Design, synthesis, and biological evaluation of cyclic-indole derivatives as anti-tumor agents via the inhibition of tubulin polymerization

Article abstract of DOI:10.1016/j.ejmech.2016.09.056

This study revealed a new attractive cyclic-indole scaffold for the discovery of mitosis-targeting anti-tumour agents. Among all of the synthesized derivatives, compound 20 displayed the most potent anti-proliferative activity (with IC₅₀values

Full text of DOI:10.1016/j.ejmech.2016.09.056

Accepted Manuscript
Design, synthesis, and biological evaluation of cyclic-indole derivatives as anti-tumor
agents via the inhibition of tubulin polymerization
Jun Yan, Jinhui Hu, Baijiao An, Li Huang, Xingshu Li
PII:
S0223-5234(16)30792-9
10.1016/j.ejmech.2016.09.056
EJMECH 8922
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 30 May 2016
Revised Date: 26 August 2016
Accepted Date: 18 September 2016
Please cite this article as: J. Yan, J. Hu, B. An, L. Huang, X. Li, Design, synthesis, and biological
evaluation of cyclic-indole derivatives as anti-tumor agents via the inhibition of tubulin polymerization,
European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.09.056.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Design, synthesis, and biological evaluation of cyclic-indole
derivatives as anti-tumor agents via the inhibition of tubulin
polymerization
∗
Jun Yan, Jinhui Hu, Baijiao An, Li Huang, Xingshu Li
Institute of Drug Synthesis and Pharmaceutical Process, School of Pharmaceutical
Sciences, Sun Yat-sen University, Guangzhou 510006, China
ABSTRACT: This study revealed a new attractive cyclic-indole scaffold for the
discovery of mitosis-targeting anti-tumour agents. Among all of the synthesised
derivatives, compound 20 displayed the most potent anti-proliferative activity (with
IC values of 22 - 56 nM against seven cancer cell lines) and tubulin polymerization
5
0
inhibition (IC50 = 0.15 ± 0.07 ꢀM), which were much better than those of the
reference compound Combretastain A-4 (CA-4). High selectivity ratios (9.68-7.61) of
compound 20 toward human normal cells and cancer cells were also observed.
Immunofluorescence assay elucidated that compound 20 disrupted the intracellular
microtubule network and interfered with cell mitosis. Cellular mechanism studies
demonstrated that compound 20 arrested the cell cycle at the G /M phase and induced
2
apoptosis in a time- and dose-dependent manner. In summary, compound 20 deserves
consideration for in vivo anti-tumour evaluation in further studies.
KEYWORDS: cyclic-indole derivatives, antiproliferative activity, tubulin
polymerization inhibitor, cell cycle arrest, apoptosis
1
. Introduction
Microtubules are long, hollow cylinders that are mainly composed of α- and β-tubulin
Abbreviations: CA-4, Combretastain A-4; VDAs, vascular disrupting agents; HMTA, hexamethylenetetramine;
HUVECs, human umbilical vein endothelial cells; SARs, sturcture and activity relationships; SD, standard error;
PI, propidium iodide; HR-MS, high resolution mass spectra.
For X. Li.: Tel.: +086-20-3994-3050; Fax: +086-20-3994-3050; e-mail: lixsh@mail.sysu.edu.cn
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dimers; and γ-tubulin plays a major role in the nucleation of microtubule assembly.[1]
These tubulin dimers polymerize end-to-end to form a protofilament.[2] Microtubules
continuously keep a state of dynamic equilibrium to exert their structural function.[3,
4
] The tubulin-microtubule system is an important intracellular framework, which
plays a pivotal role in the maintenance of cell structure, regulation of motility, and cell
division.[2, 5, 6] Consequently, it has become an attractive target for the design of
new antimitotic agents for cancer therapy.[7-9] Antimitotic agents currently used in
the clinic are mainly classified into two broad categories: vinca alkaloids (represented
by vincristine and vinorelbine), which inhibit microtubule polymerization and toxoids
(represented by a family of taxanes), which promote microtubule polymerization.[10]
Colchicine was the first drug reported to function as the tubulin polymerization
inhibitor in the late 1930s.[11] Combretastatin A-4 (CA-4, Figure 1) is another potent
antimitotic agent, which was first isolated from the African bush willow Combretum
caffrum, and exhibits strong cytotoxicity against a variety of cancer cells.[12] The
encouraging antitumour profile of CA-4 has inspired many medicinal chemists’
extensive interest in the design and synthesis of CA-4 analogues.[13-16] Most of the
reported modifications involved stabilizing the conformation of the olefinic bridge
using different substitutions or replacing the 3-hydroxy-4-methoxyphenyl moiety of
CA-4 with other biologically active entities.[17] The cis double bond of CA-4 is easy
to isomerize to the more thermodynamically stable, but less bioactive, trans isomers.
In this case, numerous modifications, which on one hand aimed to stabilize the
conformation of CA-4 by replacing the olefinic bridge with different cyclic moieties
and on the other hand to retain its biological activity, were under extensive
investigation.[18, 19] Pinney and co-workers described the discovery of the
benzosuberene analog 1 (Figure 1) as a potent tubulin polymerization inhibitor,[20]
and then established various functionalized benzosuberene analogues for further
evaluation as promising anti-cancer agents.[21-23] Meantime, Maderna and other
groups also obtained new benzosuberene analogues with structural modifications on
the B-ring as cytotoxicity agents.[24, 25] Considering the extensive pharmacological
activities of the indole-based compounds, Pinney and other groups developed novel
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indole-based inhibitors of tubulin polymerization[26-29] and compound Oxi8006[30]
was one of the most representative agent which demonstrated pronounced interference
with tumor vasculature in a preliminary in vivo study.[31] In the previously work, the
seven membered ring of compound 1 was substituted with heterocyclic part to obtain
compound 2 (Figure 1) which displayed excellent anti-tumor activity in vitro and in
vivo.[32] In recent years, our group has also been committed to the design and
synthesis some indole-based compound 3[33] (Figure 1) and indole-chalcone
derivative 4[34] (Figure 1) as novel tubulin polymerization inhibitors. Inspired by the
work of Pinney and others, we fused benzosuberenes with indole-based part to obtain
a novel series of cyclic-indole derivatives. Preliminary biochemical evaluation in
terms of inhibition of tubulin polymerization along with anti-proliferative activity
study toward various types of cancer cells, analysis of structure-activity relationships,
and elucidation of the molecular mechanism are presented.
Figure 1. Structures of the representative tubulin polymerization inhibitors and
vascular disrupting agents (VDAs).
2
. Results and Discussion
.1 Chemistry
2
The synthetic routes for the cyclic-indole derivatives are outlined in Schemes 1-3.
Using the N-benzenesulfonyl-protected starting material 6, the Wittig reaction in the
presence of a strong base (NaHMDS) produced the aryl pentenoic acid 7.[35, 36] The
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intermediate 7 was then subjected to a hydrogenation and intramolecular
Friedel–Crafts acylation afforded cyclohepta-indolone 9. The reaction of 9 with a
lithium reagent in situ prepared from 5-bromo-1,2,3-trimethoxybenzene and n-BuLi
provides cyclohepta-indolol 10. A good yield of cyclohepta-indole derivative 11 was
easily obtained from the dehydration of 10 in the presence of an acid.[20]
Deprotection of the cyclohepta-indole 11 in the solution of KOH in methanol afforded
cyclohepta-indole 12. The modification of intermediate 12 by N-alkylation produced
compounds 13a - 13c. Treatment intermediate 12 with sodium cyanoborohydride in
acid conditions yielded the indoline 14. The methylation of indoline 14 with
iodomethane afforded methyl indoline 15. Indole-aldehyde 16 was obtained from
indole 12 using the Duff reaction in the presence of hexamethylenetetramine (HMTA)
(Scheme 1).
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Scheme 1. Reagents and conditions: (a) benzenesulfonyl chloride, KOH, TBAHS, CH Cl , room
2
2
temperature; (b) (i) (3-carboxypropyl)triphenylphosphonium bromide, NaHMDS, THF, -20 °C; (ii)
aldehyde, 6, -78 °C, 18 h; (c) Pd/C, H , MeOH; (d) Eaton's Reagent: 7.7% (w/w) P O in
2
2
5
methanesulfonic acid, room temperature; (e) (i) 5-bromo-1,2,3-trimethoxybenzene, n-BuLi, THF;
ii) ketone, 9, -78 °C, 12 h; (f) p-toluenesulfonic acid, CHCl ; (g) 1 M KOH, MeOH; (h) CH I /
(
3
3
C H Br / C H Br, NaH, THF; (i) NaBH CN, CH COOH; (j) Hexamethylenetetramine, CH COOH,
2
5
3
7
3
3
3
N .
2
Under the same conditions, aldehyde 17 was readily produced from N-methylated
indole 13a. Reduction of aldehyde 17 with sodium borohydride afforded indolyl
methanol 18. 13a was treated with oxalyl chloride to afford intermidate oxoacetyl
chloride. Then the mixture was immediately quenched by methanol to obtain
oxoacetate 19. Reduction of the double bond of olefin 13a gave alkane 20 (Scheme 2)
with a good yield.
Scheme 2. Reagents and conditions: (a) Hexamethylenetetramine, CH COOH, N ; (b) (i) oxalyl
3
2
chloride, Et O; (ii) CH OH, Et N; (c) H , Pd/C, CH OH; (d) NaBH , CH OH.
2
3
3
2
3
4
3
Simultaneously with the synthesis of cyclohepta-indoles, the hepta-cyclic nucleus was
also replaced by the five-cyclic skeleton. Similarly, acrylic acid 21 was prepared with
good yield using the Knoevenagel condensation reaction with malonic acid and the
starting material 6. The following hydrogenation and cyclization produced the
indolone 23 with an acceptable yield. Cyclopenta-indolone 25 was easily obtained by
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deprotection and then methylation of the indolone 23. Treating 23 with a lithium
