Design, synthesis and preliminary bioactivity studies of indomethacin derivatives as Bcl-2/Mcl-1 dual inhibitors

Article abstract of DOI:10.1016/j.bmc.2019.05.003

Bcl-2 family proteins, which divides into pro-apoptosis proteins and anti-apoptosis proteins, are involved in cell apoptosis progression. As numerous studies illustrated, targeting Bcl-2 family proteins is more and more attractive and practicable to cancer treatment. In this work, we designed and synthesized a series of indomethacin derivatives as new inhibitors for Bcl-2 family proteins. Our results of binding assay to Bcl-2 proteins, MTT assay and apoptotic assay indicated that some compounds had potent binding affinity to Bcl-2/Mcl-1 but not Bcl-X_L. Furthermore, compound 8j showed improved anti-proliferative activity than known Bcl-2 inhibitor WL-276.

Full text of DOI:10.1016/j.bmc.2019.05.003

Accepted Manuscript
Design, synthesis and preliminary bioactivity studies of indomethacin deriva-
tives as Bcl-2/Mcl-1 dual inhibitors
Chen Chen, Yiming Nie, Guangsen Xu, Xinying Yang, Hao Fang, Xuben Hou
PII:
S0968-0896(18)32172-2
https://doi.org/10.1016/j.bmc.2019.05.003
BMC 14890
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 January 2019
22 April 2019
4 May 2019
Please cite this article as: Chen, C., Nie, Y., Xu, G., Yang, X., Fang, H., Hou, X., Design, synthesis and preliminary
bioactivity studies of indomethacin derivatives as Bcl-2/Mcl-1 dual inhibitors, Bioorganic & Medicinal
Chemistry (2019), doi: https://doi.org/10.1016/j.bmc.2019.05.003
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Design, synthesis and preliminary bioactivity studies of indomethacin
derivatives as Bcl-2/Mcl-1 dual inhibitors
Chen Chen, Yiming Nie, Guangsen Xu, Xinying Yang, Hao Fang, Xuben Hou*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education),
School of Pharmaceutical Science, Shandong University, 44 West Wenhua Road, Jinan, Shandong
250012, P.R. China
Abstract
Bcl-2 family proteins, which divides into pro-apoptosis proteins and anti-apoptosis proteins, are
involved in cell apoptosis progression. As numerous studies illustrated, targeting Bcl-2 family
proteins is more and more attractive and practicable to cancer treatment. In this work, we designed
and synthesized a series of indomethacin derivatives as new inhibitors for Bcl-2 family proteins.
Our results of binding assay to Bcl-2 proteins, MTT assay and apoptotic assay indicated that some
compounds had potent binding affinity to Bcl-2/Mcl-1 but not Bcl-X . Furthermore, compound 8j
L
showed improved anti-proliferative activity than known Bcl-2 inhibitor WL-276.
Key words: apoptosis, anticancer, Bcl-2/Mcl-1 dual inhibitor, indomethacin derivatives.
1. Introduction
Cell apoptosis is a process of programmed cell death remove the damaged or unwanted cells,
which is strictly controlled by multi-genes. It should be mentioned that one of the causes of
tumorigenesis and tumor metastasis is insufficient apoptosis.1,2 Nowadays, regulating cell apoptosis
3
has become a promising strategy for treatment of cancer. Bcl-2 family of proteins are critical
regulators of cell apoptosis, which divides into anti-apoptosis proteins and pro-apoptosis proteins.
Anti-apoptosis proteins, including Bcl-2, Bcl-X , Bcl-W, Mcl-1, and A1, share the BH 1-4 domains.
L
On the other hand, pro-apoptosis proteins can be subdivided into BH3-only proteins and BH1-3
proteins. BH3-only proteins, whose BH3 region is the only homology, include Bad, Bim, Bid, Puma,
Noxa, Bmf, Hrk/DP5, Bik/Blk, Beclin-1 and Mule. BH1-3 proteins, which are multi-region proteins,
include Bax, Bak and Bok.4,5 In most cancer cells, the reason why there is resistance to apoptosis is
the high expression of anti-apoptosis Bcl-2 proteins.2,6 Therefore, the inhibition of anti-apoptosis
Bcl-2 proteins expression is able to overcome the drug resistance to conventional anticancer agents.
Heretofore, varieties of small molecule inhibitors have been reported for Bcl-2 family proteins
(
Fig. 1). For example, WL-276 is a Bcl-2 protein inhibitor, which can overcome the drug resistance
7
due to its pan-inhibition of Bcl-2 family protein. Obatoclax (GX015-070, pan Bcl-2 family
inhibitor) is on phase II clinical trials for previously untreated Acute Myeloid Leukemia (AML).8
Navitoclax (ABT-263) is a dual Bcl-X
L
/Bcl-2 inhibitor which was banned because of transient
thrombocytopenia with the inhibition of Bcl-X
L
, despite the promising phase I/II clinical trial data
for various cancers.3,9 Based on Navitoclax, Venetoclax (ABT-199, Bcl-2-selective inhibitor),
which has been approved by FDA for treatment of chronic lymphocytic leukemia (CLL) who harbor
a 17p chromosomal deletion, was designed to avoid the side effects.10–12 Due to the side effects of
1
/ 27

Bcl-X
L
inhibition and the up-regulation of Mcl-1 and Bcl-X , the drug resistance to ABT-199
L
13
emerged. Therefore, developing a dual inhibitor of Bcl-2 and Mcl-1 is fundamental.
Screening of our “in-house” compound library lead to the identification of compound 4a (Fig. 2),
which shows 94% inhibition against Bcl-2 protein at the concentration of 100µM. Target compounds
were designed according to our previous study on the WL-276 derivatives. According previous SAR
results, we found that the benzyl group is essential for the inhibitory activities of WL-276 derivatives.
1
4–16
Thus, we designed new indomethacin derivatives by introducing different side chains (R
1
) and
different substituted benzene sulfonamide (R ) (Fig. 2). In this paper, we will describe the synthesis
2
and the preliminary biological evaluations of these new compounds.
Fig. 1. Representative small-molecule inhibitors of anti-apoptotic Bcl-2 proteins.
9
4% inhibition against Bcl-2
protein at 100µM
The scaffold of the
indomethacin derivatives
Fig. 2. The structure of lead compound 4a and newly designed indomethacin derivatives.
2.Chemistry
The synthesis of scaffolds 4 and 8 are listed in scheme 1 and 2.15,17 Briefly, the condensation
reaction of N-Boc-amino acid 1a-1i and benzene sulfonamide derivatives 2a-2c was performed to
obtain the intermediates 3a-3k, then the Boc group was deprotected in acid condition. Finally
2
/ 27

compounds 4a-4l were gained through coupling reaction. The intermediates 6a-6n were prepared
by Williams reaction. Compounds 8a-8q were obtained by the same method as what 4a-4l were
prepared.
1
a-1i
2a-2c
3a-3k
4a-4l
Scheme 1. Synthesis of target compounds 4a-4l. Reagents and conditions: (a) benzene sulfonamide derivatives,
isobutyl chloroformate, NMM, NaH, 0℃ to rt, overnight, ethyl acetate saturated with HCl, rt, overnight; (b)
2 2
Indomethacin, TBTU, DIEA, CH Cl , 0℃to rt, 8 h.
8
a-8q
5
6a-6n
2a-2b
7a-7q
Scheme 2. Synthesis of target compounds 8a-8q. Reagents and conditions: (a) benzene sulfonamide derivatives,
isobutyl chloroformate, NMM, NaH, 0℃ to rt, overnight, ethyl acetate saturated with HCl, rt, overnight; (b)
Indomethacin, TBTU, DIEA, CH
2
Cl
2
, 0℃to rt, 8 h. (c) Boc-Tyrosine, Iodide or benzyl bromides, K
2
CO , DMF, 90℃,
3
4
h. NaOH(aq) rt ,6h.
3
.Results and discussion
We synthesized twenty-nine new indomethacin derivatives. The fluorescence polarization assay
FPA) was performed to evaluate the binding affinities of these target compounds to Bcl-2 protein.
Results were listed in Table 1 as K values.
For compounds 4a-4l, most of them exhibited low binding affinity or even no activity, but
surprisingly, compounds 4k (K =0.59±0.14µM) showed good binding affinity. This result suggested
that the binding affinity gets better when the benzyl group was introduced into R . In order to obtain
more potent compounds, benzyl derivatives were introduced into R to form compounds 8a-8q.
(
i
i
1
1
Compared to 8a-8d, compounds 8e-8q, which contained two benzyl group, showed more favorable
inhibitory activity and even 2-3 times better than WL-276 and 5-6 times better than UMI-77. For
the substitution in the benzyl moiety (R ), the meta-substitution showed more potent than para- and
3
ortho-substitution (e.g. 8l vs 8m vs 8n). In addition, the introduction of electron withdrawing group
or electron donating group did not show obvious impact on the inhibitory activity (e.g. 8h vs 8m).
As for the impact of the substitution in benzene sulfonamide moiety (R
substituted exerted higher potency than 4-Br substituted (e.g. 8o vs 8p).
2
), the 3-NO -4-Cl
2
In order to figure out the way that these indomethacin derivatives interact with Bcl-2 protein and,
we performed a docking study of 8h(K =0.43±0.03μM) to Bcl-2 protein (PDB code: 4MAN) by
Surflex-Dock software (Fig. 3A). According to our docking result of compound 8h (Fig. 3A), the
-chlorobenzene group of 8h occupied the p2 pocket in Bcl-2 protein, and the 3-methyl-benzyloxy
i
4
3
/ 27

group of 8h occupied the p3 pocket. As the docking result indicates, compound 8h could bind
properly in the active site of Bcl-2 protein (Fig. 3B). The sulfonyl group of compound 8h could
form a hydrogen bond with Arg140 and Arg143 and the nitro-group could form a hydrogen bond
with Gly142. The 3-methyl-benzyl group could interact with Tyr199.
Furthermore, we choose five compounds to evaluate their binding affinities on Bcl-X
, aiming to confirm if these compounds could also bind to other anti-apoptotic Bcl-2 family
proteins. As the result depicts in Table 2, the five compounds had similar binding affinities on Mcl-
as on Bcl-2, but not on Bcl-X . It is promising to avoid the transient thrombocytopenia which
resulted by the inhibition of Bcl-X . Based on these data, these compounds can serve as new Bcl-
/Mcl-1 dual inhibitors.
Compounds 8h, 8i, 8j, 8k, 8l and 8m were selected to evaluate their anti-proliferative activities
L
and Mcl-
1
1
L
L
2
in vitro because of their good inhibition on both Bcl-2 and Mcl-1. Jurkat (Acute T-cell leukemia cell)
and K562 (chronic myelogenous leukemia cell), which belong to leukemia cancer cell line, showed
high levels of Bcl-2 and Mcl-1 expression and PC-3 (prostatic cancer cell), which belongs to prostate
cancer cell line, showed high level of Mcl-1 expression but low level of Bcl-2 expression.19–22 Then
Jurkat, K562 and PC-3 were chosen to be evaluated by MTT assay. As the MTT assay results (Table
3) illustrate, compared to WL-276, the anti-proliferative activities to the three tumor cell lines of the
most compounds in test are improved. Compound 8j displayed most potent anti-proliferative
activities in PC-3, Jurkat and K562 cell lines. Especially, the anti-proliferative activities to Jurkat
cell lines is 2-fold better than WL-276. As shown in Table 3 and Figure 4, the most active
compound 8j (Bcl-2 K
i
=0.44±0.02µM and Mcl-1 K =0.44±0.03µM), could significantly inhibit the
i
proliferation and induce apoptosis of PC-3, Jurkat and K562. The good anti-tumor activity of 8j may
due to the high expression of Bcl-2 and Mcl-1 protein in these cell lines.
To evaluate the ability to induce apoptosis in Jurkat cell line of compound 8j, annexin-V and
propidium iodide (PI) double staining was performed by flow cytometry (Fig.4). As the depicted, it
is dose-dependent for compound 8j to induce apoptosis. Besides, treating Jurkat cell line for 20 µM
and 40 µM of 8j for 48 h leads 43.1% and 56.8% of cell apoptosis (early + late) respectively,
compared to 13.3% of cell apoptosis in DMSO control. Compound 8j showed better ability to induce
apoptosis in Jurkat cell line than WL-276, which leads 29.1% and 34.2% of cell apoptosis under 20
µM and 40 µM of WL-276 for 48 h.
Table 1
The structures and Bcl-2 inhibitory activities of 4a-4l, 8a-8q.
Compound
R
1
R
2
R
3
K
i
a(µM)
N.A.b
4a
H
3-NO
2
-4-Cl
—
4
/ 27

