticancer drug development. A number of natural products, like
paclitaxel, vincristine, and colchicine, have been demonstrated to
bind to microtubule and exerted disruptive effect to tubulin
dynamic process.17 Specifically, paclitaxel functions as micro-
tubule-stabilizer, while colchicine behaves as microtubule-
destabilizer.
to remove this moiety in E28, a nitrogen atom was introduced to
replace the sulfonyl group in the five member ring. Consequently,
A number of N-arylsulfonylsubstituted-1H indoles (Fig. 2) were
designed as a novel type of STAT3 inhibitors. Interestingly, this
new skeleton is structurally similar to a reported microtubule
inhibitors ABT-75115,20 (Fig. 1). Therefore, we further evaluated
the compounds biological activity as tubulin polymerization
inhibitors.
In the course of searching potent STAT3 inhibitors, our labora-
tory conducted structural modification based on the reported
compound Stattic (Fig. 1), and discovered one of our most potent
STAT3 inhibitor E28.18 E28 inhibited STAT3 Tyr705 phosphoryla-
tion and induced tumor cell apoptosis. Unfortunately, E28 struc-
2. Results and discussion
2.1. Chemistry
The synthetic routes of the designed compounds 4a–4x, 7a–7c,
11a–11d were shown in Scheme 1. To prepare the reaction
intermediates 2b, 9a and 9b, the chlorine was introduced to the
turally bears the
a, b-unsaturated Michael acceptor like
moiety19, which could result in a broad spectrum toxicity. Aiming
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3
Scheme 1. Synthetic route of N-arylsulfonylsubstituted-1H indoles. Reagents and conditions: (a) NCS, 1 h, room temperature, DMF; (b) substituted benzene sulfonyl chloride,
NaOH, TEBAC, DCM; (c) corresponding boronic acid, PdCl2(dppf), K2CO3. 86 °C, 4–12 h; (d) Fe, CH3COOH/H2O, 65 °C; (e) Pd2(dpa)3, BINAP, sodium tert-butoxide, 80 °C; (f) 4-
cyanoaniline, EDC, pyridine, room temperature.
b-position of the indole ring by reaction of indole with N-chlorosuc-
cinimide (NCS) in DMF. The sulfonamides 3a–3f, 6a, 6b and 10a–
10d were obtained through reactions of benzenesulfonyl chloride
with the corresponding substituted indoles. The final target com-
pounds 4a–4x, 11a–11d were achieved through Suzuki coupling
reaction of bromo-substituted arylsulfonyl 1H-indole with the
corresponding boronic acids at moderate yields. Reduction of nitro
group to amino in 6a gave 6c, which was reacted with 4-bromo-2-
fluorobenzonitrile or 4-bromobenzonitrile to furnish the synthesis
of 7a or 7b. The reaction of carboxylic acid 6b with 4-cyanoaniline
using coupling reagent EDC provided compound 7c. All target com-
pounds were characterized by 1H NMR, 13C NMR and HRMS (ESI).
High pressure liquid chromatography was performed to guarantee
the compound purity is greater than 98% before further biological
evaluation.
2.2. Biological study
2.2.1. MTT assay and SAR analysis
To study the compound inhibitory activity against cancer cell
proliferation, MTT assay was performed and the result was shown
in Table 1. Cell lines with continuous activation of STAT3 were
selected, including A549, DU145, MCF-7, MDA-MB-231, KB and
HepG2. One of our reported highly potent compound E28 was used
as positive control. The IC50 values for all prepared compounds
were shown in Table 1. For those compounds displaying less than
50% inhibition at 20 lM, their IC50 values were not further deter-
mined. Otherwise, compounds were diluted and tested to calculate
IC50 values. Overall, six compounds (4a, 4f, 4n, 4o, 4v, and 7c) dis-
played potent inhibitory activity against cell proliferation for all six
cancer cells, with IC50 values less than 10 lM. Compounds 4a and
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Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Table 1
Antiproliferative activity of the designed compounds.
Comp
MTT, IC50 SD(l
M)a
Cells
4a
4b
4c
4d
4e
A549
MCF-7
3.20 0.09
5.33 0.29
>10
3.01 0.14
>10
DU145
KB
HepG2
3.29 0.86
5.51 0.64
5.94 0.96
4.68 0.73
>10
MDA-231
2.23 0.12
>10
>10
2.69 0.25
>10
3.31 1.64
10.25 1.40
10.61 1.12
5.30 0.34
>10
8.88 0.51
10.66 4.41
14.14 1.36
7.06 0.53
>10
6.17 0.12
8.35 2.03
>10
>10
>10
4f
5.30 1.11
4.97 0.96
4.61 0.72
7.29 1.51
11.42 1.01
>20
11.67 0.01
3.01 1.28
2.89 0.75
3.32 1.03
8.44 1.06
4.68 0.24
>10
7.31 0.176
>10
>10
>10
>10
>10
>10
>10
4.75 0.97
1.27 0.277
>10
>10
>10
4.44 1.45
12.43 1.26
6.01 0.38
>10
>10
6.21 0.58
>20
10.43 0.69
7.25 0.24
6.16 1.98
10.66 0.21
8.49 0.23
>10
4.78 0.13
>10
2.31 0.36
>10
9.59 0.15
>10
7.56 0.35
5.86 0.54
5.85 1.65
2.63 0.27
>10
9.07 3.14
>10
5.10 0.28
4.17 0.14
6.21 0.25
>10
5.38 0.68
>10
3.65 0.74
2.65 0.12
>10
8.86 0.36
>10
>10
>10
>10
>10
>10
9.23 1.02
3.39 0.89
2.54 0.85
>10
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
>10
>10
>10
>10
>10
>10
4s
4t
8.96 1.26
>10
>10
4.83 0.94
10.19 3.27
9.39 1.72
4.06 0.56
3.64 0.89
3.47 0.32
>20
6.63 2.10
>10
6.25 1.50
>10
>10
>10
4.82 0.83
>10
>10
2.77 0.11
7.15 0.42
>20
8.36 0.95
19.3 0.67
>10
2.75 0.15
4.30 0.43
>10
5.51 0.23
2.28 1.22
7.75 0.58
>10
3.7 1.35
7.31 0.48
6.55 0.09
ND
ND
>10
7.22 0.07
4.42 0.22
ND
>10
N
4u
4v
4w
4x
7a
6.95 2.17
5.58 0.39
8.36 2.63
2.46 0.95
10.50 2.47
7.59 0.96
3.08 0.77
>20
>10
>10
>10
0.91 0.07
8.77 0.29
NDb
ND
8.75 0.62
>10
8.19 0.51
ND
>20
N
>20
1.03 0.29
>10
7b
7c
8.64 1.19
2.91 0.07
>10
>10
>10
11a
11b
11c
11d
E28
>20
>10
>20
1.70 0.29
>10
>10
>10
0.70 0.34
>10
ND
>10
ND
a
Cell proliferation experiment determined by MTT method, The data are the mean SD from at least three independent experiments.
Not determined.
b
4v displayed similar inhibitory activity for all cell lines, indicating
chlorine at 3-position of indole had no favorable effect. Compared to
4u (without methoxy group at the para- position of sulfonamide),
compound 4a (para-methoxy) displayed higher potency against
all cancer cell lines, suggesting electron-donating methoxy group
at sulfonamide para- position could increase the compound inhibi-
tory ability. However, for compound 4s, with two methoxy groups
at meta- and para- position, low solubility in the assay solution
was observed, which may lead to the reduced potency compared
to 4a. Apparently, the position of the aryl group on the phenyl ring
of indole was critical for the compound potency, compound 4a, with
Ar at 7-position, demonstrated much higher inhibitory activity
compared to 11b (Ar at position 6) and 11d (Ar at position 5) as indi-
cated by their IC50 values. In addition, heterocycles at position 7
were tolerated. For example, compounds 4c (3-thienyl), 4d
(4-pyridyl-) and 4f (2-furanyl) displayed similar IC50 values with
compound 4b (phenyl), while compound 4g (2-thienyl) exhibited
the lowest activity. A variety of substitutes at R3 position were
investigated, and it was observed that the electron withdrawing
cyano group (4o) or methylsulfonyl (4n) was more favorable for
compound inhibitory activity comparing to chloro- (4s) or fluoro-
(4j). Both 4s and 4j were more potent than compound 4l, with elec-
tron donation methoxy- substitute. Additionally, introduction of
substituent at 3-(4i) or 4-position (4j) position of the phenyl ring
decreased the compound inhibitory activity. Finally, insertion of
amino linkage (7a, 7b) or amide linkage (7c) would not add favor-
able effect to the compound potency.
was performed to detect the amount of p-STAT3 at Tyr 705 and the
T-STAT3 using MDA-MB-231 breast cancer cell, E28 was used as
control compound. As shown in Fig. 3A, after 12 h treatment, all
three compounds (4a, 4n and 7a) reduced the amount of p-STAT3
in a concentration depended manner, while the amount of T-STAT3
was not changed at the selected concentration range, indicating
these compounds exerted inhibitory effect against STAT3 phospho-
rylation, and the increase of p-STAT3 was hardly related to the
reduction of the T-STAT3. This inhibitory manner is similar to that
of E28, though the activity is slightly weaker.
