1-Phenyl-1H-indole derivatives as a new class of Bcl-2/Mcl-1 dual inhibitors: Design, synthesis, and preliminary biological evaluation

Article abstract of DOI:10.1016/j.bmc.2017.08.024

Bcl-2 proteins, such as B-cell lymphoma (Bcl-2) protein, myeloid cell leukemia sequence 1 (Mcl-1) protein, has been implicated in the progression and survival of multiple tumor types and become a validated and attractive target for cancer therapy. In this work, a series of 1-phenyl-1H-indole derivatives has been designed and synthesized. The preliminary biological studies (binding assay for Bcl-2 proteins and MTT assay) suggested that some active compounds showed potent inhibitory activities on Bcl-2/Mcl-1 without binding on Bcl-X_L. Furthermore, Compound 9c and 9h showed better anti-proliferative activity than WL-276.

Full text of DOI:10.1016/j.bmc.2017.08.024

Accepted Manuscript
1-Phenyl-1H-Indole Derivatives as a New Class of Bcl-2/Mcl-1 Dual Inhibitors:
Design, Synthesis, and Preliminary Biological Evaluation
Guangsen Xu, Tingting Liu, Yi Zhou, Xinying Yang, Hao Fang
PII:
S0968-0896(17)31210-5
DOI:
http://dx.doi.org/10.1016/j.bmc.2017.08.024
BMC 13927
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 June 2017
24 July 2017
12 August 2017
Please cite this article as: Xu, G., Liu, T., Zhou, Y., Yang, X., Fang, H., 1-Phenyl-1H-Indole Derivatives as a New
Class of Bcl-2/Mcl-1 Dual Inhibitors: Design, Synthesis, and Preliminary Biological Evaluation, Bioorganic &
Medicinal Chemistry (2017), doi: http://dx.doi.org/10.1016/j.bmc.2017.08.024
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1-Phenyl-1H-Indole Derivatives as a New Class of
Bcl-2/Mcl-1 Dual Inhibitors: Design, Synthesis, and
Preliminary Biological Evaluation
Guangsen Xu, Tingting Liu, Yi Zhou, Xinying Yang, Hao Fang *
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of
Education), School of Pharmacy, Shandong University, 44 West Wenhua Road, Jinan,
Shandong 250012, PR China
Abstract
Bcl-2 proteins, such as B-cell lymphoma (Bcl-2) protein, myeloid cell leukemia
sequence 1(Mcl-1) protein, has been implicated in the progression and survival of
multiple tumor types and become a validated and attractive target for cancer therapy.
In this work, a series of 1-phenyl-1H-indole derivatives has been designed and
synthesized. The preliminary biological studies (binding assay for Bcl-2 proteins and
MTT assay) suggested that some active compounds showed potent inhibitory
activities on Bcl-2/Mcl-1 without binding on Bcl-XL. Furthermore, Compound 9c and
9h showed better anti-proliferative activity than WL-276.
Key Words
apoptosis， Bcl-2/Mcl-1 dual inhibition, 1-phenyl-1H-indole derivatives, anti-tumor
1. Introduction
Apoptosis or programmed cell death gives a major push to physiological growth
control and regulation of tissue homeostasis, while a function of such progress is one
of main reasons of cancer and contributes to resistance.1-3 Herein, it is prospective to
target key apoptosis regulators for cancer treatment. Bcl-2 protein family, the first
identified anti-apoptosis family, plays a pivotal role in the regulation of apoptosis
process.4,5 The Bcl-2 protein family includes pro-apoptotic and anti-apoptotic
members. Bcl-2, Bcl-XL, Bcl-W, Mcl-1 and A1 are anti-apoptotic proteins and they
share common BH regions. While, for pro-apoptotic ones, they are divided into two

groups. The multi-region pro-apoptotic proteins, such as Bax, Bak and Bok, were
conventionally thought to have BH1–3 regions, whereas the BH3-only proteins were
proposed to share homology in the BH3 region only. Members of BH3-only proteins
include Bad, Bim, Bid, Noxa, Puma, Bik/Blk, Bmf, Hrk/DP5, Beclin-1 and Mule.6
Elevated anti-apoptotic Bcl-2 proteins expression level confers cancer cells resistance
to apoptosis and exists in many cancer cells. As a result, inhibiting the expression of
Bcl-2 proteins may restore apoptosis process and overcome the drug resistance of
cancer cell lines to conventional anticancer agents.
Currently, different series of small molecule inhibitors such as Venetoclax
9
(ABT-199, selective inhibitor of Bcl-2)7,8, S63845（Mcl-1 inhibitor） and Obatoclax
(GX015-070, pan Bcl-2 family inhibitor)10 have been reported (Fig.1).
It is encouraging that Venetoclax (ABT-199) has been approved in the United States
for the treatment of chronic lymphocytic leukemia (CLL) with 17p deletion.11
However, prolonged monotherapy tends to beget resistance to ABT-199 by
7
upregulation of Mcl-1 and Bcl-XL, and inhibition of Bcl-XL could result in the
reduction of platelet.12 Hence, it is essential to develop the dual inhibition of Bcl-2
and Mcl-1.

Figure 1. Representative small-molecule inhibitors of anti-apoptotic Bcl-2 proteins.
In 2008, professor Xing designed and synthesized a novel Bcl-2 inhibitor
WL-276 (Fig.1) based on the scaffold of 2-thioxo-4-thiazolidinone.13 Recently, we
developed different types of Bcl-2 proteins inhibitors according to the SAR studies of
WL-276.14-19 In our recent studies, we found a new 1-phenyl-1H-indole derivative 1
showing 71% inhibition against Bcl-2 protein at 100 μM and 54% inhibition against
Mcl-1 protein at 50 μM. Such findings triggered our further research and a series of
1-phenyl-1H-indole derivatives were designed based on the structure 1 (Fig. 2). In
order to achieve higher potency, we introduced amino acid side chains (R1) and
biphenyl (R4) to structure 1 on the basis of recent work. Besides, for the benzene
sulfonamide moiety, we changed different substitutions on the phenyl ring (R2 and R3).
In this paper, we will describe the synthesis, binding affinities to Bcl-2 family
proteins and anti-proliferative activity against some cancer cell lines of these
compounds.

Figure 2. Design of 1-phenyl-1H-indole derivatives.
2. Chemistry
Target compounds were synthesized by the experiment process in Scheme 1.
Firstly, the p-chlorlbenzoyl group on indomethacin 2 were deprotected by
potassium carbonate and methyl alcohol solution. Then, the aryl groups were
introduced by Ullmann reaction to get the key intermediates 4.20 The intermediates 7
were afforded through the condensation reaction of N-Boc-amino acid with benzene
sulfonamide derivatives and the Boc group was removed by ethyl acetate saturated
with HCl gas. Finally, the target compounds 8 and 9 were obtained by the coupling
reactions.
Scheme 1. Synthesis of target compounds

Reagents and conditions: (a) K2CO3 , MeOH, rt, 1h; (b) 4-iodo-1,1'-biphenyl or
1-iodo-4-methylbenzene, KOH, CuO, DMF, N2, reflux, 5h; (c) benzene sulfonamide
o
derivatives, isobutyl chloroformate, NMM, NaH, 0 C, rt, overnight; (d) benzene
o
sulfonamide derivatives, isobutyl chloroformate, NMM, NaH, 0 C, rt, overnight,
ethyl acetate saturated with HCl, rt, overnight ;(e) 4a-4i, HATU, DIEA, DCM, 0 oC, rt,
8h .
3. Results and discussion
We synthesized a series of 1-phenyl-1H-indole derivatives according to the
structure of compound 1. The inhibitory activities of all the target compounds against
Bcl-2 protein were evaluated by fluorescence polarization assays (FPAs). The binding
affinities Ki values was listed in Table 1.
For compounds 8a–8m, most of them showed poor binding affinities. On the
contrary, more than half of target compounds 9a–9l exhibited potent binding affinities
with target proteins. In this work, the target compounds were mainly modified in parts,
the N-1 position of indole (R4), sulfonamide moiety (R2 and R3) and amino acid side
chain (R1).
For the N-1 position of indole (R4), almost all of target compounds that exhibited
moderate binding activities contained the biphenyl part, such as 8h, 9e, 9k, etc. The
possible reason may be due to forming the terphenyl-type structure when
1-phenyl-1H-indole scaffold links with phenyl group in R4 directly. It was reported
that the terphenyl-type structure could mimic α-helix peptide21-23 and enhance the
interaction between target compounds and Bcl-2 protein. For the R1 position,
introducing aromatic amino acid could obtain higher potency of target compounds (eg.
9b, 9d, 9e, 9f, 9k, etc.), especially phenylalanine. The increased hydrogen bond
interaction and hydrophobic effect might contribute to the improving of binding
activities. For the substitution in benzene sulfonamide moiety (R2 and R3), large
substituents (eg. phenyl, -N-benzyl amine, -N-morpholine, etc.) have no benefit on
affinities for Bcl-2 protein, such as compound 8j, 8k, 8l, 8m, 9g, etc. But the
compounds exerted higher potency with small substituents (eg. -Cl, methoxyl, nitro),
such as compound 8h, 9d, 9e.

