Synthesis, biological evaluation, and molecular docking investigation of 3-amidoindoles as potent tubulin polymerization inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.11.038

A series of novel 3-amidoindole derivatives possessing 3,4,5-trimethoxylphenyl groups were synthesized and evaluated for their antiproliferative and tubulin polymerization inhibitory activities. Some of them demonstrated moderate to potent activities in vitro against six cancer cell lines including MCF-7, MDA-MB-231, BT549, T47D, MDA-MB-468, and HS578T. The most active compound 27 inhibited the growth of T47D, BT549, and MDA-MB-231 cell lines with IC₅₀ values at 0.04, 3.17, and 6.43 μM, respectively. Moreover, the flow cytometric analysis clearly revealed that compound 27 significantly inhibited growth of breast cancer cells through arresting cell cycle in G2/M phase via a concentration-dependent manner. In addition, the compound also exhibited the most potent anti-tubulin activity with IC₅₀ values of 9.5 μM, which was remarkable, compared to CA-4. Furthermore, molecular docking analysis demonstrated the interaction of the compound 27 at the colchicine-binding site of tubulin. These preliminary results suggest that compound 27 is a very promising tubulin-binding agent and is worthy of further investigation aiming to the development of new potential anticancer agents.

Full text of DOI:10.1016/j.ejmech.2018.11.038

Accepted Manuscript
Synthesis, biological evaluation, and molecular docking investigation of 3-
amidoindoles as potent tubulin polymerization inhibitors
Peng Chen, Yu-Xin Zhuang, Peng-Cheng Diao, Fang Yang, Shao-Yu Wu, Lin Lv,
Wen-Wei You, Pei-Liang Zhao
PII:
S0223-5234(18)30997-8
DOI:
https://doi.org/10.1016/j.ejmech.2018.11.038
EJMECH 10895
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 15 July 2018
Revised Date: 29 October 2018
Accepted Date: 16 November 2018
Please cite this article as: P. Chen, Y.-X. Zhuang, P.-C. Diao, F. Yang, S.-Y. Wu, L. Lv, W.-W. You,
P.-L. Zhao, Synthesis, biological evaluation, and molecular docking investigation of 3-amidoindoles as
potent tubulin polymerization inhibitors, European Journal of Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.ejmech.2018.11.038.
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ACCEPTED MANUSCRIPT
Synthesis, biological evaluation, and molecular docking investigation of
3-amidoindoles as potent tubulin polymerization inhibitors
Peng Chen#, Yu-Xin Zhuang#, Peng-Cheng Diao, Fang Yang, Shao-Yu Wu, Lin Lv*, Wen-Wei You*
Pei-Liang Zhao*
Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China

ACCEPTED MANUSCRIPT
Synthesis, biological evaluation, and molecular docking investigation of
3-amidoindoles as potent tubulin polymerization inhibitors
Peng Chen#, Yu-Xin Zhuang#, Peng-Cheng Diao#, Fang Yang, Shao-Yu Wu, Lin Lv*, Wen-Wei
You*, Pei-Liang Zhao*
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Guangdong Provincial Key Laboratory of New Drug Screening, School of Pharmaceutical
Science, Southern Medical University, Guangzhou 510515, P.R.China
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ABSTRACT: A series of novel 3-amidoindole derivatives possessing 3,4,5-trimethoxylphenyl
groups were synthesized and evaluated for their antiproliferative and tubulin polymerization
inhibitory activities. Some of them demonstrated moderate to potent activities in vitro against six
cancer cell lines including MCF-7, MDA-MB-231, BT549, T47D, MDA-MB-468, and HS578T.
The most active compound 27 inhibited the growth of T47D, BT549, and MDA-MB-231 cell lines
with IC50 values at 0.04, 3.17, and 6.43 µM, respectively. Moreover, the flow cytometric analysis
clearly revealed that compound 27 significantly inhibited growth of breast cancer cells through
arresting cell cycle in G2/M phase via a concentration-dependent manner. In addition, the
compound also exhibited the most potent anti-tubulin activity with IC50 values of 9.5 µM, which
was remarkable, compared to CA-4. Furthermor, molecular docking analysis demonstrated the
interaction of the compound 27 at the colchicine-binding site of tubulin. These preliminary results
suggest that compound 27 is a very promising tubulin-binding agent and is worthy of further
investigation aiming to the development of new potential anticancer agents.
Keywords: Microtubules; 3-Amidoindoles; Antiproliferative activity; Tubulin polymerization
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——————————
Corresponding author. E-mail: plzhao@smu.edu.cn (P.-L. Zhao), youww@smu.edu.cn (W.-W.
You), lynnlv@smu.edu.cn (L. Lv)
# These authors contributed equally to this work.
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1. Introduction
Microtubules are the basic components of cell structure and have been an important therapeutic
target in tumour cells due to their crucial roles in cell proliferation, trafficking, signaling and
migration in eukaryotic cells [1-3]. Microtubule-targeting agents that are able to modulate the
microtubule assembly by inhibiting tubulin polymerization or blocking microtubule disassembly
have attracted considerable interest in anti-cancer therapy [4-7].
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In the last decade, a large number of structurally diverse tubulin polymerization inhibitors have
been identified [8-12]. Among them, combretastatin A-4 (CA-4, Figure 1) is one of the most
well-known antitubulin agents and its corresponding prodrug salt CA-4P has shown promising
results in human cancer clinical trials [13]. Most interestingly, the replacement of cis-olefin moiety
of CA-4 with indole core led to discover a 3-arylindole (2) with potent antitubulin activity.
Subsequently, by introducing methylene, sulfur and carbonyl bridges, 3-benzylindole (3),
3-arylthioindole (4) and 3-aroylindole (5) have been developed as significant inhibitors of tubulin
polymerization with potent antiproliferative activities [14-17]. However, chemical modification of
incorporating amido bridge into 3-arylindole was not exhaustively explored. Inspired by these
observations and as a part of our continuing effort on the development of nitrogen-containing
heterocycles as novel antitumor agents [18-22], we designed a series of novel 3-amidoindole
derivatives 6–34 by incorporating an amido bridge into 3-position of indole core (Fig. 1), which
have, to our knowledge, not been reported so far. In the present study, we described the detailed
synthetic routes, antiproliferative and tubulin polymerization inhibitory activities of these
compounds.
<Insert figure 1 here>
2. Chemistry
The synthetic route for target compounds 6–34 was illustrated in Scheme 1. Substituted
2-aminobenzonitriles 35 were converted to the corresponding N-ethoxycarbonylanilines 36
by treatment with neat ethyl chloroformate (ClCO2Et) at reflux. The subsequent
condensation, followed by Thorpe–Ziegler cyclization with various α-bromoketones using
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potassium carbonate as base in dimethylformamide (DMF) provided the common intermediates
N-1-ethoxycarbonyl-2-substituted-3-aminoindoles 37. These intermediates were subsequently
converted into the corresponding N-unsubstituted indole 38 after alkaline hydrolysis using sodium
hydroxide (NaOH) in aqueous ethanol, which had been illustrated in detail in our previously
reported approach [23]. Subsequently, the direct nucleophilic substitution of commercially
available 3,4,5-trimethoxybenzoyl chloride and 3-amino-1H-indoles 38 in the presence of
triethylamine in anhydrous tetrahydrofuran solution gave the desired 3-amidoindole derivatives
6–34 in yields of 54.5–92.1%, respectively. The structures of target compounds 6–34 were
characterized with spectroscopic techniques including 1H NMR, 13C NMR, and HRMS
spectroscopic techniques, and the spectral data agree with the proposed structures.
