Improved binding affinities of pyrrolidine derivatives as Mcl-1 inhibitors by modifying amino acid side chains

Article abstract of DOI:10.1016/j.bmc.2016.10.020

As an important member of anti-apoptotic Bcl-2 protein, myeloid cell leukemia sequence 1 (Mcl-1) protein is an attractive target for cancer therapy. In this study, a new series of pyrrolidine derivatives as Mcl-1 inhibitors were developed by mainly modifying the amino acid side chain of compound 1. Among them, compound 18 (K_i= 0.077 μM) exhibited better potent inhibitory activities towards Mcl-1 protein compared to positive control Gossypol (K_i= 0.18 μM). In addition, compound 40 possessed good antiproliferative activities against PC-3 cells (K_i= 8.45 μM), which was the same as positive control Gossypol (K_i= 7.54 μM).

Full text of DOI:10.1016/j.bmc.2016.10.020

Bioorganic & Medicinal Chemistry xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Improved binding affinities of pyrrolidine derivatives as Mcl-1
inhibitors by modifying amino acid side chains
⇑
Yichao Wan, Tingting Liu, Xiaoxian Li, Chen Chen, 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
a r t i c l e i n f o
a b s t r a c t
Article history:
As an important member of anti-apoptotic Bcl-2 protein, myeloid cell leukemia sequence 1 (Mcl-1)
protein is an attractive target for cancer therapy. In this study, a new series of pyrrolidine derivatives
as Mcl-1 inhibitors were developed by mainly modifying the amino acid side chain of compound 1.
Received 1 September 2016
Revised 16 October 2016
Accepted 17 October 2016
Available online xxxx
Among them, compound 18 (Ki = 0.077 lM) exhibited better potent inhibitory activities towards Mcl-1
protein compared to positive control Gossypol (Ki = 0.18
lM). In addition, compound 40 possessed good
antiproliferative activities against PC-3 cells (Ki = 8.45
Gossypol (Ki = 7.54 lM).
l
M), which was the same as positive control
Keywords:
Bcl-2
Mcl-1
Ó 2016 Elsevier Ltd. All rights reserved.
Cancer
Pyrrolidine
Target
1. Introduction
Among these anti-apoptotic proteins, Mcl-1 protein is one of
the overexpressed proteins that are present in cancer cells.6 And
its distinct role in tumorgenesis and development has been proved
in many Mcl-1 transgenic mice models.7 And the specific downreg-
ulation of Mcl-1 by certain gene silencing approaches can induce
some cancer cell types to apoptosis.8–11 Consequently, suppressing
the function of Mcl-1 protein may be an attractive approach for
cancer therapy.
As we all know, apoptosis which can be characterized by some
morphological changes, such as cells shrink, DNA cleavage, is crit-
ical for embryogenesis, tissue homeostasis and defense against
pathogens.1 However, deregulation of apoptosis contributes to
tumorgenesis, autoimmune and degenerative diseases.2 Therefore,
a better understanding of its regulation in normal and tumor cells
will lay a foundation for developing more potent and selective
anti-tumor agents.
The B-cell lymphoma-2 (Bcl-2) family proteins are essential
regulators in the regulation of the intrinsic apoptotic pathway,
including anti-apoptotic and pro-apoptotic proteins. Anti-apop-
totic (pro-survival) proteins such as Bcl-2, Bcl-XL and Mcl-1 could
inhibit the activity of pro-apoptotic members by binding the BH3
domain of pro-apoptotic members (Bax, Bak and BH3-only pro-
teins).3 The BH3-only proteins such as Bim, Bid and Noxa, are an
important subgroup of pro-apoptotic members, which could neu-
tralize the inhibitory activity of anti-apoptotic proteins and also
directly contributes to the activation of Bax and Bak.4 The relative
ratio of anti-apoptotic and pro-apoptotic members is in a dynamic
equilibrium in normal cells. However, cancer cells can evade
apoptosis by overexpressing one or more anti-apoptotic proteins.5
In previous study, based on the lead compound WL-276 and
scaffold hopping strategy, we designed compound 1 which had a
new scaffold, pyrrolidine. It displayed the best inhibitory activity
to Mcl-1 protein, with a Ki value of 0.53 l
M.12 However, the inhi-
bitory activity for Mcl-1 protein should be improved compared
to positive Gossypol and other reported lead compounds. Given
that the interfaces between the anti-apoptotic and pro-apoptotic
proteins are large and flexible, it is more difficult to target than
enzyme/substrate interactions that are involved in smaller and
more defined active sites.13 Therefore, in order to enhance the
binding affinity for Mcl-1 protein, we used many non-classical
amino acids obtaining by etherification and arylation on the aryl
ring of L-4-Br phenylalanine to undertake a new round of struc-
tural modifications. In addition, to explore if the Boc protecting
group on the pyrrolidine ring was essential for activities, we
removed the Boc protecting group to synthesize some derivatives
(Fig. 1). Herein, we will introduce synthesis, preliminary bioactiv-
ity and structure–activity relationships of the new pyrrolidine
derivatives as Mcl-1 inhibitors in detail.