2.2.3. Compound 4a inhibited IL-6 induced STAT3 phosphorylation
To further study the compound inhibitory effect toward STAT3
phosphorylation, MDA-MB-231 cell was starved by cultivating in
serum-free medium overnight, then treated with 4a for 2 h before
being stimulated by IL-6 (25 ng/mL). The result is shown in Fig. 3B.
IL-6 stimulation significantly elevated the level of phosphorylated
STAT3, while 4a treatment markedly reduced the overexpression
of STAT3 phosphorylation. This result further demonstrated 4a
could inhibit STAT3 phosphorylation.
2.2.4. Compound 4a could induce cell apoptosis
To determine whether compound 4a possessed the ability to
induce cell apoptosis, MDA-MB-231 cell was treated with different
concentration of 4a, and the level of full PARP and cleaved PARP
were characterized through western blot analysis. Consistent with
the expectation, 4a treatment significantly elevated the level of
cleaved PARP in time- and concentration-dependent manner
(Fig. 3C). While PARP is the characteristic protein for cell apoptosis,
this result demonstrated that the antiproliferative activity of 4a
was partially attributed to its ability to induce cell apoptosis.
2.2.2. Inhibition the phosphorylation of STAT3 (Tyr705)
To investigate the compound interaction with STAT3 protein,
compounds 4a, 4n and 7a were selected and western blot analysis
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Fig. 3. Compounds affected STAT3-Y705 phosphorylation in MDA-MB-231 cell.
2.2.5. Compound 4a could block the cell cycle
dependent manner, which is similar to that of colchicine, both 4a
and colchicine destroyed tubulin assembly process, consistent with
the results from immunofluorescence staining experiment.
Based on the above observed results from cell apoptosis exper-
iment, the effect of compound 4a on cell cycle was examined by
flow cytometry, and the result was shown in the Fig. 4. Treatment
of A549 cell with 4a for 12 h at different concentration resulted in a
concentration-dependent accumulation of A549 cells in the G2/M
phase, accompanied by losses of the G1 and S phases, especially
the reduction of G1 phase. These results indicate cell cycle arrested
in G2/M phase was induced by 4a.
3. Conclusion
In summary, a class of N-arylsulfonyl substituted indole deriva-
tives were synthesized and evaluated as dual inhibitors of STAT3
and tubulin. We have demonstrated that this series of compounds
retained inhibitory activity to STAT3 phosphorylation, while
empowered inhibitory ability to tubulin polymerization. Several
compounds demonstrated strong antiproliferative activity
in vitro. It was also confirmed the representative compound
blocked the cell cycle in A549 with cell accumulation at G2/M
phase. Additionally, immunofluorescence staining and inhibition
of tubulin polymerization experiments confirmed the compound
microtubule-destabilizing effect. Further study should be carried
out to investigate these compounds in vivo antitumor activity, as
well as the pharmacokinetic properties.
2.2.6. Fluorescence images of microtubules morphology
To investigate the compound effect on the cellular microtubule,
the morphology of microtubules in A549 cells was examined by
confocal microscope. Paclitaxel and colchicine were used as refer-
ence compounds. The results were shown in Fig. 5. As reported,
paclitaxel promoted b-tubulin polymerization, while colchicine
inhibited microtubule polymerization process. At 5 lM, com-
pounds 4a and 7c exhibited similar behavior with colchicine by
destroying the b-tubulin assembly. This result revealed that com-
pound 4a and 7c could bind to b-tubulin and exert the inhibitory
effect against microtubule polymerization. By comparison, com-
pound E28 exerted no effect to tubulin polymerization/
depolymerization.
4. Materials and methods
4.1. Chemical synthesis
2.2.7. Inhibition of tubulin polymerization in vitro
To further examine the compound inhibitory activity against
tubulin polymerization, 4a was evaluated for inhibition of tubulin
polymerization using tubulin polymerization assay. Paclitaxel and
colchicine were chosen as control compounds. The result was
shown in Fig. 6. Contrary to the paclitaxel depolymerization effect,
compound 4a inhibited tubulin polymerization in a concentration-
All chemicals were commercially available from J&K Scientific
LTD, Sinopharm Chemical Reagent Co, Ltd and Energy Chemical.
The chemical reaction process was monitored by thin layer chro-
matography (TLC) and observed under the UV light (254 nm).
Reaction of post-processing contains silica gel chromatograph,
recrystallization, salting out methods and so on. 1H NMR and 13C
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Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
Fig. 4. Concentration effect of compound 4a on cell cycle progress of A549 cells.
NMR spectra were measured on a Agilent 400 MHz or 600 MHz
spectrometer and referenced to TMS. All final compounds were
examined by HPLC analysis to assure the purity was higher than
98%. Mass spectra were obtained on an Agilent HRMS (ESI)
spectrometer.
reaction. The mixture was extracted with DCM (3 Â 20 mL). The
combined organic layers were dried with Na2SO4, the solvent
was removed by rotary evaporators to afford reaction intermedi-
ates 3a–3f, 6a, 6b, and 10a–10d.
A mixture of compound 6a (0.541 mmol) and Fe (3.63 mmol)
was stirred in 5 mL of EtOH:H2O = 3:1 for 10 min. To this mixture
was added AcOH (7.31 mmol) slowly, and the resulting reaction
mixture was stirred at 75 °C until the reaction was complete. The
mixture was filtered through a pad of Celite, and the filtrate was
extracted with EA (3 Â 20 mL). The combined organic layers were
washed with saline for three times, dried with Na2SO4, concen-
trated, and purified by silica gel chromatography to give 6c.
7-Bromo-1-((4-methoxyphenyl)sulfonyl)-1H-indole (3a). Yield
72%, white solid; 1H NMR (400 MHz,CDCl3) d 7.89 (d, J = 4.0 Hz,
4.1.1. The synthesis of intermediates: 2b, 9a, and 9b
The solution of 5-, 6- or 7-bromo substituted indole (0.26
mmol) and NCS (0.31 mmol) in 5 mL DMF was stirred for 1 h, then
water was added to terminate the reaction. The aqueous layer was
extracted with DCM (3 Â 15 mL). The combined organic layers
were dried with Na2SO4, DCM was removed by rotary evaporator
to afford intermediates 2b, 9a, and 9b.
7-Bromo-3-chloro-1H-indole (2b). Yield 89%, liquid. 1H NMR
1H, CH@CH-N), 7.74 (d, J = 8.0 Hz, 2H, Ar-H), 7.51 (d, J = 8.0 Hz,
1H, Ar-H), 7.45 (d, J = 8.0 Hz, 1H, Ar-H), 7.06 (d, J = 4.0 Hz, 1H,
Ar-H), 6.92 (d, J = 12.0 Hz, 2H, Ar-H), 6.70 (d, J = 4.0 Hz, 1H,
(400 MHz, CDCl3) d 8.39 (s, 1H, NH), 7.59 (d, J = 8.0 Hz, 1H,
CH@CH-N), 7.40 (d, J = 8.0 Hz, 1H, Ar-H), 7.25 (d, J = 8.0 Hz, 1H,
Ar-H), 7.07 (m, 1H, Ar-H).
6-Bromo-3-chloro-1H-indole (9a). Yield 92%, liquid. 1H NMR
CH@CH-N), 3.83 (s, 3H, CH3)
7-Bromo-3-chloro-1-((4-methoxyphenyl)sulfonyl)-1H-indole (3b).
(400 MHz, CDCl3) d 8.34 (s, 1H, NH), 7.63 (d, J = 7.8 Hz, 1H,
Yield 62%, white solid; 1H NMR (400 MHz,CDCl3) d 9.13 (d, J =
CH@CH-N), 7.39 (d, J = 7.6 Hz, 1H, Ar-H), 7.04 (t, J = 7.7 Hz, 1H,
Ar-H), 6.66 (s, 1H, Ar-H).
3.8 Hz, 1H, CH@CH-N), 8.36 (d, J = 6.7 Hz, 1H, Ar-H), 7.70 (dd, J =
7.8, 4.7 Hz, 1H, Ar-H), 7.55 (t, J = 7.7 Hz, 2H, Ar-H), 7.45 (t, J = 7.4
5-Bromo-3-chloro-1H-indole (9b),21 Yield 93%. 1H NMR (400
Hz, 1H, Ar-H), 7.28 (s, 1H, Ar-H), 6.42 (s, 1H, CH@CH-N), 3.59 (s,
3H, CH3).