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<Insert scheme 1 here>
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3. Results and Discussion
3.1 In vitro antiproliferative activity
All the synthesized 3-amidoindole derivatives 6–34 were evaluated for their in vitro
antiproliferative activities against a panel of six human cancer cell lines (MCF-7, MDA-MB-231,
BT549, T47D, MDA-MB-468, and HS578T) by using MTT assay. The assay results expressed as
IC50 (µM) were summarized in Table 1 and compared with the inhibitory activities of a reference
compound, CA-4, one of the most potent natural tubulin-binding anticancer agents. Here, the IC50
value represents the concentration of one compound resulting in a 50% inhibition in cell growth
after 72 h incubation, and is the average of three independent experiments.
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<Insert table 1 here>
Table 1 summarizes the growth inhibitory effects of 3-amidoindole derivatives 6–34 against
six human cancer cell lines, with CA-4 as a reference compound. In most cases, the antiproliferative
activities of the compounds were greater against the T47D, BT549, and MDA-MB-231 cells
compared with the other three cell lines. Among the target 3-amidoindoles, 5 compounds
demonstrated potent antiproliferative activities against T47D cell lines with the IC50 value of < 7
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µM. While the most active compound identified in this study was derivative 27, which inhibited the
growth of T47D, BT549, and MDA-MB-231 cancer cell lines with IC50 values at 0.04, 3.17, and
6.43 µM, respectively.
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Further analysis clearly revealed that different antiproliferative activities were observed when
various substituents R1, R2 groups were introduced into the indole ring. Concerning R1 substituents
at the C-6 position of the indole ring, it is clear that compounds bearing methoxy groups (32–34),
exhibit generally lower potency than the corresponding derivatives substituted with methyl, chloro,
and hydrogen moieties. For the substituent R2, compounds with a bulky 4-phenylphenyl or
naphthalenyl group (10, 17, 12) resulted in a drastic reduction in antiproliferative activities against
tested cancer cell lines. Futhermore, replacement of the phenyl ring (R2) with a heterocyclic ring
such as a thiazole or a furan moietyled to an almost complete loss in the activity (11, 19, 28), which
indicated that the phenyl substitution on the indole ring might played a crucial role in modulating
the antitumor activity. In addition, a comparison of substituent effects revealed that replacement of
the electron-donating groups on the phenyl ring with electron-withdrawing atoms strongly increased
growth inhibitory properties (6, 9 versus 7, 8 and 16 versus 14, 15, 18, 20 and 26, 27 versus 22, 23,
25).
To investigate the mechanism of action of these compounds on cancer cells, one represented
analogue 27 was evaluated for the influence on the cell cycle progression. In this study, BT549 cells
were treated with 2, 4 and 8 µM concentrations of compound 27 for 24 h. As shown in Fig. 2,
analogue 27 displayed 26.29% (2 µM), 35.92% (4 µM), and 56.58% (8 µM) of cell accumulation in
G2/M phase respectively, whereas in control (untreated cells) 8.88% and CA-4 (positive control, 8
µM) 72.90% of G2/M phase were observed. These data clearly suggested that compound 27
induced a significant cell cycle arrest at the G2/M phase in a concentration-dependent manner,
compared to untreated cells.
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<Insert figure 2 here>
3.2. Immunocytochemistry
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To confirm the mechanism of action of these compounds, we further investigated the effect of
the most active compound 27 on tubulin organization in intact cells, and the microtubule structure
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of cells was visualized via immunocytochemistry. Confocal images depicted in Fig. 3, displayed
that both α-tubulin and β-tubulin microtubules formed an intact network with fine filaments in the
untreated BT549 cells. However, cells treated with 10 µM of 27 for 6 h resulted in significant
disruption of both tubulin subtypes, loss of cellular structure, and formation of cell membrane
rounding. Furthermore, a 6 h exposure of BT549 cells to 20 µM of compound 27 led to an absolute
loss of microtubule formation (microtubules became short and wrapped around the nucleus) and
remarkably affected the cell shape (turned round). These results reveal that compound 27 acts as an
antitubulin inhibitor by blocking cell cycle progression and metaphase by disrupting spindle
assembly.
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<Insert figure 3 here>
3.3. Inhibition of in vitro tubulin polymerization
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To investigate whether the antiproliferative activities of these derivatives were related to an
interaction with the microtubule system, eleven representative active compounds were selected for
the evaluation of their in vitro inhibitory activities against tubulin polymerization at 10 µM
concentration, and CA-4 was also used as the reference. The results were summarized in Table 2. It
was clear that there was a positive correlation between the inhibition of tubulin polymerization and
antiproliferative activity of the tested compounds. The most potent compound 27 displayed
anti-tubulin activity with an IC50 value of 9.5 µM, which was in the same range as the reference
compound CA-4 (IC50 = 4.2 µM).
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<Insert table 2 here>
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3.4. Molecular studies
To investigate the possible binding mode for this series of compounds, we selected the most
active compound 27 to perform the molecular docking simulations on the colchicine binding site of
tubulin. As shown in Fig. 4, the results obtained are similar to those reported for arylindole
derivatives, with the trimethoxyphenyl ring in proximity of βCys241 and the 2-chlorophenyl ring at
C-2 position of the indole setting up hydrophobic interactions with the βLys254, αVal181, βAsn258
and βMet259 side chains [15]. On the other hand, three hydrogen bonds was observed: (i) The
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1-NH of the indole ring established a H-bond with αThr179 (2.2 Å); (ii) The carbonyl group of the
4-chlorobenzoyl moiety forms a hydrogen bond with βAsn258 (2.7 Å). (iii) One of the oxygens of
trimethoxyphenyl ring established a H-bond with βCys241 (2.8 Å).
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<Insert figure 4 here>
4. Conclusion
In the present study, 3,4,5-trimethoxylphenyl groups were introduced in 3-position of indole
cores to generate a series of novel 3-amidoindole derivatives. The antiproliferative activities of the
synthesized compounds in vitro were evaluated against MCF-7, MDA-MB-231, BT549, T47D,
MDA-MB-468, and HS578T cell lines. Most compounds demonstrated significant antiproliferative
activity against six human cancer cell lines employed in this study. The IC50 values of the most
promising compound 27 were 0.04, 3.17, and 6.43 µM against T47D, BT549, and MDA-MB-231
cell lines, respectively. Moreover, an immunofluorescence study of compound 27 revealed that its
target was most likely tubulin. In addition, the compound 27 also exhibited the most potent
anti-tubulin activity with IC50 values of 9.5 µM, which was remarkable, compared to CA4. These
preliminary results demonstrate that compound 27 is a very promising tubulin-binding agent and is
worthy of further investigation aiming to the development of new potential anticancer agents.
5. Experimental protocols
5.1 Chemistry
13
1H NMR and C NMR spectra were recorded on a Mercury-Plus 400 spectrometer in CDCl3 or
DMSO-d6 solution and chemical shifts were recorded in parts per million (ppm) with TMS as the
internal reference. High-resolution mass spectra (HRMS) performed on an Agilent
QTOF 6540 mass spectrometer. Melting points (mp) were taken on a Buchi B-545 melting point
apparatus and are uncorrected. Unless otherwise noted, reagents were purchased from commercial
suppliers and used without further purification while all solvents were redistilled before use.
General procedure for the preparation of 3-amidoindole derivatives 6–34.