⇑
Corresponding author. Tel.: +86 531 88382731; fax: +86 531 88382548.
E-mail address: haofangcn@sdu.edu.cn (H. Fang).
http://dx.doi.org/10.1016/j.bmc.2016.10.020
0968-0896/Ó 2016 Elsevier Ltd. All rights reserved.
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2
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
by the methods which were previously reported.12 Then com-
R2
Br
pounds 1, 10–12 were removed the protecting group of Boc to yield
the target compounds 48–51.
O
O
S
O
O
O
S
O
R3
NO2
Cl
N
N
2.2. Binding assay to Mcl-1 protein
H
NH
H
NH
O
N
O
N
R4
Boc
The binding affinities for Mcl-1 protein of these pyrrolidine
derivatives were examined by using fluorescence polarization
assays (FPAs). We modified compound 1 by four parts, that is,
amino acid side chain, biphenyl group, sulfonamide moiety and
Boc protecting group on the scaffold of pyrrolidine. The results
were summarized in Table 1. According to the results of binding
assays, some important SAR information could be described as
the following: (1) when there were no substituents on R1 and R3
position, the activities for Mcl-1 protein were poor except com-
pound 33, such as 13, 19–20. (2) when there were bulky sub-
stituents on R2 position, compounds with no substituents on R1
position displayed better inhibitory activities, such as 18, 28. (3)
When there were ether bonds on R2 position and 3-NO2-4-Cl group
on R3 position, the binding affinities for Mcl-1 protein of com-
pounds with Cl group on R1 position were better except com-
pounds 30, 40. However, when the ether bonds were removed
and directly replaced by aryl groups, the activities were reduced,
such as 43, 45. The ether may afford a suitable change in the direc-
tion of the aryl ring of L-4-Br phenylalanine, which was a possible
explanation for the activities. (4) When we removed the Boc pro-
tecting group, the binding affinities for Mcl-1 protein were reduced
significantly, such as 48–50. The results indicated that the Boc
group was necessary for binding affinities. It was that the Boc
O
R1
O
1
Figure 1. The design of target compounds.
2. Results and discussion
2.1. Chemistry
Schemes 1 and 2 showed the synthetic route of the pyrrolidine
derivatives as Mcl-1 inhibitors. -Tyrosine was as the starting
material to synthesis intermediate 2 by amino and carboxyl pro-
tections. Then intermediate 2 was reacted with different halohy-
drocarbons by Williamson reaction and hydrolyzed by the NaOH
L
solution to obtain compounds 4a–4h. In addition, L-tyrosine was
successively protected by Cu2+ cation, reacted with different sub-
stituted benzyl bromides and protected by Boc group to yield com-
pounds 4i–4k. Compound 4k and N-Boc-4-Br-phenylanine were
treated by protection of carboxyl group and Suzuki reaction to
get intermediate 4l–4n. The target compounds 1, 10–47 were got
OR'
OH
O
O
b
a
OH
O
HN
HN
2
Boc
4a-4h
Boc
O
OH
O
CH2Br
OH
HN
O
Boc
4i
OH
O
NH2
NO2
O
O2N
CH2Br
c
OH
HN
Boc
4j
O
O
Br
CH2Br
R''
Br
d
OCH3
O
O
Br
OH
Boc
4n
OH
Boc
HN
HN
4k
e
O
O
OH
Boc
OH
Boc
HN
HN
4l-4m
3
R'=n-Pr,i-Pr,i-Bu,Hex,Ph-(CH2)2,Naph-2-CH2,4-NO2-Ph,TBDMS;
R''= Ph, 4-OCH3-Ph;
Scheme 1. Reagents and conditions: (a) (i) MeOH, CH3COCl; (ii) (Boc)2O, Et3N, CH2Cl2; (b) (i) K2CO3, DMSO, 30 °C, overnight or imidazole, TBDMSCl, DMF, 0 °C–rt; (ii) 1 M
NaOH, THF, 60–80 °C, 1.5 h; (c) (i) CuSO4ꢀ5H2O, NaOH, EDTAꢀ2Na, CH3OH, H2O; (ii) (Boc)2O, Et3N, CH2Cl2; (d–e) (i) MeOH, CH3COCl; (ii) (Boc)2O, Et3N, CH2Cl2; (iii) Pd(OAc)2,
Na2CO3, DMF, H2O, TBAB.