It is noteworthy that the most active target compounds, 9c (Ki=0.35±0.03µM),
9d (Ki = 0.47±0.03µM) and 9h (Ki = 0.58±0.06µM), show better inhibitory activities
than WL-276 (Ki = 0.94±0.14µM).
Table 1 The structure and in vitro Bcl-2 inhibitory activity of 1, 8a–8m, 9a-9l.
Bcl-2
Kia (µM)
R1
-
R2
NO2
H
R3
Cl
R4
Cmpd
1
CH3
CH3
CH3
CH3
CH3
CH3
＞10
-
NO2
OCH3
8a
8b
8c
＞10
＞10
-
H
-
NO2
NO2
NO2
H
＞10
-
8d
8e
＞10
-
＞10
-
H
OCH3
Cl
8f
＞10
-
H
8g
8h
8i
＞10
-
NO2
NO2
NO2
0.72±0.06
＞10
-
-
8j
＞10

-
-
-
NO2
NO2
NO2
H
8k
8l
＞10
＞10
8m
9a
＞10
H
Cl
CH3
CH3
＞10
NO2
H
0.72±0.04
0.35±0.03
0.47±0.03
0.91±0.01
0.91±0.05
＞10
9b
H
9c
H
OCH3
Cl
9d
H
9e
H
NO2
9f
H
9g
NO2
H
Cl
H
0.58±0.06
＞10
9h
9i
H
OCH3
Cl
9j
＞10
NO2
NO2
0.83±0.13
＞10
9k
Cl
9l
WL-276
0.94±0.14
a Each value was reproduced in three independent assays and expressed with standard deviations.
In order to understand the interaction between these 1-phenyl-1H-indole-based
derivatives and Bcl-2 protein, docking studies were performed using the most active
target compound 9c using with Surflex-Dock software (Fig. 3). The result suggested
that compound 9c could bind to the active pocket of Bcl-2 protein through Van der
Waals forces, hydrogen bond, etc. The amide group of compound 9c could form one
hydrogen bond with Arg143. In addition, there is π-π interaction between aromatic of

benzene sulfonamide fragment and Phe109. Meanwhile, indole scaffold could bind to
Phe101 by π-π interaction.
Figure 3 Docking results of 9c and Bcl-2 protein
(a) Docked compound 9c with Bcl-2 protein. (b) The docking mode of compound
9c in the active site of Bcl-2 protein. (c) The interplays between compound 9c and
Bcl-2 protein in the active site by software: Molecular Operating Environment
To identify if these compounds could also inhibit other anti-apoptotic Bcl-2
proteins, three compounds were selected to evaluate the binding affinities for Mcl-1
and Bcl-XL proteins by FPAs. According to the data in Table 2, all the active
compounds exhibit similar binding affinities on Mcl-1 without inhibition on Bcl-XL,
which indicated that these compounds could be regarded as new Bcl-2/Mcl-1 dual
inhibitors.

Table 2 The binding affinities of representative target compounds to three Bcl-2 proteins
Bcl-2
Bcl-XL
Kia (µM)
N.A.b
N.A.b
N.A.b
Mcl-1
Cmpd
Kia (µM)
0.35±0.03
0.47±0.03
0.58±0.06
0.94±0.14
Kia (µM)
0.96±0.01
0.88±0.06
0.48±0.02
0.47±0.03
9c
9d
9h
WL-276
1.2±0.10
a Each value was reproduced in three independent assays and expressed with standard deviations.
b No activity
It was reported that Bcl-2 proteins were found to be over-expressed in various
cancer cells, such as breast, prostate carcinoma andleukemias.24-27 Therefore,
compound 9c, 9d and 9h that possessed better Bcl-2/Mcl-1 dual inhibition than other
compounds were chosen to test their anti-proliferative activities in vitro. MTT
(3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazoliumbromide) assay were using
to evaluated three different tumor cell lines which include K562 (chronic
myelogenous leukemia cell), PC-3 (prostatic cancer cell) and MDA-MB-231 (breast
cancer cell). The results of IC50 values were listed in Table 3 and suggested that all
the active compounds display better anti-proliferative activities against in three tumor
cell lines compared with WL-276. Especially, compound 9h exhibited most potent
activities on PC-3 and K562 cell lines.
Table 3 Antiproliferative activities of representative compounds
IC50 (μM)a
Cmpd
MDA-MB-231
33.55±5.01
38.62±2.81
42.06±3.26
45.89±1.34
PC-3
K562
17.49±4.22
23.48±1.34
13.90±0.24
30.13±4.52
18.75±2.88
27.53±1.94
13.02±1.52
43.97±1.31
9c
9d
9h
WL-276
a
Inhibitory data are means of no fewer than two independent determinations and expressed with standard
deviations.

4. Conclusions
In conclusion, a series of 1-phenyl-1H-indole-based derivatives as Bcl-2/Mcl-1
inhibitors were designed and synthesized. According to the data of protein binding
assay, compounds 9c, 9d and 9h displayed the promising inhibitory activity against
Bcl-2/Mcl-1 proteins without Bcl-XL inhibition. Compound 9h showed better
anti-proliferative activity, compared with WL-276. These results could be a good
starting point for the development of more potent Bcl-2/Mcl-1 dual inhibitors in the
future.
5. Experiment section
5.1. General chemistry information
Only if otherwise mentioned, all solvents (dichloromethane, tetrahydrofuran, etc.)
and starting materials (indomethacin, benzene sulfonamide, etc.) were from
commercial suppliers and utilized without further purification. Entire reactions were
monitored by thin-layer chromatography (TLC) on 0.25mm silica gel plates (60
GF-254) and visualized with under ultraviolet light（254 nM or 365 nM）or iodine
vapor. Most target compounds were purified by recrystallization or normal phase
column chromatography. Proton NMR (1H NMR) spectrums were determined on a
Brucker DRX spectrometer (400 MHz), δ in parts per million and J in Hertz, using
TMS as an internal standard. Measurements were made in DMSO-d6 or CDCl3
solutions. High-resolution mass spectral (HRMS) data were displayed as m/z on an
Agilent 6510 Quadrupole Time-of-Flight LC/MS deliver. Melting points were
determined on an electrothermal melting point apparatus and were uncorrected.
5.1.1. 2-(5-methoxy-2-methyl-1H-indol-3-yl)acetic acid (3)
Under the condition of ice bath, potassium carbonate powder (3.7 g, 27 mmol) was
dissolved in methyl alcohol (100 mL). After 2-(1-(4-chlorobenzoyl)-5-methoxy-
2-methyl-1H-indol-3-yl) acetic acid (3.0 g, 9 mmol) was added drop by drop, the
mixture was stirred at room temperature for 1 h (detected by TLC). The methyl
alcohol was distillated under reduced pressure and dissolved in water (200ml).

The white precipitates were separated and the solid particles were filtered from
water. When the filtrates was regulated to acidityby HCl (2mol/L), the white
precipitates were separated. The white solid, the compound 2 was filtered with suction,
pressed to remove excess water, and allowed to dry in air. White solid. Yield: 72%,
o
mp: 158–160 C.1H NMR (400 MHz, DMSO-d6) δ 12.03 (s, 1H), 10.64 (s, 1H),
7.17–7.00 (m, 1H), 6.88 (d, J = 2.4 Hz, 1H), 6.73–6.55 (m, 1H), 3.72 (s, 3H), 3.53 (d,
J = 9.5 Hz, 2H), 2.29 (s, 3H).
5.1.2. 2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-indol-3-yl)acetic acid (4a)
To a solution of 3 (1.5g, 7.5 mmol), 1-iodo-4-methylbenzene (1.8 g, 8.3 mmol) KOH
(0.87g, 16.5mmol) and CuO (0.26g, 3.2mmol) in 20 mL of DMF. After refluxing at
154 oC under N2 protection for 5h, the solution was poured into 300 mL of distilled
water with off white solid precipitation. The solid were filtered from water. When the
filtrates was regulated to acidity by HCl (2mol/L), the brown precipitates were
separated. The brown solid, compound 4a was filtered with suction, pressed to
remove excess water, and allowed to dry in air. Brown solid. Yield: 62%, mp:
130–133 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.19 (s, 1H), 7.38 (d, J = 8.1 Hz, 2H),
7.26 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 2.4 Hz, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.68 (dd, J
= 8.8, 2.4 Hz, 1H), 3.76 (s, 3H), 3.65 (s, 2H), 2.41 (s, 3H), 2.19 (s, 3H).
5.1.2.1 2-(1-([1, 1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetic acid
(4b)
To a solution of 3 (1.5 g, 7.5 mmol), 4-iodo-1, 1'-biphenyl (1.8 g, 8.3 mmol),
KOH (0.87 g, 16.5 mmol) and CuO (0.26 g, 3.2 mmol) in 20 mL of DMF. After
refluxing at 154 oC under N2 protection for 5h, the solution was poured into 300 mL
of HCl (0.5 mol/L) with off white solid precipitation. The solid particles were filtered
from water, the solid was then dissolved using EtOAc. The insoluble impurities were
filtered from solution and the EtOAc was distillated under reduced pressure. Finally,
the brown oil was purified by silica gel chromatography (PE / EA = 5:1, 0.3% HOAc)
to generate compound 7b as a yellow powder solid. Yield: 41%, mp: 160–162 oC. 1H