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To a solution of 3-amidoindole derivatives 6–34 (1.50 mmol) in THF (10 mL), triethylamine
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(0.227 g, 2.25 mmol) was added at 0°C. At this temperature, the reaction mixture was stirred for 20
min before the addition of 3,4,5-trimethoxybenzoyl chloride (2.25 mmol) in dry THF (5 mL). The
resulting mixture was stirred at room temperature for 6 h and was then concentrated on a rotary
evaporator. The crude product obtained was poured into 20 mL of water and extracted with
dichloromethane. The combined organic extracts was dried over anhydrous MgSO4 and
concentrated in vacuo. The crude residue was purified by column chromatography using a mixture
of petroleum ether and acetone as an eluent to give the white solid compounds in yields of
54.5–92.1%.
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5.1.1 N-(2-(4-fluorobenzoyl)-6-methyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (6). Yield, 78.0%;
mp: 251.2–251.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 3.71 (s, 3H, CH3O), 3.79
(s, 6H, 2×CH3O), 6.92-6.97 (m, 3H, ArH), 7.25 (dd, J1 = 8.8 Hz, J2 = 5.2 Hz, 3H, ArH), 7.55 (d, J =
8.0 Hz, 1H, ArH), 7.81 (dd, J1 = 4.0 Hz, J2 = 6.0 Hz, 2H, ArH), 10.01 (s, 1H, NH), 11.68 (s, 1H,
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13
NH). C NMR (100 MHz, DMSO-d6) δ: 186.63, 165.43, 152.82, 140.45, 136.85, 135.90, 131.99,
129.78, 122.55, 121.88, 121.66, 119.80, 115.60, 115.39, 112.49, 105.24, 60.48, 56.32, 22.07.
HRMS (ESI) m/z: calcd for C26H23FN2O5 (M+H+) 463.1591 found 463.1752.
5.1.2 3,4,5-trimethoxy-N-(6-methyl-2-(4-methylbenzoyl)-1H-indol-3-yl)benzamide (7). Yield, 92.1%;
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mp: 252.2–252.6°C; H NMR (400 MHz, DMSO-d6) δ: 2.26 (s, 3H, CH3), 2.44 (s, 3H, CH3), 3.71
(s, 3H, CH3O), 3.79 (s, 6H, 2×CH3O), 6.93 (d, J = 4.0 Hz, 2H, ArH), 7.22 (d, J = 7.6 Hz, 2H ,ArH),
7.27 (s, 1H, ArH), 7.42 (s, 1H, ArH), 7.55 (d, J = 8.8 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 2H, ArH),
9.99 (s, 1H, NH), 11.61 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.69, 165.41, 152.78,
142.47, 140.37, 136.73, 136.12, 135.65, 129.90, 129.47, 122.43, 121.89, 119.76, 112.47, 108.15,
105.31, 60.48, 56.32, 22.07. HRMS (ESI) m/z: calcd for C27H26N2O5 (M+H+) 459.1842 found
459.2006.
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5.1.3 N-(2-benzoyl-6-methyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (8). Yield, 79.5%; mp:
250.1–250.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 3.71 (s, 3H, CH3O), 3.78 (s,
6H, 2×CH3O), 6.90 (s, 2H, ArH), 6.96 (d, J = 8.4 Hz, 1H, ArH), 7.28 (s, 1H, ArH), 7.42 (t, J = 14.8
Hz, 2H, ArH), 7.49 (d, J = 7.2 Hz, 1H, ArH), 7.55 (d, J = 8.4 Hz, 1H, ArH), 10.01 (s, 1H, NH),
11.65 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.98, 165.38, 152.78, 140.38, 138.77,
136.84, 135.82, 132.32, 129.30, 122.48, 121.81, 119.93, 112.49, 105.27, 60.48, 56.35, 22.08.
HRMS (ESI) m/z: calcd for C26H24N2O5 (M+H+) 445.1685 found 445.1847.
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5.1.4 N-(2-(4-chlorobenzoyl)-6-methyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (9). Yield,
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77.7%; mp: 252.2–252.8°C; H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 3.70 (s, 3H,
CH3O), 3.80 (s, 6H, 2×CH3O), 6.90 (s, 2H, ArH), 6.96 (d, J = 8.4 Hz, 1H, ArH), 7.27 (d, 1H, ArH),
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7.46 (s, J = 8.4 Hz, 2H, ArH), 7.55 (d, J = 8.0 Hz, 2H, ArH), 7.72 (d, J = 8.4 Hz, 2H, ArH), 10.00 (s,
1H, NH), 11.70 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.82, 165.49, 152.82, 140.43,
137.55, 136.96, 136.07, 131.03, 129.72, 128.56, 127.00, 122.62, 121.66, 120.04, 112.50, 105.19,
60.45, 56.30, 22.09. HRMS (ESI) m/z: calcd for C26H23ClN2O5 (M+H+) 479.1295 found 479.1403.
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5.1.5 N-(2-(biphenylcarbonyl)-6-methyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (10). Yield,
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83.0%; mp: 267.2–267.6°C; H NMR (400 MHz, DMSO-d6) δ: 2.45 (s, 3H, CH3), 3.61 (s, 3H,
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CH3O), 3.70 (s, 6H, 2×CH3O), 6.96 (d, J = 12.4 Hz, 3H, ArH), 7.29 (s, 1H, ArH), 7.39-7.46 (m, 3H,
ArH), 7.55 (dd, J1 = 7.6 Hz, J2 = 8.8 Hz, 3H ,ArH), 7.67 (d, J = 7.6 Hz, 2H ,ArH), 7.84 (d, J = 8.0
Hz, 2H, ArH), 10.02 (s, 1H, NH), 11.70 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.51,
165.42, 152.75, 139.54, 137.63, 136.88, 135.88, 129.99, 129.36, 128.54, 127.28, 127.12, 126.71,
122.53, 122.02, 120.08, 105.29, 60.36, 56.21, 22.09. HRMS (ESI) m/z: calcd for C32H28N2O5
(M+H+) 521.1998 found 521.2107.
5.1.6 3,4,5-trimethoxy-N-(6-methyl-2-(thiophene-2-carbonyl)-1H-indol-3-yl)benzamide (11). Yield,
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69.0%; mp: 244.2–244.5°C; H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 3H, CH3O), 3.83 (s, 6H,
2×CH3O), 7.14(d, J = 6.0 Hz, 2H, ArH), 7.19 (t, J = 8.4 Hz, 2H, ArH), 7.51 (s, 1H, ArH), 7.73 (d, J
= 8.4 Hz, 1H, ArH), 7.84 (d, J = 3.6 Hz, 1H, ArH), 8.04 (d, J = 4.8 Hz, 1H, ArH), 10.32 (s, 1H, NH),
11.95 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 179.28, 165.44, 153.00, 143.45, 140.68,
136.16, 135.34, 134.30, 130.41, 129.70, 128.78, 123.78, 122.30, 120.95, 119.03, 112.47, 105.47,
60.52, 56.43. HRMS (ESI) m/z: calcd for C23H19ClN2O5S (M+H+) 471.1093 found 471.0816.