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Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
3
O
OH
Boc
R1
OH
N
R2
R2
5a-5c
f
O
O
O
O
O
O
O
S
OH
O
O O
S
Boc
e
R1
N
N
H
N
H
HN
O
NH
O
N
NH
8a-8c
R3
R3
OH
OH
Boc
7
h
6
HN
i
R2
R2
O
O
g
O
O
O
O
R1
R1
S
N
OH
H
NH
NH
Boc
Boc
R3
1,10-47
48-51
9a-9u
3,4a-4o
R1= H, CH3, Cl
R2= H, Br, Ph, 4-OCH3-Ph,n-Pr-O,i-Pr-O,i-Bu-O,Hex-O,Ph-(CH2)2-O,Naph-2-CH2-O,
4-NO2-Ph-O,TBDMS-O,Bn-O,4-Br-Bn-O,4-NO -Bn-O,4-(4-OCH3-Ph)-Bn-O
2
R3=H, 4-NO2,3-NO2-4-Cl
Scheme 2. Reagents and conditions: (e) (i) MeOH, CH3COCl; (ii) (Boc)2O, Et3N,CH2Cl2; (f) (i) PPh3, DEAD, various biphenols, THF, rt, 12 h; (ii) 1 M NaOH, THF, rt, overnight; (g)
HATU, DIEA, DCM, 0 °C–rt; (h) (i) ethyl acetate saturated with HCl, rt, overnight; ii. NMM, isobutyl chloroformate, NaH, different benzenesulfonamides, ꢁ20 °C to rt, 4 h; (i)
ethyl acetate saturated with HCl, rt, overnight, saturated NaHCO3.
group may display certain interactions with some amino acid resi-
dues or make the whole scaffold enrich the active pocket of Mcl-1
protein better. Among these pyrrolidine derivatives, compound 18
had the most potent inhibitory activities for Mcl-1 protein
(Ki = 77 nM) which displayed approximately 10 times more potent
against Bcl-XL protein. Therefore, other four compounds could be
potent Bcl-2/ Mcl-1 inhibitors.
2.5. Cell antiproliferative assay
than compound 1 (Ki = 0.53
the positive control Gossypol (Ki = 0.18
l
M) and over two times potent than
M). As we all know, the
As aberrant expression of anti-apoptotic Bcl-2 proteins could be
observed in many human malignancies, such as breast cancer,
prostate and lemkemia,14,15 three human cancer cells including
MDA-MB-231 (breast cancer cell), PC-3 (prostatic cancer cell)
and K562 (chronic myelogenous leukemia cell) were selected to
test their antiproliferative activities of compounds 18, 27, 34, 36,
40 by using MTT assay and Gossypol was as the reference com-
pound. The antiproliferative activities were tabulated as IC50 values
in Table 3. The data in Table 3 showed that all of them had poorer
inhibitory activities against MDA-MB-231 and K562 cells com-
pared to Gossypol. To our delight, they could inhibit the growth
of PC-3 cells effectively. Especially, compound 40 displayed similar
antiproliferative activities against PC-3 cells with Gossypol.
l
interactions between proteins and proteins involved large and
flexible interfaces. The structure of compound 18 was bigger than
compound 1 so that it may occupied more space of Mcl-1 protein
active pocket, which maybe a reason for the improved inhibitory
activities for Mcl-1 protein.