reagent, prepared as described above at -78 °C, yielded cyclopenta-indole 26 by
sequentially treating the reaction mixture with aqueous NH Cl at room temperature
4
(Scheme 3).
Scheme 3. Reagents and conditions: (a) malonic acid, piperidine, pyridine; (b) Pd/C, H , MeOH;
2
(c) methanesulfonic acid, P O , room temperature; (d) 1 M KOH, MeOH; (e) CH I, NaH, THF; (f)
2 5 3
(i)5-bromo-1,2,3-trimethoxybenzene, n-BuLi, THF; (ii) ketone, 25, -78 °C, 12 h.
2
.2 In vitro antiproliferative activity
In the initial screening of the antiproliferative activity, seven human cancer cell
lines derived from various tissues and organs were chosen including the non-small
cell lung cancer cell line A549, human epithelial cervical cancer cell line HeLa,
human prostate cancer line PC-3, human liver carcinoma cell line Bel-7402, human
colon cancer cell line Lovo, human ovarian cancer cell line A2780, and human breast
carcinoma cell line MCF-7. Evaluation of the antiproliferative activity was carried out
using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay.
The data in Table 1 indicate that the length of the alkyl chain at the N-1 position of the
indole ring was obviously related to the anti-proliferative activity. Comparing the
activity of compounds 12 (IC range from 0.45 to 1.56 ꢀΜ) and 13a (IC range from
5
0
50
0
.044 to 0.093 ꢀΜ), the N-methylation was indeed favourable for the anti-proliferative
activity. Moreover, when the R group was replaced by a methyl group or an ethyl
1
group, both compounds 13a and 13b displayed similar potent activities. However,
extending the carbon chain to a propyl group resulted in a 7.27- to 17.7-fold decrease
in the anti-proliferative activities.
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Next, the effects of different substitutions on the 3-position of the indole ring were
also investigated. As shown in Table 1, when R was replaced by an aldehyde group
2
or hydroxymethyl group, compounds 17 and 18 nearly retained the excellent
anti-proliferative activities towards all of the tested cancer cell lines, while the
replacement with oxoacetyl group (compound 19) led to the absolute loss of activity.
Moreover, the difference in anti-proliferative activities between compounds 16 and 17
further evidenced the importance of the N-methylation.
To elucidate the sturcture and activity relationships (SARs) in more detail, we
investigated whether the double bond of the indole ring and hepta-cyclic ring were
indispensable for the anti-proliferative activity. After the double bond of the indole
ring and hepta-cyclic ring were reduced, the targeted compounds 15 and 20 also
displayed potent anti-proliferative activities. However, reducing the size of the
hepta-cyclic ring to the five-cyclic skeleton (compound 26) caused nearly a 3.04- to
7
.19-fold decrease in the anti-proliferative activities compared to the corresponding
compound 13a.
Table 1. Antiproliferative activity of compounds against seven human cancer cell
a
lines
b
IC50 mean±SD (ꢀΜ)
Compd.
A549
HeLa
PC-3
Bel-7402
0.62±0.29
Lovo
A2780
MCF-7
1
1
1
1
1
1
1
1
1
1
2
0.451±0.13
0.79±0.08
0.98±0.13
0.52±1.36
1.01±1.29
1.56±1.46
3a
3b
3c
4
0.049±0.023 0.056±0.017 0.093±0.025 0.078±0.031 0.047±0.013 0.044±0.012 0.069±0.021
0.056±0.031 0.061±0.014 0.085±0.036 0.066±0.014 0.039±0.015 0.032±0.023 0.079±0.026
0.52±0.03
0.60±0.02
0.69±0.12
0.48±0.23
0.69±0.17
0.24±0.09
0.78±0.31
0.312±0.242
0.701±0.16
0.741±0.09
0.797±0.321 1.123±0.231 0.597±0.159 0.812±0.452
5
0.056±0.009 0.045±0.014 0.077±0.021 0.061±0.011 0.079±0.037 0.069±0.038 0.058±0.029
0.47±0.57 1.26±0.74 1.13±0.15 1.02±0.39 2.06±1.17
0.86±0.12 0.95±0.23
6
7
0.029±0.014 0.041±0.027 0.031±0.013 0.049±0.029 0.052±0.021 0.033±0.011 0.059±0.019
0.043±0.002 0.033±0.015 0.049±0.021 0.046±0.009 0.032±0.017 0.031±0.019 0.063±0.022
8
9
>10
>10
>10
>10
>10
>10
>10
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2
2
0
6
0.022±0.013 0.056±0.019 0.047±0.035 0.039±0.009 0.049±0.033 0.037±0.025 0.051±0.041
0.149±0.091 0.326±0.129 0.412±0.014 0.561±0.138 0.297±0.112 0.186±0.021 0.449±0.127
0.010±0.001 0.012±0.001 0.026±0.009 0.013±0.006 0.017±0.002 0.016±0.002 0.029±0.004
CA-4
a
Cell lines were treated with different concentrations of the compounds for 48 h. Cell viability was measured by
b
MTT assay as described in the Experimental Section. IC50 values are indicated as the mean±SD (standard error)
of at least three independent experiments.
2
.3 In vitro inhibition of tubulin polymerization
Considering the critical role of microtubules in cell architecture, such as cell mitosis,
cell shape maintenance and protein transportation, we also evaluated the inhibitory
efficacy of these cyclic-indole derivatives on microtubule assembly in vitro.
According to the method originally described by Bonne et al.,[37] the tested
compounds were incubated with un-polymerized pure porcine tubulin, and the
polymerization was monitored by the change of fluorescence intensity. In the control
group, tubulin polymerization had indeed occurred as evidenced by an enhancement
of the fluorescence intensity along with time extension. When incubated with the
tested compounds at the indicated concentrations, the increased tendency of the
fluorescence intensity was slowed down or absolutely suppressed at higher
concentrations. On the base of the antiproliferative activity screening, compounds 13a,
1
5, 17, 18, 20, which beared representative structures and displayed excellent
antiproliferative activities, were primarily choosed for the tubulin polymerization
evaluation. The IC values of compounds 13a, 15, 17, 18, and 20 listed in Table 2
5
0
showed that these compounds are potent tubulin polymerization inhibitors and more
potent than reference compound CA-4. Since compound 20 displayed excellent
anti-proliferative activity and the best tubulin polymerization inhibitory activity in the
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initial screening, we selected it as the optimized compound for the following further
studies.
Table 2. Effects of the selected compounds on tublin polymerization inhibition
Compd.
13a
15
17
18
20
CA-4
a
IC50 /ꢀM
0.58 ± 0.12
1.04 ± 0.18
0.47 ± 0.02
0.41 ± 0.14
0.15 ± 0.07
1.12 ± 0.05
a
IC values are indicated as the mean±SD (standard error) of three independent experiments.
5
0
2
.4 Selectivity of compound 20 towards normal cells and cancer cells.
Considering the high cytotoxicity of chemotherapeutic agents, their selectivity
towards normal cells and cancer cells is particularly important. To evaluate the
selectivity of the optimal compound 20 towards human normal cells and cancer cells,
four types of non-tumorigenic cell lines from different origins were used, including
HUVECs (human umbilical vein endothelial cells), MCF-10A (human mammary
epithelial cell line), BJ (human dermal fibroblasts), and HLF (human embryonic lung
fibroblast cell line). The results summarized in Table 3 showed that 20 was associated
with a high cytotoxicity towards A549 cells and a relatively low toxicity towards
human normal cells, which exhibited 9.68-, 30.5-, 55.0-, and 76.1-fold selectivity
ratios for quiescent HUVECs, MCF-10A, BJ, and HLF cells, respectively. In order to
elucidate the results of the reduced cytotoxicity against HUVECs, we performed a
contemporaneous microscopy study using A549 cells and HUVECs cells treated with
compound 20 at 20 nM and 200nM (IC concentrations). As shown in Figure S ꢀ
5
0
(supporting information), treated with compound 20 nM for 48 h, the A549 cells
displayed obvious cell morphology alteration such as cell rounding, cell shrinkage,
membrane blebbing and loss of adherent property, which well indicated the
cytotoxicity of compound 20. Moreover, this morphology changes were more apprent
at 200 nM treatment. While, treated at the same condition, the HUVECs cells did not
exhibit any cell damges at 20 nM and displayed a certain extent of damage at 200 nM.
Along with the results of MTT assay (Table 3), these results well demonstrated the
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reduced cytotoxicity against HUVECs.The tremendous role of microtubules in cell
division makes it a fascinating target for anticancer drugs. Compared to the normal
cells, cancer cells have a rapid proliferation characteristics of division which highly
depend on microtubule dynamic equilibrium.[38] Thus, cancer cells are more
sensitive to microtubules-destabilizing agents, which might explain the apparent
selectivity of compound 20 toward A549 cells and four normal cell lines.
Table 3. The selectivity ratio of compound 20 toward human normal cells and cancer
cells.
a
c
IC , mean ± SD (ꢀM)
Selectivity ratio
50
A549
Quiescent HUVECs
MCF-10A
BJ
0.022 ± 0.013
0.213 ± 0.017
0.672 ± 0.156
1.211 ± 0.224
1.674 ± 0.538
b
9.68
30.5
55.0
76.1
HLF
a
b
Data are presented as the mean ± SE from the dose-response cures of at least three independent experiments. For
quiescent growth conditions, HUVECs were cultured in endothelial cell medium containing 0.5% fetal calf serum
c
and in absence of growth factor. Selectivity ratio = (IC50 human normal cells) / (IC50 A549).
2
.5 Disruption of intracellular microtubule dynamics
Microtubules, which consist of α-, β-tubulin heterodimers, are key components of the
mitotic spindle and always sustain the dynamic balance to maintain its biological
function.[39] Since the tubulin polymerization inhibitory effect of 20 has been
verified in vitro, we subsequently performed the immunofluorescence assay to
observe its effect on the intracellular microtubule system. As shown in Figure 2,
compound 20 disrupted the cellular microtubule network and disturbed the cell
mitosis. The nucleus and microtubules in the vehicle-treated group appeared as
normal state as characterized by the slim and fibrous microtubules, well-organized
structure surrounding the uncondensed nucleus, and spindles present during bipolar
1
0