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5.1.7 N-(2-(2-naphthoyl)-6-methyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (12). Yield, 79.7%;
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mp: 257.7–258.8°C; H NMR (400 MHz, DMSO-d6) δ: 2.44 (s, 3H, CH3), 3.68 (s, 9H, 3×CH3O),
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6.70 (s, 2H, ArH), 6.95 (d, J = 8.0 Hz, 1H, ArH), 7.28 (s, 1H, ArH), 7.45 (t, J = 15.2 Hz, 1H, ArH),
7.55-7.59 (m, 3H, ArH), 7.73 (d, J = 6.8 Hz, 1H, ArH), 7.93-7.87 (m, 2H, ArH), 8.16 (d, J = 8.0 Hz,
1H, ArH), 9.93 (s, 1H, NH), 11.68 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 188.68, 165.37,
152.65, 140.35, 136.34, 133.66, 131.25, 129.65, 128.54, 127.90, 127.29, 126.55, 125.11, 122.60,
121.24, 112.57, 105.13, 60.47, 56.22, 22.12. HRMS (ESI) m/z: calcd for C30H26N2O5 (M+H+)
495.1842 found 495.1947.
5.1.8 N-(2-(4-fluorobenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (13). Yield, 88.3%; mp:
255.2–255.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 3H, CH3O), 3.79 (s, 6H, 2×CH3O), 6.92
(s, 2H, ArH), 7.12 (t, J = 14.8 Hz, 1H, ArH), 7.26 (t, J = 16.0 Hz, 2H, ArH), 7.35 (t, J = 15.2 Hz, 1H,
ArH), 7.50 (d, J = 8.4 Hz, 1H, ArH), 7.65 (d, J = 8.4 Hz, 1H, ArH), 7.81-7.84 (m, 2H, ArH), 10.03
(s, 1H, NH), 11.84 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.85, 165.62, 152.80, 140.48,
137.43, 137.06, 136.46, 131.15, 130.67, 129.51, 128.62, 128.23, 123.57, 122.65, 121.01, 119.51,
112.48, 105.30, 60.45, 56.33. HRMS (ESI) m/z: calcd for C25H21FN2O5 (M+H+) 448.1434 found
448.1577.
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5.1.9 3,4,5-trimethoxy-N-(2-(4-methylbenzoyl)-1H-indol-3-yl)benzamide (14). Yield, 75.3%; mp:
241.5–241.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 2.26 (s, 3H, CH3), 3.70 (s, 3H, CH3O), 3.78 (s,
6H, 2×CH3O), 6.92 (s, 2H, ArH), 7.12 (d, J = 7.2 Hz, 1H, ArH), 7.28 (d, J = 7.6 Hz, 2H, ArH), 7.33
(t, J = 14.8 Hz, 2H, ArH), 7.50 (d, J = 6.6 Hz, 1H, ArH), 7.64 (d, J = 8.0 Hz, 1H, ArH), 7.69 (d, J =
7.6 Hz, 2H, ArH), 10.00 (s, 1H, NH), 11.77 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.83,
165.54, 152.78, 142.67, 140.37, 136.12, 135.95, 129.92, 127.85, 125.86, 123.94, 121.86, 120.26,
119.23, 113.10, 105.32, 60.46, 56.32, 21.42. HRMS (ESI) m/z: calcd for C26H24N2O5 (M+H+)
445.1685 found 445.1888.
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5.1.10 N-(2-benzoyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (15). Yield, 74.5%; mp:
248.6–248.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 3H, CH3O), 3.78 (s, 6H, 2×CH3O), 6.90
(s, 2H, ArH), 7.12 (t, J = 14.8 Hz, 1H, ArH), 7.34 (t, J = 15.2 Hz, 1H, ArH), 7.43 (t, J = 14.8 Hz, 2H,
ArH), 7.51 (d, J = 8.4 Hz, 2H, ArH), 7.65 (d, J = 8.0 Hz, 1H ,ArH), 10.02 (s, 1H, NH), 11.80 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 188.14, 165.52, 152.77, 138.59, 136.24, 132.48, 129.87,
129.36, 129.14, 128.73, 128.51, 127.71, 126.01, 123.90, 121.88, 119.42, 105.28, 60.48, 56.36.
HRMS (ESI) m/z: calcd for C25H22N2O5 (M+H+) 431.1529 found 431.1704.
260
5.1.11 N-(2-(4-bromobenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (16). Yield, 81.0%; mp:
253.3–253.8°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.72 (s, 3H, CH3O), 3.81 (s, 6H, 2×CH3O), 6.91
(s, 2H, ArH), 7.13 (t, J = 15.2 Hz, 1H, ArH), 7.36 (t, J = 15.2 Hz, 2H, ArH), 7.50 (d, J = 8.4 Hz, 1H,
ArH), 7.61 (d, J = 7.6 Hz, 2H, ArH), 7.67 (d, J = 7.6 Hz, 3H, ArH), 10.01 (s, 1H, NH), 11.85 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.17, 165.63, 152.82, 140.43, 137.70, 136.36, 131.52,
131.20, 129.71, 127.52, 126.25, 123.90, 121.76, 120.44, 119.59, 113.16, 105.18, 60.45, 56.32.
HRMS (ESI) m/z: calcd for C25H21BrN2O5 (M+H+) 509.0634 found 509.10735.
265
270
5.1.12 N-(2-(biphenylcarbonyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (17). Yield, 74.3%; mp:
266.6–267.1°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.61 (s, 3H, CH3O), 3.70 (s, 6H, 2×CH3O), 6.94
(s, 2H, ArH), 7.13 (t, J = 14.8 Hz, 1H, ArH), 7.35-7.47 (m, 4H, ArH), 7.53 (t, J = 16.4 Hz, 3H, ArH),
7.64-7.69 (m, 2H, ArH), 7.85 (d, J = 8.0 Hz, 2H, ArH), 10.02 (s, 1H, NH), 11.84 (s, 1H, NH). 13C
NMR (100 MHz, DMSO-d6) δ: 187.68, 165.55, 152.75, 139.51, 137.45, 136.27, 130.05, 129.80,
129.38, 128.58, 127.86, 127.14, 126.74, 124.03, 119.58, 113.16, 105.31, 60.37, 56.22. HRMS (ESI)
m/z: calcd for C31H26N2O5 (M+H+) 507.1842 found 507.1927.
275
280
5.1.13 3,4,5-trimethoxy-N-(2-(3-methoxybenzoyl)-1H-indol-3-yl)benzamide (18). Yield, 70.0%; mp:
256.1–256.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.71 (d, J = 6.8 Hz, 6H, 2×CH3O), 3.78 (s, 6H,
2×CH3O), 6.91 (s, 2H, ArH), 7.02-7.12 (m, 2H, ArH), 7.26 (s, 1H, ArH), 7.33 (d, J = 8.0 Hz, 2H,
ArH), 7.50 (d, J = 8.4 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H, ArH), 10.13 (s, 1H, NH), 11.82 (s, 1H,
9

ACCEPTED MANUSCRIPT
NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.76, 165.49, 159.24, 152.80, 139.68, 136.37, 130.59,
129.78, 129.61, 123.75, 122.57, 121.64, 118.88, 113.79, 112.46, 105.23, 60.50, 56.30, 55.48.
HRMS (ESI) m/z: calcd for C26H24N2O6 (M+H+) 461.1634 found 461.1855.