MHz, CDCl3) d 8.13 (s, 1H, NH), 7.78 (d, J = 1.8 Hz, 1H, CH@CH-N),
7.33 (dd, J = 8.7, 1.8 Hz, 1H, Ar-H), 7.24 (d, J = 8.7 Hz, 1H, Ar-H),
7.19 (d, J = 2.6 Hz, 1H, Ar-H).
7-Bromo-1-((3,4-dimethoxyphenyl)sulfonyl)-1H-indole (3c). Yield
66%, white solid; 1H NMR (400 MHz,CDCl3) d 8.14 (s, 1H, CH@CH-
N), 7.46 (t, J = 6.0 Hz, 2H, Ar-H), 7.31 (t, J = 10.2 Hz, 2H, Ar-H), 7.22
(d, J = 12.0 Hz, 1H, Ar-H), 6.79 (d, J = 8.5 Hz, 1H, Ar-H), 6.55
4.1.2. The synthesis of intermediates: 3a-3f, 6a-6c and 10a-10d
A mixture of 2a (0.765 mmol), or 2b, 5a, 5b, 9a, 9b, TEBAC
(0.383 mmol) and NaOH (3.06 mmol) in 10 mL THF was stirred
for 30 min, then 4-methoxybenzenesulfonyl chloride (1.15 mmol)
was slowly added under ice bath condition, and the reaction was
stirred for another 30 min before adding water to terminate the
(d, J = 3.3 Hz, 1H, CH@CH-N), 3.81 (s, 6H, CH3).
7-Bromo-3-chloro-1-((3,4-dimethoxyphenyl)sulfonyl)-1H-indole
(3d). Yield 68%, white solid; 1H NMR (400 MHz,CDCl3) d 8.19 (s, 1H,
CH@CH-N), 7.52 (d, J = 3.3 Hz, 1H, Ar-H), 7.38 (d, J = 8.3 Hz, 1H,
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Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
7
Fig. 5. Effect of 4a and 7c on the cellular microtubule network.
Fig. 6. Compound 4a inhibits tubulin polymerization.
Ar-H), 7.35–7.28 (m, 2H, Ar-H), 6.85 (d, J = 8.5 Hz, 1H, Ar-H), 6.60
(d, J = 7.7 Hz, 1H, Ar-H), 7.47 (dd, J = 17.6, 8.0 Hz, 3H, Ar-H), 7.06
(d, J = 3.3 Hz, 1H, CH@CH-N), 3.86 (s, 6H, CH3).
(t, J = 7.7 Hz, 1H, Ar-H), 6.73 (d, J = 3.5 Hz, 1H, CH@CH-N).
7-Bromo-3-chloro-1-(phenylsulfonyl)-1H-indole (3f). Yield 81%,
white solid; 1H NMR (400 MHz, CDCl3) d 7.88 (d, J = 8.7 Hz, 2H,
7-Bromo-1-(phenylsulfonyl)-1H-indole (3e). Yield 89%, white
solid; 1H NMR (400 MHz,CDCl3) d 7.92 (d, J = 3.5 Hz, 1H, CH@CH-N),
7.80 (d, J = 7.5 Hz, 2H, Ar-H), 7.59 (t, J = 7.4 Hz, 1H, Ar-H), 7.53
Ar-H, CH@CH-N), 7.86 (d, J = 1.2 Hz, 1H, Ar-H), 7.68 (d, J = 1.8 Hz,
Please cite this article in press as: Zhou Q., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.023

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Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
1H, Ar-H), 7.58 (dd, J = 13.7, 6.3 Hz, 2H, Ar-H), 7.49–7.45 (m, 3H,
Ar-H).
1-((4-Methoxyphenyl)sulfonyl)-7-phenyl-1H-indole (4b). Yield
70%, white solid. 1H NMR (400 MHz, CDCl3) d 7.69 (d, J = 3.8 Hz,
1-((4-Methoxyphenyl)sulfonyl)-7-nitro-1H-indole (6a). Yield 87%,
1H, CH@CH-N), 7.48 (dd, J = 7.7, 1.1 Hz, 1H, Ar-H), 7.32 (dt, J =
17.0, 7.3 Hz, 5H, Ar-H), 7.21 (dd, J = 13.4, 8.3 Hz, 3H, Ar-H), 7.07
(d, J = 7.4 Hz, 1H, Ar-H), 6.74–6.72 (m, 2H, Ar-H), 6.71 (s, 1H,
white solid; 1H NMR (400 MHz, CDCl3) d 7.86 (d, J = 9.0 Hz, 2H, Ar-
H), 7.76 (d, J = 7.8 Hz, 1H, CH@CH-N), 7.72 (d, J = 3.7 Hz, 1H, Ar-H),
7.65 (d, J = 7.8 Hz, 1H, Ar-H), 7.31 (t, J = 7.8 Hz, 1H, Ar-H), 6.98 (d, J
CH@CH-N), 3.79 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3)
d
= 9.0 Hz, 2H, Ar-H), 6.80 (d, J = 3.7 Hz, 1H, CH@CH-N), 3.86 (s, 3H,
CH3).
163.17, 140.99, 133.61, 133.48, 131.05, 130.82, 129.64, 129.48,
128.81, 128.65, 127.45, 127.03, 123.75, 120.46, 113.81, 110.25,
55.57. HRMS (ESI) 386.0828 m/z [M+Na+] (calcd for 363.0929,
1-((4-Methoxyphenyl)sulfonyl)-1H-indole-7-carboxylic acid (6b).
Yield 65%, white solid; 1H NMR (400 MHz, CDCl3) d 8.97 (m, 1H,
C21H17NO3S).
1-((4-Methoxyphenyl)sulfonyl)-7-3-thienyl-1H-indole (4c). Yield
COOH), 8.23–8.17 (m, 1H, CH@CH-N), 7.54 (dd, J = 7.8, 4.7 Hz, 1H,
Ar-H), 7.40 (t, J = 7.7 Hz, 2H, Ar-H), 7.30 (t, J = 7.4 Hz, 1H, Ar-H),
56%, white solid. 1H NMR (400 MHz, CDCl3): d 7.75 (d, J = 3.6 Hz,
1H, CH@CH-N), 7.49 (d, J = 4.0 Hz, 1H, Ar-H), 7.40 (d, J = 1.2 Hz,
1H, Ar-H), 7.30 (d, J = 8.8 Hz, 2H, Ar-H), 7.20 (t, J = 7.6 Hz, 1H, Ar-
H), 7.09–7.06 (m, 2H, Ar-H), 6.78 (d, J = 8.0 Hz, 2H, Ar-H), 6.71 (d,
7.12 (d, J = 7.6 Hz, 2H, Ar-H), 6.26 (s, 1H, CH@CH-N), 3.44 (s, 3H,
CH3).
1-((4-Methoxyphenyl)sulfonyl)-7-amino-1H-indole (6c). Yield
59%, yellow solid; 1H NMR (400 MHz, CDCl3) d7.78 (d, J = 8.6 Hz,
J = 3.6 Hz, 1H, Ar-H), 6.48 (d, J = 0.8 Hz, 1H, CH@CH-N), 3.81 (s,
3H, CH3). 13C NMR (151 MHz, CDCl3) d 163.24, 141.10, 140.35,
134.19, 133.29, 130.45, 130.27, 128.83, 128.56, 124.39, 123.42,
121.13, 120.83, 113.88, 113.28, 109.22, 55.59. HRMS (ESI)
370.0475 m/z [M+H+] (calcd for 369.0493, C19H15NO3S2).
2H, Ar-H), 7.55 (d, J = 3.7 Hz, 1H, CH@CH-N), 7.01 (t, J = 7.6 Hz,
1H, Ar-H), 6.88–6.83 (m, 3H, Ar-H, CH@CH-N), 6.57 (d, J = 7.7 Hz,
2H, Ar-H), 5.06 (s, 2H, NH2), 3.77 (s, 3H, CH3).
6-Bromo-1-((4-methoxyphenyl)sulfonyl)-1H-indole (10a). Yield
1-((4-Methoxyphenyl)sulfonyl)-7-4-pyridinyl-1H-indole
(4d).
86%, white solid; 1H NMR (400 MHz,CDCl3) d 7.90 (d, J = 3.4 Hz,
Yield 34%, white solid: 1H NMR (600 MHz, CDCl3) d 8.55 (s, 2H,
1H, CH@CH-N), 7.74 (d, J = 8.7 Hz, 2H, Ar-H), 7.51 (d, J = 7.7 Hz,
1H, Ar-H), 7.45 (d, J = 7.7 Hz, 1H, Ar-H), 7.04 (t, J = 7.7 Hz, 1H, Ar-
Ar-H), 7.67 (d, J = 3.7 Hz, 1H, CH@CH-N), 7.57 (d, J = 7.8 Hz, 1H,
Ar-H), 7.30 (t, J = 5.9 Hz, 3H, Ar-H), 7.20 (d, J = 8.7 Hz, 2H, Ar-H),
H), 6.91 (d, J = 8.7 Hz, 2H, Ar-H), 6.70 (d, J = 3.5 Hz, 1H, CH@CH-
N), 3.82 (s, 3H, CH3).