NMR (400 MHz, DMSO-d6) δ 12.22 (s, 1H), 7.88 (d, J = 8.5 Hz, 2H), 7.82–7.70 (m,
2H), 7.57–7.46 (m, 4H), 7.42 (t, J = 7.4 Hz, 1H), 7.04 (d, J = 2.4 Hz, 1H), 7.01 (d, J =
8.8 Hz, 1H), 6.71 (dd, J = 8.8, 2.4 Hz, 1H), 3.79 (s, 3H), 3.68 (s, 2H), 2.26 (s, 3H).
5.1.3. (S)-2-amino-3-phenyl-N-(phenylsulfonyl)propanamide hydrochloride (7a)
(tert-butoxycarbonyl)-L-phenylalanine (1.3 g, 5 mmol) was dissolved in 30 mL
o
anhydrous THF at 0 C. 5 min later, NMM (0.6 ml, 5.5 mmol) and
isobutylchloroformate (0.7 ml, 5.5 mmol) were added successively. After stirred for 1
h, the mixture of benzenesulfonamide (0.86 ml, 5.5 mmol) and NaH (0.5 g, 12.5
mmol), which stirred for 4h at room temperature, was added and the reaction was
warmed slowly to room temperature and stirred overnight. The solvent was removed
under low pressure. The resulting residue was extracted with EtOAc and washed with
0.5 M citric acid and brine, respectively. After dried over MgSO4 and concentrated,
the obtained oil was dissolved in EtOAc saturated with HCl gas and stirred at room
temperature overnight. Then the generated hydrochloride precipitate was filtered and
dried. White solid. Yield: 78%, mp: 229–230 oC. 1H NMR (400 MHz, DMSO-d6) δ
8.27 (s, 2H), 7.95–7.90 (m, 1H), 7.83 (dd, J = 7.9, 1.7 Hz, 1H), 7.76 (t, J = 7.4 Hz,
1H), 7.65 (t, J = 7.7 Hz, 1H), 7.61–7.52 (m, 2H), 7.38–7.17 (m, 4H), 4.06 (brs, 1H),
3.86 (s, 3H), 3.10–3.01 (dd, J = 14.1, 6.8 Hz, 1H), 2.96 (dd, J = 14.1, 6.8 Hz, 1H).
Compounds 7b–7l were synthesized following the procedure described above.
5.1.3.1 (S)-2-amino-N-((4-methoxyphenyl)sulfonyl)-3-phenylpropanamide
hydrochloride (4b)
White solid. Yield: 75%, mp: 209–210 oC.1H NMR (400 MHz, DMSO-d6) δ 8.42 (s,
3H), 7.83 (d, J = 18.9 Hz, 2H), 7.24–7.11 (m, 5H), 7.07 (t, J = 10.5 Hz, 2H),
4.03–3.99 (m, 1H), 3.86 (s, 3H), 3.10 (dd, J = 14.0, 6.0 Hz, 1H), 3.02 (dd, J = 14.0,
6.0 Hz, 1H).
5.1.3.2 (S)-2-amino-N-((4-nitrophenyl)sulfonyl)-3-phenylpropanamide
hydrochloride (7c)

o
White solid. Yield: 82%, mp: 236 – 237 C.1H NMR (400 MHz, DMSO-d6) δ
8.46–8.37 (m, 2H), 8.30 (s, 2H), 8.21 (s, 1H), 8.16–8.09 (m, 2H), 7.26–7.16 (m, 3H),
7.11 (d, J = 6.8 Hz, 2H), 4.13–3.99 (m, 1H), 3.08 (dd, J = 13.8, 5.5 Hz, 1H), 2.98 (dd,
J = 14.1, 7.1 Hz, 1H).
5.1.3.3 (S)-N-([1,1'-biphenyl]-4-ylsulfonyl)-2-amino-3-phenylpropanamide
hydrochloride (7d)
White solid. Yield: 62%, mp: 240–242 oC.1H NMR (400 MHz, DMSO-d6) δ 8.38 (s,
3H), 8.12–7.93 (m, 4H), 7.82–7.70 (m, 2H), 7.55 (t, J = 7.4 Hz, 2H), 7.48 (t, J = 7.3
Hz, 1H), 7.20 –7.13 (m, 3H), 7.07 (d, J = 6.5 Hz, 2H), 4.18–3.95 (m, 1H), 3.11 (dd, J
= 14.2, 5.9 Hz, 1H), 3.03 (dd, J = 14.2, 6.8 Hz, 1H).
5.1.3.4(S)-2-amino-N-((4-chlorophenyl)sulfonyl)-3-phenylpropanamide
hydrochloride (7e)
White solid. Yield: 71%, mp: 246–249 oC.1H NMR (400 MHz, DMSO-d6) δ 8.41 (s,
3H), 7.91 (d, J = 8.6 Hz, 2H), 7.73 (d, J = 8.6 Hz, 2H), 7.28–7.14 (m, 3H), 7.07 (d, J
= 6.8 Hz, 2H), 4.23–3.98 (m, 1H), 3.10 (dd, J = 14.0, 6.0 Hz, 1H), 3.02 (dd, J = 14.0,
6.7 Hz, 1H).
5.1.3.5 (S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-phenylpropanamide
hydrochloride (7f)
White solid. Yield: 59%, mp: 228–229 oC.1H NMR (400 MHz, DMSO-d6) δ 8.50 (d, J
= 2.0 Hz, 1H), 8.32 (s, 2H), 8.21 (s, 1H),, 8.14 (dd, J = 8.5, 2.0 Hz, 1H), 8.03–7.98 (m,
1H), 7.28–7.14 (m, 3H), 7.14–7.01 (m, 2H), 4.17–4.01 (m, 1H), 3.16–2.98 (m, 2H).
5.1.3.6 (S)-2-amino-3-(1H-indol-3-yl)-N-(phenylsulfonyl)propanamide
hydrochloride (7g)
White solid. Yield: 68%, mp: 230–232 oC.1H NMR (400 MHz, DMSO-d6) δ 13.08 (s,
1H), 11.04 (d, J = 1.6 Hz, 1H), 8.27 (s, 3H), 7.99–7.86 (m, 2H), 7.81–7.68 (m, 2H),
7.64 (t, J = 7.7 Hz, 2H), 7.37 (d, J = 8.1 Hz, 1H), 7.10 (dd, J = 11.1, 4.0 Hz, 1H),
7.05–6.89 (m, 2H), 4.23–3.99 (m, 1H), 3.25 (dd, J = 14.6, 5.8 Hz, 1H), 3.08 (dd, J =

14.6, 7.8 Hz, 1H).
5.1.3.7 (S)-2-amino-3-(1H-indol-3-yl)-N-((4-methoxyphenyl)sulfonyl)propanamide
hydrochloride (7h)
White solid. Yield: 73%, mp: 236–237 oC.1H NMR (400 MHz, DMSO-d6) δ 12.94 (s,
1H), 11.06 (s, 1H), 8.28 (s, 3H), 7.90–7.77 (m, 2H), 7.69 (d, J = 7.9 Hz, 1H), 7.37 (d,
J = 8.1 Hz, 1H), 7.13 (d, J = 9.0 Hz, 2H), 7.09 (d, J = 8.0 Hz, 1H), 7.03 (d, J = 2.3 Hz,
1H), 6.98 (t, J = 7.5 Hz, 1H), 4.03 (dd, J = 14.3, 7.1 Hz, 1H), 3.87 (s, 3H), 3.23 (dd, J
= 14.6, 5.9 Hz, 1H), 3.08 (dd, J = 14.6, 7.6 Hz, 1H).
5.1.3.8
(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(1H-indol-3-yl)propanamide
hydrochloride (7i)
White solid. Yield: 82%, mp: 238 oC (dec).1H NMR (400 MHz, DMSO-d6) δ 11.01 (s,
1H), 8.48 (d, J = 2.1 Hz, 1H), 8.07 (dd, J = 8.5, 2.2 Hz, 4H), 7.93 (d, J = 8.5 Hz, 1H),
7.56 (d, J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.10–7.03
(m, 1H), 6.98–6.88 (m, 1H), 4.05–3.89 (m, 1H), 3.25 (dd, J = 14.8, 5.5 Hz, 1H), 3.09
(dd, J = 14.8, 7.7 Hz, 1H).
5.1.3.9
(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((4-nitrobenzyl)oxy)phenyl)
propanamide hydrochloride (7j)
White solid. Yield: 43%, mp: 218–220 oC.1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J
= 2.0 Hz, 1H), 8.44–8.23 (m, 5H), 8.03 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.7 Hz, 2H),
7.12–7.00 (m, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.25 (s, 2H), 4.09–3.93 (m, 1H), 3.12 –
2.87 (m, 2H).
5.1.3.10
(S)-2-amino-3-(4-(benzyloxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)propana
mide hydrochloride (7k)