285
290
5.1.14 3,4,5-trimethoxy-N-(2-(thiophene-2-carbonyl)-1H-indol-3-yl)benzamide (19). Yield, 80.1%;
mp: 245.2–246.0°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 3H, CH3O), 3.83 (s, 6H, 2×CH3O),
7.14 (d, J = 5.2 Hz, 3H, ArH), 7.20 (t, J = 8.4 Hz, 1H, ArH), 7.35 (t, J = 15.2 Hz, 1H ,ArH), 7.50 (d,
J = 8.0 Hz, 1H, ArH), 7.70 (d, J = 8.0 Hz, 1H, ArH), 7.86 (d, J = 7.2 Hz, 1H, ArH), 8.02 (d, J = 4.8
Hz, 1H, ArH), 10.28 (s, 1H, NH), 11.79 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 179.45,
165.46, 152.99, 143.70, 136.06, 135.05, 134.18, 129.91, 128.72, 125.88, 123.61, 122.03, 119.11,
113.15, 105.45, 60.51, 56.42. HRMS (ESI) m/z: calcd for C23H20N2O5S (M+H+) 437.1093 found
437.1213.
5.1.15 3,4,5-trimethoxy-N-(2-(4-methoxybenzoyl)-1H-indol-3-yl)benzamide (20). Yield, 64.9%; mp:
256.0–256.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.72 (d, J = 3.2 Hz, 6H, 2×CH3O), 3.78 (s, 6H,
2×CH3O), 6.94 (s, 1H, ArH), 6.96 (s, 3H, ArH), 7.12 (d, J = 8.4 Hz, 1H, ArH), 7.33 (t, J = 15.2 Hz,
1H, ArH), 7.49 (d, J = 8.0 Hz, 1H, ArH), 7.63 (d, J = 8.0 Hz, 1H, ArH), 7.79 (d, J = 8.4 Hz, 2H,
ArH), 10.00 (s, 1H, NH), 11.75 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.80, 165.59,
162.96, 152.81, 140.39, 135.99, 131.79, 131.11, 129.99, 128.03, 125.66, 124.01, 121.78, 120.21,
118.71, 113.93, 113.05, 105.35, 60.48, 56.30, 55.81. HRMS (ESI) m/z: calcd for C26H24N2O6
(M+H+) 461.1634 found 461.1858.
295
300
5.1.16 N-(6-chloro-2-(4-fluorobenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (21). Yield,
1
76.2%; mp: 254.1–254.6°C; H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.79 (s, 6H,
2×CH3O), 6.92 (s, 2H, ArH), 7.16 (d, J = 8.8 Hz, 1H, ArH), 7.26 (t, J = 15.6 Hz, 2H, ArH), 7.51 (s,
1H, ArH), 7.69 (d, J = 8.8 Hz, 1H, ArH), 7.83 (dd, J1 = 5.6 Hz, J2 = 5.6 Hz, 2H, ArH), 10.08 (s, 1H,
NH), 11.98 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.85, 165.62, 152.83, 140.53, 136.36,
132.20, 132.11, 129.57, 128.26, 123.53, 122.57, 121.00, 119.26, 115.73, 115.51, 105.27, 60.49,
56.34. HRMS (ESI) m/z: calcd for C25H20ClFN2O5 (M+H+) 483.1045 found 483.1143.
305
5.1.17 N-(6-chloro-2-(4-methylbenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (22). Yield,
1
310
315
74.6 %; mp: 255.7–256.5°C; H NMR (400 MHz, DMSO-d6) δ: 2.26 (s, 3H, CH3), 3.70 (s, 3H,
CH3O), 3.78 (s, 6H, 2×CH3O), 6.91 (s, 2H, ArH), 7.14 (d, J = 8.4 Hz, 1H, ArH), 7.23 (d, J = 7.6 Hz,
2H, ArH), 7.50 (s, 1H, ArH), 7.68 (d, J = 8.0 Hz, 3H, ArH), 10.05 (s, 1H, NH), 11.92 (s, 1H, NH).
13C NMR (100 MHz, DMSO-d6) δ: 187.67, 165.57, 152.86, 142.98, 140.52, 136.20, 135.73, 129.60,
129.20, 127.85, 123.69, 122.65, 120.83, 119.21, 112.47, 105.39, 60.52, 56.38, 21.43. HRMS (ESI)
m/z: calcd for C26H23ClN2O5 (M+H+) 479.1295 found 479.1388.
5.1.18 N-(6-chloro-2-benzoyl-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (23). Yield, 85.1%; mp:
10

ACCEPTED MANUSCRIPT
259.2–259.9°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 3H, CH3O), 3.77 (s, 6H, 2×CH3O), 6.89
(s, 2H, ArH), 7.15 (d, J = 8.8 Hz, 1H, ArH), 7.43 (t, J = 15.2 Hz, 2H ,ArH), 7.52 (d, J = 6.0 Hz, 2H,
ArH), 7.69 (d, J = 8.8 Hz, 1H, ArH), 7.78 (d, J = 7.6 Hz, 2H, ArH), 10.07 (s, 1H, NH), 11.95 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.85, 165.52, 152.83, 140.53, 136.36, 132.20, 132.11,
130.61, 129.57, 128.26, 123.53, 122.57, 121.00, 119.26, 115.73, 115.51, 111.24, 105.27, 60.49,
56.34. HRMS (ESI) m/z: calcd for C25H21ClN2O5 (M+H+) 465.1139 found 465.1256.
320
325
5.1.19 N-(6-chloro-2-(4-methoxybenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (24). Yield,
87.0%; mp: 256.0–256.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.72 (d, J = 4.4 Hz, 6H, 2×CH3O),
3.78 (s, 6H, 2×CH3O), 6.96 (t, J = 8.4 Hz, 4H, ArH), 7.14 (d, J = 8.8 Hz, 1H, ArH), 7.51 (s, 1H,
ArH), 7.67 (d, J = 8.4 Hz, 1H, ArH), 7.79 (d, J = 8.4 Hz, 2H, ArH), 10.05 (s, 1H, NH), 11.91 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.61, 165.55, 163.06, 152.82, 140.48, 136.13, 131.82,
130.83, 130.21, 129.77, 128.67, 123.54, 122.70, 120.82, 118.71, 113.99, 112.38, 105.38, 60.48,
56.31, 55.83. HRMS (ESI) m/z: calcd for C26H23ClN2O6 (M+H+) 495.1245 found 495.1353.
330
335
5.1.20 N-(6-chloro-2-(3-methoxybenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (25). Yield,
65.3%; mp: 254.2–254.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.71 (d, J = 2.4 Hz, 6H, 2×CH3O),
3.78 (s, 6H, 2×CH3O), 6.91 (s, 2H, ArH), 7.07 (d, J =4.8 Hz, 1H, ArH), 7.14 (d, J = 8.8 Hz, 1H,
ArH), 7.26 (s, 1H, ArH), 7.34 (d, J = 4.8 Hz, 2H, ArH), 7.51 (s, 1H, ArH), 7.67 (d, J = 8.8 Hz, 1H,
ArH), 10.07 (s, 1H, NH), 11.96 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.76, 165.49,
159.24, 152.80, 139.68, 136.37, 130.59, 129.78, 129.61, 123.75, 122.57, 121.64, 118.88, 113.79,
112.46, 105.23, 60.50, 56.30, 55.48. HRMS (ESI) m/z: calcd for C26H23ClN2O6 (M+H+) 495.1245
found 495.1353.