7.05 (d, J = 7.4 Hz, 1H, CH@CH-N), 6.78–6.75 (m, 3H, Ar-H), 3.81
(s, 3H, CH3). 13C NMR (151 MHz, CDCl3) d 163.50, 150.33, 147.53,
133.71, 132.86, 130.94, 129.31, 128.63, 127.82, 127.66, 124.70,
124.01, 121.96, 114.06, 110.51, 55.66. HRMS (ESI) 365.0905 m/z
[M+H+] (calcd for 364.0882, C20H16N2O3S).
1-((4-Methoxyphenyl)sulfonyl)-7-5-pyrimidinyl-1H-indole (4e),
Yield 61%,. 1H NMR (400 MHz, CDCl3) d 9.24 (s, 1H, N = CH-N),
5-Bromo-1-((4-methoxyphenyl)sulfonyl)-1H-indole (10b). Yield
90%, white solid; 1H NMR (400 MHz,CDCl3) d7.64 (d, J = 3.4 Hz,
1H, CH@CH-N), 7.49 (s, 1H, Ar-H), 7.47 (s, 1H, Ar-H), 7.26–7.16
(m, 2H, Ar-H), 6.78 (t, J = 7.7 Hz, 1H, Ar-H), 6.65 (d, J = 8.7 Hz, 2H,
Ar-H), 6.44 (d, J = 3.5 Hz, 1H, CH@CH-N), 3.57 (s, 3H, CH3).
6-Bromo-3-chloro-1-((4-methoxyphenyl)sulfonyl)-1H-indole (10c).
Yield 68%, white solid; 1H NMR (400 MHz,CDCl3) d 7.85 (d, J =
8.64 (s, 2H, CH = N), 7.72 (d, J = 1.7 Hz, 1H, CH@CH-N), 7.59 (d, J
= 7.3 Hz, 1H, Ar-H), 7.41–7.29 (m, 1H, Ar-H), 7.12 (d, J = 6.2 Hz,
8.8 Hz, 1H, CH@CH-N), 7.79 (d, J = 9.0 Hz, 2H, Ar-H), 7.64 (s, 1H,
Ar-H), 7.55 (d, J = 3.6 Hz, 1H, Ar-H), 7.38 (d, J = 8.8 Hz, 1H, Ar-H),
2H, Ar-H), 7.00 (d, J = 6.1 Hz, 1H, CH@CH-N), 6.77 (s, 3H, Ar-H),
3.77 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3) d 163.24, 141.10,
140.35, 134.19, 133.29, 130.45, 130.27, 128.83, 128.56, 124.39,
123.42, 121.13, 120.83, 113.88, 113.28, 109.22, 55.59. HRMS (ESI)
366.0817 m/z [M+H+] (calcd for 365.0834, C19H15N3O3S).
6.88 (d, J = 9.0 Hz, 2H, Ar-H), 6.57 (d, J = 3.6 Hz, 1H, CH@CH-N),
3.79 (s, 3H, CH3).
5-Bromo-3-chloro-1-((4-methoxyphenyl)sulfonyl)-1H-indole (10d).
Yield 61%, white solid; 1H NMR (400 MHz,CDCl3) d 7.84 (d, J =
7-2-Furanyl-1-((4-methoxyphenyl)sulfonyl)-1H-indole (4f). Yield
68%, white solid. 1H NMR (400 MHz, CDCl3): d 7.61 (d, J = 3.7 Hz,
8.8 Hz, 1H, CH@CH-N), 7.77 (d, J = 9.0 Hz, 2H, Ar-H), 7.61 (d, J =
1.6 Hz, 1H, Ar-H), 7.52 (s, 1H, Ar-H), 7.42 (dd, J = 8.8, 1.7 Hz,
1H, CH@CH-N), 7.50–7.44 (m, 2H, Ar-H), 7.41 (d, J = 9.0 Hz, 2H,
Ar-H), 7.29 (d, J = 7.5 Hz, 1H, Ar-H), 7.23 (d, J = 7.5 Hz, 1H, Ar-H),
6.77 (d, J = 9.0 Hz, 2H, Ar-H), 6.69 (d, J = 3.7 Hz, 1H, Ar-H), 6.52–
1H, CH@CH-N), 6.85 (d, J = 9.0 Hz, 2H, Ar-H), 3.74 (s, 3H, CH3).
6.48 (m, 1H, Ar-H), 6.43 (d, J = 3.2 Hz, 1H, CH@CH-N), 3.78 (s, 3H,
4.1.3. Synthesis of the final compounds 4a–4x, and 11a–11d
General procedure. A solution of 3a–3f (0.3 mmol), PdCl2(dppf)
(0.003 mmol), phenylboronic acid (0.446 mmol), and potassium
carbonate (1.04 mmol) in 5 mL of DME:H2O = 3.5:1 was heated at
86 °C for 2 h. Then, water was added to terminate the reaction,
and the mixture was extracted with DCM (3 Â 15 mL). Combined
organic layers were washed with saline for three times, dried with
Na2SO4, concentrated and purified by silica gel chromatography to
afford the target compounds.
CH3). 13C NMR (151 MHz, CDCl3)
d 163.24, 141.10, 140.35,
134.19, 133.29, 130.45, 130.27, 128.83, 128.56, 124.39, 123.42,
121.13, 120.83, 113.88, 113.28, 109.22, 55.59. HRMS (ESI)
354.0798 m/z [M+H+] (calcd for 353.0722, C19H15NO4S).
1-((4-Methoxyphenyl)sulfonyl)-7-2-thienyl-1H-indole (4g). Yield
61%, white solid. 1H NMR (400 MHz, CDCl3) d 7.76 (d, J = 3.5 Hz,
1H, CH@CH-N), 7.49 (d, J = 7.7 Hz, 1H, Ar-H), 7.40 (d, J = 2.8 Hz,
1H, Ar-H), 7.30 (dd, J = 8.7, 3.0 Hz, 2H, Ar-H), 7.22–7.19 (m, 1H,
Ar-H), 7.09–7.06 (m, 2H, Ar-H), 6.79 (dd, J = 8.7, 2.9 Hz, 2H, Ar-
N-(4-Methoxyphenyl)sulfonyl-7-(3-fluoro-4-cyano)-phenyl-1H-
indole (4a). Yield 77%, white solid; 1H NMR (400 MHz,CDCl3) d 8.08
H), 6.72 (d, J = 3.7 Hz, 1H, Ar-H), 6.48 (s, 1H, CH@CH-N), 3.80 (s,
3H, CH3). 13C NMR (151 MHz, CDCl3) d 163.26, 141.12, 140.37,
134.21, 133.31, 130.47, 130.29, 128.85, 128.58, 124.42, 123.45,
121.15, 120.85, 113.90, 113.30, 109.24, 55.61. HRMS (ESI)
368.0422 m/z [MÀH+] (calcd for 369.0493, C19H15NO3S2).
(d, J = 8.6 Hz, 1H, CH@CH-N), 7.85 (d, J = 8.8 Hz, 2H, Ar-H), 7.73 (s,
1H, Ar-H), 7.65 (dd, J = 13.4, 5.6 Hz, 2H, Ar-H), 7.49 (dd, J = 13.5,
8.7 Hz, 2H, Ar-H), 7.41 (d, J = 10.3 Hz, 1H, Ar-H), 6.90 (d, J = 8.8 Hz,
2H, Ar-H), 6.72 (d, J = 3.2 Hz, 1H, Ar-H), 3.79 (s, 3H, CH3).13C NMR
(151 MHz, CDCl3) d 163.63, 163.33, 161.61, 148.62 (d, J = 8.5 Hz),
133.89, 133.06, 132.42, 131.14, 129.09, 128.55, 127.97, 127.72,
126.05 (d, J = 2.9 Hz), 124.21, 122.11, 117.21 (d, J = 20.0 Hz), 114.29,
114.08, 110.96, 99.77 (d, J = 15.4 Hz), 55.71. HRMS (ESI) m/z
429.0700 [M+Na+] (calcd for 406.0787 C22H15FN2O3S).
1-((4-Methoxyphenyl)sulfonyl)-7-(1-methyl-1H-imidazol-2-yl)-
1H-indole (4h). Yield 57%, white solid. 1H NMR (400 MHz, CDCl3) d
8.04 (s, 1H, CH@CH-N), 7.99–7.89 (m, 1H, Ar-H), 7.76 (d, J = 8.8
Hz, 2H, Ar-H), 7.72 (d, J = 9.1 Hz, 1H, Ar-H), 7.51 (d, J = 7.7 Hz,
1H, Ar-H), 7.39 (m, 2H, Ar-H), 7.12 (s, 1H, Ar-H), 6.95 (s, 1H,
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Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
9
CH@CH-N), 6.90 (s, 2H, Ar-H), 3.82 (s, 6H, CH3). 13C NMR (151
MHz, CDCl3) d 163.10, 158.62, 133.81, 133.39, 133.18, 130.65
(d, J = 10.1 Hz), 130.42, 129.49, 128.62 (d, J = 12.5 Hz), 123.74,
120.16, 115.96, 114.56, 113.73, 112.73, 110.25, 55.52, 55.11.