4
4
4
4
4
4
4
4
4
4
4
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
8
b
c
d
e
f
H
4-Br
—
—
—
—
—
—
—
—
—
—
—
CH
CH
N.A.
Me
i-Pr
s-Bu
i-Bu
3-NO
3-NO
3-NO
3-NO
3-NO
H
2
2
2
2
2
-4-Cl
-4-Cl
-4-Cl
-4-Cl
-4-Cl
N.A.
N.A.
4.9±0.4
＞5
g
h
i
CH
Bn
Bn
Bn
3
S-(CH
)
2 2
-
＞5
N.A.
4-Br
＞5
j
3-NO
3-NO
3-NO
4-Br
2
2
2
-4-Cl
-4-Cl
-4-Cl
＞5
k
l
4-Br-Bn-
0.59±0.14
＞5
Indolyl-3-CH -
2
a
b
c
d
e
f
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
3
-
-
＞5
3-NO
3-NO
3-NO
4-Br
2
2
2
-4-Cl
-4-Cl
-4-Cl
3
＞5
i-Pr-
n-Pr-
Bn-
＞5
1.3±0.10
0.53±0.10
0.52±0.05
0.56±0.06
0.43±0.03
0.48±0.04
0.44±0.02
0.45±0.01
0.47±0.04
0.44±0.06
0.67±0.08
0.63±0.15
0.51±0.07
0.56±0.01
1.5±0.25
2.90±0.10
3-NO
3-NO
3-NO
3-NO
3-NO
3-NO
3-NO
3-NO
3-NO
4-Br
2
2
2
2
2
2
2
2
2
-4-Cl
-4-Cl
-4-Cl
-4-Cl
-4-Cl
-4-Cl
-4-Cl
-4-Cl
-4-Cl
Bn-
g
h
i
4-CH
3-CH
3-CH
3
3
3
-Bn-
-Bn-
O-Bn-
j
4-Br-Bn-
2-Br-Bn-
4-CN-Bn-
3-CN-Bn-
2-CN-Bn-
k
l
m
n
o
p
q
4-NO
4-NO
2
-Bn-
-Bn-
3-NO
3-NO
2
2
-4-Cl
-4-Cl
2
Naph-2-CH -
2
WL-276
UMI-77
a
Each value was expressed with standard deviations and reproduced in three independent assays.
No activity.
b
5
/ 27

6
/ 27

Fig. 3. Docking results of 8h with Bcl-2 protein. (A) Docked compound 8h with Bcl-2 protein (PDB
code: 4MAN). (B) The interactions between compound 8h and Bcl-2 protein.
Table 2
The binding affinities of representative compounds against three Bcl-2 proteins.
a
a
a
Compound
Bcl-2 Ki (µM) Bcl-XL Ki (µM)
Mcl-1 Ki (µM)
8
8
8
8
8
h
j
0.43±0.03
0.44±0.02
0.45±0.01
0.47±0.04
0.44±0.06
1.50±0.25
2.90±0.10
N.A.b
N.A.b
N.A.b
N.A.b
0.49±0.08
0.44±0.03
0.52±0.11
0.53
k
l
m
N.A.b
0.48±0.11
1.10±0.02
0.18±0.11
WL-276
1.30±0.15
7.85±2.15
UMI-77
a
Each value was expressed with standard deviations and reproduced in three independent assays.
No activity.
b
Table 3
Antiproliferative activities of representative compounds.
Compound
IC50 (µM)a
PC-3
Jurkat
K562
8
8
8
8
8
8
h
i
28.58±4.16
31.70±4.08
25.79±0.58
36.08±3.18
39.31±0.69
38.23±0.36
39.62±1.60
30.32±8.34
35.92±0.13
24.69±0.76
33.98±1.74
83.48±0.92
55.25±3.39
31.84±4.98
38.82±0.53
36.18±1.99
35.90±1.69
41.46±2.87
49.25±5.78
51.62±1.31
42.26±6.70
j
k
l
m
WL-276
a
Inhibitory data are means of no fewer than three independent determinations and expressed with
standard deviations.
7
/ 27

Fig.4. Analysis of apoptosis induced by 8j in the Jurkat cell line.
4.Conclusion
In summary, we synthesized a series of new indomethacin derivatives as Bcl-2/Mcl-1 dual
inhibitors. Based on the result of binding assay, compounds 8h, 8i, 8j, 8l and 8m exhibited favorable
inhibitions on Bcl-2/Mcl-1 proteins and no inhibition on Bcl-X . Compared to WL-276, compound
j displayed a better antiproliferative activity. In the future, these new inhibitors can serve as lead
compound to develop more potent Bcl-2/Mcl-1 dual inhibitors.
L
8
5.Experimental Section
5.1. General chemistry information
All solvents and starting materials were purchased without further purification, only if otherwise
mentioned. Reactions were all detected by thin-layer chromatography (TLC) on 0.25 mm silica gel
plates (60 GF-254) and observed by the UV light (254 nM or 365 nM) or iodine vapor. Most target
compounds were purified by silica gel chromatography (200–300mesh) or recrystallization. Proton
1
13
NMR ( H-NMR) and carbon NMR ( C-NMR) spectrums were recorded by a Brucker DRX
spectrometer (400 MHz) in the indicated solvent, which using TMS as an internal standard. δ
(chemical shift) were given in parts per million and J were expressed in Hertz. High-resolution mass
spectral (HRMS) was conducted on an Agilent 6510 Quadrupole Time-of-Flight LC/MS deliver and
the data were showed as m/z. To measure the melting points, the RY-1 electrothermal melting point
apparatus was used and uncorrected.
5.1.1. 2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)acetamide hydrochloride (3a)
To a solution of (tert-Butoxycarbonyl)-glycine (1.1 g, 6.5mmol) in 30 mL anhydrous THF at 0℃,
8
/ 27

NMM (0.8 ml, 7.1 mmol) and isobutylchloroformate (0.9 ml, 7.1 mmol) were added. Then after
stirred for 1 h, the solution of 4-chloro-3-nitrobenzenesulfonamide (1.7 g, 7.1 mmol) in THF, which
was pretreated with NaH (0.65 g, 16.1 mmol) for 4 h at room temperature aforehand, was added in
drops. This reaction was recovered slowly to room temperature and stirred overnight. Then the
solvent was evaporated in vacuum. The resulting residue was neutralized by 1M HCl solution and
extracted with EtOAc, then the organic phase was washed by 0.5 M citric acid solution and brine.
After dried by MgSO and concentrated, use EtOAc which was saturated with HCl gas to dissolve
4
the obtained oil and stirred at room temperature overnight. Then the generated precipitate was
1
filtered and dried. white powder. Yield:13%, mp: 220-223℃. H NMR (400 MHz, DMSO-d
6
) δ 8.56
(d, J = 2.2 Hz, 1H), 8.27 – 8.17 (m, 4H), 8.07 (d, J = 8.5 Hz, 1H), 3.68 (t, J = 5.5 Hz, 2H).
Compounds 3b–3l were synthesized following the procedure described above.
5.1.1.1. 2-amino-N-((4-bromophenyl)sulfonyl)acetamide hydrochloride (3b) white solid. Yield:85%,
1
mp: 208-211℃. H NMR (400 MHz, DMSO-d
6
) δ 8.18 (s, 3H), 7.88 (s, 4H), 3.69 (s, 2H).
5.1.1.2.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)propanamide hydrochloride (3c) light
1
yellow solid. Yield:73%, mp: 218-220℃. H NMR (400 MHz, DMSO-d
.27 (s, 3H), 8.19 (dd, J = 8.5, 2.1 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 3.90 – 3.82 (m, 1H), 1.35 (d,
J = 7.1 Hz, 3H).
6
) δ 8.55 (d, J = 2.1 Hz, 1H),
8
5.1.1.3.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-methylbutanamide hydrochloride (3d)
1
white solid. Yield:46%, mp: 234-236℃. H NMR (400 MHz, DMSO-d
6
) δ 8.51 (d, J = 1.7 Hz, 1H),
8
.15 (dd, J = 8.5, 1.9 Hz, 1H), 8.11 – 8.01 (m, 3H), 7.99 (d, J = 8.5 Hz, 1H), 3.61 – 3.51 (m, 1H), 2.15
(
dq, J = 13.7, 6.6 Hz, 1H), 0.88 (d, J = 6.9 Hz, 3H), 0.82 (d, J = 6.9 Hz, 3H).
5.1.1.4.(2S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-methylpentanamide
hydrochloride
1
(
3e) white solid. Yield:33%, mp: 217-223℃. H NMR (400 MHz, DMSO-d
6
) δ 8.56 (d, J = 2.1 Hz,
1
5
H), 8.32 (d, J = 5.6 Hz, 3H), 8.20 (dd, J = 8.5, 2.2 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H), 3.75 (t, J =
.3 Hz, 1H), 1.94 – 1.82 (m, 1H), 1.34 (dtd, J = 14.7, 7.4, 4.2 Hz, 1H), 1.17 – 1.03 (m, 1H), 0.85 (d,
J = 6.9 Hz, 3H), 0.81 (t, J = 7.4 Hz, 3H).
5.1.1.5.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-4-methylpentanamide hydrochloride (3f)
1
white solid. Yield:44%, mp: 197-202℃. H NMR (400 MHz, DMSO-d
6
) δ 8.45 (d, J = 4 Hz, 1H),
8
1
.11 (dd, J = 8.4, 1.8 Hz, 1H), 7.96 – 7.94 (m, 4H), 3.58 (s, 1H), 1.64 (dt, J = 12.8, 6.4 Hz, 1H),
.60 – 1.52 (m, 1H), 1.45 (ddd, J = 13.8, 8.3, 6.0 Hz, 1H), 0.84 (d, J = 6.3 Hz, 6H).
5.1.1.6.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-4-(methylthio)butanamide hydrochloride
1
(
3g) white solid. Yield:77%, mp: 204-208℃. H NMR (400 MHz, DMSO-d
6
) δ 8.52 (d, J = 2.0 Hz,
1
2
H), 8.30 (s, 3H), 8.17 (dd, J = 8.5, 2.1 Hz, 1H), 8.02 (d, J = 8.5 Hz, 1H), 3.91 – 3.80 (m, 1H), 2.50 –
.43 (m, 1H), 2.38 (ddd, J = 13.5, 9.4, 5.7 Hz, 1H), 2.09 – 1.91 (m, 5H).
5.1.1.7.(S)-2-amino-3-phenyl-N-(phenylsulfonyl)propanamide hydrochloride (3h) white solid.
1
Yield:11%, mp: 214-217℃. H NMR (400 MHz, DMSO-d
6
) δ 13.10 (s, 1H), 8.39 (s, 3H), 7.92 (d, J
=
7.4 Hz, 2H), 7.77 (t, J = 7.4 Hz, 1H), 7.65 (t, J = 7.7 Hz, 2H), 7.21 (dt, J = 14.0, 6.8 Hz, 3H), 7.05 (d,
9
/ 27

J = 6.8 Hz, 2H), 4.11 (s, 1H), 3.12 – 2.95 (m, 2H).
5.1.1.8.(S)-2-amino-N-((4-bromophenyl)sulfonyl)-3-phenylpropanamide hydrochloride (3i) white
1
solid. Yield:94%, mp: 222-223℃. H NMR (400 MHz, DMSO-d
6
) δ 8.44 (s, 3H), 7.85 (q, J = 8.7
Hz, 4H), 7.21 (dt, J = 14.1, 6.8 Hz, 3H), 7.07 (d, J = 7.0 Hz, 2H), 4.14 (s, 1H), 3.07 (qd, J = 14.0,
6.4 Hz, 2H).
5.1.1.9.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-phenylpropanamide hydrochloride (3j)
1
light yellow solid. Yield:97%, mp: 239-240℃. H NMR (400 MHz, DMSO-d
6
) δ 8.47 (d, J = 1.9
Hz, 1H), 8.16 (s, 3H), 8.11 (dd, J = 8.5, 2.0 Hz, 1H), 8.00 (d, J = 8.5 Hz, 1H), 7.19 (q, J = 6.0 Hz,
3H), 7.08 (d, J = 6.4 Hz, 2H), 4.00 – 3.91 (m, 1H), 3.03 (qd, J = 14.1, 6.4 Hz, 2H).
5.1.1.10.(S)-2-amino-3-(4-bromophenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)propanamide
1
hydrochloride (3k) white solid. Yield:70%, mp: 212-217℃. H NMR (400 MHz, DMSO-d
6
) δ 8.51
(d, J = 2.0 Hz, 1H), 8.28 (s, 3H), 8.15 (dd, J = 8.5, 2.1 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.38 (d, J
=
8.3 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H), 4.08 – 4.00 (m, 1H), 3.05 (ddd, J = 42.9, 14.1, 6.2 Hz, 2H).
5.1.1.11(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(1H-indol-3-yl)propanamide
1
hydrochloride (3l) brown solid. Yield:33%, mp: 235-237℃. H NMR (400 MHz, DMSO-d
6
) δ 11.03
(s, 1H), 8.49 (s, 1H), 8.17 (s, 3H), 8.08 (dd, J = 8.5, 2.0 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.58 (d,
J = 7.9 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.13 (d, J = 2.0 Hz, 1H), 7.07 (t, J = 7.5 Hz, 1H), 6.93 (t,
J = 7.5 Hz, 1H), 4.00 (s, 1H), 3.26 (dd, J = 14.7, 5.6 Hz, 1H), 3.11 (dd, J = 14.8, 7.6 Hz, 1H).
5
2
.1.2. N-(2-((4-chloro-3-nitrophenyl) sulfonamido)-2-oxoethyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-
-methyl-1H-indol-3-yl) acetamide (4a)
To the solution of intermediate 3a (0.2 g, 0.51 mmol) in dry dichloromethane (25 mL),
ethyldiisopropylamine (0.26 ml, 1.5 mmol) was added in ice bath. After stirred for 10 min, O-
Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU, 0.22 g, 0.67 mmol)
(
was added and stirred for 30 min in 0℃, then the indomethacin (0.22 g, 0.6 mmol) was added and
stirred overnight at room temperature. The solvent was removed in vacuum and then dissolved in
EtOAc. 0.5 M citric acid and brine were used to wash the organic phase, then it was concentrated
to yield yellow oil. Purification by column chromatography (PE/EA = 10:7) to generate compound
1
4
a, a white powder. Yield:35%, mp: 156-159℃. H NMR (400 MHz, DMSO-d
6
) δ 8.54 (d, J = 2.2
Hz, 1H), 8.26 (t, J = 5.8 Hz, 1H), 8.17 (dd, J = 8.5, 2.3 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.66 (dd, J =
8
.6 Hz, 4H), 7.07 (d, J = 2.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.70 (dd, J = 9.0, 2.6 Hz, 1H), 3.83 (d, J
13
=
5.7 Hz, 2H), 3.75 (s, 3H), 3.54 (s, 2H), 2.19 (s, 3H). C NMR (101 MHz, DMSO) δ 170.80, 170.51,
1
1
1
69.47, 168.29, 156.00, 147.68, 139.72, 138.04, 135.68, 134.66, 133.44, 132.75, 131.64, 131.18, 131.09,
30.69, 129.49, 125.45, 114.95, 114.33, 111.95, 102.19, 60.22, 55.81, 49.07, 42.87, 31.15, 21.23, 14.56,
-
3.79. HRMS (AP-ESI) m/z Calcd for C27
H
2
22Cl N
4
O
8
S [M-H] 631.04572. Found: 631.0456.
Compounds 4a–4l were synthesized following the procedure described above.
5.1.2.1.N-(2-((4-bromophenyl)sulfonamido)-2-oxoethyl)-2-(1-(4-chlorobenzoyl)-5-methoxy-2-
1
methyl-1H-indol-3-yl) acetamide (4b) yellow solid. Yield:57%, mp: 216-219℃. H NMR (400 MHz,
DMSO-d ) δ 12.38 (s, 1H), 8.21 (t, J = 5.7 Hz, 1H), 7.83 (s, 4H), 7.66 (q, J = 8.6 Hz, 4H), 7.07 (d,
6
1
0 / 27