5.1.21 N-(6-chloro-2-(3-bromobenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (26). Yield,
1
65.4%; mp: 241.2–241.9°C; H NMR (400 MHz, DMSO-d6) δ: 3.70 (s, 3H, CH3O), 3.79 (s, 6H,
340
345
2×CH3O), 6.95 (s, 2H, ArH), 7.14 (d, J = 8.4 Hz, 1H, ArH), 7.36 (t, J = 15.2 Hz, 1H, ArH), 7.52 (s,
1H, ArH), 7.70 (dd, J1 = 8.4 Hz, J2 = 12.4 Hz, 3H, ArH), 7.85 (s, 1H, ArH), 10.27 (s, 1H, NH),
12.05 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.76, 165.49, 159.24, 152.80, 139.68,
136.37, 130.59, 129.78, 129.61, 123.75, 122.57, 121.64, 120.95, 119.53, 118.88, 113.79, 112.46,
105.23, 60.50, 56.30. HRMS (ESI) m/z: calcd for C25H20ClBrN2O5 (M+H+) 543.0244 found
543.0329.
5.1.22 N-(6-chloro-2-(4-chlorobenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (27). Yield,
1
67.8%; mp: 248.1–248.9°C; H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.80 (s, 6H,
2×CH3O), 6.93 (s, 2H, ArH), 7.14 (d, J = 8.8 Hz, 1H, ArH), 7.46 (d, J = 8.0 Hz, 2H, ArH), 7.53 (s,
1H, ArH), 7.70 (d, J = 8.8 Hz, 1H, ArH), 7.75 (d, J = 8.0 Hz, 2H, ArH), 10.20 (s, 1H, NH), 12.03 (s,
1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.85, 165.62, 152.80, 140.48, 137.33, 137.06,
136.46, 131.15, 130.67, 129.51, 128.62, 128.23, 123.57, 122.57, 121.01, 119.51, 105.30, 60.45,
350
11

ACCEPTED MANUSCRIPT
56.33. HRMS (ESI) m/z: calcd for C25H20Cl2N2O5 (M+H+) 499.0749 found 499.0849.
5.1.21 N-(6-chloro-2-(furan-2-carbonyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (28). Yield,
1
62.7%; mp: 241.2–241.8°C; H NMR (400 MHz, DMSO-d6) δ: 3.75 (s, 3H, CH3O), 3.85 (s, 6H,
355
2×CH3O), 6.95 (s, 1H, ArH), 7.15 (d, J = 8.8 Hz, 1H, ArH), 7.24 (s, 2H, ArH), 7.42 (s, 1H, ArH),
7.54 (s, 1H, ArH), 7.81 (d, J = 8.8 Hz, 1H, ArH), 8.03 (s, 1H, ArH), 10.52 (s, 1H, NH), 11.87 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 165.62, 153.09, 152.01, 140.80, 140.48, 136.41, 130.70,
129.86, 126.62, 124.29, 121.64, 120.87, 120.47, 119.81, 113.14, 112.54, 105.55, 60.54, 56.48.
HRMS (ESI) m/z: calcd for C23H19ClN2O6 (M+H+) 455.0932 found 455.1026.
360
365
5.1.22 N-(6-chloro-2-(thiophene-2-carbonyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (29). Yield,
1
66.0%; mp: 244.2–244.5°C; H NMR (400 MHz, DMSO-d6) δ: 3.73 (s, 3H, CH3O), 3.83 (s, 6H,
2×CH3O), 7.14(d, J = 6.0 Hz, 2H, ArH), 7.19 (t, J = 8.4 Hz, 2H, ArH), 7.51 (s, 1H, ArH), 7.73 (d, J
= 8.4 Hz, 1H, ArH), 7.84 (d, J = 3.6 Hz, 1H, ArH), 8.04 (d, J = 4.8 Hz, 1H, ArH), 10.32 (s, 1H, NH),
11.95 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 179.28, 165.44, 153.00, 143.45, 140.68,
136.16, 135.34, 134.30, 130.41, 129.70, 128.78, 123.78, 122.30, 120.95, 119.03, 112.47, 105.47,
60.52, 56.43. HRMS (ESI) m/z: calcd for C23H19ClN2O5S (M+H+) 471.1093 found 471.0816.
5.1.21 N-(6-chloro-2-(3,4-difluorobenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide (30). Yield,
1
76.8%; mp: 254.3–254.8°C; H NMR (400 MHz, DMSO-d6) δ: 3.72 (s, 3H, CH3O), 3.80 (s, 6H,
2×CH3O), 6.96 (s, 2H, ArH), 7.16 (dd, J1 = 0.4 Hz, J2 = 0.4 Hz, 1H, ArH), 7.47-7.52 (m, 2H, ArH),
7.60 (s, 1H, ArH), 7.74 (dd, J1 = 0.8 Hz, J2 = 0.8 Hz, 2H, ArH), 10.15 (s, 1H, NH), 12.04 (s, 1H,
NH). 13C NMR (100 MHz, DMSO-d6) δ: 185.54, 165.62, 152.87, 140.66, 136.53, 130.90, 129.34,
127.88, 123.48, 122.53, 121.15, 119.61, 118.68, 118.50, 117.94, 117.76, 112.49, 105.21, 60.51,
56.30. HRMS (ESI) m/z: calcd for C25H19ClF2N2O5 (M+H+) 501.0951 found 501.1049.
370
5.1.22
N-(6-chloro-2-(3-hydroxy-4-methoxybenzoyl)-1H-indol-3-yl)-3,4,5-trimethoxybenzamide
375
380
(31). Yield, 54.5%; mp: 247.7–248.0°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.72 (d, J = 4.4 Hz, 6H,
2×CH3O), 3.79 (s, 6H, 2×CH3O), 6.89 (d, J = 8.8 Hz, 1H, ArH), 6.98 (s, 2H, ArH), 7.14 (dd, J1 =
2.0 Hz, J2 = 1.6 Hz, 1H, ArH), 7.29 (d, J = 6.8 Hz, 2H, ArH), 7.49 (d, J = 1.6 Hz, 1H, ArH), 7.68 (d,
J = 8.8 Hz, 1H, ArH), 10.06 (s, 1H, NH), 11.84 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ:
186.76, 165.54, 158.62, 152.84, 152.00, 146.66, 140.45, 136.01, 131.02, 129.97, 128.48, 123.62,
122.52, 120.72, 118.72, 116.04, 112.33, 114.45, 105.37, 60.48, 56.30, 56.08. HRMS (ESI)
m/z: calcd for C27H26N2O8 (M+K+) 544.1689 found 544.4191.
5.1.22 3,4,5-trimethoxy-N-(6-methoxy-2-(4-methoxybenzoyl)-1H-indol-3-yl)benzamide (32). Yield,
86.0%; mp: 258.8–259.7°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.72 (s, 6H, 2×CH3O), 3.79 (s, 6H,
2×CH3O), 3.83 (s, 3H, CH3O), 6.77 (dd, J1 = 1.6 Hz, J2 = 2.0 Hz, 1H, ArH), 6.91-6.95 (m, 3H,
ArH), 6.98 (s, 2H, ArH), 7.57 (d, J = 8.8 Hz, 1H, ArH), 7.76 (d, J = 8.4 Hz, 2H, ArH), 10.08 (s, 1H,
385
12

ACCEPTED MANUSCRIPT
NH), 11.55 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 186.22, 165.33, 162.66, 159.04, 152.84,
137.47, 131.88, 131.41, 129.93, 126.63, 123.32, 120.32, 120.32, 118.13, 112.21, 105.36, 60.49,
56.31, 55.77, 55.62. HRMS (ESI) m/z: calcd for C27H26N2O7 (M+H+) 491.1740 found 491.1912.