HRMS (ESI) 368.0975 m/z [M+H+] (calcd for 367.0991,
7.55 (d, J = 7.8 Hz, 1H, Ar-H), 7.44 (d, J = 8.0 Hz, 2H, Ar-H), 7.28
(d, J = 7.7 Hz, 1H, Ar-H), 7.17 (d, J = 8.8 Hz, 2H, Ar-H), 7.02 (d, J =
7.4 Hz, 1H, CH@CH-N), 6.75 (dd, J = 5.7, 3.0 Hz, 3H, Ar-H), 3.82 (s,
3H, CH3), 3.12 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3) d 171.09,
163.40, 146.60, 138.81, 133.65, 133.21, 130.89, 130.21, 129.45,
128.84, 128.41, 128.0, 126.48, 123.87, 121.53, 114.13, 110.28,
60.35, 55.67. HRMS (ESI) 464.0712 m/z [M+Na+] (calcd for
441.0705, C22H19NO5S2).
C
19H17N3O3S).
7-(3-Fluorophenyl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole (4i).
Yield 50%, white solid. 1H NMR (600 MHz, CDCl3) d 7.73 (d, J =
3.8 Hz, 1H, CH@CH-N), 7.53 (dd, J = 7.8, 1.0 Hz, 1H, Ar-H), 7.28–
7.26 (m, 1H, Ar-H), 7.24 (t, J = 1.7 Hz, 1H, Ar-H), 7.23 (d, J = 1.1
Hz, 2H, Ar-H), 7.22 (s, 1H, Ar-H), 7.18 (d, J = 8.4 Hz, 2H, Ar-H),
N-(4-Methoxyphenyl)sulfonyl-7-(4-cyano)-phenyl-1H-indole (4o).
Yield 76%, white solid. 1H NMR (400 MHz, CDCl3): d 7.65 (d, J = 3.8
Hz, 1H, CH@CH-N), 7.59 (d, J = 8.2 Hz, 2H, Ar-H), 7.53 (d, J = 7.7 Hz,
1H, Ar-H), 7.41 (d, J = 8.1 Hz, 2H, Ar-H), 7.29 (d, J = 7.5 Hz, 1H, Ar-
7.03 (dd, J = 7.3, 0.8 Hz, 1H, CH@CH-N), 6.77 (t, J = 6.9 Hz, 3H,
Ar-H), 3.83 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3) d 163.31,
162.81, 161.19, 142.97 (d, J = 8.0 Hz), 133.50, 133.29, 130.84,
129.56 (d, J = 4.0 Hz), 128.83 (d, J = 8.3 Hz), 128.67, 128.32,
125.53 (d, J = 2.4 Hz), 123.72, 120.96, 116.57, 116.43, 114.15–
113.71, 110.07, 55.60. HRMS (ESI) 404.0736 m/z [M+Na+] (calcd
for 381.0835, C21H16FNO3S).
H), 7.19 (d, J = 8.9 Hz, 2H, Ar-H), 7.04 (d, J = 7.4 Hz, 1H, CH@CH-
N), 6.77–6.73 (m, 3H, Ar-H), 3.81 (s, 3H, CH3). 13C NMR (151
MHz, CDCl3) d 163.45, 145.79, 133.76, 133.26, 131.33, 131.02,
129.99, 129.33, 129.15, 128.57, 128.03, 124.07, 121.54, 119.09,
113.93, 110.77 (d, J = 5.7 Hz), 55.65. HRMS (ESI) 411.0781 m/z
[M+Na+] (calcd for 388.0882, C22H16N2O3S).
7-(4-Fluorophenyl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole (4j).
N-4-Methoxyphenylsulfonyl-7-(4-hydroxyl)phenyl-1H-indole (4p).
Yield 55%, white solid. 1H NMR (600 MHz, CDCl3): d 7.71 (d, J =
Yield 63%, white solid. 1H NMR (600 MHz, CDCl3): d 8.41 (s, 1H,
3.7 Hz, 1H, CH@CH-N), 7.50 (d, J = 7.7 Hz, 1H, Ar-H), 7.25 (d, J =
7.5 Hz, 1H, Ar-H), 7.21 (dd, J = 11.5, 3.8 Hz, 4H, Ar-H), 7.03 (d, J
= 7.4 Hz, 1H, Ar-H), 6.97 (t, J = 8.7 Hz, 2H, Ar-H), 6.75 (d, J = 6.0
OH), 7.82 (d, J = 8.8 Hz, 2H, Ar-H), 7.66 (d, J = 7.7 Hz, 1H,
CH@CH-N), 7.55 (d, J = 8.3 Hz, 2H, Ar-H), 7.20 (dd, J = 9.4, 5.2 Hz,
2H, Ar-H), 7.14 (dd, J = 13.8, 7.9 Hz, 3H, Ar-H), 7.00 (d, J = 8.7
Hz, 2H, Ar-H), 6.74 (d, J = 3.2 Hz, 1H, CH@CH-N), 3.79 (s, 3H,
CH3). 13C NMR (151 MHz, CDCl3): d 163.33, 162.83, 161.21,
142.98 (d, J = 8.0 Hz), 133.51, 133.30, 130.85, 129.57 (d, J = 4.0
Hz), 128.85 (d, J = 8.3 Hz), 128.68, 128.33, 125.54 (d, J = 2.4 Hz),
123.74, 120.97, 116.59, 116.44, 114.16, 113.72, 110.09, 55.61.
HRMS (ESI) 404.0739 m/z [M+Na+] (calcd for 381.0835,
Hz, 2H, Ar-H), 6.62 (s, 1H, CH@CH-N), 3.89 (s, 3H, CH3). 13C
NMR (151 MHz, CDCl3) d 164.21, 148.78, 138.27, 133.45, 130.77,
129.35, 128.47, 126.64, 124.71, 124.12, 123.13, 121.94, 120.48,
120.27, 114.47, 103.11, 55.79. HRMS (ESI) 378.0805 m/z [MÀH+]
(calcd for 379.0878, C21H17NO4S).
7-(2-Fluorophenyl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole (4q).
C
21H16FNO3S).
Yield 69%, white solid. 1H NMR (600 MHz, CDCl3): d 7.75 (d, J =
7-(3,4-Dichlorophenyl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole
3.6 Hz, 1H, CH@CH-N), 7.49 (d, J = 7.7 Hz, 1H, Ar-H), 7.40 (d, J =
1.3 Hz, 1H, Ar-H), 7.30 (d, J = 8.8 Hz, 2H, Ar-H), 7.20 (t, J = 7.6
Hz, 1H, Ar-H), 7.08–7.07 (m, 2H, Ar-H), 6.78 (d, J = 8.9 Hz, 3H,
Ar-H), 6.71 (d, J = 3.6 Hz, 1H, Ar-H), 6.48 (d, J = 0.7 Hz, 1H,
(4k). Yield 60%, white solid. 1H NMR (400 MHz, CDCl3): d 7.75 (d, J =
3.6 Hz, 1H, CH@CH-N), 7.55 (d, J = 7.8 Hz, 1H, Ar-H), 7.37 (d, J = 8.2
Hz, 1H, Ar-H), 7.23 (d, J = 7.7 Hz, 1H, Ar-H), 7.21–7.15 (m, 3H, Ar-H),
7.06 (s, 1H, Ar-H), 6.97 (d, J = 7.3 Hz, 1H, Ar-H), 6.79 (s, 1H, CH@CH-
N), 6.76 (d, J = 5.2 Hz, 2H, Ar-H), 3.84 (s, 3H, CH3). 13C NMR (151
MHz, CDCl3) d 163.43, 140.54, 133.41, 133.01, 131.38, 131.18 (d, J
= 12.9 Hz), 130.70, 129.75, 129.57, 129.21, 129.03, 128.37, 128.17,
127.91, 123.59, 121.35, 114.05, 109.46, 55.62. HRMS (ESI)
454.0044 m/z [M+Na+] (calcd for 431.0150, C21H15Cl2NO3S).
7-(4-Methoxyphenyl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole (4l).
Yield 64%, white solid. 1H NMR (400 MHz, CDCl3): d 7.70 (d, J =
CH@CH-N), 3.81 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3) d
163.26, 141.11, 140.37, 134.21, 133.31, 130.47, 130.29, 128.85,
128.57, 124.41, 123.44, 121.15, 120.85, 113.90, 113.29, 109.23,
55.61. HRMS (ESI) 404.0739 m/z [M+Na+] (calcd for 381.0835,
C
21H16FNO3S).