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mitotic division. However, with increasing compound concentrations, the meshy
microtubule networks were heavily shrunk around the nucleus, and monopolarization
or multipolarization of the spindle and multinucleation phenomena were easily
observed, which indicated that compound 20 disrupted microtubule organizations and
interfered
with
the
mitosis
of
A549
cells.
Figure 2. Disruption effect of compound 20 on the cellular microtubule network (A) and cell
mitosis (B) visualized by immunofluorescence. A549 cells were plated in confocal dishes and
exposed to 20 at the indicated concentrations for 24 h. Then, the cells were fixed and processed to
study the immunofluorescence of microtubules (stained with primary β-tubulin mouse antibody
and Alexa Fluor 488 goat anti-mouse IgG antibody, green) and nuclei (stained with Hochest 33342,
blue) using an LSM 570 laser confocal microscope (Carl Zeiss, Germany) as described in the
Experimental Section. Magnification: ×64 (A), ×100 (B). The experiments were performed three
times, and the results of representative experiments are shown.
2
.6 Cell cycle analysis
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As most microtubule polymerization inhibitors disrupt cell mitosis and exert cell
cycle arrest effects,[40] we applied flow cytometry to analyse the cell cycle arrest
effect of compound 20. As shown in Figure 3, 20 caused significant cell cycle arrest at
the G /M phase in a dose- and time-dependent manner with concomitant losses of G
2
1
phase cells. When treated with 20 at 10, 25, and 50 nM for 24 h, the percentages of
cells arrested at the G /M phase were 41.0%, 55.1%, and 72.1%, respectively. After a
2
4
8 h treatment, the remarkable accumulation of cells arrested in the G /M phase was
2
more obvious. These results demonstrated that 20, similar to most tubulin
polymerization inhibitors, disrupted the dynamic balance of the tubulin-microtubule
system and further blocked the cell cycle distribution at the G /M phase.
2
Figure 3. Compound 20 arrested cell cycle progression at the G /M phase in a dose- and
2
1
2

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time-dependent manner. The A549 cells were treated with compound 20 at 10, 20, or 50 nM for 24
h (A) or 48 h (B). Quantitative analysis of the percentage of cells in each cell cycle phase were
analysed by EXPO32 ADC analysis software. The experiments were performed three times, and the
results of representative experiments are shown.
2
.7 Cell apoptosis analysis
According to the cell cycle analysis results above, it was assumed that compound 20
treatment induces cell apoptosis, which was evidenced by the appearance of the
characteristic hypodiploid DNA content peak (sub-G1) (Figure 5B). To confirm this
hypothesis, A549 cells were treated with various concentrations of compound 20 for
2
4 or 48 h and then were harvested, stained with Annexin V-FITC/PI and monitored
by flow cytometry. As shown in Figure 4, compound 20 induced A549 cell apoptosis
in a dose- and time-dependent manner. The percentage of apoptotic cells after the 24 h
treatment was only 0.63% in the control grouup. The early and late apoptotic cells
increased to 22.8%, 39.6%, and 59.8% after 24 h of 10, 20, and 50 nM of compound
2
0 treatment, respectively, whereas after a 48 h incubation, the percentages of the
early and late apoptotic cells were strikingly increased to 41.4%, 58.4%, and 60.2%,
respectively. Moreover, after the 48 h incubation, most cells treated at the highest
concentration underwent late apoptosis. These data demonstrate that compound 20
effectively induced cell apoptosis in A549 cells in a dose- and time-dependent manner,
eventually leading to cell death.
1
3

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Figure 4. Compound 20 induced A549 cell apoptosis in a dose- and time-dependent manner. The
A549 cells were treated with compound 20 at 10, 20, or 50 nM for 24 h (A) or 48 h (B). The
percentages of cells in each stage of cell apoptosis were quantified by flow cytometry: (upper left
quadrant) necrosis cells; (upper right quadrant) late-apoptotic cells; (bottom left quadrant) live
cells; and (bottom right quadrant) early apoptotic cells. The experiments were performed three
times, and the results of representative experiments are shown.
3
. Conclusions
In summary, we have designed, synthesized, and evaluated a series of novel
cyclic-indole derivatives as tubulin polymerization inhibitors for cancer therapy. All
of the designed compounds possessed potent anti-proliferative activities against seven
1
4