2.3. Docking study
To further understand the interactions between these com-
pounds and Mcl-1 protein, we chose the most potent compound
18 to dock with Mcl-1 protein in the active pocket by using Sur-
flex-Dock in silico. As shown in Figure 2, biphenyl group of com-
pound 18 could mimetic the
a-helix of Bim protein (Fig. 2a).
3. Conclusions
Acyl-sulfonyl group could form three hydrogen bonds with
Arg263 and Trp261. In addition, the Boc protecting group on pyrro-
lidine ring could form another one hydrogen bond with Thr266.
The result may be a reason for that the Boc protection group was
essential for binding affinities for Mcl-1 protein (Fig. 2b).
In brief, through mainly extending the aryl ring of amino acid
side chain on compound 1, we had designed and synthesized a
new series of pyrrolidine derivatives as Mcl-1 inhibitors. Some of
them displayed better binding affinities for Mcl-1 protein than
compound 1. Moreover, the most active compound 18 had a Ki
value of 77 nM, which was over two times potent than the positive
control Gossypol. In addition, compound 40 could effectively inhi-
bit the growth of PC-3 cells. Notably, we also found that the Boc
protection group of pyrrolidine scaffold was essential for binding
for Mcl-1 protein. These data may be significance of guidance to
find more potent Mcl-1 inhibitor.
2.4. Binding assays for other anti-apoptotic Bcl-2 proteins
In order to explore whether the new series of pyrrolidine
derivatives also could bind to other anti-apoptotic Bcl-2 proteins,
five active compounds (18, 27, 34, 36, 40) were also chosen to eval-
uate their inhibitory activities against Bcl-XL and Bcl-2 proteins by
FPAs. As demonstrated in Table 2, these compounds exhibited good
binding affinities for Bcl-2 and Mcl-1 proteins almost as the same
as the positive control Gossypol. However these compounds dis-
played better potency in inhibiting Mcl-1 protein than the other
two proteins, especially compound 18. To our surprise, these com-
pounds had no inhibitory activities against Bcl-XL protein except
18, whereas Gossypol displayed certain degree inhibitory potency
4. Experiment section
4.1. General chemistry information
All chemicals and solvent were purchased from commercial
suppliers and used directly unless other stated. Thin-layer
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4
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
Table 1
The binding affinities of pyrrolidine derivatives for Mcl-1 protein
R2
O
O
O
S
N
H
O
N
NH
R3
R4
O
R1
R3
Compd
R1
R2
R4
Mcl-1 Ki (l
M)a
1
–H
–CH3
–Cl
–CH3
–H
–CH3
–H
–CH3
–Cl
–H
–H
–H
–Br
–Br
–Br
–H
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
–H
4-NO2
4-NO2
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
–H
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–Boc
–H
0.53 0.09
0.63 0.20
0.58 0.07
0.56 0.18
4.0 0.67
0.90 0.11
4.7 1.1
0.74 0.09
0.72 0.14
0.077 0.0042
8.1 2.1
2.0 0.021
0.50 0.04
0.71 0.08
0.79 0.15
0.43 0.10
0.78 0.14
0.41 0.03
0.26 0.02
0.34 0.05
0.50 0.07
>10
0.54 0.05
0.78 0.18
0.59 0.15
0.22 0.017
0.80 0.27
0.32 0.03
0.46 0.05
0.33 0.03
0.58 0.06
0.30 0.09
0.68 0.06
0.77 0.10
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
Gossypol
n-Pr-O
n-Pr-O
n-Pr-O
n-Pr-O
n-Pr-O
TBDMS-O
i-Pr-O
i-Bu-O
i-Bu-O
i-Bu-O
i-Bu-O
Hex-O
Hex-O
Ph-(CH2)2-O
Ph-(CH2)2-O
Naph-2-CH2-O
Naph-2-CH2-O
Naph-2-CH2-O
4-NO2-Ph-O
Bn-O
Bn-O
Bn-O
Bn-O
–H
–Cl
3-NO2-4-Cl
3-NO2-4-Cl
4-NO2
–CH3
–CH3
–Cl
–CH3
–CH3
–Cl
–H
–CH3
–Cl
–Cl
–H
–H
–Cl
–CH3
–H
–CH3
–Cl
–H
–CH3
–Cl
–CH3
–Cl
–CH3
–Cl
–CH3
–Cl
–H
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
–H
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
3-NO2-4-Cl
4-Br-Bn-O
4-Br-Bn-O
4-Br-Bn-O
4-NO2-Bn-O
4-NO2-Bn-O
4-NO2-Bn-O
Ph
Ph
4-OCH3-Ph
4-OCH3-Ph
4-(4-OCH3-Ph)-Bn-O
0.50 0.03
>10
0.56 0.08
0.48 0.04
N.A.b
4-(4-OCH3-Ph)-Bn-O
–Br
–Br
–Br
–H
–CH3
–Cl
–CH3
–H
–H
–H
N.A.b
N.A.b
1.3 0.26
0.18 0.01
a
Each value is the result of three separate experiments.