J = 2.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.70 (dd, J = 9.0, 2.4 Hz, 1H), 3.79 (d, J = 5.8 Hz, 2H),
3
1
.75 (s, 3H), 3.53 (s, 2H), 2.18 (s, 3H). 13C NMR (100 MHz, DMSO) δ 170.48, 168.90, 168.29,
56.00, 138.91, 138.03, 135.68, 134.66, 132.74, 131.66, 131.19, 130.68, 129.97, 129.49, 128.26,
114.96, 114.34, 111.96, 102.19, 55.83, 42.65, 31.19, 13.83. HRMS (AP-ESI) m/z Calcd for
-
C
27
H
23BrClN
3
O
6
S [M-H] 632.0082. Found: 632.0074.
5
1
.1.2.2.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1
H-indol-3-yl) acetamido) propenamide (4c) light yellow solid. Yield:27%, mp: 188-191℃. H
) δ 12.75 (s, 1H), 8.61 – 8.49 (m, 2H), 8.13 (dd, J = 8.5, 2.1 Hz, 1H),
.94 (d, J = 8.5 Hz, 1H), 7.75 – 7.61 (m, 4H), 7.05 (d, J = 2.3 Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H),
.69 (dd, J = 9.0, 2.4 Hz, 1H), 4.23 (p, J = 7.0 Hz, 1H), 3.74 (s, 3H), 3.51 (q, J = 15.4 Hz, 2H), 2.18
NMR (400 MHz, DMSO-d
6
7
6
13
(
s, 3H), 1.21 (d, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSO-d
6
) δ 172.90, 170.07, 168.25, 155.96,
1
1
47.58, 139.57, 138.05, 135.54, 134.65, 133.40, 132.54, 131.60, 131.21, 131.10, 130.61, 129.47,
25.40, 114.94, 114.58, 111.91, 102.11, 55.77, 49.41, 30.93, 17.27, 13.77. HRMS (AP-ESI) m/z
-
Calcd for C28
H
2
24Cl N
4
O
8
S [M-H] 645.0614. Found:645.0611.
5
1
.1.2.3.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1
H-indol-3-yl)acetamido)-3-methylbutanamide (4d) yellow solid. Yield:46%, mp: 154-156℃. H
) δ 12.80 (s, 1H), 8.55 (d, J = 2.2 Hz, 1H), 8.41 (d, J = 7.9 Hz, 1H), 8.15
dd, J = 8.5, 2.2 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.72 – 7.60 (m, 4H), 7.10 (d, J = 2.5 Hz, 1H), 6.92 (d,
J = 8.9 Hz, 1H), 6.68 (dd, J = 9.0, 2.5 Hz, 1H), 4.18 (t, J = 7.1 Hz, 1H), 3.67 – 3.50 (m, 2H), 2.20 (s, 3H),
NMR (400 MHz, DMSO-d
6
(
1
3
2
1
1
.00 (d, J = 7.6 Hz, 1H), 0.79 (d, J = 6.8 Hz, 6H). C NMR (100 MHz, DMSO-d ) δ 172.04, 170.57,
6
68.26, 155.98, 147.61, 139.62, 138.04, 135.48, 134.68, 133.39, 132.61, 131.58, 131.20, 131.09, 130.59,
29.49, 125.47, 114.94, 112.06, 101.99, 60.21, 58.61, 55.78, 30.84, 30.45, 19.36, 18.24, 13.72. HRMS
-
(
AP-ESI) m/z Calcd for C30
H
2
28Cl N
4
O
8
S [M-H] 673.0927. Found:673.0933.
5
1
.1.2.4.(2S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1
H-indol-3-yl)acetamido)-3-methylpentanamide (4e) yellow solid. Yield:10%, mp: 130-134℃. H
NMR (400 MHz, DMSO-d
dd, J = 8.5, 2.3 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.70 – 7.60 (m, 4H), 7.08 (d, J = 2.6 Hz, 1H), 6.91 (d,
J = 8.9 Hz, 1H), 6.68 (dd, J = 9.0, 2.6 Hz, 1H), 4.17 (t, J = 7.4 Hz, 1H), 3.73 (s, 3H), 3.66 – 3.45 (m, 2H),
.18 (s, 3H), 1.72 (d, J = 8.1 Hz, 1H), 1.28 (s, 1H), 1.07 (dq, J = 14.8, 7.7 Hz, 1H), 0.73 (dt, J = 7.6, 4.0
6
) δ 12.83 (s, 1H), 8.52 (d, J = 2.3 Hz, 1H), 8.41 (d, J = 7.7 Hz, 1H), 8.13
(
2
13
Hz, 6H). C NMR (100 MHz, DMSO-d
6
) δ 172.29, 170.50, 168.27, 155.99, 147.60, 138.02, 135.47,
1
5
34.70, 133.35, 132.59, 131.58, 131.18, 130.97, 130.59, 129.50, 125.41, 114.95, 114.91, 112.09, 101.98,
7.83, 55.78, 36.63, 31.43, 30.85, 24.69, 22.53, 15.59, 13.71, 11.14. HRMS (AP-ESI) m/z Calcd for
-
C
31
H
2
30Cl N
4
O
8
S [M-H] 687.1083. Found:687.1093.
5
1
.1.2.5.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
1
H-indol-3-yl)acetamido)-4-methylpentanamide (4f) white solid. Yield:13%, mp: 168-174℃. H
) δ 12.82 (s, 1H), 8.51 (d, J = 2.2 Hz, 1H), 8.46 (d, J = 7.2 Hz, 1H), 8.13
dd, J = 8.5, 2.3 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H), 7.66 (t, J = 6.3 Hz, 4H), 7.05 (d, J = 2.5 Hz, 1H), 6.93
NMR (400 MHz, DMSO-d
6
(
(
(
d, J = 9.0 Hz, 1H), 6.68 (dd, J = 9.0, 2.5 Hz, 1H), 4.26 (s, 1H), 3.73 (s, 3H), 3.59 – 3.44 (m, 2H), 2.17
s, 3H), 1.58 (p, J = 6.6 Hz, 1H), 1.41 (dqd, J = 13.7, 9.0, 5.2 Hz, 2H), 0.86 (d, J = 6.6 Hz, 3H), 0.77 (d,
13
J = 6.5 Hz, 3H). C NMR (100 MHz, DMSO-d
6
) δ172.99, 170.37, 168.27, 155.99, 147.63, 138.02,
1
1 / 27

1
1
35.48, 134.70, 133.40, 132.53, 131.59, 131.18, 131.02, 130.61, 129.49, 125.35, 114.94, 114.71, 112.05,
01.99, 55.77, 52.21, 30.95, 24.75, 23.36, 21.52, 13.72. HRMS (AP-ESI) m/z Calcd for
-
C
31
H
2
30Cl N
4
O
8
S [M-H] 687.1083. Found:687.1090.
5
1
.1.2.6.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-4-(methylthio)butanamide (4g) white solid. Yield:51%, mp: 184-186℃.
1H NMR (400 MHz, DMSO-d
) δ 12.73(s, 1H), 8.56 – 8.46 (m, 2H), 8.14 (dd, J = 8.5, 2.3 Hz, 1H),
.97 (d, J = 8.5 Hz, 1H), 7.71 – 7.61 (m, 4H), 7.07 (d, J = 2.6 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H),
.69 (dd, J = 9.0, 2.5 Hz, 1H), 4.30 (ddd, J = 9.6, 7.3, 4.3 Hz, 1H), 3.74 (s, 3H), 3.57 (dd, J = 15.3
6
7
6
Hz, 2H), 2.35 (dt, J = 8.7, 5.8 Hz, 2H), 2.18 (s, 3H), 1.95 (s, 3H), 1.74 (dtd, J = 14.2, 9.0, 5.6 Hz,
1
1
5
H). 13C NMR (100 MHz, DMSO-d
35.56, 134.67, 133.44, 132.64, 131.60, 131.16, 130.61, 129.50, 125.48, 114.99, 114.59, 112.04, 101.96,
6
) δ 171.90, 170.44, 168.27, 156.01, 147.62, 139.47, 138.04,
5.81, 53.02, 31.04, 30.84, 29.91, 15.00, 13.78. HRMS (AP-ESI) m/z Calcd for C30
H
28Cl
2
N
4
O
8
S [M-
2
-
H] 705.0647. Found:706.0655.
5.1.2.7.(S)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)-3-phenyl-N-
1
(
phenylsulfonyl)propanamide (4h) white solid. Yield:54%, mp: 193-195℃. H NMR (400 MHz,
DMSO-d
.11 (s, 5H), 7.03 (d, J = 2.4 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 9.0, 2.4 Hz, 1H), 4.50
td, J = 9.5, 4.4 Hz, 1H), 3.73 (s, 3H), 3.52 – 3.41 (m, 2H), 2.91 (dd, J = 13.6, 4.3 Hz, 1H), 2.67 (dd,
J = 13.6, 9.9 Hz, 1H), 2.03 (s, 3H). 13C NMR (100 MHz, DMSO-d
) δ 171.13, 170.10, 168.25,
56.01, 139.57, 137.99, 137.15, 135.47, 134.67, 134.18, 131.57, 131.25, 130.57, 129.59, 129.49,
6
) δ 12.53 (s, 1H), 8.33 (d, J = 7.2 Hz, 1H), 7.90 (d, J = 7.5 Hz, 2H), 7.77 – 7.55 (m, 7H),
7
(
6
1
1
28.43, 127.87, 126.88, 114.89, 114.49, 111.97, 102.16, 55.85, 54.87, 36.90, 30.97, 13.71. HRMS
-
(
AP-ESI) m/z Calcd for C34
H
30ClN
3
O
6
S [M-H] 642.1466. Found:642.1481.
5
3
.1.2.8.(S)-N-((4-bromophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-
1
-yl)acetamido)-3-phenylpropanamide (4i) white solid. Yield:50%, mp: 198-200℃. H NMR (400
MHz, DMSO-d
6
) δ 12.64 (s, 1H), 8.38 (d, J = 7.7 Hz, 1H), 7.81 (s, 4H), 7.63 (s, 4H), 7.12 (s, 5H),
.03 (d, J = 2.2 Hz, 1H), 6.93 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 9.0, 2.2 Hz, 1H), 4.49 (td, J = 9.1,
.7 Hz, 1H), 3.74 (s, 3H), 3.53 – 3.41 (m, 2H), 2.92 (dd, J = 13.5, 4.4 Hz, 1H), 2.70 (dd, J = 13.5,
7
4
9
1
13
.8 Hz, 1H), 2.05 (s, 3H). C NMR (100 MHz, DMSO-d
37.09, 135.50, 134.67, 132.69, 131.57, 131.23, 130.57, 129.93, 129.59, 129.50, 128.42, 126.91,
14.89, 114.48, 111.97, 102.16, 55.85, 54.92, 36.85, 30.95, 13.71. HRMS (AP-ESI) m/z Calcd for
6
). δ 171.29, 170.14, 168.26, 156.00, 138.00,
1
-
C
34
H
29BrClN
3
O S [M-H] 722.0553. Found:722.0550.
6
5
1
.1.2.9.(S)-N-((4-chloro-3-nitrophenyl) sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-phenylpropanamide (4j) light yellow solid. Yield:9%, mp: 196-200℃.
1H NMR (400 MHz, DMSO-d
.11 (dd, J = 8.5, 2.1 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.64 (s, 4H), 7.12 (s, 5H), 7.03 (d, J = 2.3
Hz, 1H), 6.92 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 9.0, 2.4 Hz, 1H), 4.53 – 4.43 (m, 1H), 3.73 (s, 3H),
.55 – 3.42 (m, 2H), 2.95 (dd, J = 13.6, 4.7 Hz, 1H), 2.74 (dd, J = 14.0, 9.0 Hz, 1H), 2.07 (s, 3H).
13C NMR (100 MHz, DMSO-d
). δ 172.03, 170.18, 168.25, 155.99, 147.54, 139.88, 138.02, 137.08,
35.52, 134.65, 133.34, 132.67, 131.57, 131.21, 130.91, 130.58, 129.61, 129.48, 128.37, 126.87, 125.43,
14.90, 114.50, 111.97, 102.10, 55.81, 55.16, 36.86, 30.94, 13.70. HRMS (AP-ESI) m/z Calcd for
) δ 12.94 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.44 (d, J = 7.0 Hz, 1H),
6
8
3
6
1
1
1
2 / 27