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human cancer cell lines derived from different tissues and organs.
Considering the
pivotal role of the tubulin-microtubule system in cell division, the in vitro tubulin
polymerization inhibitory activities were also evaluated. We discovered that these
compounds, which demonstrated good anti-proliferative activities in vitro, also
exhibited excellent tubulin polymerization inhibitory activities. The relatively low
cytotoxicity of compound 20 further revealed the possibility for clinical application.
Moreover, detection of the intracellular microtubule immunofluorescence confirmed
that the potent antiproliferative activity of compound 20 was strongly related to its
massive disruption and interference effect on microtubule network organization and
cell mitosis. Furthermore, the mechanism study elucidated that compound 20
triggered cell cycle arrest at the G /M phase and induced cell apoptosis in a dose- and
2
time-dependent manner. Taken altogether, compound 20 effectively inhibited tubulin
polymerization, disrupted the intracellular tubulin-microtubule balance, and interfered
with cell mitosis resulting in prolonged G /M cell cycle arrest and thus ultimately led
2
to apoptosis of cancer cells. As a promising new tubulin-targeting agent, compound
2
0 is worth further in vivo antitumour evaluation as a potential chemotherapeutic
agent.
4
4
. Experimental Section
.1 Chemistry
1
13
H NMR and C NMR spectra were acquired on a Bruker AvanceIII
spectrometer with TMS as the internal standard. High resolution mass spectra
HR-MS) were recorded on an Agilent LC–MS 6120 instrument with an ESI mass
(
selective detector in positive ion mode. Melting points were determined on an
SRS-OptiMelt automated melting point instrument. The purity of the synthesized
compounds was determined by high-performance liquid chromatography (HPLC)
with a TC-C18 column (4.6 × 250 mm, 5 ꢀm), an acetonitrile/water or methanol/water
mobile phase, and a flow rate of 1.00 mL/min.
Synthesis of 1-(phenylsulfonyl)-1H-indole-4-carbaldehyde (6)[35]
1
5

ACCEPTED MANUSCRIPT
After a suspension of 1H-indole-4-carbaldehyde 5 (2.0 g, 14 mmol), TBAHS
(
0.72 g, 2.1 mmol), and KOH (1.6 g, 28 mmol) in CH Cl (100 mL) was stirred for 20
2
2
min, benzenesulfonyl chloride (2.6 mL, 21 mmol) was added, and the mixture was
stirred at room temperature for 16 h. The reaction was quenched with water, and
extracted with CH Cl (50 mL × 3). The combined organic layer was dried over
2
2
anhydrous MgSO and concentrated under reduced pressure to give a yellow residue,
4
which was purified by silica gel chromatography (ethyl acetate / petroleum = 1:8) to
1
afford the 1-phenylsulfonyl indole (3.5 g, 12 mmol) as a white solid. Yield: 89%. H
NMR (400 MHz, CDCl3) δ 10.17 (s, 1H), 8.28 (d, J = 8.3 Hz, 1H), 7.88 (d, J = 8.1
Hz, 2H), 7.77 (d, J = 2.8 Hz, 1H), 7.72 (d, J = 7.4 Hz, 1H), 7.59 – 7.42 (m, 6H).
Synthesis of (E)-5-(1-(phenylsulfonyl)-1H-indol-4-yl)pent-4-enoic acid (7)[36]
To a a suspension of (3-carboxypropyl)triphenylphosphonium bromide (0.92 g,
2
.2 mmol) in THF (12 mL), NaHMDS (2.2 mL, 4.3 mmol) was added dropwise at
temperature of -20 °C and the suspension and further stirred for 20 min. The mixture
was cooled to -78 °C and then aldehyde 6 (0.57 g, 2.0 mmol) was added. After stirred
for 18 h, the solvent was removed in vacuo and H O was added. The residue was
2
extracted with diethyl ether and the H O layer was acidified to pH 2 using HCl (1 M).
2
The acidified aqueous layer was extracted with ethyl acetate and the combined
organic layers were dried over sodium sulfate, filtered and concentrated to dryness.
The product was purified over silica gel using ethyl acetate: petroleum (1:1) to obtain
1
the alkenoic acid (1.4 mmol, 0.48 g) as yellow oil. Yield: 68%. H NMR (400 MHz,
Chloroform-d) δ 7.92 – 7.82 (m, 3H), 7.58 (d, J = 3.7 Hz, 1H), 7.53 – 7.46 (m, 1H),
7
.40 (t, J = 7.8 Hz, 2H), 7.32 – 7.21 (m, 2H), 6.81 (d, J = 3.5 Hz, 1H), 6.76 – 6.65 (m,
H), 6.27 (dt, J = 15.9, 6.3 Hz, 1H), 2.56 (q, J = 5.6, 4.5 Hz, 4H).
1
Synthesis of 5-(1-(phenylsulfonyl)-1H-indol-4-yl)pentanoic acid (8)[35]
To a stirred suspension of 7 (0.71 g, 2.0 mmol) in methanol (10 mL), Pd / C
(
0.10 g) was added and the mixture was stirred under H atmosphere at room
2
temperature for 3 h. Then, the mixture was filtered and vacuumed to get colorless oil
1
(
0.21 g, 0.60 mmol). Yield: 30%. H NMR (400 MHz, Chloroform-d) δ 7.88 (dd, J =
8
.2, 1.0 Hz, 2H), 7.83 (d, J = 8.3 Hz, 1H), 7.57 (d, J = 3.7 Hz, 1H), 7.52 (t, J = 7.4 Hz,
1
6

ACCEPTED MANUSCRIPT
1
H), 7.43 (t, J = 7.7 Hz, 2H), 7.25 – 7.19 (m, 1H), 7.03 (d, J = 7.4 Hz, 1H), 6.70 (d, J
=
3.7 Hz, 1H), 2.81 (t, J = 7.1 Hz, 2H), 2.36 (t, J = 6.9 Hz, 2H), 1.74 – 1.67 (m, 4H).
Synthesis of 3-(phenylsulfonyl)-7,8,9,10-tetrahydrocyclohepta[e]indol-6(3H)-one
9)[41]
To a rapidly stirred solution of phosphorus pentoxide:methanesulfonic acid (1:10,
0 g), compound 8 (1.0 g, 2.8 mmol) was added in small portions. After the mixture
(
1
was stirred at room temperature for 4.0 h, 40 mL of water was added in drop wise and
the aqueous mixture was stirred rapidly for 5-10 min to ensure the hydrolysis of
methanesulfonic anhydride. The mixture was extracted with ethyl acetate, washed
subsequently with dilute aqueous sodium bicarbonate and water, dried over anhydrous
sodium sulfate, and concentrated in vacuum to provide the crude product, which was
purified by chromatography on silica gel (4:1 of petroleum/ethyl acetate as an eluent)
1
to afford the product (1.2 mmol, 0.42 g) as pink oil in 44% yield. H NMR (400 MHz,
Chloroform-d) δ 7.89 (d, J = 3.0 Hz, 3H), 7.74 (d, J = 8.7 Hz, 1H), 7.63 (d, J = 3.8 Hz,
1
H), 7.55 (d, J = 7.5 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 6.81 (d, J = 3.7 Hz, 1H), 3.10
d, J = 6.4 Hz, 2H), 2.78 – 2.74 (m, 2H), 1.91 (p, J = 6.5 Hz, 2H), 1.85 – 1.79 (m,
H).
Synthesis
,6,7,8,9,10-hexahydrocyclo- hepta[e]indol -6-ol (10)[20]
(
2
of
3-(phenylsulfonyl)-6-(3,4,5-trimethoxyphenyl)-
3
Under the argon atmosphere, the solution of 5-bromo-1,2,3-trimethoxybenzene
o
(
0.49 g, 2.0 mmol) in anhydrous THF (10 mL) was cooled to 78 C, and then added
−
n-BuLi (2.5 M , 0.80 mL, 2.0 mmol,) in drop wise via syringe. The mixture was
stirred for 0.5 h at the same temperature and followed by the addition of the solution
of indolone 9 (0.34 g, 1.0 mmol) in THF (20 mL). The reaction mixture was stirred at
−7
8 °C overnight. The reaction was carefully quenched by saturated aqueous NH Cl
4
and was stirred at room temperature for another 2 h. The organic layers were
separated and washed with water and brine respectively, dried over Na SO , filtered,
2
4
concentrated under reduced pressure. The crude product was chromatographed on
silica gel (petroleum/ethyl acetate, 3:1 as an eluent) to afford the product (0.55 g, 1.1
1
mmol) as yellow oil. Yield: 54%. H NMR (400 MHz, Chloroform-d) δ 7.93 – 7.88
1
7