White solid. Yield: 61%, mp: 212–213 oC.1H NMR (400 MHz, DMSO-d6) δ 8.52 (d, J
= 2.1 Hz, 1H), 8.28 (s, 3H), 8.14 (dd, J = 8.5, 2.2 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H),
7.45–7.32 (m, 4H), 7.23–7.14 (m, 1H), 6.99 (t, J = 8.3 Hz, 2H), 6.82 (d, J = 8.7 Hz,
2H), 5.07 (d, J = 12.1 Hz, 2H), 4.03–4.15 (s, 1H), 3.10–2.90 (m, 2H).
5.1.3.11
(S)-2-amino-3-(4-(benzyloxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)propana
mide hydrochloride (7l)
White solid. Yield: 77%, mp: 219–222 oC. 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d,
J = 2.0 Hz, 1H), 8.15–8.06 (m, 1H), 8.00 (s, 2H), 7.96 (d, J = 8.5 Hz, 1H), 7.38 (d, J =
12.6 Hz, 2H), 7.03 (d, J = 12.6 Hz, 2H), 4.13–3.97 (m, 1H), 3.11–2.99 (m, 1H), 2.95
(dd, J = 14.2, 6.9 Hz, 1H).
5.1.4.N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-in
dol-3-yl)acetamide (1)
N-methylmorpholine (0.09 g, 0.89 mmol) and isobutyl chloroformate (0.12 g, 0.89
mmol) was added into a solution of intermediate 7a (0.24 g, 0.81 mmol) in 10 mL
o
anhydrous THF at 0 C in order. After stirred for 1 h, a solution of
4-chloro-3-nitrobenzenesulfonamide (0.21g, 0.89mmol) and NaH (0.08 g, 2 mmol) in
10 mL anhydrous THF was added drop by drop and stirred for 8h at room temperature.
Then the solvent was removed by vacuum distillation and dissolved in amount of
EtOAc. The solution was washed by 0.5 M citric acid, brine and dried over anhydrous
MgSO4. After removal of the solvent, the crude product was further purified by
column chromatography (DCM / MeOH = 90:1) to yield pure compound 1 as yellow
powder. White solid. Yield: 37%, mp: 160–162 oC.1H NMR (400 MHz, DMSO-d6) δ
12.73 (s, 1H), 8.52 (d, J = 2.1 Hz, 1H), 8.16 (dd, J = 8.5, 2.2 Hz, 1H), 8.01 (d, J = 8.5
Hz, 1H), 7.37 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 6.89 (d, J = 2.3 Hz, 1H),
6.85 (d, J = 8.8 Hz, 1H), 6.65 (dd, J = 8.8, 2.4 Hz, 1H), 3.70 (s, 3H), 3.69 (s, 2H),
2.40 (s, 3H), 2.12 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.02, 154.34, 147.58,
139.82, 137.60, 135.86, 135.15, 133.43, 132.87, 132.33, 131.13, 130.60, 128.48,

127.75, 125.49, 110.83, 110.63, 104.83, 101.04, 55.88, 32.47, 21.15, 11.42. HRMS
(AP-ESI) m/z Cacld for C25H22ClN3O6S [M-H]- 526.0845. Found: 526.0847.
Compounds 8a – 8m were synthesized following the procedure described above.
5.1.4.1
2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-indol-3-yl)-N-((4-nitrophenyl)sulfonyl)aceta
mide (8a)
Yellow solid. Yield: 31%, mp: 168–170 oC.1H NMR (400 MHz, DMSO-d6) δ 12.67 (s,
1H), 8.56–8.27 (m, 2H), 8.27–8.00 (m, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.2
Hz, 2H), 6.85 (t, J = 5.6 Hz, 2H), 6.64 (dd, J = 8.8, 2.4 Hz, 1H), 3.69 (s, 5H), 2.40 (s,
3H), 2.11 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 170.66, 154.32, 150.68, 144.88,
137.61, 135.93, 135.13, 132.33, 130.60, 129.71, 128.46, 127.75, 124.88, 110.77,
110.61, 104.72, 101.09, 55.89, 32.33, 21.15, 11.42. HRMS (AP-ESI) m/z Cacld for
C25H23N3O6S [M-H]- 492.1235. Found: 492.1239.
5.1.4.2
2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-indol-3-yl)-N-((4-methoxyphenyl)sulfonyl)ac
etamide (8b)
White solid. Yield: 26%, mp: 170–173 oC.1H NMR (400 MHz, DMSO-d6) δ 12.22 (s,
1H), 7.97–7.82 (m, 2H), 7.37 (d, J = 8.1 Hz, 2H), 7.32–7.19 (m, 2H), 7.12–7.06 (m,
2H), 6.93 (d, J = 2.3 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 6.65 (dd, J = 8.8, 2.4 Hz, 1H),
3.83 (s, 3H), 3.71 (s, 3H), 3.64 (s, 2H), 2.40 (s, 3H), 2.11 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 170.13, 163.55, 154.35, 137.58, 135.77, 135.18, 132.31, 131.27, 130.60,
130.40, 128.56, 127.75, 114.66, 110.95, 110.61, 105.19, 101.04, 56.21, 55.91, 32.16,
21.16, 11.40. HRMS (AP-ESI) m/z Cacld for C26H26N2O5S [M-H]- 477.1490. Found:
477.1493.
5.1.4.3
2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-indol-3-yl)-N-((4-(4-methylpiperazin-1-yl)-3-
nitrophenyl)sulfonyl)acetamide (8c)

Yellow solid. Yield: 21%, mp: 230–232 oC.1H NMR (400 MHz, CDCl3) δ 8.36 (d, J =
2.2 Hz, 1H), 7.96 (dd, J = 8.9, 2.3 Hz, 1H), 7.35 (d, J = 8.1 Hz, 2H), 7.22 (d, J = 8.2
Hz, 2H), 7.08 (d, J = 9.0 Hz, 1H), 7.00 (d, J = 8.9 Hz, 1H), 6.77 (dd, J = 8.9, 2.3 Hz,
1H), 6.69 (d, J = 2.2 Hz, 1H), 3.76 (s, 3H), 3.72 (s, 2H), 3.30–3.16 (m, 4H), 2.69–2.53
(m, 4H), 2.47 (s, 3H), 2.38 (s, 3H), 2.20 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
154.23, 147.50, 139.01, 137.44, 135.32, 132.94, 132.29, 130.58, 128.81, 127.72,
126.78, 121.13, 110.79, 110.46, 106.56, 101.29, 55.89, 53.97, 49.78, 44.89, 33.56,
21.16, 11.46. HRMS (AP-ESI) m/z Cacld for C30H33N5O6S [M-H]- 590.2079. Found:
590.2079.
5.1.4.4
2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-indol-3-yl)-N-((4-morpholino-3-nitrophenyl)s
ulfonyl)acetamide (8d)
Yellow solid. Yield: 15%, mp: 132–135 oC.1H NMR (400 MHz, DMSO-d6) δ 12.42 (s,
1H), 8.26 (dd, J = 7.8, 2.3 Hz, 1H), 7.93 (td, J = 8.8, 2.3 Hz, 1H), 7.47–7.39 (m, 1H),
7.37 (d, J = 8.1 Hz, 2H), 7.23 (d, J = 8.2 Hz, 2H), 6.94 (d, J = 2.4 Hz, 1H), 6.85 (t, J =
8.1 Hz, 1H), 6.65 (dd, J = 8.8, 2.4 Hz, 1H), 3.75–3.62 (m, 9H), 3.24–3.10 (m, 4H),
13
2.42 (d, J = 11.0 Hz, 3H), 2.12 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 170.52,
154.37, 148.54, 138.18, 137.60, 135.82, 135.15, 133.00, 132.31, 130.60, 128.89,
128.52, 127.75, 127.53, 120.95, 110.94, 110.64, 105.00, 101.03, 66.11, 55.91, 50.81,
32.25, 21.15, 11.42. HRMS (AP-ESI) m/z Cacld for C29H30N4O7S [M-H]- 577.1762.
Found: 577.1759.
5.1.4.5
N-((4-(benzylamino)-3-nitrophenyl)sulfonyl)-2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-
indol-3-yl)acetamide (8e)
Yellow solid. Yield: 22%, mp: 187–189 oC.1H NMR (400 MHz, DMSO-d6) δ 12.31 (s,
1H), 9.20 (t, J = 6.2 Hz, 1H), 8.56 (d, J = 2.3 Hz, 1H), 7.80 (dd, J = 9.2, 2.2 Hz, 1H),
7.34 (dt, J = 14.8, 7.9 Hz, 6H), 7.24 (dd, J = 10.0, 7.7 Hz, 3H), 7.04 (d, J = 9.3 Hz,
1H), 6.89 (d, J = 2.4 Hz, 1H), 6.85 (d, J = 8.8 Hz, 1H), 6.64 (dd, J = 8.8, 2.4 Hz, 1H),