7-(Furan-3-yl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole
(4r).
Yield 81%, white solid. 1H NMR (600 MHz, CDCl3) d 7.75 (s, 1H,
CH@CH-N), 7.49 (d, J = 7.7 Hz, 1H, Ar-H), 7.40 (d, J = 2.8 Hz, 1H,
Ar-H), 7.30 (dd, J = 8.7, 3.0 Hz, 2H, Ar-H), 7.21–7.18 (m, 1H, Ar-
H), 7.09–7.05 (m, 2H, Ar-H), 6.78 (dd, J = 8.7, 2.9 Hz, 2H, Ar-H),
3.7 Hz, 1H, CH@CH-N), 7.46 (d, J = 7.7 Hz, 1H, Ar-H), 7.20 (dd, J
= 17.3, 8.3 Hz, 5H, Ar-H), 7.04 (d, J = 7.3 Hz, 1H, CH@CH-N),
6.82 (d, J = 8.5 Hz, 2H, Ar-H), 6.74–6.70 (m, 3H, Ar-H), 3.87 (s,
3H, CH3), 3.79 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3):d
163.10, 158.62, 151.06, 133.81, 133.39, 133.18, 130.65 (d, J =
10.1 Hz), 130.42, 129.49, 128.62 (d, J = 12.5 Hz), 123.74, 120.16,
115.96, 114.56, 113.73, 112.73, 110.25, 55.52, 55.11. HRMS
(ESI) 416.0934 m/z [M+Na+] (calcd for 393.1035, C22H19NO4S).
1-((4-Methoxyphenyl)sulfonyl)-7-(4-(trifluoromethoxy)phenyl)-
1H-indole (4m). Yield 57% white solid. 1H NMR (400 MHz, CDCl3): d
6.72 (d, J = 3.7 Hz, 1H, Ar-H), 6.47 (s, 1H, CH@CH-N), 3.80 (s, 3H,
CH3). 13C NMR (151 MHz, CDCl3)
d 163.00, 140.86, 140.11,
133.96, 133.06, 130.21, 130.03, 128.60, 128.32, 124.16, 123.19,
120.89, 120.60, 113.65, 113.04, 108.98, 55.36. HRMS (ESI)
354.0784 m/z [M+H+] (calcd for 353.0722, C19H15NO4S).
3-Chloro-N-4-methoxylbenzenesulfonyl-7-(4-chlorophenyl)-1H-
indole (4s). Yield 74%, white solid. 1H NMR (400 MHz, CDCl3) d 7.70
(d, J = 3.7 Hz, 1H, CH@CH-N), 7.51 (d, J = 7.7 Hz, 1H, Ar-H), 7.24–
7.18 (m, 5H, Ar-H), 7.15 (d, J = 8.4 Hz, 2H, Ar-H), 7.01 (d, J = 7.3 Hz,
7.73 (d, J = 3.7 Hz, 1H, CH@CH-N), 7.53 (d, J = 7.7 Hz, 1H, Ar-H), 7.24–
7.19 (m, 3H, Ar-H), 7.16 (d, J = 8.9 Hz, 2H, Ar-H), 7.10 (d, J = 8.3 Hz,
2H, Ar-H), 7.01 (d, J = 7.4 Hz, 1H, Ar-H), 6.75 (d, J = 2.4 Hz, 2H, Ar-H),
6.73 (s, 1H, CH@CH-N), 3.80 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3)
d 167.67, 164.26, 162.55, 147.98 (d, J = 8.1 Hz), 137.58, 134.35 (d, J =
15.3 Hz), 134.13, 133.77, 132.29, 130.88, 129.56, 129.12, 128.81,
126.86, 125.02, 123.71–123.36, 117.90, 115.05, 114.91, 114.50,
114.00 (d, J = 12.0 Hz), 99.89 (d, J = 12.8 Hz), 65.54. HRMS (ESI)
470.0651 m/z [M+Na+] (calcd for 447.0752, C22H16F3NO4S).
1H, CH@CH-N), 6.77–6.73 (m, 3H, Ar-H), 3.81 (s, 3H, CH3). 13C NMR
(151 MHz, CDCl3) d 163.27, 139.18, 133.49 (d, J = 3.0 Hz), 132.99,
130.79 (d, J = 3.0 Hz), 129.84, 129.56, 128.55, 128.37, 127.51,
123.74, 120.84, 113.86, 109.97, 55.63. HRMS (ESI) 455.0027 m/z [M
+Na+] (calcd for 455.0042, C21H16ClNO3S).
N-3,4-Dimethoxylbenzenesulfonyl-7-(4-cyano-3-fluorophenyl)-
1H-indole (4t). Yield 79%, white solid. 1H NMR (400 MHz, CDCl3): d
7.64 (d, J = 3.2 Hz, 1H, CH@CH-N), 7.58 (dd, J = 13.1, 7.2 Hz, 2H, Ar-
H), 7.33–7.27 (m, 2H, Ar-H), 7.08 (dd, J = 13.3, 8.8 Hz, 2H, Ar-H),
6.88 (d, J = 8.3 Hz, 1H,), 6.74 (dd, J = 12.3, 6.0 Hz, 2H, CH3), 6.69 (s,
1-((4-Methoxyphenyl)sulfonyl)-7-(4-(methylsulfonyl)phenyl)-1H-
indole (4n). Yield 50%, white solid. 1H NMR (400 MHz, CDCl3): d
7.82 (d, J = 7.9 Hz, 2H, Ar-H), 7.68 (d, J = 3.7 Hz, 1H, CH@CH-N),
Please cite this article in press as: Zhou Q., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.023

10
Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
1H, CH@CH-N), 3.87 (s, 3H, CH3), 3.74 (s, 3H, CH3). 13C NMR (151 MHz,
CDCl3) d 163.34, 161.63, 153.37, 148.78, 148.54 (d, J = 8.4 Hz), 133.90,
133.22, 132.43, 131.26, 129.08, 128.05, 127.68, 125.94 (d, J = 2.9 Hz),
124.28, 122.13, 120.50, 117.12 (d, J = 20.0 Hz), 114.17, 111.08,
110.18, 108.47, 99.77 (d, J = 15.3 Hz), 56.21, 56.05. HRMS (ESI)
459.0795 m/z [M+Na+] (calcd for 436.0893, C23H17FN2O4S).
N-(4-Methoxybenzenesulfonyl)-6-(4-cyano-3-fluorophenyl)-1H-
indole (11b). Yield 76%, white solid. 1H NMR(400 MHz, CDCl3): d 8.20
(s, 1H, CH@CH-N), 7.84 (d, J = 6.9 Hz, 2H, Ar-H), 7.70 (s, 1H, Ar-H),
7.64 (d, J = 8.1 Hz, 2H, Ar-H), 7.53 (d, J = 6.5 Hz, 1H, Ar-H), 7.46 (d,
J = 7.9 Hz, 2H, Ar-H), 6.91 (d, J = 6.8 Hz, 2H, Ar-H), 6.70 (s, 1H,
CH@CH-N), 3.80 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): d 163.82,
163.52, 161.80, 148.81 (d, J = 8.5 Hz), 134.08, 133.25, 132.61,
131.33, 129.28, 128.74, 128.16, 127.91, 126.24 (d, J = 2.9 Hz),
124.40, 122.30, 117.40 (d, J = 20.0 Hz), 114.48, 114.27, 111.15,
99.96 (d, J = 15.4 Hz), 55.90. HRMS (ESI) m/z 424.1129 [M+NH+4]
(calcd for 406.0787, C22H15FN2O3S).
N-Benzenesulfonyl-7-(4-cyano-3-fluorophenyl)-1H-indole
(4u)
Yield 86%, white solid; 1H NMR (600 MHz, CDCl3): d 7.67 (d, J =
3.7 Hz, 1H, CH@CH-N), 7.57 (d, J = 7.8 Hz, 1H, Ar-H), 7.55–7.51
(m, 2H, Ar-H), 7.35 (t, J = 7.9 Hz, 2H, Ar-H), 7.30 (dd, J = 11.2, 3.9
Hz, 3H, Ar-H), 7.22 (dd, J = 7.9, 1.1 Hz, 1H, Ar-H), 7.04 (t, J = 9.5
Hz, 2H, Ar-H), 6.78 (d, J = 3.8 Hz, 1H, CH@CH-N). 13C NMR (151
MHz, CDCl3): d 163.26, 161.55, 148.39 (d, J = 8.4 Hz), 137.62,
133.76 (d, J = 8.7 Hz), 133.00, 132.44, 130.98, 128.94, 127.83 (d, J
= 13.6 Hz), 126.15, 126.01 (d, J = 3.0 Hz), 124.28, 122.14,117.25
(d, J = 20.0 Hz), 114.20, 111.06, 99.81 (d, J = 15.4 Hz), HRMS (ESI)
m/z 375.0626 [MÀH+] (calcd for 376.0682 C21H13FN2O2S).