-
C
34
H
2
30Cl N
4
O S [M-H] 721.0927. Found:721.0926.
8
5.1.2.10.(S)-3-(4-bromophenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl)acetamido)propenamide (4k) light yellow solid. Yield:30%, mp:
1
1
37-141℃. H NMR (400 MHz, DMSO-d
6
) δ 12.68 (s, 1H), 8.54 (d, J = 2.2 Hz, 1H), 8.43 (d, J = 7.7
Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.64 (s, 4H), 7.27 (d, J = 7.9 Hz, 2H),
7
4
1
1
1
.08 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 2.5 Hz, 1H), 6.94 (d, J = 9.0 Hz, 1H), 6.70 (dd, J = 9.0, 2.5 Hz, 1H),
.47 (td, J = 8.7, 4.7 Hz, 1H), 3.74 (s, 3H), 2.94 (dd, J = 13.8, 4.5 Hz, 1H), 2.70 (dd, J = 13.7, 9.7 Hz,
13
H), 2.08 (s, 3H). C NMR (100 MHz, DMSO-d ). δ 171.60, 170.23, 168.24, 155.98, 147.62, 138.04,
6
36.52, 135.56, 134.67, 133.42, 132.66, 131.80, 131.57, 131.24, 131.18, 131.11, 130.60, 129.49, 125.48,
20.22, 114.90, 114.41, 111.83, 102.22, 55.84, 54.83, 36.13, 30.95, 19.11, 13.71. HRMS (AP-ESI) m/z
-
Calcd for C34
H
27BrCl
2
N
4
O
8
S [M-H] 801.0012. Found:801.0013.
5
1
1
.1.2.11.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(1H-indol-3-yl)propenamide (4l) light yellow solid. Yield:37%, mp:
1
40-144℃. H NMR (400 MHz, DMSO-d
6
) δ 12.95 (s, 1H), 10.84 (s, 1H), 8.51 – 8.44 (m, 2H), 8.00
(dd, J = 8.5, 2.1 Hz, 1H), 7.82 (d, J = 8.5 Hz, 1H), 7.68 – 7.60 (m, 5H), 7.57 (d, J = 7.9 Hz, 1H),
7
–
1
1
1
1
5
.30 (d, J = 8.1 Hz, 1H), 7.11 – 7.08 (m, 1H), 7.04 (t, J = 7.5 Hz, 1H), 6.99 (d, J = 2.3 Hz, 1H), 6.96
6.88 (m, 2H), 6.67 (dd, J = 9.0, 2.4 Hz, 1H), 4.55 (q, J = 7.3 Hz, 1H), 3.69 (s, 3H), 3.47 (d, J =
6.6 Hz, 3H), 3.38 (q, J = 7.0 Hz, 2H), 3.09 (dd, J = 14.5, 5.5 Hz, 1H), 2.91 (dd, J = 14.5, 8.8 Hz,
1
3
H), 2.05 (s, 3H), 1.09 (t, J = 7.0 Hz, 1H). C NMR (100 MHz, DMSO-d ) δ 172.34, 170.27, 168.23,
6
55.95, 147.41, 139.40, 138.01, 136.40, 135.48, 134.66, 133.37, 132.36, 131.58, 131.24, 131.16, 130.56,
29.48, 127.41, 125.43, 124.50, 121.38, 118.84, 118.70, 114.93, 114.57, 112.06, 111.75, 109.01, 101.92,
-
5.74, 54.52, 30.85, 27.26, 13.60. HRMS (AP-ESI) m/z Calcd for C36
H
2
29Cl N
5
O
8
S [M-H] 760.1036.
Found:760.1044.
5.1.3.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-methoxyphenyl)propanoic acid (6a)
L – tyrosine (2.8 g, 10 mmol) was dissolved in 50 mL DMF, then K CO (4.15 g, 30 mmol) and
2
3
iodomethane (1.37 mL, 22mmol) were added in a row. Stirred at room temperature till the reactant
was run out, detected by TLC. Then, 150 mLwater was added into the solution and extracted with
EtOAc. After dried by MgSO and concentrated, the obtained oil was dissolved in 30mL methanol
4
and NaOH(1.3mmol/mL) was added. After stirred for 5 h, the solution was neutralized by 1M HCl
solution and extracted with EtOAc. 0.5 M citric acid and brine were used to wash the organic phase,
and concentrated to colorless oil. Finally, purification by column chromatography (PE/EA = 10:3)
1
to generate compound 6a, colorless oil. Yield:13%. H NMR (400 MHz, Chloroform-d) δ 7.12 (d,
J = 8.2 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 4.68 – 4.49 (m, 1H), 3.81 (s, 3H), 3.11 (ddd, J = 34.8,
13.6, 5.3 Hz, 2H), 1.45 (s, 9H).
Compounds 6b–6n were synthesized following the procedure described above.
5.1.3.1.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-isopropoxyphenyl)propanoic acid (6b) white solid.
1
Yield:27%, mp: 120-124℃. H NMR (400 MHz, Chloroform-d) δ 7.10 (d, J = 8.0 Hz, 2H), 6.85 (d,
J = 8.4 Hz, 2H), 4.55 (tq, J = 12.0, 6.2 Hz, 2H), 3.10 (ddd, J = 37.7, 13.9, 5.6 Hz, 2H), 1.44 (s, 6H),
1.34 (d, J = 6.1 Hz, 9H).
1
3 / 27

5.1.3.2.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-propoxyphenyl)propanoic acid (6c) colorless oil.
1
Yield:60%. H NMR (400 MHz, Chloroform-d) δ 7.08 (d, J = 8.1 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H),
4.64 – 4.51 (m, 1H), 3.90 (t, J = 6.6 Hz, 2H), 3.24 – 2.93 (m, 2H), 1.79 (h, J = 7.1 Hz, 2H), 1.38 (d,
J = 36.4 Hz, 9H), 1.03 (t, J = 7.4 Hz, 3H).
5.1.3.3.(S)-3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (6d) white solid.
1
Yield:34%, mp: 110-113℃. H NMR (400 MHz, Chloroform-d) δ 10.69 (s, 1H), 7.34 (m, 5H), 7.09 (d,
J = 8.2 Hz, 2H), 6.90 (d, J = 8.1 Hz, 2H), 5.00 (s, 2H), 4.58 (q, J = 6.6 Hz, 1H), 3.13 (dd, J = 14.3, 5.6
Hz, 1H), 3.02 (dd, J = 14.4, 6.2 Hz, 1H), 1.30 (s, 9H).
5.1.3.4.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-methylbenzyl)oxy)phenyl)propanoic acid (6e)
1
yellow oil. Yield: 41%. H NMR (400 MHz, DMSO-d
6
) δ 12.54 (s, 1H), 7.31 (d, J = 7.8 Hz, 2H), 7.16
(
(
dd, J = 14.4, 7.9 Hz, 4H), 7.04 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.2 Hz, 2H), 5.00 (s, 2H), 4.08 – 3.98
m, 1H), 2.93 (dd, J = 13.9, 4.6 Hz, 1H), 2.75 (dd, J = 13.9, 10.2 Hz, 1H), 2.30 (s, 3H), 1.32 (s, 9H).
5.1.3.5.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((3-methylbenzyl)oxy)phenyl)propanoic acid (6f)
1
yellow oil. Yield: 38%. H NMR (400 MHz, DMSO-d
6
) δ 12.48 (s, 1H), 7.29 – 7.19 (m, 3H), 7.14 (m,
3
1
H), 7.05 (d, J = 8.3 Hz, 1H), 6.91 (d, J = 8.5 Hz, 2H), 5.01 (s, 2H), 4.07 – 3.98 (m, 1H), 2.93 (dd, J =
3.8, 4.6 Hz, 1H), 2.75 (dd, J = 13.9, 10.2 Hz, 1H), 2.31 (s, 3H), 1.32 (s, 9H).
5.1.3.6.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((3-methoxybenzyl)oxy)phenyl)propanoic acid (6g)
1
yellow oil. Yield: 35%. H NMR (400 MHz, DMSO-d
6
) δ 12.54 (s, 1H), 7.30 (t, J = 8.0 Hz, 1H), 7.15
(
d, J = 8.2 Hz, 2H), 7.04 – 6.96 (m, 3H), 6.90 (t, J = 8.5 Hz, 3H), 5.04 (s, 2H), 4.02 (td, J = 8.8, 8.0, 4.5
Hz, 1H), 3.76 (s, 3H), 2.94 (dd, J = 13.7, 4.6 Hz, 1H), 2.76 (dd, J = 13.8, 10.0 Hz, 1H), 1.33 (s, 9H).
5.1.3.7.(S)-3-(4-((4-bromobenzyl)oxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (6h)
1
yellowy oil. Yield: 37%. H NMR (400 MHz, DMSO-d
6
) δ 12.54 (s, 1H), 7.60 – 7.55 (m, 2H), 7.39 (d,
J = 8.3 Hz, 2H), 7.16 (d, J = 8.2 Hz, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.94 – 6.87 (m, 2H), 5.05 (s, 2H),
4
1
.03 (ddd, J = 10.1, 8.3, 4.5 Hz, 1H), 2.94 (dd, J = 13.9, 4.6 Hz, 1H), 2.75 (dd, J = 13.9, 10.3 Hz, 1H),
.32 (s, 9H).
5.1.3.8.(S)-3-(4-((2-bromobenzyl)oxy)phenyl)-2-((tert-butoxycarbonyl)amino)propanoic acid (6i)
1
yellowy oil. Yield: 34%. H NMR (400 MHz, DMSO-d
6
) δ 12.47 (s, 1H), 7.67 (dd, J = 8.0, 1.2 Hz, 1H),
7
2
2
.57 (dd, J = 7.6, 1.7 Hz, 1H), 7.46 – 7.38 (m, 1H), 7.31 (td, J = 7.7, 1.7 Hz, 1H), 7.17 (d, J = 8.1 Hz,
H), 6.92 (d, J = 8.3 Hz, 2H), 5.08 (s, 2H), 4.03 (d, J = 9.5 Hz, 1H), 2.97 (d, J = 13.6 Hz, 1H), 2.83 –
.71 (m, 1H), 1.33 (s, 9H).
5.1.3.9.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-cyanobenzyl)oxy)phenyl)propanoic acid (6j)
1
green solid. Yield: 45%, mp: 127-130℃. H NMR (400 MHz, DMSO-d
6
) δ 12.58 (s, 1H), 7.85 (d, J =
8
.0 Hz, 2H), 7.63 (d, J = 8.1 Hz, 2H), 7.18 (d, J = 8.3 Hz, 2H), 7.05 (d, J = 8.4 Hz, 1H), 6.93 (d, J = 8.2
Hz, 2H), 5.19 (s, 2H), 4.11 – 4.01 (m, 1H), 2.96 (dd, J = 13.9, 4.7 Hz, 1H), 2.77 (dd, J = 13.9, 10.2 Hz,
1
H), 1.32 (s, 9H).
5.1.3.10.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((3-cyanobenzyl)oxy)phenyl)propanoic acid (6k)
1
4 / 27