ACCEPTED MANUSCRIPT
(
m, 2H), 7.81 (d, J = 8.9 Hz, 1H), 7.58 (d, J = 3.8 Hz, 1H), 7.56 – 7.50 (m, 2H), 7.45
t, J = 7.7 Hz, 2H), 6.77 (d, J = 3.8 Hz, 1H), 6.45 (s, 2H), 3.85 (s, 3H), 3.69 (s, 6H),
(
3.10 (dd, J = 15.1, 6.7 Hz, 1H), 2.79 – 2.68 (m, 1H), 2.57 (ddd, J = 14.1, 7.2, 2.8 Hz,
1H), 2.37 (s, 1H), 2.18 (ddd, J = 13.8, 10.3, 3.1 Hz, 1H), 1.94 (dt, J = 14.5, 7.2 Hz,
1H), 1.85 – 1.73 (m, 2H), 1.65 – 1.56 (m, 1H).
Synthesis
of
3-(phenylsulfonyl)-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-
tetrahydrocyclo-hepta[e] indole (11)[20]
Indolol 10 (0.20 g, 0.39 mmol) and p-toluenesulfonic acid (6.7 mg, 0.039 mmol)
were added in CHCl (5.0 mL) at room temperature for 30 min. The reaction was
3
quenched by saturated aqueous NaHCO , and extracted with CH Cl . The combined
3
2
2
organic layers were washed with saturated aqueous NaCl, dried over Na SO , filtered
2
4
and concentrated under reduced pressure. The crude product was chromatographed on
silica gel (8:1 of petroleum/ethyl acetate as an eluent) to afford the product
1
(
0.17g ,0.35 mmol) as white solid in 90% yield. H NMR (400 MHz, Chloroform-d) δ
7.97 – 7.86 (m, 2H), 7.80 (d, J = 8.6 Hz, 1H), 7.61 (d, J = 3.7 Hz, 1H), 7.59 – 7.51 (m,
1H), 7.46 (td, J = 7.1, 1.6 Hz, 2H), 7.00 (d, J = 8.6 Hz, 1H), 6.80 (dd, J = 3.7, 0.6 Hz,
1H), 6.44 (d, J = 3.0 Hz, 3H), 3.86 (s, 3H), 3.76 (s, 6H), 2.84 (t, J = 7.0 Hz, 2H), 2.23
(
q, J = 7.1 Hz, 2H), 1.92 (q, J = 7.2 Hz, 2H).
Synthesis of 6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]indole
12)[35]
To a stirred suspension of cyclohepta-indole derivatives 11 (0.49 g, 1.0 mmol) in
(
methanol (5.0 mL), 1 M KOH (aq) (1.2 mL, 1.2 mmol) was added and the mixture
was stirred at refluxed for 1 h. The reaction was quenched by water, and extracted
with ethyl acetate. The organic layer was collected and dried over anhydrous MgSO4
and concentrated in vacuo to yield colorless oil. The residue was purified by flash
column chromatography over silica gel (petroleum/ethyl acetate = 2:1) to afford 8
1
(
0.28 g, 0.81 mmol) as colorless oil. Yield: 81%; H NMR (400 MHz, DMSO) δ
11.10 (s, 1H), 7.34 (d, J = 3.1 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 6.69 (d, J = 8.4 Hz,
1
H), 6.57 (dd, J = 3.0, 0.6 Hz, 1H), 6.50 (s, 2H), 6.44 (t, J = 7.3 Hz, 1H), 3.68 (s, 6H),
.67 (s, 3H), 2.86 (t, J = 6.9 Hz, 2H), 2.27 – 2.15 (m, 2H), 1.88 (q, J = 7.1 Hz, 2H).
3
1
8

ACCEPTED MANUSCRIPT
1
3
C NMR (101 MHz, CDCl3) δ 152.84, 144.01, 139.09, 137.13, 134.80, 134.57,
1
2
3
30.82, 127.30, 127.03, 124.16, 124.04, 108.51, 105.29, 101.14, 60.98, 56.13, 35.78,
+
8.18, 25.91. HRMS (ESI) (m/z) [M+Na] calcd. for C H N O , 350.1751; found,
22
23
3
50.1751. Purity: 98.5% (by HPLC).
General procedure for the preparation of 13[34]
To a solution of 12 (0.35 g, 1.0 mmol) in anhydrous THF, NaH (1.2 mmol) was
added at 0 °C. After the the mixture was stirred for 15 minutes, a corresponding
iodohydrocarbon or bromohydrocarbon (1.0 mmol) in THF was added in drop wise.
The mixture was stirred at room temperature for 4 h. The organic layer was washed
with brine and then dried (Na SO ). The solvents were removed under reduced
2
4
pressure to obtain the crude product, which was purified by silica gel column
chromatography (petroleum ether/ ethyl acetate =4:1) to provide 13.
3
-methyl-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]indole
13a)
Colorless oil. (0.31 g, 0.85 mmol) Yield 85%. H NMR (400 MHz, Chloroform-d)
δ 7.15 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 3.1 Hz, 1H), 6.93 (d, J = 8.5 Hz, 1H), 6.63 –
(
1
6
.60 (m, 1H), 6.53 (s, 2H), 6.45 (t, J = 7.3 Hz, 1H), 3.87 (s, 3H), 3.81 (s, 3H), 3.78 (s,
13
6
H), 2.96 (t, J = 7.0 Hz, 2H), 2.30 (p, J = 7.1 Hz, 2H), 1.98 (q, J = 7.2 Hz, 2H). C
NMR (101 MHz, CDCl3) δ 152.83, 143.98, 138.97, 137.22, 135.68, 134.71, 130.41,
1
2
3
3
28.49, 127.50, 127.12, 123.79, 106.63, 105.28, 99.49, 60.92, 56.12, 35.70, 32.99,
+
8.04, 25.87. HRMS (ESI) (m/z) [M+Na] calcd. for C H N O , 364.1907; found,
2
3
25
3
64.1912. Purity: 95.1% (by HPLC).
-ethyl-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]indole (13b)
1
Colorless oil. (0.33 g, 0.87 mmol) Yield 87%. H NMR (400 MHz, Chloroform-d)
δ 7.20 – 7.14 (m, 2H), 6.91 (d, J = 8.5 Hz, 1H), 6.62 (d, J = 3.1 Hz, 1H), 6.54 (s, 2H),
.44 (t, J = 7.3 Hz, 1H), 4.18 (q, J = 7.3 Hz, 2H), 3.86 (s, 3H), 3.78 (s, 6H), 2.96 (t, J
6
=
7.0 Hz, 2H), 2.37 – 2.25 (m, 2H), 1.98 (q, J = 7.2 Hz, 2H), 1.50 (t, J = 7.3 Hz, 3H).
1
3
C NMR (101 MHz, CDCl ) δ 152.82, 144.03, 139.04, 137.19, 134.83, 134.73,
3
1
30.38, 127.62, 127.19, 126.67, 123.66, 106.72, 105.31, 99.58, 60.93, 56.15, 41.11,
1
9

ACCEPTED MANUSCRIPT
+
3
3
3
5.75, 28.09, 25.91, 15.52. HRMS (ESI) (m/z) [M+Na] calcd. for C H N O3,
24
27
78.2064; found, 378.2067. Purity: 96.3% (by HPLC).
-propyl-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]indole (13c)
1
Colorless oil. (0.35 g, 0.90 mmol) Yield 90%. H NMR (400 MHz, Chloroform-d)
δ 7.15 (d, J = 10.4 Hz, 2H), 6.89 (d, J = 8.5 Hz, 1H), 6.60 (s, 1H), 6.54 (s, 2H), 6.43 (t,
J = 7.2 Hz, 1H), 4.07 (t, J = 7.0 Hz, 2H), 3.86 (s, 3H), 3.78 (d, J = 2.1 Hz, 6H), 2.96 (t,
J = 6.8 Hz, 2H), 2.30 (p, J = 6.4 Hz, 2H), 1.98 (q, J = 6.9 Hz, 2H), 1.88 (dt, J = 13.0,
1
3
6
1
1
.6 Hz, 2H), 0.98 (td, J = 7.5, 2.1 Hz, 3H). C NMR (101 MHz, CDCl3) δ 152.83,
44.05, 139.05, 137.27, 135.05, 134.76, 130.32, 127.60, 127.54, 127.14, 123.61,
06.82, 105.39, 99.39, 60.92, 56.16, 48.27, 35.74, 28.10, 25.92, 23.61, 11.64. HRMS
+
(
ESI) (m/z) [M+Na] calcd. for C H N O , 392.2220; found, 392.2237. Purity: 96.2%
2
5
29
3
(by HPLC).
Synthesis
-(3,4,5-trimethoxyphenyl)-1,2,3,8,9,10-hexahydrocyclohepta[e]indole (14)[42]
To a stirred solution of cyclohepta-indole derivatives 12 (0.17 g, 0.5 mmol) in
of
6
AcOH (10 mL), NaBH CN (94 mg, 1.5 mmol) was added. After the mixture was
3
further stirred for 4.0 h under N , the reaction was quenched by adding of water. The
2
solvent was removed in vacuo and the product was extracted with ethyl acetate. The
organic layer was washed with saturated aqueous NaHCO , dried with Na SO and
3
2
4
concentrated under reduced pressure to give crude product as colourless oil. The crude
product was purified by silica gel column chromatography (petroleum ether/ ethyl
1
acetate =2:1) to provide 14 (0.13 g, 0.36 mmol) as colorless oil. Yield: 71%. H NMR
(
400 MHz, Chloroform-d) δ 6.73 (d, J = 8.0 Hz, 1H), 6.52 (s, 2H), 6.48 (d, J = 8.0 Hz,
H), 6.26 (t, J = 7.3 Hz, 1H), 3.85 (s, 3H), 3.80 (s, 6H), 3.63 (t, J = 8.4 Hz, 2H), 3.09
t, J = 8.4 Hz, 2H), 2.57 (t, J = 6.9 Hz, 2H), 2.13 (p, J = 7.1 Hz, 2H), 1.98 (q, J = 7.1
1
(
13
Hz, 2H). C NMR (101 MHz, CDCl3) δ 152.78, 150.27, 143.49, 138.92, 138.29,
1
30.98, 128.99, 128.10, 126.68, 125.84, 106.78, 105.45, 60.88, 56.15, 47.13, 33.96,
+
2
8.75, 28.52, 25.76. HRMS (ESI) (m/z) [M+Na] calcd. for C H N O , 352.1907;
2
2
25
3
found, 352.1907. Purity: 97.2% (by HPLC).
2
0