3-Chloro-N-(4-methoxybenzenesulfonyl)-5-(4-cyano-3-fluoro-
phenyl)-1H-indole (11c). Yield 76%, white solid. 1H NMR (600 MHz,
CDCl3) d 8.09 (d, J = 8.6 Hz, 1H, CH@CH-N), 7.85 (d, J = 9.0 Hz, 2H, Ar-
H), 7.72 (s, 1H, Ar-H), 7.71–7.66 (m, 1H, Ar-H), 7.61 (s, 1H, Ar-H),
7.58 (d, J = 8.7 Hz, 1H, Ar-H), 7.53–7.49 (m, 1H, Ar-H), 7.45 (d, J =
10.1 Hz, 1H, CH@CH-N), 6.92 (d, J = 8.9 Hz, 2H, Ar-H), 3.81 (s, 3H,
CH3). 13C NMR (151 MHz, CDCl3) d 164.24 (d, J = 6.7 Hz), 162.55,
148.08 (d, J = 8.0 Hz), 134.25, 133.93, 133.76, 129.24, 129.08, 128.91,
124.85, 123.69–123.38, 117.82, 115.02, 114.89, 114.72, 114.48,
113.98, 113.63, 99.82 (d, J = 15.6 Hz), 55.72. HRMS (ESI) m/z
458.0736 [M+NH4+] (calcd for 440.0398, C22H14ClFN2O3S).
3-Chloro-N-4-methoxylbenzenesulfonyl-7-(4-cyano-3-fluorophenyl)-
1H-indole (4v). Yield 76%, white solid; 1H NMR (600 MHz, CDCl3)
d 7.64 (s, 1H, CH-N), 7.62–7.57 (m, 2H, Ar-H), 7.38 (t, J = 7.6 Hz,
1H, Ar-H), 7.29 (dd, J = 8.0, 1.1 Hz, 1H, Ar-H), 7.24 (d, J = 8.9 Hz,
2H, Ar-H), 7.14 (d, J = 7.4 Hz, 1H, Ar-H), 7.08 (d, J = 9.6 Hz, 1H,
CH@CH-N), 6.80 (d, J = 8.9 Hz, 2H, Ar-H), 3.82 (s, 3H, CH3). 13C
NMR (151 MHz, CDCl3) d 163.85, 163.34, 161.63, 147.75 (d, J =
8.4 Hz), 132.55 (d, J = 5.1 Hz), 131.54, 128.89, 128.67, 128.35
(d, J = 15.6 Hz), 126.96, 125.97 (d, J = 3.0 Hz), 124.73, 119.76,
117.23, 117.10, 115.68, 114.14 (d, J = 16.9 Hz), 100.14 (d, J =
15.3 Hz), 55.72. HRMS (ESI) m/z 458.0740 [M+NH+4] (calcd for
440.0398, C22H14ClFN2O3S)
N-(4-Methoxybenzenesulfonyl)-5-(4-cyano-3-fluorophenyl)-1H-
indole (11d). Yield 86%, white solid. 1H NMR (600 MHz, CDCl3) d 7.64
(d, J = 3.7 Hz, 1H, CH@CH-N), 7.59–7.54 (m, 2H, Ar-H), 7.29 (t, J = 7.7
Hz, 2H, Ar-H), 7.22 (d, J = 8.9 Hz, 2H, Ar-H), 7.06 (dd, J = 15.3, 8.5 Hz,
2H, Ar-H), 6.76 (dd, J = 12.2, 6.3 Hz, 2H, Ar-H; 1H, CH@CH-N), 3.80 (s,
3H, CH3). 13C NMR (151 MHz, CDCl3) d 163.60, 163.30, 161.59, 148.59
(d, J = 8.5 Hz), 133.87, 133.04, 132.40, 131.11, 129.07, 128.52, 127.95,
127.70, 126.03 (d, J = 2.9 Hz), 124.18, 122.08, 117.18 (d, J = 20.0 Hz),
114.26, 114.06, 110.94, 99.74 (d, J = 15.4 Hz), 55.69. HRMS (ESI) m/
z 424.1138 [M+NH4+] (calcd for 406.0787, C22H15FN2O3S).
3-Chloro-N-3,4-dimethoxylbenzenesulfonyl-7-(4-cyano-3-fluo-
rophenyl)-1H-indole (4w) Yield 78%, white solid. 1H NMR (600 MHz,
CDCl3) d 7.63–7.57 (m, 3H, Ar-H), 7.39 (t, J = 7.7 Hz, 1H, CH@CH-N),
7.29 (d, J = 7.9 Hz, 1H, Ar-H), 7.15 (dd, J = 12.4, 8.6 Hz, 2H, Ar-H),
4.1.4. Synthesis of 7a and 7b
6.91 (dd, J = 8.5, 1.2 Hz, 1H, CH@CH-N), 6.73 (dd, J = 17.2, 4.8 Hz, 2H,
Ar-H), 3.88 (s, 3H, CH3), 3.76 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3)
d 163.41, 161.70, 153.66, 148.87, 147.74 (d, J = 8.4 Hz), 132.80, 132.63,
131.65, 128.88, 128.48, 128.23, 127.17, 125.84 (d, J = 3.1 Hz), 124.93,
120.77, 119.84, 117.13, 117.00, 116.06, 114.02, 110.25, 108.48,
100.14 (d, J = 15.4 Hz), 56.18, 56.12. HRMS (ESI) 471.0471 m/z [M
+H+] (calcd for 470.0503, C22H14ClFN2O3S).
A mixture of 6c (0.25 mmol), Pd2(dba)3 (0.005 mmol), 4-bromo-
2-fluorobenzonitrile or 4-bromobenzonitrile (0.25 mmol), t-BuONa
(0.35 mmol), BINAP (0.015 mmol) in toluene (10 mL) was stirred
under N2 at 80 °C until the reaction was complete. The mixture
was extracted with DCM (3 Â 15 mL), and the combined organic
layers were washed with saline for three times, dried with Na2SO4,
concentrated and purified by silica gel chromatography to afford
compound 7a or 7b.
3-Chloro-N-benzenesulfonyl-7-(4-cyano-3-fluorophenyl)-1H-indole
(4x). Yield 73%, white solid: 1H NMR(400 MHz, CDCl3): d 7.67 (s,
1-((4-Methoxyphenyl)sulfonyl)-7-(N-(4-cyano-3-fluoro)phenyl)
1H, CH-N), 7.59 (dd, J = 11.3, 4.3 Hz, 1H, Ar-H), 7.55 (dd, J = 11.5,
4.6 Hz, 2H, Ar-H), 7.38 (dd, J = 16.0, 8.2 Hz, 3H, Ar-H), 7.33–7.30
(m, 2H, Ar-H), 7.23 (dd, J = 7.9, 1.1 Hz, 1H, Ar-H), 7.14 (d, J = 7.4
Hz, 1H, Ar-H), 7.05 (d, J = 9.5 Hz, 1H, Ar-H). 13C NMR (151 MHz,
CDCl3):d 163.30, 161.58, 147.55 (d, J = 8.4 Hz), 137.04, 134.05,
132.54 (d, J = 19.6 Hz), 131.48, 129.04 (d, J = 11.6 Hz), 128.23,
126.80, 126.25, 125.95 (d, J = 3.1 Hz), 124.85, 119.83, 117.32,
117.24 (d, J = 20.1 Hz), 115.89, 114.03, 100.18 (d, J = 15.4 Hz),
HRMS (ESI) m/z 428.0643 [M+NH+4] (calcd for 410.0292,
amino-1H-indole (7a). Yield 79%, yellow solid. 1H NMR (600 MHz,
CDCl3) d 7.79 (d, J = 3.6 Hz, 1H, NH), 7.47–7.42 (m, 2H, Ar-H), 7.30–
7.27 (m, 1H, CH@CH-N), 7.24 (t, J = 8.6 Hz, 3H, Ar-H), 7.17 (d, J =
7.7 Hz, 1H, Ar-H), 6.76 (d, J = 3.7 Hz, 1H, Ar-H), 6.57 (d, J = 8.9 Hz,
2H, Ar-H), 6.49 (dd, J = 8.6, 1.7 Hz, 1H, Ar-H), 6.30 (dd, J = 11.7, 1.6
Hz, 1H, CH@CH-N), 3.72 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3) d
134.43, 133.91 (d, J = 1.9 Hz), 129.66, 128.45, 127.75, 125.87,
124.34, 120.43, 118.83, 115.06, 114.18, 110.89, 108.39, 100.41,
100.25, 89.52 (d, J = 16.1 Hz), 55.64. HRMS (ESI) 422.0971 m/z [M
+H+] (calcd for 421.0896, C22H16FN3O3S).
C
21H12ClFN2O2S).