No activity.
b
chromatography (TLC) was undertook on 0.25 mm silica gel
plates (60 GF-254) to monitor the completion of all the reactions
and visualize the spots under UV light (254 nM or 365 nM) or
iodine vapor. All the melting points were determined on an elec-
trothermal melting point apparatus without correction. ESI-MS
were measured on an Aglient-1100 series LC/MSD trap spectrom-
eter. HRMS spectra were performed on an Agilent 6510 Quadru-
pole Time-of-Flight LC/MS deliver. 1H NMR and 13C NMR spectra
were measured with a Bruker DRX spectrometer (600 MHz) and a
Bruker Avance spectrometer (300 MHz or 400 MHz). Chemical
shifts are given in parts per million (ppm) with tetramethylsilane
(TMS) as an internal standard, and coupling constants (J values)
are given in hertz (Hz). The splitting patterns were assigned as
s (singlet), d (doublet), dd (doublet doublet), t (triplet), q (quar-
tet), m (multiplet), and br s (broad singlet). All products were
purified by recrystallization or column chromatography (silica
gel 200–300 mesh). The following yields were generated after
purification.
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Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
5
Figure 2. (a) Compound 18 docked with Mcl-1 protein complexed with Bim protein in ribbon. (b) Surface representations of compound 18 in the hydrophobic groove of
Mcl-1 protein.
Table 2
The binding affinities of representative pyrrolidine derivatives for three Bcl-2 proteins
4.1.2. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-propoxyphenyl)
propanoic acid (4a)
Compd
Bcl-XL
Ki (
M)a
Bcl-2
Ki (
M)a
Mcl-1
Ki (
M)a
To a solution of compound 2 (4.43 g, 15 mmol) and K2CO3
(4.14 g, 30 mmol) in 80 mL DMSO, 1-iodopropane (4.37 mL,
45 mmol) was added into the mixture slowly. After stirred over-
night at 30 °C, 200 mL H2O was added into the solution and
extracted with EtOAc. The combined EtOAc layer was washed by
1 N NaOH solution and brine. The organic layer was dried over
anhydrous MgSO4 and concentrated to get yellow oil. Then the yel-
low oil was dissolved in 60 mL THF and 60 mL 1 N NaOH solution.
After stirred for 1.5 h in 60–80 °C, the solvent was removed under
reduced pressure. The residue was acidified with 1 N HCl until pH
2–3 and then extracted with EtOAc. The organic layer was dried
over anhydrous MgSO4 and concentrated to give crude residue.
The residue was purified by silica gel chromatography (petroleum
ether–ethyl acetate = 20:1, 0.2% HOAc) to generate 2.72 g com-
pound 8a as a white powder. Yield: 56%, mp: 76–78 °C.1H NMR
(DMSO-d6, 300 MHz), d 12.54 (s, 1H), 7.15 (d, J = 8.7 Hz, 2H), 7.05
(d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.7 Hz, 2H), 4.06–3.95 (m, 1H), 3.90
(t, J = 6.3 Hz, 2H), 2.96–2.90 (m, 1H), 2.78–2.70 (m, 1H), 1.76–
1.64 (m, 2H), 1.32 (s, 9H), 0.99 (t, J = 7.5 Hz, 3H).
l
l
l
18
27
34
36
40
Gossypol
1.3 0.65
N.A.b
0.93 0.07
0.67 0.08
0.51 0.031
0.68 0.04
0.75 0.09
0.45 0.03
0.077 0.0042
0.26 0.02
0.22 0.017
0.32 0.03
0.30 0.09
0.18 0.01
N.A.b
N.A.b
N.A.b
1.4 0.54
a
Each value was reproduced in three independent assays and expressed with
standard deviations.
b
No activity.