4.68 (d, J = 6.2 Hz, 2H), 3.68 (s, 3H), 3.63 (s, 2H), 2.41 (s, 3H), 2.08 (s, 3H). 13C
NMR (100 MHz, DMSO-d6) δ 170.43, 154.34, 147.71, 138.05, 137.59, 135.78,
135.18, 134.24, 132.31, 130.59, 130.55, 129.10, 128.50, 128.17, 127.74, 127.70,
127.37, 125.02, 115.97, 110.82, 110.59, 105.04, 101.07, 55.88, 46.33, 32.25, 21.16,
11.38. HRMS (AP-ESI) m/z Cacld for C32H30N4O6S [M-H]- 597.1813. Found:
597.1814.
5.1.4.6
2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-(phenylsulfonyl)a
cetamide (8f)
White solid. Yield: 32%, mp: 178–180 oC.1H NMR (400 MHz, CDCl3) δ 8.14 (s, 1H),
8.02–7.96 (m, 2H), 7.82–7.73 (m, 2H), 7.71–7.61 (m, 3H), 7.59–7.47 (m, 4H), 7.42
(dd, J = 11.7, 5.3 Hz, 3H), 7.10 (d, J = 8.9 Hz, 1H), 6.81 (dd, J = 8.9, 2.4 Hz, 1H),
6.63 (d, J = 2.3 Hz, 1H), 3.73 (d, J = 2.9 Hz, 5H), 2.25 (s, 3H). 13C NMR (100 MHz,
DMSO-d6) δ 170.30, 154.50, 139.83, 139.72, 139.68, 137.06, 135.73, 134.14, 132.12,
129.58, 129.54, 128.75, 128.31, 128.23, 127.96, 127.24, 111.16, 110.76, 105.61,
101.03, 55.93, 32.18, 11.51. HRMS (AP-ESI) m/z Cacld for C30H26N2O4S [M-H]-
509.1541. Found: 509.1541.
5.1.4.7
2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-((4-methoxypheny
l)sulfonyl)acetamide (8g)
White solid. Yield: 17%, mp: 220–221 oC.1H NMR (400 MHz, CDCl3) δ 8.11 (s, 1H),
7.98–7.85 (m, 2H), 7.81–7.72 (m, 2H), 7.74–7.60 (m, 2H), 7.51 (dd, J = 10.3, 4.8 Hz,
2H), 7.48–7.34 (m, 3H), 7.10 (d, J = 8.9 Hz, 1H), 7.05–6.89 (m, 2H), 6.81 (dd, J = 8.9,
2.4 Hz, 1H), 6.63 (d, J = 2.3 Hz, 1H), 3.89 (s, 3H), 3.73 (d, J = 10.5 Hz, 5H), 2.25 (s,
3H). 13C NMR (100 MHz, DMSO-d6) δ 170.11, 163.56, 154.50, 139.72, 139.68,
137.08, 135.72, 132.15, 131.29, 130.42, 129.54, 128.77, 128.31, 128.30, 128.23,
127.24, 114.67, 111.11, 110.73, 105.70, 101.14, 56.21, 55.92, 32.17, 11.52. HRMS
(AP-ESI) m/z Cacld for C31H28N2O5S [M-H]- 539.1646. Found: 539.1647.

5.1.4.8
2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-((4-chloro-3-nitro
phenyl)sulfonyl)acetamide (8h)
White solid. Yield: 13%, mp: 206–208 oC.1H NMR (400 MHz, DMSO-d6) δ 12.74 (s,
1H), 8.54 (d, J = 2.2 Hz, 1H), 8.18 (dd, J = 8.5, 2.2 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H),
7.86 (d, J = 8.4 Hz, 2H), 7.76 (d, J = 7.3 Hz, 2H), 7.51 (t, J = 7.6 Hz, 2H), 7.46 (d, J =
8.4 Hz, 2H), 7.41 (t, J = 7.3 Hz, 1H). 6.97 (d, J = 8.8 Hz, 1H), 6.91 (d, J = 2.4 Hz,
1H), 6.69 (dd, J = 8.8, 2.4 Hz, 1H), 3.75 (d, J = 15.0 Hz, 2H), 3.72 (s, 3H), 2.20 (s,
13
3H). C NMR (100 MHz, DMSO-d6) δ 170.88, 154.48, 147.60, 139.74, 139.66,
137.03, 135.84, 133.47, 132.90, 132.15, 131.23, 129.53, 128.67, 128.31, 128.23,
127.23, 125.53, 110.99, 110.76, 105.25, 101.11, 55.88, 32.40, 11.55. HRMS (AP-ESI)
m/z Cacld for C30H24ClN3O6S [M-H]- 588.1002. Found: 588.1006.
5.1.4.9
2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-((4-(cyclohexylam
ino)-3-nitrophenyl)sulfonyl)acetamide (8i)
Yellow solid. Yield: 32%, mp: 218–220 oC.1H NMR (400 MHz, CDCl3) δ 8.71 (d, J =
2.2 Hz, 1H), 8.51 (d, J = 7.3 Hz, 1H), 7.94 (d, J = 7.8 Hz, 1H), 7.76 (d, J = 8.2 Hz,
2H), 7.71–7.60 (m, 2H), 7.50 (dd, J = 10.3, 4.7 Hz, 2H), 7.42 (dd, J = 11.6, 4.6 Hz,
3H), 7.08 (d, J = 8.9 Hz, 1H), 6.91 (d, J = 9.3 Hz, 1H), 6.78 (dd, J = 8.9, 2.3 Hz, 1H),
6.67 (s, 1H),δ 3.74 (s, 3H), 3.71 (s, 2H), 3.56 (s, 1H), 2.26 (s, 3H), 2.07 (d, J = 8.2 Hz,
2H), 1.84 (d, J = 9.9 Hz, 2H), 1.69 (d, J = 12.0 Hz, 1H), 1.48–1.27 (m, 5H).13C NMR
(100 MHz, DMSO-d6) δ 170.73, 154.45, 146.81, 139.69, 139.67, 137.09, 135.67,
134.45, 132.14, 129.91, 129.53, 128.74, 128.28, 128.24, 127.22, 125.21, 115.89,
110.93, 110.67, 105.80, 101.21, 65.39, 55.89, 51.29, 32.52, 32.10, 25.37, 24.44, 15.64,
11.55. HRMS (AP-ESI) m/z Cacld for C36H36N4O6S [M-H]- 651.2283. Found:
651.2277.
5.1.4.10 2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-

((4-(benzylamino)-3-nitrophenyl)sulfonyl)acetamide (8j)
Yellow solid. Yield: 24%, mp: 210–212 oC.1H NMR (400 MHz, CDCl3) δ 8.79 (s, 1H),
8.73 (s, 1H), 8.14 (s, 1H), 7.94 (d, J = 8.8 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.67 (d, J
= 7.4 Hz, 2H), 7.50 (t, J = 7.5 Hz, 2H), 7.46–7.29 (m, 7H), 7.07 (d, J = 8.9 Hz, 1H),
6.90 (d, J = 8.9 Hz, 1H), 6.77 (d, J = 8.7 Hz, 1H), 6.64 (s, 1H), 4.59 (d, J = 3.9
Hz,2H), 3.71 (d, J = 6.4 Hz, 5H), 2.25 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
170.82, 154.45, 147.59, 139.69, 138.11, 137.10, 135.62, 134.31, 132.13, 130.51,
129.54, 129.09, 128.77, 128.30, 128.23, 128.04, 127.69, 127.37, 127.23, 125.62,
115.87, 110.96, 110.68, 105.86, 101.20, 55.88, 46.32, 32.50, 11.52. HRMS (AP-ESI)
m/z Cacld for C37H32N4O6S [M-H]- 659.1970. Found: 659.1967.
5.1.4.11 2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-
((3-nitro-4-((3-phenylpropyl)amino)phenyl)sulfonyl)acetamide (8k)
Yellow solid. Yield: 39%, mp: 204–206 oC.1H NMR (400 MHz, CDCl3) δ 8.70 (d, J =
2.3 Hz, 1H), 8.49 (t, J = 5.1 Hz, 1H), 8.15 (s, 1H), 7.96 (dd, J = 9.1, 2.1 Hz, 1H),
7.84–7.73 (m, 2H), 7.68 (dd, J = 5.2, 3.3 Hz, 2H), 7.51 (dd, J = 10.3, 4.8 Hz, 2H),
7.47–7.37 (m, 3H), 7.33 (dd, J = 10.0, 4.5 Hz, 2H), 7.25–7.17 (m, 3H), 7.08 (d, J =
8.9 Hz, 1H), 6.82 (t, J = 7.1 Hz, 1H), 6.78 (dd, J = 8.9, 2.4 Hz, 1H), 6.62 (d, J = 2.3
Hz, 1H), 3.72 (s, 3H), 3.72 (s, 2H), 3.38 (dd, J = 12.5, 7.0 Hz, 2H), 2.80 (t, J = 7.4 Hz,
2H), 2.26 (s, 3H), 2.19–2.03 (m, 2H). 13C NMR (100 MHz, DMSO-d6) δ 170.40,
154.47, 147.76, 141.65, 139.70, 139.65, 137.06, 135.74, 134.30, 132.16, 130.11,
129.51, 128.77, 128.70, 128.28, 128.21, 127.20, 126.31, 124.56, 115.53, 110.95,
110.69, 105.56, 101.17, 55.87, 42.66, 32.82, 32.33, 30.08, 11.53. HRMS (AP-ESI)
m/z Cacld for C39H36N4O6S [M-H]- 687.2283. Found: 687.2277.
5.1.4.12
2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-((3-nitro-4-((2-(ph
enylthio)ethyl)amino)phenyl)sulfonyl)acetamide (8l)
Yellow solid. Yield: 43%, mp: 186–188 oC.1H NMR (400 MHz, CDCl3) δ 8.71 (t, J =
5.5 Hz, 1H), 8.68 (d, J = 2.3 Hz, 1H), 8.17 (s, 1H), 7.94 (dd, J = 9.1, 2.2 Hz, 1H),