1
yellowy oil. Yield: 32%. H NMR (400 MHz, DMSO-d
6
) δ 7.90 (q, J = 2.5, 1.7 Hz, 1H), 7.79 (t, J = 7.8
Hz, 2H), 7.61 (t, J = 7.8 Hz, 1H), 7.17 (d, J = 8.0 Hz, 2H), 6.95 (dd, J = 13.5, 8.2 Hz, 3H), 5.13 (s, 2H),
4
.02 (td, J = 9.1, 4.4 Hz, 1H), 2.99 – 2.91 (m, 1H), 2.76 (dd, J = 13.7, 9.8 Hz, 1H), 1.32 (s, 9H).
5.1.3.11.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((2-cyanobenzyl)oxy)phenyl)propanoic acid (6l)
1
white solid. Yield: 46%. H NMR (400 MHz, DMSO-d
6
) δ 12.59 (s, 1H), 7.91 (d, J = 7.6 Hz, 1H), 7.74
(q, J = 7.8 Hz, 2H), 7.57 (t, J = 8.1 Hz, 1H), 7.19 (d, J = 8.4 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.96 (d,
J = 8.5 Hz, 2H), 5.21 (s, 2H), 4.05 (t, J = 9.2 Hz, 1H), 2.96 (dd, J = 13.8, 4.4 Hz, 1H), 2.77 (dd, J = 13.7,
1
0.4 Hz, 1H), 1.32 (s, 9H).
5.1.3.12.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-nitrobenzyl)oxy)phenyl)propanoic acid (6m)
1
brown oil. Yield: 30%. H NMR (400 MHz, Chloroform-d) δ 8.23 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.5
Hz, 2H), 7.12 (d, J = 8.4 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H), 5.14 (s, 2H), 4.66 – 4.46 (m, 1H), 3.09 (ddd,
J = 43.4, 13.8, 5.3 Hz, 2H), 1.38 (d, J = 30.5 Hz, 9H).
5.1.3.13.(S)-2-((tert-butoxycarbonyl)amino)-3-(4-(naphthalen-2-ylmethoxy)phenyl)propanoic acid
1
(
6n). white solid. Yield: 23%, mp: 145-147℃. H NMR (400 MHz, DMSO-d
6
) δ 12.57 (s, 1H), 8.01 –
7
2
1
.88 (m, 4H), 7.60 – 7.48 (m, 3H), 7.17 (d, J = 8.1 Hz, 2H), 7.06 (d, J = 8.3 Hz, 1H), 6.97 (d, J = 8.4 Hz,
H), 5.24 (s, 2H), 4.03 (ddd, J = 10.2, 8.2, 4.6 Hz, 1H), 2.94 (dd, J = 13.8, 4.6 Hz, 1H), 2.75 (dd, J =
3.9, 10.3 Hz, 1H), 1.31 (s, 9H).
5.1.4.(S)-2-amino-N-((4-bromophenyl)sulfonyl)-3-(4-methoxyphenyl)propanamide hydrochloride
(7a)
To a solution of 6a (1.1 g, 6.5mmol) in 30 mL anhydrous THF at 0℃, NMM (0.3 ml, 2.5 mmol)
and isobutylchloroformate (0.32 ml, 7.3 mmol) in THF, which was pretreated with NaH (0.23 g, 6.4
mmol) for 4 h at room temperature aforehand, were added in drops. This reaction was recovered
slowly to room temperature and stirred overnight. Then the solvent was evaporated in vacuum. The
resulting residue was neutralized by 1M HCl solution and extracted with EtOAc, then the organic
phase was washed by 0.5 M citric acid solution and brine. After dried by MgSO and concentrated,
4
use EtOAc which was saturated with HCl gas to dissolve the obtained oil and stirred at room
temperature overnight. Then the generated precipitate was filtered and dried. white solid. Yield:66%,
1
mp: 227-229℃. H NMR (400 MHz, DMSO-d
6
) δ 13.61 (s, 1H), 8.42 (s, 3H), 7.87 (q, J = 8.7 Hz, 4H),
13
6
.95 (d, J = 8.5 Hz, 2H), 6.72 (d, J = 8.5 Hz, 2H), 4.10 (s, 1H), 3.73 (s, 3H), 3.08 – 2.94 (m, 2H). C
NMR (101 MHz, DMSO) δ 169.88, 158.79, 140.79, 133.02, 131.06, 130.32, 126.46, 125.62, 114.16,
5
5.44, 54.95, 35.58. ESI-MS m/z: 411.4 [M-H]-.
Compounds 7b–7q were synthesized following the procedure described above.
5.1.4.1.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-methoxyphenyl)propanamide
1
hydrochloride (7b) white solid. Yield:65%, mp: 214-216℃. H NMR (400 MHz, DMSO-d
6
) δ 10.86
(
s, 1H), 8.52 (s, 1H), 8.27 (s, 3H), 8.15 (dd, J = 8.5, 1.6 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 6.98 (d, J =
8
.4 Hz, 2H), 6.72 (d, J = 8.5 Hz, 2H), 4.06 – 3.96 (m, 1H), 3.71 (s, 3H), 3.01 (ddq, J = 20.3, 14.0, 6.6
1
3
Hz, 2H). C NMR (101 MHz, DMSO) δ 169.90, 158.82, 147.45, 140.98, 133.07, 133.00, 131.07, 130.26,
26.42, 125.55, 114.13, 55.44, 54.95, 35.58. ESI-MS m/z: 412.4 [M-H]-.
1
1
5 / 27

5.1.4.2.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-isopropoxyphenyl)propanamide
1
hydrochloride (7c) white solid. Yield:34%, mp: 210-212℃. H NMR (400 MHz, DMSO-d
6
) δ 10.53
(
s, 1H), 8.53 (d, J = 2.0 Hz, 1H), 8.28 (s, 3H), 8.17 (dd, J = 8.5, 2.0 Hz, 1H), 8.05 (d, J = 8.5 Hz, 1H),
6
.96 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.5 Hz, 2H), 4.53 (hept, J = 6.0 Hz, 1H), 4.07 – 3.96 (m, 1H), 3.00
13
(
ddt, J = 20.7, 14.2, 6.1 Hz, 2H), 1.25 (d, J = 2.6 Hz, 3H), 1.23 (d, J = 2.6 Hz, 3H). C NMR (101 MHz,
DMSO) δ 169.84, 157.06, 147.48, 140.86, 133.10, 131.13, 130.34, 126.09, 125.59, 115.72, 69.45, 54.91,
3
5.55, 22.30. ESI-MS m/z: 440.5 [M-H]-.
5.1.4.3.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-propoxyphenyl)propenamide
1
hydrochloride (7d) white solid. Yield:49%, mp: 210-214℃. H NMR (400 MHz, DMSO-d
6
) δ 11.82
(
s, 1H), 8.53 (s, 1H), 8.35 (s, 3H), 8.17 (dd, J = 8.5, 2.0 Hz, 1H), 8.06 (d, J = 8.5 Hz, 1H), 6.97 (d, J =
8
.5 Hz, 2H), 6.71 (d, J = 8.5 Hz, 2H), 4.04 (dd, J = 9.0, 5.2 Hz, 1H), 3.87 (t, J = 6.5 Hz, 2H), 3.02 (qd, J
13
=
14.2, 6.2 Hz, 2H), 1.71 (h, J = 7.1 Hz, 2H), 0.98 (t, J = 7.4 Hz, 3H). C NMR (101 MHz, DMSO) δ
1
3
69.60, 158.27, 147.48, 140.46, 133.20, 133.04, 131.06, 130.56, 126.13, 125.61, 114.61, 69.27, 54.78,
5.50, 22.50, 10.88. ESI-MS m/z: 440.5 [M-H]-.
5
.1.4.4.(S)-2-amino-3-(4-(benzyloxy)
phenyl)-N-((4-bromophenyl)
sulfonyl)
propenamide
) δ
1
hydrochloride (7e) white powder. Yield:55%, mp: 228-230℃. H NMR (400 MHz, DMSO-d
6
1
3.09 (s, 1H), 8.35 (s, 3H), 7.91 – 7.81 (m, 4H), 7.49 – 7.31 (m, 6H), 6.96 (d, J = 8.5 Hz, 2H), 6.82 (d, J
13
=
8.6 Hz, 2H), 5.07 (s, 2H), 4.10 – 4.02 (m, 1H), 3.07 – 2.92 (m, 2H). C NMR (101 MHz, DMSO) δ
1
5
68.58, 157.99, 138.74, 137.51, 132.66, 131.11, 130.29, 128.93, 128.33, 128.12, 126.34, 115.09, 69.58,
4.46, 35.46. ESI-MS m/z: 487.4 [M-H]-.
5.1.4.5.(S)-2-amino-3-(4-(benzyloxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)propanamide
1
hydrochloride (7f) gray powder. Yield:43%, mp: 219-222℃. H NMR (400 MHz, DMSO-d
6
) δ 8.54
(d, J = 2.1 Hz, 1H), 8.38 – 8.25 (m, 3H), 8.16 (dd, J = 8.5, 2.2 Hz, 1H), 8.04 (d, J = 8.5 Hz, 1H),
7
1
1
.47 – 7.32 (m, 5H), 7.01 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.3 Hz, 2H), 5.06 (s, 2H), 3.02 (qd, J =
1
3
4.2, 6.2 Hz, 2H). C NMR (101 MHz, DMSO) δ 168.58, 157.99, 137.51, 132.66, 131.11, 130.29,
28.93, 128.33, 128.12, 126.34, 115.09, 69.58, 54.46, 35.46. ESI-MS m/z: 488.4 [M-H]-.
5.1.4.6.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((4-methylbenzyl)oxy)phenyl)
1
propanamide hydrochloride (7g) white solid. Yield:71%, mp: 184-186℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.50 (d, J = 2.1 Hz, 1H), 8.13 (h, J = 4.4 Hz, 3H), 8.00 (d, J = 8.5 Hz, 1H), 7.32 (d, J
=
7.8 Hz, 2H), 7.20 (d, J = 7.8 Hz, 2H), 6.97 (d, J = 8.3 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 5.00 (s,
13
2
1
6
H), 2.97 (qd, J = 14.2, 6.1 Hz, 2H), 2.31 (s, 3H). C NMR (101 MHz, DMSO) δ 168.56, 157.99,
47.46, 140.46, 137.45, 136.86, 133.16, 133.05, 131.10, 130.54, 128.87, 128.09, 126.32 125.60, 115.06,
9.56, 54.43, 35.46, 20.88. ESI-MS m/z: 502.4 [M-H]-.
5.1.4.7.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((3-methylbenzyl)oxy)phenyl)
1
propanamide hydrochloride (7h) white solid. Yield:94%, mp: 208-210℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.52 (d, J = 2.1 Hz, 1H), 8.23 (s, 3H), 8.14 (dd, J = 8.5, 2.2 Hz, 1H), 8.02 (d, J = 8.5
Hz, 1H), 7.31 – 7.19 (m, 3H), 7.14 (d, J = 7.4 Hz, 1H), 6.99 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 8.3 Hz,
13
2
H), 5.01 (s, 2H), 3.99 (q, J = 6.1 Hz, 1H), 3.00 (qd, J = 14.3, 6.2 Hz, 2H), 2.32 (s, 3H). C NMR
101 MHz, DMSO) δ 168.60, 157.98, 147.51, 140.49, 138.15, 137.48, 133.22, 133.08, 131.16, 130.56,
6 / 27
(
1

1
28.43, 128.36, 126.35, 125.68, 125.31, 115.08, 69.58, 54.46, 35.51, 20.86. ESI-MS m/z: 502.1 [M-H]-.
5.1.4.8.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((3-methoxybenzyl)oxy)phenyl)
1
propanamide hydrochloride (7i) pink solid. Yield:13%, mp: 200-202℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.51 (d, J = 2.2 Hz, 1H), 8.18 (d, J = 5.1 Hz, 3H), 8.13 (dd, J = 8.4, 2.2 Hz, 1H), 8.01 (d,
J = 8.4 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.04 – 6.97 (m, 4H), 6.93 – 6.87 (m, 1H), 6.85 – 6.78 (m, 2H),
13
5
.03 (s, 2H), 3.96 (q, J = 5.4, 5.0 Hz, 1H), 2.99 (qd, J = 14.2, 6.2 Hz, 2H). C NMR (101 MHz, DMSO)
δ 168.60, 159.68, 157.99, 139.51, 132.66, 131.14, 130.32, 129.46, 126.38, 121.62, 115.12, 113.45, 113.16,
6
9.63, 55.24, 54.52, 35.48. ESI-MS m/z: 518.1 [M-H]-.
5.1.4.9.(S)-2-amino-3-(4-((4-bromobenzyl)oxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)
1
propanamide hydrochloride (7j) white solid. Yield:61%, mp: 230-232℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.50 (d, J = 2.7 Hz, 1H), 8.24 – 8.10 (m, 3H), 8.01 (dd, J = 8.5, 4.0 Hz, 1H), 7.60 (d, J =
8
2
1
.1 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.00 (dd, J = 8.6, 2.4 Hz, 2H), 6.81 (d, J = 8.3 Hz, 2H), 5.04 (s,
13
H), 2.99 (tdd, J = 20.2, 13.9, 5.4 Hz, 2H). C NMR (101 MHz, DMSO) δ 168.58, 157.96, 132.66,
31.53, 131.14, 130.32, 129.64, 126.36, 122.80, 115.12, 69.60, 54.51, 35.48. ESI-MS m/z: 568.0 [M-H]-.
5.1.4.10.(S)-2-amino-3-(4-((2-bromobenzyl)oxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)
1
propanamide hydrochloride (7k) white solid. Yield:41%, mp: 202-204℃. H NMR (400 MHz,
DMSO-d ) δ 8.41 (s, 1H), 8.06 (d, J = 10.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.76 (s, 3H), 7.69 (d,
6
J = 7.9 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 6.97 (d,
J = 8.4 Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 5.06 (s, 2H), 3.73 – 3.61 (m, 1H), 3.02 – 2.84 (m, 2H).
13C NMR (101 MHz, DMSO) δ 168.59, 157.94, 136.18, 132.71, 131.14, 130.84, 130.55, 130.31, 127.82,
1
27.25, 126.32, 115.14, 69.54, 54.43, 35.45. ESI-MS m/z: 568.1 [M-H]-.
5.1.4.11.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((4-cyanobenzyl)oxy)phenyl)
1
propanamide hydrochloride (7l) white powder. Yield:55.2%, mp: 210-212℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.52 (d, J = 2.1 Hz, 1H), 8.27 (d, J = 5.4 Hz, 3H), 8.14 (dd, J = 8.5, 2.2 Hz, 1H), 8.03 (d,
J = 8.5 Hz, 1H), 7.88 (d, J = 8.0 Hz, 2H), 7.64 (d, J = 8.1 Hz, 2H), 7.02 (d, J = 8.3 Hz, 2H), 6.83 (d, J =
8
1
5
.3 Hz, 2H), 5.18 (s, 2H), 4.01 (s, 1H), 3.01 (qd, J = 14.2, 6.2 Hz, 2H). 13C NMR (101 MHz, DMSO) δ
68.60, 158.02, 144.31, 132.63, 132.46, 131.15, 130.26, 129.67, 126.33, 119.34, 115.14, 111.18, 69.55,
4.41, 35.52. ESI-MS m/z: 513.2 [M-H]-.
5.1.4.12.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((3-cyanobenzyl)oxy)phenyl)
1
propanamide hydrochloride (7m) white solid. Yield:52%, mp: 182-184℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.47 (d, J = 2.1 Hz, 1H), 8.10 (dd, J = 8.5, 2.1 Hz, 1H), 8.07 – 7.94 (m, 4H), 7.90 (d, J =
1
5
1
.8 Hz, 1H), 7.85 – 7.76 (m, 2H), 7.63 (t, J = 7.8 Hz, 1H), 6.99 (d, J = 8.5 Hz, 2H), 6.88 – 6.80 (m, 2H),
1
3
.13 (s, 2H), 3.04 – 2.87 (m, 2H). C NMR (101 MHz, DMSO) δ 168.56, 157.94, 138.11, 132.59, 131.04,
30.74, 130.23, 130.06, 129.06, 128.08, 126.29, 118.53, 115.07, 111.93, 69.52, 54.46, 35.46. ESI-MS
m/z: 513.3 [M-H]-.
5.1.4.13.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((2-cyanobenzyl)oxy)phenyl)
1
propanamide hydrochloride (7n) white solid. Yield:72%, mp: 210-212℃. H NMR (400 MHz,
1
7 / 27