4.1.1. (S)-Methyl-2-((tert-butoxycarbonyl)amino)-3-(4-hydroxy-
phenyl)propanoate (2)
In an ice bath, acetyl chloride (21.32 mL, 300 mmol) was added
into 200 mL anhydrous methanol slowly. After stirred for 30 min,
-tyrosine (18.1 g, 100 mmol) was added into the solution. Then
the solution was refluxed for 4 h in an oil bath and the solvent
was removed under reduced pressure. The residue was added
some acetone and filtered to give 22.78 g of L-tyrosine methyl ester
hydrochloride as a white powder, yield: 99%. Di-tert-butyl dicar-
bonate (11.34 g, 52 mmol) in 50 mL 1,4-dioxane was slowly added
L
Compounds 4b–4h were synthesized following the procedure
described above.
into the solution of L-tyrosine methyl ester hydrochloride (9.26 g,
4.1.2.1.
(S)-2-((tert-Butoxycarbonyl)amino)-3-(4-isopropoxy-
40 mmol), K2CO3 (8.28 g, 60 mmol) in 160 mL mixture solvent
(1,4-dioxane–H2O = 1:1). After stirred overnight at room tempera-
ture, the reaction mixture was concentrated under reduced pres-
sure and EtOAc was added. The EtOAc layer was washed by 1 N
citric acid, saturated NaHCO3 and brine. The organic layer was
dried over anhydrous MgSO4 and concentrated to give compound
2 as a white powder. It could be directly used in the further steps
without purification.
phenyl)propanoic acid (4b).
White powder, yield: 51%, mp:
88–90 °C. 1H NMR (DMSO-d6, 300 MHz), d 12.55 (br s, 1H), 7.14
(d, J = 8.4 Hz, 2H), 7.05 (d, J = 8.1 Hz, 1H), 6.82 (d, J = 8.4 Hz, 2H),
4.60–4.48 (m, 1H), 4.07–3.96 (m, 1H), 2.96–2.90 (m, 1H), 2.77–
2.69 (m, 1H), 1.32 (s, 9H), 1.24 (d, J = 6.0 Hz, 6H).
4.1.2.2.
henyl)propanoic acid (4c).
(S)-2-((tert-Butoxycarbonyl)amino)-3-(4-isobutoxyp-
White powder, yield: 29%, mp: 93–
95 °C. 1H NMR (DMSO-d6, 300 MHz), d 12.54 (br s, 1H), 7.15 (d,
J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 1H), 6.84 (d, J = 8.4 Hz, 2H), 4.07–
4.00 (m, 1H), 3.70 (d, J = 6.6 Hz, 2H), 2.97–2.91 (m, 1H), 2.79–2.71
(m, 1H), 2.05–1.91 (m, 1H), 1.33 (s, 9H), 0.97 (d, J = 6.6 Hz, 6H).
Table 3
Antiproliferative activities of representative compounds
Compd
IC50
(l
M)a
MDA-MB-231
PC-3
K562
4.1.2.3. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-(hexyloxy)phe-
18
27
34
36
40
Gossypol
23.4 0.80
25.2 1.3
20.6 2.8
21.3 2.5
16.9 0.54
9.23 1.2
10.2 2.4
13.2 1.3
9.06 0.46
10.6 1.4
8.45 1.8
7.54 0.28
17.3 1.2
17.8 2.1
17.0 2.1
16.4 2.5
16.6 2.4
5.60 0.29
nyl)propanoic acid (4d).