5.1.21 3,4,5-trimethoxy-N-(6-methoxy-2-benzoyl-1H-indol-3-yl)benzamide (33). Yield, 84.2%; mp:
259.3–259.6°C; 1H NMR (400 MHz, DMSO-d6) δ: 3.71 (s, 3H, CH3O), 3.79 (s, 6H, 2×CH3O), 3.83
(s, 3H, CH3O), 6.77 (dd, J1 = 1.6 Hz, J2 = 2.0 Hz, 1H, ArH), 6.91-6.93 (m, 3H, ArH), 7.43 (t, J =
14.8 Hz, 2H, ArH), 7.50 (d, J = 7.2 Hz, 1H, ArH), 7.60 (d, J = 9.2 Hz, 1H, ArH), 10.11 (s, 1H, NH),
11.59 (s, 1H, NH). 13C NMR (100 MHz, DMSO-d6) δ: 187.43, 165.29, 159.29, 152.82, 140.47,
390
395
138.94, 137.79, 132.15, 129.81, 129.20, 128.49, 126.34, 123.47, 121.11, 118.00, 112.34,
105.30,
94.12, 60.50, 56.37, 55.63. HRMS (ESI) m/z: calcd for C26H24N2O6 (M+H+) 461.1634 found
461.1740.
5.1.22 3,4,5-trimethoxy-N-(6-methoxy-2-(4-methylbenzoyl)-1H-indol-3-yl)benzamide (34). Yield,
1
88.9%; mp: 253.2–253.7°C; H NMR (400 MHz, DMSO-d6) δ: 2.27 (s, 3H, CH3), 3.71 (s, 3H,
CH3O), 3.80 (s, 6H, 2×CH3O), 3.83 (s, 3H, CH3O), 6.77 (dd, J1 = 2.0 Hz, J2 = 0.8 Hz, 1H, ArH),
6.91 (d, J = 1.6 Hz, 1H, ArH), 6.96 (s, 2H, ArH), 7.22 (d, J = 8.0Hz, 2H, ArH), 7.59 (d, J = 8.8 Hz,
1H, ArH), 7.66 (d, J = 8.0 Hz, 2H, ArH), 10.09 (s, 1H, NH), 11.56 (s, 1H, NH). 13C NMR (100 MHz,
DMSO-d6) δ: 187.17, 165.27, 159.18, 152.83, 142.26, 140.45, 137.66, 136.27, 129.85, 129.37,
400
405
129.06, 126.43, 123.47, 120.94, 118.01, 112.25,
105.33, 94.13, 60.47, 56.33, 55.62, 21.40.
HRMS (ESI) m/z: calcd for C27H26N2O6 (M+H+) 475.1791 found 475.1990.
5.2 Biological evaluation
5.2.1 Antitumor activity
The antitumor activities of compounds 6–34 were evaluated with MCF-7, MDA-MB-231,
BT549, T47D, MDA-MB-468, and HS578T cell lines by the standard MTT
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay in vitro. The cancer cell lines
were cultured in RPMI-1640 medium supplemented with 10% FBS. Tested compounds were
prepared by dissolving in dimethyl sulfoxide (DMSO) at 100 mM and diluted with the medium into
a series of concentrations. Exponentially growing cells were plated in 96-well plates (2×103
cells/well) and incubated at 37 °C for 24 h for attachment. The culture medium was then changed,
and cells grew in medium with the tested compounds. DMSO (0.1%) and CA-4 were used as
negative and positive control, respectively. Cells were incubated at 37 °C for 72 h. After the
treatment period, 10 µL of MTT solution (5 mg/mL) was added to each well, and the plates were
incubated for 4 h at 37°C. The medium was then aspirated and formazan crystals were dissolved in
410
415
13

ACCEPTED MANUSCRIPT
DMSO (150 µL) for about 10 mins. The absorbance at 570 nm (Abs) of the suspension was
measured by a microplate reader (Bio-Rad laboratories, USA). The inhibition percentage was
calculated using the following formula: % inhibition = (Abscontrol－Abscompound)/Abscontrol×100%.
The IC50 values of the tested compounds and CA-4 were measured by treating cells with drugs of
various concentrations and analyzed by use of the prism statistical package (GraphPad Software,
San Diego, CA, U.S.A.).
420
5.2.2 Flow-activating cell sorting (FACS) analysis
425
430
435
440
445
Flow cytometric analysis analysis was performed to estimate the effect of compound 27 on cell
cycle phase distribution of human breast cancer cell lines (BT549). When the cells grew to about
70% confluence in 60 mm dishs over night, they were incubated with compound 27 at 2, 4, and 8
µM concentrations for 24 h. Control and treated cells were harvested, washed with PBS, and fixed
in 75% ice-cold ethanol at 4 °C overnight. They were then washed with PBS, incubated with 50
µg/mL of RNase at 37 °C for 30 min, stained with 50 µg/mL of propidium iodide, and then
subjected to flow cytometry (Beckman Coulter).
5.2.3 Immunocytochemistry
BT549 cells (2 × 104 cells/well) were plated on glass coverslips in 12-well plates and allowed
to attach overnight. After treatment with compound 27 (10 and 20 µM) or 0.1% DMSO for 6 h,
cells were rinsed twice, fixed with 4% paraformaldehyde, and permeabilized with 0.1% Triton
X-100. After blocking with 5% BSA for 60 min, the coverslips were incubated overnight at 4°C
with primary antibodies (α-tubulin mouse monoclonal antibody and β-tubulin rabbit monoclonal
antibody). After washing, the cells were incubated with the following secondary antibodies: Alexa
Fluor® 555 conjugate anti-rabbit IgG and FITC AffiniPure goat anti-mouse IgG. Nuclei were
visualized by inclusion of DAPI. Images were taken using a laser scanning confocal microscope
(Carl Zeiss LSM 880).
5.2.4 In vitro tubulin polymerization assay
The in vitro tubulin polymerization activity assay was performed following our previously
reported method [25].
5.2.5 Molecular modeling
Molecular docking simulation studies were carried out by using the SURFLEX module of
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SYBYL 7.3 package [26], and the tubulin structure was down loaded from the PDB data bank
(http://www.rcsb.org/-PDB code: 5lyj) [27]. The studied molecule was initially built in Sybyl 7.3.
Structural energy minimization process was performed using the Tripos force field with a
distance–dependent dielectric and powell gradient algorithm with a convergence criterion of 0.005
kcal/mol Å. Meanwhile, partial atomic charges were calculated using Gasteiger–Hückel method.
The best of the 20 conformations with different scores were obtained. During the docking process,
all the other parameters were assigned their default values. The best ranking pose was visualized
with PyMOL [24].
450
455
Acknowledgments
We should thank Dr. Hong-Bing Zhao (Shanghai TANG Bioscience Co., Ltd.) for the test of
tubulin polymerisation activity. The present work was supported by the National Natural Science
Foundation of China (21372113), and the Science and Technology Program of Guangzhou, China
(201707010198).
460
Appendix A. Supplementary data
Supplementary
data
related
to
this
article
can
be
found
at
http://dx.doi.org/10.1016/j.ejmech.2018.xx.xxx.
465
470
475
480
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Figure captions
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Fig. 1. The structures of CA-4, representative 3-substituted indole derivatives with potent anti-tubulin activity
and the general structure of target compounds 6-34.
Fig. 2. Effect of compound 27 and CA-4 on cell cycle and apoptosis in BT549 cells. Flow cytometry analysis of
BT549 cells treated with 27 for 24 h. (A) Control; (B) 27, 2 µM; (C) 27, 4 µM; (D) 27, 8 µM; (E) CA-4, 8 µM.