DMSO-d
6
) δ 8.41 (s, 1H), 8.06 (d, J = 10.2 Hz, 1H), 7.88 (d, J = 8.4 Hz, 1H), 7.76 (s, 3H), 7.69 (d, J =
7
.9 Hz, 1H), 7.57 (d, J = 7.5 Hz, 1H), 7.43 (t, J = 7.5 Hz, 1H), 7.33 (d, J = 7.6 Hz, 1H), 6.97 (d, J = 8.4
13
Hz, 2H), 6.80 (d, J = 8.5 Hz, 2H), 5.06 (s, 2H), 3.71 – 3.64 (m, 1H), 3.00 – 2.86 (m, 2H). C NMR (101
MHz, DMSO) δ 168.64, 157.97, 139.66, 132.62, 131.86, 131.09, 130.25, 128.67, 128.34, 128.31, 126.33,
1
17.21, 115.06, 112.58, 69.55, 54.43, 35.41. ESI-MS m/z: 513.3 [M-H]-.
5.1.4.14.(S)-2-amino-N-((4-bromophenyl)sulfonyl)-3-(4-((4-nitrobenzyl)oxy)phenyl)propanamide
1
hydrochloride (7o) light yellow solid. Yield:47%, mp: 232-234℃. H NMR (400 MHz, DMSO-d
6
)
δ 8.36 (s, 3H), 8.28 (d, J = 8.7 Hz, 2H), 7.85 (q, J = 8.8 Hz, 4H), 7.73 (d, J = 8.6 Hz, 2H), 6.99 (d, J =
13
8
.5 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.26 (s, 2H), 4.13 – 4.04 (m, 1H), 3.11 – 2.93 (m, 2H). C NMR
(
101 MHz, DMSO) δ 168.60, 157.54, 147.45, 145.48, 139.04, 138.80, 132.63, 131.19, 130.27, 128.63,
1
28.28, 126.76, 124.10, 115.17, 68.42, 54.48, 35.53. ESI-MS m/z: 532.2 [M-H]-.
5.1.4.15.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-((4-nitrobenzyl)oxy)phenyl)
1
propanamide hydrochloride (7p) light yellow solid. Yield:54%, mp: 218-220℃. H NMR (400 MHz,
DMSO-d
6
) δ 8.50 (d, J = 2.7 Hz, 1H), 8.24 – 8.11 (m, 3H), 8.01 (dd, J = 8.5, 4.0 Hz, 1H), 7.60 (d,
J = 8.1 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 7.00 (dd, J = 8.6, 2.4 Hz, 2H), 6.81 (d, J = 8.3 Hz, 2H),
1
3
5
1
.04 (s, 2H), 2.99 (tdd, J = 20.2, 13.9, 5.4 Hz, 2H). C NMR (101 MHz, DMSO) δ 168.56, 157.52,
47.48, 147.43, 145.44, 140.47, 138.77, 133.22, 133.06, 131.16, 130.55, 128.62, 126.72, 125.59, 124.08,
1
15.16, 68.41, 54.44, 35.47. ESI-MS m/z: 533.2 [M-H]-.
5.1.4.16.(S)-2-amino-N-((4-chloro-3-nitrophenyl)sulfonyl)-3-(4-(naphthalen-2-ylmethoxy)phenyl)
1
propanamide hydrochloride (7q) light yellow solid. Yield:92%, mp: 201-204℃. H NMR (400 MHz,
DMSO-d
H), 6.99 (d, J = 8.3 Hz, 2H), 6.87 (d, J = 8.3 Hz, 2H), 5.23 (s, 2H), 3.05 – 2.89 (m, 2H). C NMR
101 MHz, DMSO) δ 168.60, 157.91, 136.43, 133.64, 133.32, 132.74, 131.19, 130.35, 128.87, 128.68,
6
) δ 8.49 (d, J = 2.1 Hz, 1H), 8.16 – 8.05 (m, 4H), 8.02 – 7.90 (m, 6H), 7.60 – 7.51 (m,
13
3
(
1
27.93, 127.47, 126.39, 126.36, 126.42, 126.17, 115.19, 69.66, 54.57, 35.53. ESI-MS m/z: 538.5 [M-H]-.
5.1.5.(S)-N-((4-bromophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-
yl)acetamido)-3-(4-methoxyphenyl)propenamide (8a)
To the solution intermediate 7a (0.76 g, 1.8 mmol) in dry dichloromethane (25 mL),
ethyldiisopropylamine (0.82 ml, 7.4 mmol) was added in ice bath. After stirred for 10 min, O-
(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTO, 0.77 g, 2.4 mmol) was
added and stirred for 30 min in 0℃, then the indomethacin (0.79 g, 2.2 mmol) was added and stirred
overnight at room temperature. The solvent was removed in vacuum and then dissolved in EtOAc.
0.5 M citric acid and brine were used to wash the organic phase, then it was concentrated to yield
yellow oil. Purification by column chromatography (PE/EA = 10:7) to generate compound 8a, a
1
white solid. Yield:54%, mp: 217-220℃. H NMR (400 MHz, DMSO-d
6
) δ 12.62 (s, 1H), 8.31 (d, J
=
7.7 Hz, 1H), 7.82 (s, 4H), 7.63 (s, 4H), 7.06 (d, J = 2.2 Hz, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.94 (d,
J = 9.0 Hz, 1H), 6.70 (dd, J = 9.0, 2.3 Hz, 1H), 6.63 (d, J = 8.4 Hz, 2H), 4.44 (td, J = 8.8, 4.8 Hz,
1
1
1
H), 3.74 (s, 3H), 3.65 (s, 3H), 3.55 – 3.42 (m, 2H), 2.85 (dd, J = 13.6, 4.3 Hz, 1H), 2.63 (dd, J =
13
3.6, 9.6 Hz, 1H), 2.07 (s, 3H). C NMR (100 MHz, DMSO-d
56.00, 138.78, 138.01, 135.53, 134.66, 132.69, 131.57, 131.23, 130.60, 129.96, 129.48, 128.80, 128.24,
14.90, 114.50, 113.77, 111.91, 102.23, 55.85, 55.29, 55.10, 36.07, 30.97, 13.75. HRMS (AP-ESI) m/z
8 / 27
6
) δ 171.27, 170.08, 168.26, 158.29,
1
1

-
3 7
Calcd for C35H31BrClN O S [M-H] 752.0659. Found:752.0664.
Compounds 8b–8q were synthesized following the procedure described above.
5
1
1
1
.1.5.1.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-methoxyphenyl)propenamide (8b) white solid. Yield:42%, mp: 190-
1
94℃. H NMR (400 MHz, DMSO-d
6
) δ 12.90 (s, 1H), 8.53 (d, J = 2.1 Hz, 1H), 8.38 (d, J = 7.3 Hz,
H), 8.13 (dd, J = 8.5, 2.1 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.64 (s, 4H), 7.07 – 7.00 (m, 3H), 6.93
(d, J = 9.0 Hz, 1H), 6.69 (dd, J = 9.0, 2.4 Hz, 1H), 6.64 (d, J = 8.5 Hz, 2H), 4.49 – 4.39 (m, 1H),
3
1
1
1
.74 (s, 3H), 3.65 (s, 3H), 3.49 (q, J = 15.4 Hz, 2H), 2.88 (dd, J = 13.7, 4.7 Hz, 1H), 2.67 (dd, J =
13
3.6, 9.4 Hz, 1H), 2.08 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 171.88, 170.17, 168.26, 158.32,
6
55.98, 147.58, 138.02, 135.55, 134.66, 133.41, 132.67, 131.57, 131.20, 131.08, 130.61, 130.59, 129.48,
28.76, 125.48, 114.90, 114.49, 113.74, 111.91, 102.17, 55.81, 55.28, 35.99, 30.93, 13.72. HRMS (AP-
-
ESI) m/z Calcd for C35
H
2
30Cl N
4
O
9
S [M-H] 751.1032. Found:751.1033.
5
1
1
.1.5.2.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-isopropoxyphenyl)propenamide (8c) white solid. Yield:52%, mp:
1
22-126℃. H NMR (400 MHz, DMSO-d
6
) δ 12.92 (s, 1H), 8.57 – 8.53 (m, 1H), 8.42 (d, J = 7.4
Hz, 1H), 8.17 – 8.11 (m, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.64 (s, 4H), 7.09 – 7.05 (m, 1H), 7.01 (d, J
=
8.3 Hz, 2H), 6.94 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 9.0, 1.9 Hz, 1H), 6.62 (d, J = 8.3 Hz, 2H), 4.50
4.40 (m, 2H), 3.75 (s, 3H), 3.49 (q, J = 15.4 Hz, 2H), 2.87 (dd, J = 13.6, 4.2 Hz, 1H), 2.69 – 2.61
–
1
3
(
m, 1H), 2.08 (s, 3H), 1.19 (t, J = 6.7 Hz, 7H). C NMR (100 MHz, DMSO-d
6
) δ 171.80, 170.24,
1
1
3
68.22, 156.56, 155.98, 147.60, 139.41, 138.05, 135.53, 134.63, 133.45, 132.68, 131.57, 131.21, 130.68,
30.59, 129.47, 128.49, 125.53, 115.43, 114.89, 114.48, 111.90, 102.16, 69.37, 55.80, 55.27, 35.96, 31.45,
-
0.93, 22.56, 22.23, 14.43, 13.71. HRMS (AP-ESI) m/z Calcd for C37
H
2
34Cl N
4
O
9
S [M-H] 779.1345.
Found:779.1346.
5
1
1
.1.5.3.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-propoxyphenyl)propenamide (8d) white solid. Yield:39%, mp: 134-
1
38℃. H NMR (400 MHz, DMSO-d
6
) δ 12.90 (s, 1H), 8.54 (d, J = 2.0 Hz, 1H), 8.40 (d, J = 7.6
Hz, 1H), 8.13 (dd, J = 8.5, 2.0 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.63 (s, 4H), 7.09 – 6.98 (m, 3H),
6
1
1
d
1
5
.94 (d, J = 9.0 Hz, 1H), 6.69 (dd, J = 9.0, 2.3 Hz, 1H), 6.62 (d, J = 8.5 Hz, 2H), 4.44 (q, J = 8.5 Hz,
H), 3.83 – 3.71 (m, 5H), 3.49 (q, J = 15.4 Hz, 2H), 2.87 (dd, J = 13.7, 4.4 Hz, 1H), 2.68 – 2.62 (m,
1
3
H), 2.09 (s, 3H), 1.67 (h, J = 7.0 Hz, 2H), 0.94 (t, J = 7.4 Hz, 3H). C NMR (100 MHz, DMSO-
) δ 171.77, 170.20, 168.24, 157.78, 155.98, 147.59, 139.44, 138.03, 135.54, 134.64, 133.45, 132.68,
31.58, 131.19, 130.60, 129.47, 128.57, 125.51, 114.89, 114.47, 114.25, 111.91, 102.18, 69.16, 55.81,
6
5.25, 38.71, 35.99, 30.94, 22.51, 13.72, 10.86. HRMS (AP-ESI) m/z Calcd for C37
H
34Cl
2
N
4
9
O S [M-
-
H] 779.1345. Found:779.1350.
5.1.5.4.(S)-3-(4-(benzyloxy)phenyl)-N-((4-bromophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-
methoxy-2-methyl-1H-indol-3-yl)acetamido)propenamide (8e) white powder. Yield:72%, mp: 196-
1
1
7
1
99℃. H NMR (400 MHz, DMSO-d
6
) δ 12.64 (s, 1H), 8.33 (d, J = 7.7 Hz, 1H), 7.81 (s, 4H), 7.67 –
.58 (m, 4H), 7.44 – 7.30 (m, 5H), 7.07 (d, J = 2.4 Hz, 1H), 7.01 (d, J = 8.5 Hz, 2H), 6.93 (d, J = 9.0 Hz,
H), 6.70 (td, J = 6.8, 3.2 Hz, 3H), 4.97 (s, 2H), 4.44 (td, J = 8.8, 4.8 Hz, 1H), 3.74 (s, 3H), 3.56 – 3.41
1
3
(
m, 2H), 2.85 (dd, J = 13.6, 4.4 Hz, 1H), 2.63 (dd, J = 13.6, 9.7 Hz, 1H), 2.08 (s, 3H). C NMR (100
1
9 / 27