ACCEPTED MANUSCRIPT
Synthesis
-methyl-6-(3,4,5-trimethoxyphenyl)-1,2,3,8,9,10-hexahydrocyclohepta[e]indole
15)[34]
To a solution of 14 (0.18 g, 0.50 mmol) in anhydrous THF, NaH (24 mg, 0.60
mmol, 60 %) was added. After the mixture was stirred for 15 minutes, iodomethane
63 ꢀL, 1.0 mmol) in THF was added in dropwise. The mixture was stirred at room
of
3
(
(
temperature. Organic layer was washed with brine and dried. The solvents were
removed under reduced pressure to obtain the crude product, which was purified by
silica gel column chromatography (petroleum ether/ ethyl acetate =4:1) to provide
1
indoline 15 (0.17 g, 0.46 mmol) as colorless oil. Yield 91%. H NMR (400 MHz,
Chloroform-d) δ 6.79 (d, J = 8.0 Hz, 1H), 6.53 (s, 2H), 6.33 (d, J = 8.1 Hz, 1H), 6.24
(t, J = 7.2 Hz, 1H), 3.86 (s, 3H), 3.80 (s, 6H), 3.39 (t, J = 8.2 Hz, 2H), 3.00 (t, J = 8.2
Hz, 2H), 2.79 (s, 3H), 2.56 (t, J = 6.9 Hz, 2H), 2.12 (d, J = 7.0 Hz, 2H), 1.98 (q, J =
1
3
7
1
3
3
.0 Hz, 2H). C NMR (101 MHz, CDCl3) δ 152.77, 152.06, 143.49, 139.06, 137.86,
37.24, 130.21, 129.16, 127.33, 125.67, 105.41, 104.52, 60.91, 56.16, 55.86, 36.17,
+
3.82, 29.71, 28.32, 27.54, 25.79. HRMS (ESI) (m/z) [M+Na] calcd. for C H NO ,
23
27
3
66.2064; found, 366.2068. Purity: 99.1% (by HPLC).
Synthesis
of
6
-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]indole-1-carbaldehy
de (16)[43]
To a stirred solution of indole derivatives 12 (0.17 g, 0.5 mmol) in AcOH (5.0
mL), hexamethylenetetramine (0.11 g, 0.75 mol) was added at room temperature.
After heated at reflux under N for 18 h, the mixture was diluted with water (10 mL),
2
neutralized with saturated aqueous NaHCO until no CO was evolved. The mixture
3
2
was extracted with ethyl acetate, and the organic layer was washed with water, brine
and dried over Na SO . After removal of solvent by evaporation, the crude product
2
4
was obtained as an orange oil, which was purified by silica gel chromatography eluted
with petroleum ether/ ethyl acetate =1: 1 to give 16 (0.11 g, 0.29 mmol) as yellow
1
solid. Yield: 58%. H NMR (400 MHz, Chloroform-d) δ 10.09 (s, 1H), 9.02 (s, 1H),
7
.96 (d, J = 3.2 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 8.4 Hz, 1H), 6.53 (d, J
2
1

ACCEPTED MANUSCRIPT
=
9.0 Hz, 3H), 3.87 (s, 3H), 3.79 (s, 6H), 3.38 (t, J = 6.9 Hz, 2H), 2.39 (p, J = 7.1 Hz,
1
3
2
1
1
H), 1.96 (q, J = 7.2 Hz, 2H). C NMR (101 MHz, CDCl3) δ 191.16, 184.34, 152.89,
43.26, 138.93, 138.25, 137.15, 136.78, 135.41, 128.92, 126.22, 123.47, 121.12,
+
09.13, 105.30, 60.99, 56.13, 36.72, 29.72, 25.66. HRMS (ESI) (m/z) [M+Na] calcd.
for C H N O , 378.1700; found, 378.1697. Purity: 97.7% (by HPLC).
2
3
23
4
Synthesis
of
3
-methyl-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e] indole-1-
carbaldehyde (17)[43]
To a stirred solution of 13a (0.18 g, 0.50 mmol) in AcOH (5.0 mL),
hexamethylenetetramine (0.11 g, 0.75 mmol) was added at room temperature. The
mixture was heated to reflux under N for 18 h, and then, diluted with water (10 mL),
2
neutralized with saturated aqueous NaHCO until no CO was evolved. The mixture
3
2
was extracted with ethyl acetate, organic layer was washed with water, brine and dried
over Na SO . After removal of solvent by evaporation, aldehyde 17 (0.12 g, 0.31
2
4
1
mmol) was obtained as yellow oil. Yield: 61%. H NMR (400 MHz, Chloroform-d) δ
0.04 (s, 1H), 7.81 (s, 1H), 7.18 (d, J = 8.5 Hz, 1H), 7.07 (d, J = 8.5 Hz, 1H), 6.52 (d,
J = 3.7 Hz, 3H), 3.88 (d, J = 3.7 Hz, 6H), 3.79 (s, 6H), 3.38 (t, J = 6.9 Hz, 2H), 2.38
1
1
3
(
p, J = 7.0 Hz, 2H), 1.95 (q, J = 7.2 Hz, 2H). C NMR (101 MHz, CDCl3) δ 183.64,
52.94, 143.22, 141.66, 138.75, 137.83, 137.32, 135.42, 128.86, 125.90, 124.27,
19.49, 107.35, 105.29, 60.96, 56.14, 36.59, 33.85, 30.23, 25.64. HRMS (ESI) (m/z)
1
1
+
[
M+Na] calcd. for C H N O , 392.1856; found, 392.1865. Purity: 95.1% (by
24 25 4
HPLC).
Synthesis
of
(3-methyl-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]indol-1-yl)
methanol (18)[44]
To a stirred solution of aldehyde 17 (0.20 g, 0.50 mmol) in CH OH (10 mL),
3
NaBH (57 mg, 1.5 mmol) was added in portions at 10 – 15 °C. After stirred at room
4
temperature for 3 h, the mixture was quenched by addition of 5.0 mL of H O,
2
extracted by ethyl acetate, washed with brine dried with NaSO and concentration
4
under reduced pressure to obtain the crude product. The crude product was purified by
2
2