7.82–7.73 (m, 2H), 7.69 (dd, J = 11.3, 10.0 Hz, 2H), 7.56–7.46 (m, 2H), 7.47 – 7.37
(m, 5H), 7.37–7.27 (m, 3H), 7.08 (d, J = 8.9 Hz, 1H), 6.78 (dd, J = 9.0, 2.2 Hz, 2H),
6.61 (d, J = 2.3 Hz, 1H), 3.72 (d, J = 2.6 Hz, 5H), 3.67–3.50 (m, 2H), 3.23 (t, J = 6.7
13
Hz, 2H), 2.26 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 170.39, 154.47, 147.51,
139.71, 139.66, 137.06, 135.76, 135.39, 134.26, 132.16, 130.25, 129.52, 129.51,
129.11, 128.68, 128.28, 128.22, 128.19, 127.22, 126.52, 124.96, 115.63, 110.96,
110.70, 105.53, 101.18, 55.90, 42.44, 32.31, 31.48, 11.55. HRMS (AP-ESI) m/z Cacld
for C38H34N4O6S2 [M-H]- 872.1202. Found: 872.1201.
5.1.4.13
2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)-N-((4-morpholino-3-
nitrophenyl)sulfonyl)acetamide (8m)
Yellow solid. Yield: 32%, mp: 136–138 oC.1H NMR (400 MHz, CDCl3) δ 8.38 (d, J =
2.3 Hz, 1H), 8.13 (s, 1H), 8.00 (dt, J = 15.6, 7.8 Hz, 1H), 7.76 (t, J = 12.5 Hz, 2H),
7.69 (t, J = 10.6 Hz, 2H), 7.51 (t, J = 7.5 Hz, 2H), 7.42 (t, J = 8.2 Hz, 3H), 7.16–7.02
(m, 2H), 6.81 (dd, J = 8.9, 2.3 Hz, 1H), 6.67 (d, J = 2.2 Hz, 1H), 3.91–3.83 (m, 4H),
13
3.77 (s, 3H), 3.74 (s, 2H), 3.27–3.18 (m, 4H), 2.27 (s, 3H). C NMR (100 MHz,
DMSO-d6) δ 170.54, 154.50, 148.52, 139.74, 139.67, 138.18, 137.06, 135.76, 133.00,
132.14, 129.55, 128.96, 128.72, 128.32, 127.52, 127.25, 120.97, 111.10, 110.77,
105.53, 101.11, 66.12, 55.92, 50.82, 32.27, 11.55. HRMS (AP-ESI) m/z Cacld for
C34H32N4O7S [M-H]- 639.1919. Found: 639.1916.
5.1.5.(S)-2-(2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-indol-3-yl)acetamido)-3-phenyl-
N-(phenylsulfonyl)propenamide (9a)
The intermediate 7b (0.23 g, 0.76 mmol) dissolved in dry dichloromethane (25
mL) was treated with ethyldiisopropylamine (0.28ml, 1.4 mmol) under ice bath
condition, after stirred for 5 min, 2-(7-Aza-1H-benzotriazole-1-yl)-1,1,3,3
tetramethyluronium hexafluorophosphate (2.28 g, 6 mmol) was added and stirred for
o
30 min in 0 C, then the mixture of 4a (0.24 g, 0.7 mmol) and ethyldiisopropylamine
(0.14 ml, 0.7 mmol) in dichloromethane (15 mL) was added. After stirred overnight at

room temperature. The solvent was removed and the concentrated solution was
dissolved in EtOAc. The oganic phase was washed by 0.5 M citric acid, brine, dried,
and concentrated to yield yellow oil. Finally, the oil was purified by column
chromatography (DCM / MeOH = 70:1) to generate compound 8a as a white powder.
Yield: 13%, mp: 177–179 oC. 1H NMR (400 MHz, DMSO-d6) δ 12.47 (s, 1H), 8.23 (d,
J = 8.0 Hz, 1H), 7.94–7.86 (m, 2H), 7.72 (t, J = 7.4 Hz, 1H), 7.61 (t, J = 7.7 Hz, 2H),
7.37 (d, J = 8.0 Hz, 2H), 7.19 (d, J = 8.2 Hz, 2H), 7.12 (s, 5H), 7.02 (d, J = 2.4 Hz,
1H), 6.84 (d, J = 8.8 Hz, 1H), 6.63 (dd, J = 8.8, 2.4 Hz, 1H), 4.62–4.49 (m, 1H), 3.73
(s, 3H), 3.47 (q, J = 15.3 Hz, 2H), 2.90 (dd, J = 13.6, 4.4 Hz, 1H), 2.68 (dd, J = 13.7,
9.7 Hz, 1H), 2.41 (s, 3H), 2.01 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ 171.16,
154.30, 139.58, 137.42, 137.19, 135.34, 135.25, 134.20, 132.24, 130.56, 129.62,
128.84, 128.47, 127.91, 127.70, 126.89, 110.99, 110.39, 106.82, 101.19, 55.93, 54.86,
36.94, 31.61, 21.17, 11.33. HRMS (AP-ESI) m/z Cacld for C34H33N3O5S [M-H]-
594.2068. Found: 594.2072.
Compounds 9b–9l were synthesized following the procedure described above.
5.1.5.1
(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(5-methoxy-2-methyl-1-(p-tolyl)-1H-i
ndol-3-yl)acetamido)-3-(4-((4-nitrobenzyl)oxy)phenyl)propanamide (9b)
1
Gray solid. Yield: 8%, mp: 148–150 oC H NMR (400 MHz, DMSO-d6) δ 12.85 (s,
1H), 8.53 (d, J = 1.9 Hz, 1H), 8.25 (d, J = 8.6 Hz, 3H), 8.13 (dd, J = 8.5, 2.0 Hz, 1H),
7.98 (d, J = 8.5 Hz, 1H), 7.67 (d, J = 8.6 Hz, 2H), 7.34 (d, J = 8.0 Hz, 2H), 7.18 (d, J
= 8.1 Hz, 2H), 7.04 (dd, J = 5.2, 2.6 Hz, 3H), 6.84 (d, J = 8.8 Hz, 1H), 6.75 (d, J = 8.4
Hz, 2H), 6.64 (dd, J = 8.8, 2.2 Hz, 1H), 5.16 (s, 2H), 4.42 (dd, J = 12.7, 8.3 Hz, 1H),
3.73 (s, 3H), 3.48 (dd, J = 31.5, 15.2 Hz, 2H), 2.88 (dd, J = 12.9, 3.5 Hz, 1H), 2.68
(dd, J = 13.5, 9.4 Hz, 1H), 2.38 (s, 3H), 2.02 (d, J = 13.6 Hz, 3H).13C NMR (100
MHz, DMSO-d6) δ 171.96, 171.22, 157.02, 154.26, 147.60, 147.41, 145.54, 139.66,
137.38, 135.28, 133.43, 132.70, 132.21, 131.08, 130.76, 130.53, 129.60, 128.79,
128.51, 127.66, 125.51, 124.06, 114.73, 110.96, 110.40, 106.81, 101.17, 68.35, 55.88,
55.24, 35.98, 31.59, 21.13, 11.34. HRMS (AP-ESI) m/z Cacld for C41H36ClN5O10S

[M-H]- 824.1799. Found: 824.1801.
5.1.5.2(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetami
do)-3-phenyl-N-(phenylsulfonyl)propanamide (9c)
White solid. Yield: 17%, mp: 120–122 oC.1H NMR (400 MHz, DMSO-d6) δ 12.51 (s,
1H), 8.28 (d, J = 6.8 Hz, 1H), 7.89 (dd, J = 13.9, 8.0 Hz, 4H), 7.80–7.74 (m, 2H), 7.72
(t, J = 7.4 Hz, 1H), 7.61 (t, J = 7.7 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.41 (dd, J = 7.9,
6.2 Hz, 3H), 7.14 (s, 5H), 7.05 (d, J= 2.3 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.67 (dd,
J = 8.8, 2.4 Hz, 1H).4.49 (dd, J = 13.5, 8.4 Hz, 1H), 3.74 (s, 3H), 3.51 (dd, J = 26.9,
15.3 Hz, 2H), 2.94 (dd, J = 13.7, 4.9 Hz, 1H). 2.77 (dd, J = 13.7, 9.2 Hz, 1H), 2.11 (s,
13
3H). C NMR (100 MHz, DMSO-d6) δ 171.20, 171.11, 154.44, 139.71, 139.63,
139.57, 137.24, 137.22, 135.18, 134.18, 132.05, 129.63, 129.61, 129.54, 129.05,
128.48, 128.27, 128.25, 127.91, 127.24, 126.91, 111.16, 110.51, 107.34, 101.28,
55.94, 54.89, 36.95, 31.60, 11.46. HRMS (AP-ESI) m/z Cacld for C39H35N3O5S
[M-H]- 656.2225. Found: 656.2224.
5.1.5.3
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(
(4-methoxyphenyl)sulfonyl)-3-phenylpropanamide (9d)
White solid. Yield: 15%, mp: 210 – 212 oC.1H NMR (400 MHz, DMSO-d6) δ 12.34 (s,
1H),8.23(s,1H) 7.87 (d, J = 8.5 Hz, 2H), 7.83 (d, J = 8.7 Hz, 2H), 7.80–7.74 (m, 2H),
7.52 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.9 Hz, 3H), 7.19–7.07 (m, 7H), 7.06 (d, J = 2.4
Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.67 (dd, J = 8.8, 2.4 Hz, 1H),4.47 (s, 1H), 3.84 (s,
3H), 3.75 (s, 3H), 3.50 (q, J = 15.4 Hz, 2H), 2.90 (dd, J = 13.6, 4.4 Hz, 1H), 2.70 (dd,
J = 13.4, 9.6 Hz, 1H), 2.09 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.01, 163.51,
154.43, 151.58, 139.71, 139.56, 137.24, 135.18, 132.05, 130.34, 129.65, 129.55,
129.05, 128.43, 128.27, 127.24, 126.87, 121.19, 114.64, 111.16, 110.51, 107.36,
101.27, 56.22, 55.95, 54.87, 37.09, 31.64, 11.46.HRMS (AP-ESI) m/z Cacld for
C40H37N3O6S [M-H]- 686.2330. Found: 686.2329.