MHz, DMSO-d
32.68, 131.58, 131.25, 130.66, 130.61, 129.95, 129.48, 129.16, 128.87, 128.25, 128.04, 114.87, 114.63,
14.54, 111.95, 102.29, 69.54, 55.90, 55.17, 36.05, 31.02, 13.76. HRMS (AP-ESI) m/z Calcd for
6
) δ 171.28, 170.09, 168.26, 157.45, 156.02, 138.80, 138.02, 137.54, 135.56, 134.65,
1
1
-
C
41
H
35BrClN
3
O
7
S [M-H] 828.0973. Found:828.0993.
5.1.5.5.(S)-3-(4-(benzyloxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)
-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)propenamide (8f) gray solid. Yield:13%, mp: 132-
1
1
35℃. H NMR (400 MHz, DMSO-d
6
) δ 12.94 (s, 1H), 8.55 (d, J = 2.2 Hz, 1H), 8.43 (d, J = 7.6
Hz, 1H), 8.14 (dd, J = 8.5, 2.3 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.67 – 7.59 (m, 4H), 7.43 – 7.32
m, 5H), 7.10 – 7.02 (m, 3H), 6.93 (d, J = 8.9 Hz, 1H), 6.74 (d, J = 8.3 Hz, 2H), 6.70 (dd, J = 9.0,
(
2.5 Hz, 1H), 4.98 (s, 2H), 4.47 (td, J = 8.1, 7.5, 4.9 Hz, 1H), 3.50 (q, J = 15.4 Hz, 2H), 2.90 (dd, J
1
3
=
13.9, 4.8 Hz, 1H), 2.68 (dd, J = 13.8, 9.4 Hz, 1H), 2.10 (s, 3H). C NMR (100 MHz, DMSO-d )
6
δ 171.77, 170.24, 168.25, 157.49, 155.98, 147.60, 139.39, 138.05, 137.51, 135.59, 134.63, 133.46,
32.67, 131.58, 131.21, 130.65, 129.47, 129.04, 128.88, 128.27, 128.06, 125.54, 114.90, 114.64, 114.47,
11.92, 102.18, 69.52, 55.82, 55.23, 35.97, 30.95, 13.71. HRMS (AP-ESI) m/z Calcd for
1
1
-
C
41
H
2
34Cl N
4
O S [M-H] 827.1345. Found:827.1355.
9
5
1
.1.5.6.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-((4-methylbenzyl)oxy)phenyl)propenamide (8g) light yellow solid.
1
Yield:25%, mp: 131-136℃. H NMR (400 MHz, DMSO-d
6
) δ 12.89 (s, 1H), 8.54 (d, J = 2.2 Hz, 1H),
8
7
.38 (d, J = 7.6 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.96 (d, J = 8.5 Hz, 1H), 7.70 – 7.55 (m, 4H),
.28 (d, J = 7.7 Hz, 2H), 7.18 (d, J = 7.7 Hz, 2H), 7.09 – 6.98 (m, 3H), 6.92 (d, J = 9.0 Hz, 1H), 6.70 (dd,
J = 8.9, 6.8 Hz, 3H), 4.92 (s, 2H), 4.45 (td, J = 8.4, 4.8 Hz, 1H), 3.74 (s, 3H), 3.49 (q, J = 15.4 Hz, 2H),
.88 (dd, J = 14.0, 4.8 Hz, 1H), 2.66 (dd, J = 14.0, 4.8 Hz , 1H), 2.30 (s, 3H), 2.10 (s, 3H). 13C NMR
100 MHz, DMSO-d ) δ 171.80, 170.20, 168.25, 157.52, 155.99, 147.60, 139.50, 138.05, 137.48,
2
(
6
1
35.58, 134.65, 134.48, 133.44, 132.66, 131.57, 131.21, 131.16, 130.61, 129.48, 129.41, 128.97, 128.14,
1
25.50, 114.88, 114.65, 114.48, 111.92, 102.22, 69.44, 55.84, 55.23, 38.71, 35.99, 30.96, 21.23, 13.70.
13C NMR (100 MHz, DMSO-d
S [M-H]-
6
) δ HRMS (AP-ESI) m/z Calcd for C42
H
36Cl N
2 4
O
9
841.1502. Found:841.1507.
5
.1.5.7.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamido)-3-(4-((3-methylbenzyl)oxy)phenyl)propenamide (8h) light yellow solid.
1
Yield:30%, mp: 134-138℃. H NMR (400 MHz, DMSO-d
6
) δ 12.89 (s, 1H), 8.55 (d, J = 2.2 Hz, 1H),
8
7
4
2
1
1
1
.40 (d, J = 7.6 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.70 – 7.59 (m, 4H),
.31 – 7.11 (m, 4H), 7.10 – 7.00 (m, 3H), 6.93 (d, J = 8.9 Hz, 1H), 6.77 – 6.66 (m, 3H), 4.93 (s, 2H),
.45 (td, J = 8.3, 4.8 Hz, 1H), 3.74 (s, 3H), 3.49 (q, J = 15.4 Hz, 2H), 2.89 (dd, J = 13.8, 4.8 Hz, 1H),
.73 – 2.64 (m, 1H), 2.32 (s, 3H), 2.10 (s, 3H). 13C NMR (100 MHz, DMSO-d
) δ 171.79, 170.20,
6
68.25, 157.53, 155.98, 147.61, 139.47, 138.04, 137.42, 135.58, 134.64, 133.45, 132.66, 131.58, 131.21,
31.17, 130.64, 129.48, 129.00, 128.90, 128.77, 128.63, 125.51, 125.16, 114.88, 114.61, 114.47, 111.93,
02.20, 69.57, 55.83, 55.22, 35.99, 30.95, 21.46, 13.71. HRMS (AP-ESI) m/z Calcd for
-
C
42
H
2
36Cl N
4
O
9
S [M-H] 841.1502. Found:841.1521.
5
.1.5.8.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamido)-3-(4-((3-methoxybenzyl)oxy)phenyl)propenamide (8i) light yellow solid.
2
0 / 27

1
Yield:32%, mp: 157-160℃. H NMR (400 MHz, DMSO-d
6
) δ 12.92 (s, 1H), 8.54 (d, J = 2.2 Hz, 1H),
8
7
6
3
2
1
1
5
.40 (d, J = 7.6 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.69 – 7.58 (m, 4H),
.30 (t, J = 8.0 Hz, 1H), 7.09 – 7.00 (m, 3H), 6.97 (dd, J = 4.3, 2.3 Hz, 2H), 6.93 (d, J = 9.0 Hz, 1H),
.89 (dd, J = 8.2, 2.5 Hz, 1H), 6.71 (dd, J = 12.4, 7.5 Hz, 3H), 4.96 (s, 2H), 4.45 (td, J = 8.3, 4.9 Hz, 1H),
.75 (d, J = 6.9 Hz, 6H), 3.49 (q, J = 15.4 Hz, 2H), 2.89 (dd, J = 13.8, 4.8 Hz, 1H), 2.73 – 2.64 (m, 1H),
13
.10 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 171.82, 170.20, 168.25, 159.78, 157.44, 155.98, 147.60,
6
39.51, 139.11, 138.04, 135.58, 134.64, 133.43, 132.66, 131.57, 131.21, 131.14, 130.60, 129.99, 129.48,
29.07, 125.51, 120.08, 114.88, 114.64, 114.48, 113.66, 113.49, 111.93, 102.20, 69.39, 55.83, 55.49,
-
5.23, 36.00, 30.95, 13.71. HRMS (AP-ESI) m/z Calcd for C42
H
2
36Cl N
4
O
10S [M-H] 857.1456.
Found:857.1450.
5.1.5.9.(S)-3-(4-((4-bromobenzyl)oxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)propenamide (8j) light yellow solid.
1
Yield:28%, mp: 164-170℃. H NMR (400 MHz, DMSO-d
6
) δ 12.89 (s, 1H), 8.54 (d, J = 2.2 Hz, 1H),
8
.39 (d, J = 7.5 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.61 (dt, J = 12.1, 8.3
Hz, 6H), 7.36 (d, J = 8.0 Hz, 2H), 7.10 – 6.99 (m, 3H), 6.92 (d, J = 9.0 Hz, 1H), 6.78 – 6.65 (m, 3H),
.97 (s, 2H), 4.44 (td, J = 8.6, 4.9 Hz, 1H), 3.74 (s, 3H), 3.48 (q, J = 15.4 Hz, 2H), 2.89 (dd, J = 13.9, 4.7
Hz, 1H), 2.67 (dd, J = 13.7, 9.3 Hz, 1H), 2.10 (s, 3H). 13C NMR (100 MHz, DMSO-d
) δ 171.84,
70.18, 168.25, 157.25, 155.97, 147.60, 138.04, 137.01, 135.57, 134.63, 133.42, 132.66, 131.81, 131.58,
31.20, 131.11, 131.09, 130.66, 130.59, 130.11, 129.48, 129.25, 125.49, 121.35, 114.88, 114.67, 114.47,
11.93, 102.19, 68.71, 55.83, 55.23, 35.98, 30.95, 13.70. HRMS (AP-ESI) m/z Calcd for
4
6
1
1
1
-
C
41
H
33BrCl
2
N
4
O
9
S [M-H] 907.0432. Found:907.0425.
5.1.5.10.(S)-3-(4-((2-bromobenzyl)oxy)phenyl)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-
chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-yl)acetamido)propenamide (8k) light yellow solid.
1
Yield:18%, mp: 162-167℃. H NMR (400 MHz, DMSO-d
6
) δ 12.81 (s, 1H), 8.55 (d, J = 2.2 Hz, 1H),
8
7
1
3
2
1
1
3
.40 (d, J = 7.6 Hz, 1H), 8.14 (dd, J = 8.5, 2.2 Hz, 1H), 7.98 (d, J = 8.5 Hz, 1H), 7.73 – 7.58 (m, 5H),
.53 (d, J = 7.7, 1H), 7.42 (t, J = 7.5, 1H), 7.31 (t, J = 7.7, 1H), 7.11 – 7.02 (m, 3H), 6.92 (d, J = 9.0 Hz,
H), 6.74 (d, J = 8.3 Hz, 2H), 6.69 (dd, J = 9.0, 2.6 Hz, 1H), 5.00 (s, 2H), 4.45 (td, J = 8.6, 4.9 Hz, 1H),
.74 (s, 3H), 3.49 (q, J = 15.2 Hz, 2H), 2.90 (dd, J = 13.8, 4.7 Hz, 1H), 2.69 (dd, J = 13.7, 9.4 Hz, 1H),
13
.10 (s, 3H). C NMR (100 MHz, DMSO-d ) δ 171.83, 170.20, 168.24, 157.26, 155.98, 147.61, 138.04,
6
36.35, 135.58, 134.63, 133.43, 133.07, 132.66, 131.58, 131.20, 131.11, 130.74, 130.59, 130.51, 130.45,
29.48, 129.44, 128.35, 125.51, 123.11, 114.88, 114.59, 114.47, 111.92, 102.21, 69.35, 55.85, 55.22,
-
5.97, 30.96, 13.71. HRMS (AP-ESI) m/z Calcd for C41
H
33BrCl
2
N
4
O
9
S [M-H] 907.0432.
Found:907.0418.
5
1
.1.5.11.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-((4-cyanobenzyl)oxy)phenyl)propenamide (8l) light yellow solid.
1
Yield:30%, mp: 172-174℃. H NMR (400 MHz, DMSO-d
6
) δ 12.87 (s, 1H), 8.53 (d, J = 2.2 Hz, 1H),
8
7
3
1
1
.39 (d, J = 7.6 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.86 (d, J = 8.0 Hz, 2H),
.61 (dd, J = 11.3, 5.5 Hz, 6H), 7.05 (dd, J = 5.6, 3.0 Hz, 3H), 6.91 (d, J = 9.0 Hz, 1H), 6.79 – 6.66 (m,
H), 5.11 (s, 2H), 4.44 (td, J = 8.5, 4.9 Hz, 1H), 3.73 (s, 3H), 3.48 (q, J = 15.4 Hz, 2H), 2.89 (dd, J =
3.8, 4.8 Hz, 1H), 2.68 (dd, J = 13.7, 9.4 Hz, 1H), 13C NMR (100 MHz, DMSO-d
71.84, 170.19, 168.24, 157.07, 155.97, 147.60, 143.38, 139.57, 138.04, 135.57, 134.62, 133.42, 132.87,
1 / 27
) δ 2.09 (s, 3H).
6
2