3-Chloro-N-(4-methoxybenzenesulfonyl)-6-(4-cyano-3-fluorophenyl)-
1H-indole (11a). Yield 62%, white solid. 1H NMR (600 MHz,
1-((4-Methoxyphenyl)sulfonyl)-7-(N-(4-cyano)phenyl)amino-1H-
indole (7b) Yield 82%, yellow solid. 1H NMR (600 MHz, CDCl3) d
CDCl3): d 8.10 (d, J = 8.7 Hz, 1H, CH@CH-N), 7.85 (d, J = 8.8 Hz,
2H,Ar-H), 7.73 (s, 1H,Ar-H), 7.68 (t, J = 7.3 Hz, 1H,Ar-H), 7.61
(s, 1H,Ar-H), 7.58 (d, J = 8.7 Hz, 1H,Ar-H), 7.51 (d, J = 8.0 Hz,
7.78 (d, J = 3.6 Hz, 1H, CH@CH-N), 7.45 (s, 1H, NH), 7.36 (dd, J =
11.7, 5.7 Hz, 3H, Ar-H), 7.28 (s, 1H, Ar-H), 7.27 (s, 1H, Ar-H),
1H,Ar-H), 7.45 (d, J = 10.1 Hz, 1H, CH@CH-N), 6.92 (d, J = 8.8
Hz, 2H, Ar-H), 3.81 (s, 3H, CH3). 13C NMR (151 MHz, CDCl3): d
164.24 (d, J = 5.5 Hz), 162.54, 148.07 (d, J = 8.1 Hz), 134.25,
133.91, 133.76, 129.24, 129.07, 128.90, 124.86, 123.73,
117.82, 115.01, 114.88, 114.73, 114.48, 113.99, 113.63, 99.80
(d, J = 15.6 Hz), 55.73. HRMS (ESI) m/z 458.0732 [M+NH+4] (calcd
for 440.0398, C22H14ClFN2O3S).
7.22–7.19 (m, 2H, Ar-H), 6.74 (d, J = 3.6 Hz, 1H, CH@CH-N), 6.71
(d, J = 8.6 Hz, 2H, Ar-H), 6.53 (d, J = 8.9 Hz, 2H, Ar-H), 3.69 (s, 3H,
CH3). 13C NMR (151 MHz, CDCl3)
d 163.37, 148.32, 134.38,
133.46, 129.56 (d, J = 18.8 Hz), 128.15, 127.94, 126.87, 124.24,
119.87, 119.47, 117.84, 114.42, 114.15, 108.46, 100.93, 55.60.
HRMS (ESI) 404.1063 m/z [M+H+] (calcd for 403.0991,
C22H17N3O3S).
Please cite this article in press as: Zhou Q., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.023

Q. Zhou et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
11
4.1.5. Synthesis of 7c
4.4. IL-6 induction of STAT3 phosphorylation
A mixture of 6b (0.165 mmol), 4-cyanoaniline (0.165 mmol),
EDC (0.248 mmol) in pyridine (5 mL) was stirred at room temper-
ature overnight. Water was added to quench the reaction, and the
mixture was adjusted to pH 3.0 by slow addition of diluted HCl.
The resulting mixture was extracted with DCM (3 Â 10 mL), and
the combined organic layers were washed with saline for three
times, dried with Na2SO4, concentrated, and purified by silica gel
chromatography to afford compound 7c.
MDA-MB-231 cells were seeded in 6-well plates and allowed to
adhere overnight. The cells were serum-starved for 12 h, then left
untreated or treated with 4a. After 12 h, the untreated and 4a trea-
ted cells were stimulated by IL-6 (25 ng/mL). The cells were har-
vested after 30 min and analyzed by western blot assay.
4.5. Immunofluorescence confocal microscopy
N-(4-Cyanophenyl)-1-((4-methoxyphenyl)sulfonyl)-1H-indole-7-
carboxamide (7c) Yield 74%, yellow solid. 1H NMR (400 MHz, CDCl3)
A549 cells were seeded sparsely in eight-well chamber slides
and treated with or without identified compounds 4a and 7c for
24 h. Following treatment, cells were fixed with cold MeOH at
À20 °C for 15 min, washed three times with PBS, and blotted with
1% PBS plus 0.1% Triton X-100 for 30 min at 37 °C. Microtubules
were detected by incubation with a monoclonal anti-b-tubulin at
37 °C for 1 h. Then, the cells were washed with PBS and incubated
with a FITC-conjugated antimouse IgG antibody. Nucleus were
stained with DAPI, and microtubule distribution images were
acquired with the Confocal Spectral Microscope.
d 10.47 (s, 1H, CONH), 8.26 (d, J = 7.2 Hz, 1H, CH@CH-N), 8.14 (d, J =
7.8 Hz, 2H, Ar-H), 7.83 (d, J = 8.0 Hz, 2H, Ar-H), 7.60 (d, J = 3.3 Hz, 1H,
Ar-H), 7.36–7.28 (m, 4H, Ar-H), 6.73–6.70 (m, 2H, Ar-H), 6.68 (s, 1H,
CH@CH-N), 3.73 (s, 3H, CH3).13C NMR (151 MHz, CDCl3) d 163.87,
163.68, 138.55, 134.33, 132.52, 129.56, 128.54, 128.39, 128.19,
126.01, 125.83, 124.95, 119.55, 118.50, 118.03, 115.44, 114.54,
111.11, 55.67. HRMS (ESI) 432.1012 m/z [M+H+] (calcd for
431.0940, C23H17N3O4S).
4.6. In vitro tubulin polymerization assay
4.2. MTT assay
The effect of compound 4a on tubulin polymerization was
determined kinetically using a kit (BK006P, Cytoskeleton Inc, Den-
ver, CO). Cold porcine tubulin protein (>99% purity) was added to
G-PEM buffer (80 mM PIPES, 2 mM MgCl2, 0.5 mM EGTA, and 1
mM GTP, pH 6.9) containing 15% glycerol with or without 4a.
The sample mixture was dotted onto a pre-warmed 96-well plate,
which was immediately transferred to a 37 °C plate reader. The
absorbance was read every minute for 30 min at 340 nm.
All cell lines were purchased from Cell Bank of Shanghai Insti-
tute of Biochemistry and Cell Biology, Chinese Academy of Sciences
(Shanghai, China), including: Human non-small cell lung carci-
noma cell (A549), human breast carcinoma cell (MCF-7) and
(MDA-MB-231), Human liver cancer cells (HePG2), Oral epider-
moid cancer cells (KB), Human prostate cancer cells (DU145). All
cells were maintained in RPMI-1640 medium, supplemented with
10% fetal bovine serum containing 50 mg/mL penicillin and 50 mg/
mL streptomycin. Cells were grown to 80% confluency in a tissue
culture flask at 37 °C in a humidified atmosphere containing 5%
CO2. Cells were then seeded in 96-well plates at a density of
3000–5000 cells per well and incubated at 37 °C for 16 h in a
humidified 5% CO2 incubator. Different concentrations of tested
Acknowledgment
We thank Yongming Zhu for the experimental guidance on
immunofluorescence confocal microscopy experiment.
compounds (The total volume of 10
lL) were added in triplicate
A. Supplementary data
to the plates in 190 L medium, the plates were incubated at 37
l
°C for 48 h. The percentage of DMSO in the medium not exceeded
0.1%. 3-(4, 5-Dimethylthiazolyl)-2, 5-diphenyltetrazoliumbromide
(MTT) was added to evaluate cell viability. The absorbance was
read by an ELISA reader (SpectraMax Plus384, Molecular Devices,
Sunnyvale, CA) at a test wavelength of 570 nm and a reference
wavelength of 570 nm.
Supplementary data associated with this article can be found, in
the online version, at https://doi.org/10.1016/j.bmc.2017.11.023.
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4.3. Western blot
MDA-MB-231 cells were incubated with various concentrations
of tested compounds for 12 h and harvested after trypsinisation.
Cells were gathered in epoxide (EP) tubes under low-speed cen-
trifuge, and then resuspended in PBS for three times, treated with
RIPA (Radio-Immunoprecipitation Assay) lysis buffer (50 mM
TrisHCl, pH 7.4, 150 mM NaCl, 0.25% deoxycholic acid, 1% NP-40,
1 mM EDTA and protease inhibitors) to extract the total proteins.
An aliquot of proteins from the total cell lysates was separated by
sodium dodecyl sulfate (10%) polyacryl amide gel electrophoresis.
The separated proteins were then transferred to PVDF membrane
and blocked with 5% skim milk for 1 h. PVDF membrane was further
washed with TBST (3 Â 20 mL), and incubated with primary anti-
bodies specific for objective stripe at 4 °C overnight. The objective
stripe was observed by enhanced chemiluminescence (ECL)
immunoblotting technique after the membrane was blotted with
HRP-labeled second antibody for 1 h at room temperature.
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Please cite this article in press as: Zhou Q., et al. Bioorg. Med. Chem. (2017), https://doi.org/10.1016/j.bmc.2017.11.023