Fig. 3. Effect of compound 27 on tubulin expresion in BT549 cell. Cells were seeded on glass coverslips,
incubated with compound 27 (10 µM, 20 µM) for 6 h, then fixed and processed for confocal microscopy.
Fig. 4. Docked pose of 27 (cyan stick) overlayed with CA-4 (magenta) in colchicine binding site of tubulin. The
main interacting residues are shown and labeled. The black dashed lines are the potential H-bond between Cys241
(2.0 Å), Thr179 (2.9 Å), Asn258 (2.2 Å). Final figure for docking pose was generated by PyMOL [24].
575
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Fig. 1. The structures of CA-4, representative 3-substituted indole derivatives with potent anti-tubulin activity and
the general structure of target compounds 6-34.
595
G0/G1: 31.07%
S: 33.01%
G2/M: 35.92%
G0/G1: 65.28%
S: 25.84%
G2/M: 8.88%
G0/G1: 32.10%
S: 41.60%
G2/M: 26.29%
A
B
C
G0/G1: 19.76%
S: 23.66%
G2/M: 56.58%
G0/G1: 8.90%
S: 18.20%
G2/M: 72.90%
600
D
E
Fig. 2. Effect of compound 27 and CA-4 on cell cycle and apoptosis in BT549 cells. Flow cytometry analysis of
BT549 cells treated with 27 for 24 h. (A) Control; (B) 27, 2 µM; (C) 27, 4 µM; (D) 27, 8 µM; (E) CA-4, 8 µM.
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605
610
Fig.3. Effect of compound 27 on tubulin expresion in BT549 cell. Cells were seeded on glass coverslips,
incubated with compound 27 (10 µM, 20 µM) for 6 h, then fixed and processed for confocal microscopy.
Fig. 4. Docked pose of 27 (cyan stick) overlayed with CA-4 (magenta) in colchicine binding site of tubulin. The
main interacting residues are shown and labeled. The black dashed lines are the potential H-bond between Cys241
(2.8 Å), Thr179 (2.2 Å), Asn258 (2.7 Å). Final figure for docking pose was generated by PyMOL [24].
615
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Scheme 1. Synthesis of the target compounds 6
–
34. Reagents and conditions: (a) ClCO2Et, reflux; (b) K2CO3,
620
625
α-bromoketones, DMF, rt.; (c) 2M NaOH, reflux; (d) 3,4,5-trimethoxybenzoyl chloride, Et3N, THF, r.t.
Table 1 Cytotoxic activities of compounds 6–34 against a panel of human cancer cell lines
In vitro cytotoxicity IC50(µM) a
Comp.
R1
R2
MCF-7
8.89±0.64
69.48±7.52
MDA-MB-231
5.17±0.49
BT549
T47D
MDA-MB-468
6.16±0.24
HS578T
8.95±0.43
43.68±4.05
6
7
CH3
CH3
4-FC6H4
8.66±0.27
5.72±0.66
4-CH3C6H4
33.43±2.71
69.75±2.09 40.16±1.07
75.10±2.20
8
CH3
CH3
CH3
CH3
CH3
H
C6H5
32.74±7.03
3.85±0.87
>100
16.60±3.33
3.22±0.43
18.21±2.23
>100
42.26±2.25
4.81±0.57
>100
>100
6.25±0.25
>100
>100
4.12±0.39
>100
49.06±3.19
3.59±0.07
>100
9
4-ClC6H4
10
11
12
13
14
15
16
17
18
19
20
21
>100
>100
>100
>100
>100
>100
>100
39.75±3.36
>100
>100
53.32±6.62
>100
4-FC6H4
4-CH3C6H4
C6H5
77.92±10.01
50.00±14.35
>100
50.76±3.86
12.61±1.78
17.22±2.04
6.47±0.95
>100
42.58±2.97 60.86±3.32
17.07±0.87 20.06±2.04
>100
H
14.52±0.99
>100
50.49±2.26
>100
H
>100
10.31±1.42
>100
>100
9.55±0.31
>100
H
4-BrC6H4
7.14±1.15
>100
7.87±0.55
>100
7.52±0.61
22.24±1.42
9.06±4.22
29.57±1.13
>100
H
H
3-CH3OC6H4
>100
>100
65.53±7.13 53.39±3.25
>100
H
>100
>100
>100
>100
>100
H
4-CH3OC6H4
4-FC6H4
10.70±0.77
61.08±17.61
34.96±2.92
46.65±7.47
10.37±0.23
5.56±0.37
7.09±0.78
>100
Cl
35.54±3.13 76.17±2.93
25.05±2.09 16.65±0.34
>100
22
Cl
4-CH3C6H4
34.33±2.98
33.65±0.61
5.79±0.09
14.19±0.54
23
24
Cl
Cl
C6H5
51.78±5.84
17.62±0.40
32.66±5.27
9.17±1.01
26.89±1.78 45.51±2.69
11.14±0.43 11.09±1.31
11.04±0.19
8.61±0.51
>100
4-CH3OC6H4
15.93±1.27
25
26
27
28
29
30
31
Cl
Cl
Cl
Cl
Cl
Cl
Cl
3-CH3OC6H4
3-BrC6H4
>100
>100
>100
8.42±1.06
3.17±0.46
>100
52.41±1.06
14.07±0.61
0.04±0.06
>100
>100
20.79±0.34
7.92±1.36
>100
>100
4.10±0.27
>100
23.02±2.47
10.87±0.87
>100
26.75±1.12
6.43±1.12
>100
4-ClC6H4
>100
47.46±3.29
10.14±1.16
32.60±0.68
>100
37.51±7.14 31.45±1.87
15.33±3.11
>100
49.23±4.61
64.57±5.73
4.35±0.14
3,4-F2C6H4
>100
51.25±5.85
7.48±0.19
>100
3-OH-4-CH3OC6H4
22.18±1.38
9.28±0.13
23.21±2.08
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32
33
34
CH3O
CH3O
CH3O
4-CH3OC6H4
C6H5
79.27±3.05
>100
48.84±2.76
>100
>100
3.17±0.57
18.20±0.13
>100
6.30±0.52
>100
26.38±1.36
>100
10.44±0.66
>100
4-CH3C6H4
>100
>100
>100
>100
>100
CA-4 (nM)
3.00±0.65
1.71±0.44
1.89±0.34
1.55±0.18
1.36±0.23
a 50% inhibitory concentration and mean ± SD of three independent experiments performed in duplicate.
630
Table 2 Tubulin polymerization inhibitory activities of representative selected compounds
Tubulin polymerization
Comp.
R1
R2
% inhibition a
IC50(µM)
b
6
9
CH3
CH3
H
H
Cl
Cl
Cl
Cl
Cl
4-FC6H4
4-ClC6H4
3-BrC6H4
4-MeOC6H4
4-CH3C6H4
C6H5
4-CH3OC6H4
3-BrC6H4
17
15
37
20
36
41
47
25
59
30
25
80
-
-
-
-
-
-
-
-
9.5
-
16
20
22
23
24
26
27
31
32
4-ClC6H4
Cl
CH3O
3-OH-4-CH3OC6H4
4-MeOC6H
CA-4
-
4.22
a Compounds were tested at a final concentration of 10µM. b -: not tested.
635
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Highlights
► Based on our previous work, a series of novel 3-amidoindole derivatives were
synthesized. ► IC50 of compound 27 were 0.04, 3.17, and 6.43 µM against T47D,
BT549, and MDA-MB-231, respectively. ► Compound 27 exhibited remarkable
anti-tubulin activity with IC50 of 9.5 µM, compared to CA-4.