5.1.5.4
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(
(4-chlorophenyl)sulfonyl)-3-phenylpropanamide (9e)
White solid. Yield: 21%, mp: 126–128 oC.1H NMR (400 MHz, DMSO-d6) δ 12.60 (s,
1H), 8.30 (d, J = 7.6 Hz, 1H), 7.88 (t, J = 9.1 Hz, 4H), 7.77 (d, J = 7.4 Hz, 2H), 7.67
(d, J = 8.6 Hz, 2H), 7.52 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 7.6 Hz, 3H), 7.14 (s, 5H),
7.06 (d, J = 2.1 Hz, 1H), 6.96 (d, J = 8.8 Hz, 1H), 6.68 (dd, J = 8.8, 2.2 Hz, 1H), 4.50
(dd, J = 12.5, 9.0 Hz, 1H), 3.75 (s, 3H), 3.51 (q, J = 15.4 Hz, 2H), 2.92 (dd, J = 13.5,
13
4.4 Hz, 1H), 2.72 (dd, J = 13.8, 10.1 Hz, 1H), 2.10 (s, 3H). C NMR (100 MHz,
DMSO-d6) δ 171.32, 171.18, 154.45, 139.71, 139.58, 139.20, 138.35, 137.24, 137.15,
135.19, 132.08, 129.95, 129.78, 129.63, 129.53, 129.05, 128.48, 128.27, 127.23,
126.94, 111.16, 110.52, 107.34, 101.30, 55.94, 54.93, 36.92, 31.59, 11.46. HRMS
(AP-ESI) m/z Cacld for C39H34ClN3O5S [M-H]- 690.1835. Found: 690.1835.
5.1.5.5
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(
(4-nitrophenyl)sulfonyl)-3-phenylpropanamide (9f)
Brown solid. Yield: 31%, mp: 118–120oC.1H NMR (400 MHz, DMSO-d6) δ 8.36 (d, J
= 8.9 Hz, 3H), 8.12 (d, J = 8.9 Hz, 2H), 7.87 (d, J = 8.5 Hz, 2H), 7.77 (d, J = 7.2 Hz,
2H), 7.52 (t, J = 7.6 Hz, 2H), 7.41 (dd, J = 7.9, 6.2 Hz, 3H), 7.15 (d, J = 7.0 Hz, 5H),
7.02 (d, J = 2.4 Hz, 1H), 6.95 (d, J = 8.8 Hz, 1H), 6.65 (dd, J = 8.8, 2.4 Hz, 1H), 4.49
(dd, J = 12.3, 9.4 Hz, 1H), 3.72 (s, 3H), 3.55–3.46 (m, 2H), 2.94 (dd, J = 13.6, 4.6 Hz,
1H), 2.74 (dd, J = 13.6, 9.7 Hz, 1H), 2.10 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ
171.75, 171.26, 154.39, 150.66, 144.87, 139.72, 139.57, 137.23, 137.16, 135.16,
132.04, 129.63, 129.57, 129.54, 129.00, 128.52, 128.27, 128.22, 127.24, 126.98,
124.89, 111.13, 110.51, 107.35, 101.21, 55.88, 55.09, 36.82, 31.54, 11.45. HRMS
(AP-ESI) m/z Cacld for C39H34N4O7S [M-H]- 701.2075. Found: 701.2077.

5.1.5.6
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(
[1,1'-biphenyl]-4-ylsulfonyl)-3-phenylpropanamide (9g)
Gray solid. Yield: 11%, mp: 170–172 oC.1H NMR (400 MHz, DMSO-d6) δ 12.49 (s,
1H), 8.23 (d, J = 7.9 Hz, 1H), 7.97 (d, J = 8.5 Hz, 2H), 7.86 (dd, J = 14.9, 8.5 Hz, 4H),
7.79–7.70 (m, 4H), 7.59–7.33 (m, 8H), 7.14 (d, J = 9.2 Hz, 5H), 7.07 (d, J = 2.3 Hz,
1H), 6.94 (d, J = 8.8 Hz, 1H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 4.55 (td, J = 9.3, 4.7 Hz,
1H), 4.03 (q, J = 7.1 Hz, 1H), 3.74 (s, 3H), 3.58–3.44 (m, 2H), 2.95 (dd, J = 13.6, 4.5
Hz, 1H), 2.74 (dd, J = 13.6, 9.6 Hz, 1H), 2.09 (s, 3H). 13C NMR (100 MHz, DMSO-d6)
δ 171.28, 171.14, 154.43, 145.62, 139.69, 139.54, 138.81, 138.35, 137.23, 137.22,
135.17, 132.04, 129.65, 129.53, 129.19, 129.05, 128.66, 128.47, 128.24, 128.22,
127.76, 127.65, 127.23, 126.91, 111.17, 110.50, 107.36, 101.27, 55.92, 54.93, 37.02,
31.62, 11.46. HRMS (AP-ESI) m/z Cacld for C45H39N3O5S [M-H]- 539.1646.Found:
539.1647.
5.1.5.7 (S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)
acetamido)-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-phenylpropanamide (9h)
White solid. Yield: 13%, mp: 126–128 oC. 1H NMR (400 MHz, DMSO-d6) δ 8.54 (s,
1H), 8.37 (d, J = 7.3 Hz, 1H), 8.14 (d, J = 8.3 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.87
(d, J = 8.1 Hz, 2H), 7.77 (d, J = 7.5 Hz, 2H), 7.51 (t, J = 7.4 Hz, 2H), 7.41 (t, J = 8.1
Hz, 3H), 7.15 (s, 5H), 7.08 (s, 1H), 6.97 (d, J = 8.8 Hz, 1H), 6.68 (d, J = 8.7 Hz,
1H).3.74 (s, 3H), 3.57–3.43 (m, 2H), 2.91 (dd, J = 13.6, 4.3 Hz, 1H), 2.69 (dd, J =
13.2, 10.2 Hz, 1H), 2.09 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.83, 171.27,
154.42, 147.64, 139.71, 139.57, 139.46, 137.23, 137.04, 135.18, 133.49, 132.71,
132.04, 131.20, 129.63, 129.54, 129.00, 128.46, 128.27, 128.24, 127.23, 126.95,
125.51, 111.18, 110.53, 107.31, 101.21, 55.90, 55.07, 36.81, 31.54, 11.45. HRMS
(AP-ESI) m/z Cacld for C39H33ClN4O7S [M-H]- 735.1686. Found: 735.1687.
5.1.5.8 (S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)
acetamido)-3-(1H-indol-3-yl)-N-(phenylsulfonyl)propanamide (9i)