ACCEPTED MANUSCRIPT
silica gel column chromatography (petroleum ether/ ethyl acetate =4:1) to provide 19
1
(
0.17 g, 0.42 mmol) as colorless oil. Yield: 83%. H NMR (400 MHz, Chloroform-d)
δ 7.12 (d, J = 8.5 Hz, 1H), 7.09 (s, 1H), 6.95 (d, J = 8.5 Hz, 1H), 6.54 (s, 2H), 6.46 (t,
J = 7.3 Hz, 1H), 4.94 (s, 2H), 3.87 (s, 3H), 3.78 (s, 6H), 3.76 (s, 3H), 3.12 (t, J = 6.8
1
3
Hz, 2H), 2.33 (p, J = 6.9 Hz, 2H), 1.95 (q, J = 7.1 Hz, 2H). C NMR (101 MHz,
CDCl3) δ 152.87, 143.79, 138.97, 137.32, 137.08, 135.32, 131.92, 129.15, 127.42,
1
2
4
24.76, 124.30, 114.94, 106.77, 105.40, 60.92, 58.51, 56.17, 36.59, 32.79, 27.82,
+
5.66. HRMS (ESI) (m/z) [M+Na] calcd. for C H27 N O , 416.1832; found,
2
4
4
16.1818. Purity: 95.5 % (by HPLC).
Synthesis
-(3-methyl-6-(3,4,5-trimethoxyphenyl)-3,8,9,10-tetrahydrocyclohepta[e]
indol-1-yl)-2-oxoacetate (19)[45]
of
methyl
2
To a solution of 13a (0.18 g, 0.50 mmol) in Et O (5.0 mL) at 0 °C, oxalyl
2
chloride (48 ꢀL, 0.55 mmol) was added. The resulting yellow slurry was stirred at
0
°C for 1.5 h and then at room temperature for 1.5 h. The organic solvent was
removed under reduced pressure to get yellow solid. The residue was cooled to 0 °C,
the, CH OH (5.0 mL) and Et N (2.0 mL) were added. The mixture was stirred at 0 °C
3
3
for 1 h and then warmed to room temperature over 4 h. The mixture was extracted
with ethyl acetate and the organic layer was washed with water, brine and dried over
Na SO . The organic layer was evaporated to dryness to provide yellow oil. The crude
2
4
product was purified by silica gel column chromatography (petroleum ether/ ethyl
1
acetate =4:1) to provide 19 (0.16 g, 0.37 mmol) as flaxen oil. Yield: 73%. H NMR
(400 MHz, Chloroform-d) δ 8.23 (s, 1H), 7.18 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 8.5 Hz,
1
H), 6.54 (d, J = 7.3 Hz, 1H), 6.50 (s, 2H), 3.96 (s, 3H), 3.86 (d, J = 3.0 Hz, 6H), 3.78
1
3
(
s, 6H), 3.29 (t, J = 6.3 Hz, 2H), 2.47 (p, J = 6.6 Hz, 2H), 1.96 (q, J = 7.1 Hz, 2H). C
NMR (101 MHz, CDCl3) δ 177.72, 165.10, 152.93, 143.11, 142.40, 138.93, 138.16,
1
5
37.55, 137.37, 136.55, 129.42, 126.55, 125.25, 113.82, 107.29, 105.40, 60.90, 56.14,
+
2.69, 37.34, 33.95, 30.25, 25.66. HRMS (ESI) (m/z) [M+Na] calcd. for C H N
2
6
27
O , 450.1911; found, 450.1929. Purity: 97.5% (by HPLC).
6
2
3

ACCEPTED MANUSCRIPT
Synthesis
-methyl-6-(3,4,5-trimethoxyphenyl)-3,6,7,8,9,10-hexahydrocyclohepta[e]indole
20)[46]
of
3
(
To a stirred suspension of 13a (0.18 g, 0.50 mmol) in methanol (5.0 mL), Pd/C
(
50 mg) was added and the mixture was stirred under H balloon at room temperature
2
for 3 h. Then, filtered and vacuumed to get the crude product which was purified by
silica gel column chromatography (petroleum ether/ ethyl acetate = 4:1) to provide the
1
title compound (0.16 g, 0.45 mmol). Yield: 89%. H NMR (400 MHz, Chloroform-d)
δ 7.06 (d, J = 3.2 Hz, 1H), 7.02 (d, J = 8.5 Hz, 1H), 6.66 (d, J = 8.5 Hz, 1H), 6.55 (d,
J = 3.1 Hz, 1H), 6.50 (s, 2H), 4.39 (d, J = 8.3 Hz, 1H), 3.90 (s, 3H), 3.83 (d, J = 4.6
Hz, 7H), 3.77 (s, 3H), 3.22 (dd, J = 13.2, 7.5 Hz, 1H), 3.17 – 3.05 (m, 1H), 2.29 –
1
3
2
1
5
.18 (m, 1H), 2.16 – 1.83 (m, 5H). C NMR (101 MHz, CDCl3) δ 153.07, 141.75,
36.01, 135.45, 134.23, 128.53, 128.47, 122.88, 106.14, 105.69, 99.98, 99.32, 60.94,
+
6.10, 49.67, 34.34, 32.89, 30.61, 30.19, 27.08. HRMS (ESI) (m/z) [M+Na] calcd.
for C H N O , 366.2064; found, 366.2068. Purity: 99.3 % (by HPLC).
2
3
27
3
Synthesis of (E)-3-(1-(phenylsulfonyl)-1H-indol-4-yl)acrylic acid (21)[47]
To a stirred suspension of aldehyde 6 (1.0 g, 3.5 mmol) in pyridine (40 mL),
malonic acid (0.73 g, 7.0 mmol) and piperidine (1.0 mL) were added and the mixture
was heated to reflux at 120 °C for 3 h. When allowed to cool to 0 °C, the mixture was
acidified by hydrochloric acid. The precipitate was filtered and the filter cake was
washed with 20 mL of H O, dried in vacuum to afford the crude product which was
2
purified by re-crystallization from a mixed-solvent (ethyl acetate: methanol = 4 : 1) to
1
give a white solid (0.89 g, 2.7 mmol). Yield: 78%. H NMR (400 MHz, DMSO-d6) δ
8
.07 – 7.99 (m, 4H), 7.95 (d, J = 3.8 Hz, 1H), 7.89 (d, J = 16.1 Hz, 1H), 7.70 (d, J =
.4 Hz, 2H), 7.61 (dd, J = 8.5, 7.1 Hz, 2H), 7.42 (d, J = 8.0 Hz, 1H), 7.21 (d, J = 3.8
7
Hz, 1H), 6.62 (d, J = 16.0 Hz, 1H).
Synthesis of 3-(1-(phenylsulfonyl)-1H-indol-4-yl)propanoic acid (22)[35]
To a stirred suspension of acrylic acid 21 (0.98 g, 3.0 mmol) in methanol (10
mL), Pd/C (0.15 g) was added and the mixture was stirred under H balloon at room
2
temperature for 3 h. Then, the mixture was filtered and vacuumed to get colorless oil.
2
4

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The crude product was used directly for the next step without purification.(0.47 g, 1.4
1
mmol) Yield: 48%. H NMR (400 MHz, DMSO-d6) δ 8.02 – 7.95 (m, 2H), 7.82 (d, J
=
3.8 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.72 – 7.66 (m, 1H), 7.59 (t, J = 7.7 Hz, 2H),
7
.31 – 7.23 (m, 1H), 7.10 (d, J = 7.3 Hz, 1H), 7.00 – 6.94 (m, 1H), 3.03 (t, J = 7.6 Hz,
H), 2.56 (t, J = 7.7 Hz, 2H).
2
Synthesis of 3-(phenylsulfonyl)-7,8-dihydrocyclopenta[e]indol-6(3H)-one (23)[41]
Propanoic acid 22 (1.0 g, 2.8 mmol) was added in small portions to 10 g of
rapidly stirred solution of phosphorus pentoxide:methanesulfonic acid (1:10). After
the reaction mixture was stirred at room temperature for 4 h, 40 m1 of water was
added in dropwise and the aqueous mixture was stirred rapidly for 5-10 min to ensure
the hydrolysis of methanesulfonic anhydride. The mixture was extracted with ethyl
acetate, washed subsequently with dilute sodium bicarbonate and water, dried over
anhydrous sodium sulfate, and concentrated in vacuum. The crude product was
chromatographed on silica gel (4:1 of petroleum/ethyl acetate as an eluent) to afford
1
the product (0.42g, 1.3 mmol) as white solid in 48% yield. H NMR (400 MHz,
Chloroform-d) δ 8.02 (d, J = 8.6 Hz, 1H), 7.91 (d, J = 8.0 Hz, 2H), 7.75 – 7.67 (m,
2
H), 7.57 (t, J = 7.4 Hz, 1H), 7.48 (t, J = 7.6 Hz, 2H), 6.82 (t, J = 2.8 Hz, 1H), 3.24 (t,
J = 4.9 Hz, 2H), 2.83 – 2.70 (m, 2H).
Synthesis of 7,8-dihydrocyclopenta[e]indol-6(3H)-one (24)[35]
To a stirred suspension of indolone derivatives 23 (0.31 g, 1.0 mmol) in
methanol (5.0 mL), 1 M KOH (aq) (1.2 mL, 1.2 mmol) was added. The mixture was
stirred at reflux for 2 h. The reaction was quenched by water, and extracted with ethyl
acetate. The organic layer was collected and dried over anhydrous MgSO4,
concentrated in vacuo to yield colorless oil. The residue was purified by flash column
chromatography over silica gel (petroleum/ethyl acetate = 1:1) to afford 24 (0.14 g,
1
0
.81 mmol). Yield: 81%; H NMR (400 MHz, Chloroform-d) δ 8.69 (s, 1H), 7.61 (d, J
=
8.5Hz, 1H), 7.39 (dq, J = 8.5, 0.8 Hz, 1H), 7.33 (dd, J = 3.3, 2.4 Hz, 1H), 6.73 (ddd,
J = 3.1, 2.0, 1.0 Hz, 1H), 3.41 – 3.25 (m, 2H), 2.84 – 2.72 (m, 2H).
Synthesis of 3-methyl-7,8-dihydrocyclopenta[e]indol-6(3H)-one (25)[34]
To a solution of 24 (0.37 g, 2.0 mmol) in anhydrous THF, NaH (80mg, 2.0 mmol,
2
5