1
32.66, 131.58, 131.19, 131.11, 130.71, 130.58, 129.47, 128.37, 125.48, 119.22, 114.88, 114.67, 114.46,
111.92, 110.91, 102.19, 68.58, 55.83, 55.22, 35.97, 30.96, 13.69. HRMS (AP-ESI) m/z Calcd for
-
C
42
H
2
33Cl N
5
O S [M-H] 852.1298. Found:852.1315.
9
5
1
.1.5.12.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-((3-cyanobenzyl)oxy)phenyl)propenamide (8m) light yellow solid.
1
Yield:17.18%, mp: 182-186℃. H NMR (400 MHz, DMSO-d
6
) δ 12.91 (s, 1H), 8.53 (d, J = 2.1 Hz,
1
H), 8.39 (d, J = 7.3 Hz, 1H), 8.12 (dd, J = 8.5, 2.1 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.87 (s, 1H), 7.81
d, J = 7.7 Hz, 1H), 7.75 (d, J = 7.9 Hz, 1H), 7.68 – 7.58 (m, 5H), 7.04 (dd, J = 5.5, 2.8 Hz, 3H), 6.91 (d,
J = 9.0 Hz, 1H), 6.74 (d, J = 8.5 Hz, 2H), 6.69 (dd, J = 9.0, 2.4 Hz, 1H), 5.05 (s, 2H), 4.44 (q, J = 8.4 Hz,
(
1
3
1
H), 3.73 (s, 3H), 2.89 (dd, J = 13.7, 4.6 Hz, 1H), 2.71 – 2.63 (m, 1H), 2.09 (s, 3H). C NMR (100
MHz, DMSO-d ) δ 171.79, 170.23, 168.24, 157.11, 155.96, 147.60, 139.42, 139.26, 138.04, 135.57,
34.61, 133.45, 132.73, 132.67, 132.08, 131.58, 131.34, 131.19, 130.71, 130.57, 130.18, 129.47, 129.41,
6
1
1
25.52, 119.15, 114.89, 114.67, 114.46, 111.90, 102.16, 68.34, 55.82, 55.21, 35.96, 30.95, 13.71. HRMS
-
(
AP-ESI) m/z Calcd for C42
H
2
33Cl N
5
O
9
S [M-H] 852.1298. Found:852.1292.
5
1
.1.5.13.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-((2-cyanobenzyl)oxy)phenyl)propenamide (8n) light yellow solid.
1
Yield:55%, mp: 150-155℃. H NMR (400 MHz, DMSO-d
6
) δ 12.94 (s, 1H), 8.54 (s, 1H), 8.44 (d, J
=
6.8 Hz, 1H), 8.13 (d, J = 8.0 Hz, 1H), 7.94 (dd, J = 21.7, 8.0 Hz, 2H), 7.65 (dtt, J = 26.8, 18.2, 7.3 Hz,
7
5
H), 7.17 – 6.97 (m, 3H), 6.92 (d, J = 8.9 Hz, 1H), 6.79 (d, J = 8.0 Hz, 2H), 6.69 (d, J = 8.2 Hz, 1H),
.14 (s, 2H), 4.45 (s, 1H), 3.74 (s, 3H), 3.49 (q, J = 15.0 Hz, 2H), 2.90 (d, J = 10.0 Hz, 1H), 2.76 – 2.63
13
(
m, 1H), 2.10 (s, 3H). C NMR (100 MHz, DMSO-d
6
) δ 171.78, 170.27, 168.24, 157.17, 155.97,
1
1
5
47.60, 140.48, 139.42, 138.03, 135.58, 134.63, 133.89, 133.74, 133.45, 132.65, 131.58, 131.20, 130.75,
30.58, 129.87, 129.67, 129.46, 125.53, 117.63, 114.90, 114.69, 114.48, 111.93, 111.61, 102.17, 67.83,
-
5.82, 55.23, 35.95, 30.93, 13.71. HRMS (AP-ESI) m/z Calcd for C42
H
2
33Cl N
5
O
9
S [M-H] 852.1298.
Found:852.1311.
5.1.5.14.(S)-N-((4-bromophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-
indol-3-yl)acetamido)-3-(4-((4-nitrobenzyl)oxy)phenyl)propenamide (8o) pink solid. Yield:18%,
1
mp: 228-230℃. H NMR (400 MHz, DMSO-d
6
) δ 12.58 (s, 1H), 8.25 (d, J = 8.6 Hz, 3H), 7.80 (s,
4
H), 7.67 (d, J = 8.5 Hz, 2H), 7.62 (d, J = 9.1 Hz, 4H), 7.03 (dd, J = 12.3, 5.1 Hz, 3H), 6.92 (d, J = 9.0
Hz, 1H), 6.78 – 6.66 (m, 3H), 5.16 (s, 2H), 4.49 – 4.40 (m, 1H), 3.73 (s, 3H), 3.53 – 3.41 (m, 2H), 2.85
1
3
(
dd, J = 13.6, 4.4 Hz, 1H), 2.64 (dd, J = 13.5, 9.8 Hz, 1H), 2.07 (s, 3H). C NMR (100 MHz, DMSO-
6
d ) δ 171.78, 170.78, 170.24, 168.23, 157.05, 155.98, 147.58, 147.41, 145.48, 139.48, 138.07, 135.58,
1
34.61, 133.44, 132.67, 131.56, 131.20, 130.74, 130.60, 129.45, 128.48, 125.50, 124.02, 114.87, 114.71,
114.45, 111.90, 102.20, 68.37, 60.22, 55.82, 55.22, 35.97, 30.98, 21.20, 14.53, 13.68. HRMS (AP-ESI)
-
m/z Calcd for C41
H
34BrClN
4
O S [M-H] 873.0824. Found:873.0824.
9
5
1
.1.5.15.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-((4-nitrobenzyl)oxy)phenyl)propenamide (8p) light yellow solid.
1
Yield:13%, mp: 182-186℃. H NMR (400 MHz, DMSO-d
6
) δ 12.94 (s, 1H), 8.55 (d, J = 2.2 Hz, 1H),
8
7
.43 (d, J = 7.6 Hz, 1H), 8.14 (dd, J = 8.5, 2.3 Hz, 1H), 7.97 (d, J = 8.5 Hz, 1H), 7.69 – 7.58 (m, 4H),
.39 (q, J = 7.3 Hz, 4H), 7.10 – 7.01 (m, 3H), 6.93 (d, J = 8.9 Hz, 1H), 6.74 (d, J = 8.3 Hz, 2H), 6.70 (dd,
2 / 27
2

J = 9.0, 2.5 Hz, 1H), 4.98 (s, 2H), 4.47 (td, J = 8.1, 7.5, 4.9 Hz, 1H), 3.50 (q, J = 15.4 Hz, 2H), 2.90 (dd,
1
3
J = 13.9, 4.8 Hz, 1H), 2.68 (dd, J = 13.8, 9.4 Hz, 1H), 2.10 (s, 3H). C NMR (100 MHz, DMSO-d
6
) δ
71.77, 170.24, 168.25, 157.49, 155.98, 147.60, 139.39, 138.05, 137.51, 135.59, 134.63, 133.46, 132.67,
31.58, 131.21, 130.65, 130.59, 129.47, 129.04, 128.88, 128.27, 128.06, 125.54, 114.90, 114.64, 114.47,
11.92, 102.18, 69.52, 55.82, 55.23, 35.97, 30.95, 13.71. HRMS (AP-ESI) m/z Calcd for
1
1
1
-
C
41
H
33Cl
2
N O11S [M-H] 872.1196. Found:872.1194.
5
5
1
.1.5.16.(S)-N-((4-chloro-3-nitrophenyl)sulfonyl)-2-(2-(1-(4-chlorobenzoyl)-5-methoxy-2-methyl-
H-indol-3-yl)acetamido)-3-(4-(naphthalen-2-ylmethoxy)phenyl)propenamide (8q) light yellow
1
solid. Yield:34%, mp: 197-199℃. H NMR (400 MHz, DMSO-d
6
) δ 12.95 (s, 1H), 8.54 (d, J = 2.1
Hz, 1H), 8.41 (d, J = 7.2 Hz, 1H), 8.13 (dd, J = 8.5, 2.1 Hz, 1H), 7.94 (td, J = 11.4, 10.4, 6.6 Hz, 5H),
.67 – 7.58 (m, 4H), 7.53 (dd, J = 8.5, 3.5 Hz, 3H), 7.10 – 7.00 (m, 3H), 6.91 (d, J = 9.0 Hz, 1H), 6.78
d, J = 8.5 Hz, 2H), 6.69 (dd, J = 9.0, 2.4 Hz, 1H), 5.15 (s, 2H), 4.44 (t, J = 10.7 Hz, 1H), 3.73 (s, 3H),
7
(
1
3
3
.56 – 3.41 (m, 2H), 2.88 (dd, J = 13.6, 4.6 Hz, 1H), 2.74 – 2.62 (m, 1H), 2.09 (s, 3H). C NMR (100
) δ 171.83, 170.21, 168.25, 157.50, 155.98, 147.60, 138.04, 135.58, 135.16, 134.63,
33.44, 133.23, 132.99, 132.67, 131.58, 131.21, 131.15, 130.68, 130.59, 129.47, 129.13, 128.52, 128.23,
MHz, DMSO-d
6
1
1
3
8
28.08, 126.79, 126.59, 126.08, 125.52, 114.89, 114.73, 114.48, 111.93, 102.19, 69.66, 55.83, 55.23,
-
5.99, 30.95, 13.72. HRMS (AP-ESI) m/z Calcd for C45
H
36Cl
2
N
4
O
9
S [M-H] 877.1502. Found:
77.1509.
5.2. In vitro binding assay for Bcl-2 proteins
A Bid-BH3 peptide marked with 5-carboxyfluorescein succinimidyl ester (5-FAM-
QEDIIRNIARHLAQVGDSMDRSIPPG) could bind to Bcl-2 family proteins and, meanwhile,
generate high polarization values (milipolarization units, mP). When the test compounds compete
with the marked Bid-BH3 peptide to bind the Bcl-2 family protein, the mP values decrease. The
IC50 values are calculated by a formula related to mP values and corresponding concentrations. The
K
i
values are calculated by a formula related to the concentrations of Bid-BH3 peptide and Bcl-2
protein, the IC50 values and the K
d
values.23,24
5.3. MTT assay
The three cancer cells (PC-3, Jurkat and K562 cells) were cultured in RPMI 1640 medium which
contains 10% FBS at 37℃in 5% CO humidified incubator. Cells were plated in a 96-well plate at
000-4000 cells per well. After culturing for 8h, different concentrations of the compounds were
2
2
added and cultured for 48h. Then 10 µLof 0.5% MTT solution was added to each well and incubated
for another 4h. After aspirating the culture medium out, the generated formazan was extracted by
200 µL DMSO. After mixing and dissolving for 15 min, the absorbance was read by a microtiter-
plate reader at 570 nm and the IC50 values were calculated according to the corresponding
concentrations and the inhibitory ratios.
5.4. Molecular docking
2
3 / 27

Molecular docking studies of compound 8h were performed using Surflex-Dock program in
Sybyl-X2.1. Compound 8h were assigned with Gasteiger-Hückel charges and other parameters were
set as default values. The Bcl-2 protein was downloaded from the Protein Data Bank (PDB code:
4MAN). The top-scored docking poses of 8h were selected for analysis.
5.5. Analysis of cell apoptosis
5
Jurkat cells were plated in 12-well plate at 2 × 10 cells per well and treated with 20 µM and 40
µM of 8j for 48 h. After washing with PBS, cells harvested and treated with annexin-V FITC and
propidium iodide (PI) using Annexin V-FITC Apoptosis Detection Kit (Beyotime). The percentage
of cells apoptosis was evaluated by flow cytometry.
Acknowledgments
Thanks for the binding assay for Bcl-2 family proteins by Prof. Renxiao Wang from Shanghai
Institute of Organic Chemistry in China. This work was supported by National Natural Science
Foundation of China (Grant No. 21672127), Key Research and Development Project of Shandong
Province (Grant No. 2017CXGC1401) and the Fundamental Research Funds of Shandong
University (Grant No. 2019GN045).
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7
Graphic abstract
Design, synthesis and preliminary bioactivity studies of indomethacin derivatives
as Bcl-2/Mcl-1 dual inhibitors
Chen Chen, Yiming Nie, Guangsen Xu, Xinying Yang, Hao Fang, Xuben Hou*
2
7 / 27