White solid. Yield: 24%, mp: 142–144 oC.1H NMR (400 MHz, DMSO-d6) δ 12.52 (s,
1H), 10.81 (d, J = 1.7 Hz, 1H), 8.23 (d, J = 7.3 Hz, 1H), 7.92–7.82 (m, 4H), 7.82–7.72
(m, 2H), 7.67 (d, J = 7.5 Hz, 1H), 7.63 (d, J = 7.9 Hz, 1H), 7.61–7.47 (m, 4H), 7.41
(dd, J = 11.8, 5.3 Hz, 3H), 7.32 (d, J = 8.1 Hz, 1H), 7.05 (dd, J = 11.2, 4.0 Hz, 1H),
7.02 (dd, J = 4.9, 2.3 Hz, 2H), 6.99–6.90 (m, 2H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 4.59
(dd, J = 13.5, 8.4 Hz, 1H), 3.70 (s, 3H), 3.50 (s, 2H), 3.05 (dd, J = 14.4, 5.0 Hz, 1H),
2.87 (dd, J = 14.5, 9.2 Hz, 1H), 2.04 (s, 3H).13C NMR (100 MHz, DMSO-d6) δ
171.76, 171.08, 154.45, 139.73, 139.55, 137.25, 136.53, 135.13, 134.09, 132.05,
129.54, 129.10, 128.25, 127.81, 127.57, 127.24, 124.43, 121.43, 119.05, 118.76,
111.76, 111.24, 110.55, 109.36, 107.42, 101.16, 55.89, 54.28, 11.37. HRMS (AP-ESI)
m/z Cacld for C41H36N4O5S [M-H]- 695.2334. Found: 695.2334.
5.1.5.9
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-3-(
1H-indol-3-yl)-N-((4-methoxyphenyl)sulfonyl)propanamide (9j)
Gray solid. Yield: 20%, mp: 158–161 oC.1H NMR (400 MHz, DMSO-d6) δ 12.35 (s,
1H), 10.81 (d, J = 1.7 Hz, 1H), 8.21 (d, J = 7.6 Hz, 1H), 7.86 (d, J = 8.5 Hz, 2H),
7.83–7.71 (m, 4H), 7.63 (d, J = 7.9 Hz, 1H), 7.52 (t, J = 7.6 Hz, 2H), 7.41 (t, J = 8.1
Hz, 3H), 7.32 (d, J = 8.1 Hz, 1H), 7.12–7.00 (m, 5H), 6.94 (dd, J = 7.9, 5.7 Hz, 2H),
6.66 (dd, J = 8.8, 2.4 Hz, 1H), 4.58 (dd, J = 13.7, 8.2 Hz, 1H), 3.82 (s, 3H), 3.71 (s,
3H), 3.51 (s, 2H), 3.04 (dd, J = 14.5, 5.1 Hz, 1H), 2.87 (dd, J = 14.5, 9.1 Hz, 1H),
2.05 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ 171.55, 171.02, 163.51, 154.43,
139.73, 139.54, 137.24, 136.50, 135.12, 132.04, 131.18, 130.25, 129.55, 129.09,
128.25, 127.58, 127.23, 124.41, 121.40, 119.05, 118.73, 114.63, 111.73, 111.24,
110.53, 109.39, 107.42, 101.15, 56.19, 55.89, 54.19, 31.62, 27.62, 11.36. HRMS
(AP-ESI) m/z Cacld for C42H38N4O6S [M-H]- 725.2439. Found: 725.2434.
5.1.5.10
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(
(3-chloro-4-nitrophenyl)sulfonyl)-3-(1H-indol-3-yl)propanamide (9k)

Brown solid. Yield: 17%, mp: 132–134 oC.1H NMR (400 MHz, DMSO-d6) δ 10.82 (d,
J = 1.6 Hz, 1H), 8.49 (d, J = 2.2 Hz, 1H), 8.32 (d, J = 6.9 Hz, 1H), 8.02 (dd, J = 8.5,
2.2 Hz, 1H), 7.85 (dd, J = 8.5, 7.1 Hz, 3H), 7.80–7.72 (m, 2H), 7.61 – 7.46 (m, 3H),
7.45–7.36 (m, 3H), 7.31 (d, J = 8.1 Hz, 1H), 7.13–7.00 (m, 3H), 6.94 (dd, J = 15.9,
8.0 Hz, 2H), 6.66 (dd, J = 8.8, 2.4 Hz, 1H), 4.56 (dd, J = 14.3, 7.2 Hz, 1H), 3.71 (s,
3H), 3.54 (q, J = 3.52 Hz, 2H), 3.10 (dd, J = 14.5, 5.6 Hz, 1H), 2.93 (dd, J = 14.5, 8.7
13
Hz, 1H), 2.08 (s, 3H). C NMR (100 MHz, DMSO-d6) δ 172.44, 171.20, 154.41,
147.46, 139.74, 139.56, 137.24, 136.43, 135.12, 133.40, 132.42, 132.03, 131.13,
129.55, 129.02, 128.26, 127.45, 127.24, 125.45, 124.52, 121.39, 118.87, 118.72,
111.75, 111.25, 110.56, 109.09, 107.37, 101.09, 55.85, 54.51, 31.51, 27.30, 11.35.
HRMS (AP-ESI) m/z Cacld for C41H34ClN5O7S [M-H]- 774.1795. Found: 774.1798.
5.1.5.11
(S)-2-(2-(1-([1,1'-biphenyl]-4-yl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-N-(
(4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((4-nitrobenzyl)oxy)phenyl)propanamide (9l)
Gray solid. Yield: 7%, mp: 204–205 oC 1H NMR (400 MHz, DMSO-d6) δ 13.34 (s,
1H), 8.53 (d, J = 2.1 Hz, 1H), 8.23 (t, J = 14.4 Hz, 3H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H),
7.98 (d, J = 8.5 Hz, 1H), 7.82 (d, J = 8.5 Hz, 2H), 7.76–7.69 (m, 2H), 7.66 (d, J = 8.8
Hz, 2H), 7.50 (dd, J = 16.2, 8.9 Hz, 2H), 7.44–7.37 (m, 3H), 7.08 (d, J = 2.4 Hz, 1H),
7.04 (d, J = 8.5 Hz, 2H), 6.96 (d, J = 8.8 Hz, 1H), 6.76 (d, J = 8.6 Hz, 2H), 6.68 (dd, J
= 8.8, 2.4 Hz, 1H), 5.24 (s, 2H), 4.43 (dd, J = 13.1, 8.4 Hz, 1H), 3.74 (s, 3H), 3.51 (dd,
J = 32.6, 15.3 Hz, 3H), 2.94–2.84 (m, 1H), 2.69 (dd, J = 13.7, 9.4 Hz, 1H), 2.09 (s,
13
3H). C NMR (100 MHz, DMSO-d6) δ 172.02, 171.15, 157.03, 154.42, 147.61,
147.39, 145.51, 139.65, 139.54, 137.21, 135.20, 133.42, 132.70, 132.06, 130.76,
129.64, 129.49, 129.01, 128.49, 128.23, 127.17, 125.50, 124.04, 114.75, 111.14,
110.52, 107.32, 101.30, 68.37, 55.92, 55.24, 36.00, 31.62, 11.45. HRMS (AP-ESI)
m/z Cacld for C46H38ClN5O10S [M-H]- 886.1955. Found: 886.1959.
5.2. In vitro binding assay for Bcl-2 proteins
Fluorescence polarization assay was used to test the activities to Bcl-2 family

proteins. A Bid-BH3 peptide was marked with 7692 Y. Wan et al. / Bioorg. Med.
Chem. 23 (2015) 7685–7693 5-carboxyfluorescein succinimidyl ester (FAM) as the
fluorescence labeled peptide (5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG). A
large number of experiments determined that the labeled Bid-BH3 peptide can bind to
Bcl-2 protein and the Kd value of it is 58 nM. For the competitive binding experiments,
these tested compounds and Bcl-2 protein were pre- cultured in the assay buffer (PBS
assay solution) for 30 min at room temperature. After the 5-FAM-Bid-BH3 peptide
was added and incubated for 20 min. The final volume of the solution was 200μM.
Finally, the total solutions were moved into black, Corning 384-well, flat-bottom
plates (Corning Inc.) with three wells per sample and 60 mL per well.
Under the condition of an excitation wavelength (485 nm) and an emission
wavelength (535 nm), the polarization values (milipolarization units) were monitored
by the Tecan GENios-Pro Injector Reader (Tecan Group Ltd). In the binding
experiments, the indole based derivatives were prepared in dimethylsulfoxide (DMSO)
at seven concentrations (1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 50 μM, 100 μM). The
final concentrations of 5-FAM-Bid-BH3 peptide and Bcl-2 protein were 10 nM and
290 nM, respectively.
The FP-based binding assay for Mcl-1 and Bcl-XL was parallel to the assay for
Bcl-2 except that the final concentration of Mcl-1 was 105 nM and of Bcl-XL was 125
nM. All the FP-based assays were tested in triplicate and the Ki value of all
compounds were calculated by the equation developed by Wang’s team.
5.3. MTT assay
The three cancer cells (MDA-MB-231, PC-3 and K562 cells were respectively
grown in RPMI 1640 medium containing 10% FBS at 37 oC in 5% CO2 humidified
incubator. Cell proliferation was determined by the MTT (3-[4,5-dimethyl-2-thiazolyl]
-2,5-diphenyl-2H-tetrazolium bromide) assay. Briefly, cells were plated in a 96-well
plate at 4000 cells per well, cultured for 8 h in complete growth medium, then treated
with serial dilutions of the compounds for 48 h. After the last 4 h, 10 μL of 0.5% MTT
solution was added to each well. After further incubation, formazan formed and

extracted by adding 200 μL of DMSO. After mixing for 15 min, optical density was
read with a microtiter-plate reader at 570 nm and the IC50 values were calculated
according to the inhibitory ratios.
5.4. Docking study
By Surflex-Dock program in Sybyl-X2.1, the molecular of 9c docking was performed.
The Gasteiger-Hückel charges were added to the structure of compound 9c and other
parameters were set at default values. The Bcl-2 protein was down loaded from the
Protein Data Bank (PDB code: 2O2F).
Acknowledgments
Thanks for the FP-based binding assay for Bcl-2 proteins and the help in docking
studies by Prof. Renxiao Wang from Shanghai Institute of Organic Chemistry in
China. This work was sponsored by National Natural Science Foundation of China
(Grant No. 21672127) and The Science and Technology Major Project of Shandong
Province (Major Key Technology, No, 2015ZDJS04001).
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