ACCEPTED MANUSCRIPT
6
0%) was added at 0 °C. After the mixture was stirred for 15 minutes, a
corresponding iodohydrocarbon (0.11 mL, 2.0 mmol) in THF was added in dropwise.
The mixture was stirred at room temperature for 4 h. Organic layer wash with brine
and dried. The solvents were removed under reduced pressure to obtain the crude
product, which was purified by silica gel column chromatography (petroleum ether/
1
ethyl acetate =8:1) to provide 25 (0.34 g, 1.8 mmol) as colorless oil. Yield 91%. H
NMR (400 MHz, Chloroform-d) δ 7.62 (dd, J = 8.5, 1.8 Hz, 1H), 7.34 – 7.28 (m, 1H),
7
.16 (t, J = 2.5 Hz, 1H), 6.65 (t, J = 2.4 Hz, 1H), 3.87 (s, 3H), 3.33 – 3.24 (m, 2H),
2
.82 – 2.71 (m, 2H).
Synthesis of 3-methyl-6-(3,4,5-trimethoxyphenyl)-3,8-dihydrocyclopenta[e]indole
26)[20]
To a solution of 5-bromo-1,2,3-trimethoxybenzene (0.49 g, 2.0 mmol) in
(
o
anhydrous THF (10 mL) at −78 C under the argon atmosphere, n-BuLi (2.5 M, 0.80
mL, 2.0 mmol) was added in dropwise via a syringe. The mixture was stirred for 0.5 h
at the same temperature and followed by addition of the solution of 21 (0.19 g, 1.0
mmol) in THF (10 mL). The reaction mixture was stirred for 1 hour at −78 °C then
allowed to warm to room temperature. After stirred for another 2 h, the reaction was
carefully quenched by saturated aqueous NH Cl and extracted with ethyl acetate. The
4
combined organic layers were and washed with water and brine respectively, dried
over Na SO , filtered and concentrated under reduced pressure. The crude product
2
4
was chromatographed on silica gel and eluted with petroleum/ethyl acetate (4:1) to
1
afford the 26 (0.36g, 1.1 mmol) as a yellow oil. Yield: 54% H NMR (400 MHz,
Chloroform-d) δ 7.49 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 8.4 Hz, 1H), 7.07 (d, J = 3.1 Hz,
1
3
1
H), 6.88 (s, 2H), 6.53 (dd, J = 3.1, 0.8 Hz, 1H), 6.42 (t, J = 2.1 Hz, 1H), 3.91 (s, 3H),
1
3
.90 (s, 6H), 3.77 (s, 3H), 3.64 (d, J = 2.1 Hz, 2H). C NMR (101 MHz, CDCl ) δ
3
53.34, 145.97, 137.45, 137.21, 135.83, 135.68, 132.87, 129.69, 126.90, 125.73,
114.62, 107.34, 104.97, 98.77, 61.02, 56.21, 37.03, 33.19. HRMS (ESI) (m/z)
+
[
M+Na] calcd. for C21 H21 N O , 336.1594; found, 336.1599. Purity: 98.2% (by
3
HPLC).
.2 Biological assays
4
2
6

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4
.2.1 Cell lines and culture
Seven types of human cancer cell lines used in this study were obtained from the
Laboratory Animal Center of Sun Yat-sen University. The non-small cell lung cancer
cell line A549, human epithelial cervical cancer cell line HeLa, human prostate cancer
line PC-3, human liver carcinoma cell line Bel-7402, human colon cancer cell line
Lovo, human ovarian cancer cell line A2780, and human breast carcinoma cell line
MCF-7 were grown in Dulbecco’s modified Eagle medium (DMEM, GIBCO)
containing 10% (v/v) heat-inactivated fetal bovine serum (FBS, GIBCO), 100 IU/mL
penicillin, and 100 ꢀg/mL streptomycin (GIBCO). The cells were incubated at 37 °C
in a 5% CO and 90% relative humidity (RH) atmosphere.
2
4
.2.2 In vitro antiproliferative activity assays
The antiproliferative activities of the synthesized compounds towards seven
human cancer cell lines (A549, HeLa, PC-3, Bel-7402, Lovo, A2780, and MCF-7)
were evaluated using the MTT assay as previously described. [24] Briefly, when
growing in the logarithmic phase, the cells were harvested and plated into 96-well
3
plates (5×10 cells/well) for 24 h and then exposed to different concentrations of the
test compounds for 48 h. Subsequently, 20 ꢀL of MTT (5 mg/mL, Sigma) and 150 ꢀL
of DMSO were added to dissolve the dark blue crystals (formazan). The absorbance at
5
70 nm was measured using a multifunction microplate reader (Molecular Devices,
Flex Station 3). The antiproliferative activities of the optimized compound 20 were
also determined by the CCK-8 assay. Briefly, after treatment, 10 µl CCK-8 solution
(Keygen Biotech, Nanjin, China) were added to each well and incubated for 2 h. The
absorbance at 450 nm was measured. All experiments were repeated at least three
times. The IC values, which represent the drug concentrations required to cause 50%
5
0
tumour cell growth inhibition, were calculated using a nonlinear regression model
GraphPad Prism version 5.0).
(
4
.2.3 In vitro tubulin polymerization inhibition
The tubulin polymerization assay was performed as previously described [22]
2
7

ACCEPTED MANUSCRIPT
using a commercial kit (cytoskeleton, cat.#BK011P) purchased from Cytoskeleton
(Danvers, MA, USA). Tubulin isolated from porcine brain tissue was used in this
tubulin polymerization assay kit. Tubulin polymerizations are followed by an increase
in fluorescence emission at 410-460 nm over a 60 minute period at 37 °C. Firstly, the
tubulin reaction mix containing 80.0 mM piperazine-N, N′-bis(2-ethanesulfonic acid)
sequisodium salt (pH 6.9), 2.0 mM MgCl , 0.5 mM EGTA, 1 mM GTP, and 10.2%
2
glycerol was prepared. Then, 5 ꢀL of the tested compounds at the indicated
concentrations were added, and the mixture was pre-warmed to 37 °C for 1 min.
Subsequently, the reaction was initiated by the addition of 55 ꢀL tubulin solution. The
fluorescence intensity was recorded every 60 sec for 90 min in a multifunction
microplate reader (Molecular Devices, Flex Station 3) (emission wavelength is 410
nm, excitation wavelength is 340 nm). The area under the curve was used to
determine the concentration that inhibited tubulin polymerization by 50% (IC ),
5
0
which was calculated with GraphPad Prism Software version 5.02 (GraphPad Inc., La
Jolla, CA, USA).
4
.2.4 Immunofluorescence microscopy.
The intracellular microtubule morphology was detected using immunofluorescence as
5
previously described. [22] Briefly, A549 cells (3 × 10 cells/well) were plated in a 10
3
mm confocal culture dish (NEST Biotechnology, China) for 24 h and then incubated
in the presence or absence of compound 20 at the indicated concentrations for another
1
2 h. After being washed with phosphate buffer solution (PBS) and fixed in 4%
paraformaldehyde for 15 min, the cells were permeabilized with 0.5% Triton X-100
2
8