White powder, yield: 46%, mp: 74–
76 °C. 1H NMR (CDCl3, 400 MHz), d 9.54 (br s, 1H), 7.09 (d,
J = 8.0 Hz, 2H), 6.84 (d, J = 8.0 Hz, 2H), 4.93 (d, J = 8.0 Hz, 1H),
4.61–4.55 (m, 1H), 3.94 (t, J = 6.4 Hz, 2H), 3.14–3.00 (m, 2H),
1.80–1.73 (m, 2H), 1.42 (s, 9H), 1.35–1.33 (m, 6H), 0.92 (t,
J = 6.8 Hz, 3H).
a
Each value was reproduced in three independent assays and expressed with
standard deviations.
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6
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.2.4. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-phenethoxy-
4.1.3.2. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-((4-nitroben-
phenyl)propanoic acid (4e).
White powder, yield: 29%, mp:
zyl)oxy)phenyl)propanoic acid (4k).
Pale yellow powder,
101–103 °C. 1H NMR (CDCl3, 400 MHz), d 7.33–7.28 (m, 4H),
7.23–7.22 (m, 1H), 7.09 (d, J = 8.4 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H),
4.91 (d, J = 8.0 Hz, 1H), 4.55–4.53 (m, 1H), 4.16 (t, J = 7.2 Hz, 1H),
3.14–3.04 (m, 4H), 1.42 (s, 9H).
yield: 33%, mp: 82–84 °C. 1H NMR (CDCl3, 400 MHz), d 8.24 (d,
J = 8.8 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.13 (d, J = 8.4 Hz, 2H),
6.90 (d, J = 8.8 Hz, 2H), 5.14 (s, 2H), 4.96 (d, J = 7.2 Hz, 1H), 4.56
(d, J = 4.8 Hz, 1H), 3.25–3.02 (m, 2H), 1.41 (s, 9H).
4.1.2.5. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-(naphthalen-
4.1.3.3.
amino)propanoic acid (4l).
(S)-3-([1,10-Biphenyl]-4-yl)-2-((tert-butoxycarbonyl)
N-Boc-4-Br-phenylanine was
2-ylmethoxy)phenyl)propanoic acid (4f).
White powder,
yield: 61%, mp: 145–147 °C. 1H NMR (CDCl3, 400 MHz), d 7.85–
7.81 (m, 4H), 7.52–7.46 (m, 3H), 7.12 (d, J = 8.0 Hz, 2H), 6.97 (d,
J = 8.0 Hz, 2H), 5.19 (s, 2H), 4.93 (d, J = 8.0 Hz, 1H), 4.57 (d,
J = 6.0 Hz, 1H), 3.15–3.02 (m, 2H), 1.42 (s, 9H).
the starting material to obtain N-Boc-4-Br-phenylanine methyl
ester by the procedure of compound 2. N-Boc-4-Br-phenylanine
methyl ester (3.58 g, 10 mmol), phenyl boronic acid (1.46 g,
12 mmol), Na2CO3 (4.24 g, 40 mmol), Pd(OAc)2 (0.03 g, 0.1 mmol),
TBAB (3.22 g, 10 mmol) were added into 60 mL mixture solvent
(DMF–H2O = 1:1). After stirred overnight at 80 °C, lots of water
was added into the reaction solution and extracted with EtOAc.
The EtOAc layer was washed by 1 N citric acid and brine. The
organic layer was dried over anhydrous MgSO4 and concentrated
to give the crude product. Finally, it was purified by silica gel chro-
matography (petroleum ether–ethyl acetate = 6:1, 0.2% HOAc) to
generate 1.6 g compound 4l as a white solid. Yield: 47%, mp:
126–128 °C. 1H NMR (CDCl3, 400 MHz), d 7.60–7.55 (m, 4H),
7.45–7.41 (m, 2H), 7.36–7.34 (m, 1H), 7.32–7.27 (m, 2H), 4.98 (d,
J = 7.6 Hz, 1H), 4.66 (d, J = 6.0 Hz, 1H), 3.27–3.11 (m, 2H), 1.42 (s,
9H).
4.1.2.6.
(S)-2-((tert-Butoxycarbonyl)amino)-3-(4-(4-nitrophe-
noxy)phenyl)propanoic acid (4g).
White powder, yield:
36%, mp: 125–127 °C. 1H NMR (CDCl3, 400 MHz), d 8.23 (d,
J = 9.2 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 7.06 (d, J = 8.0 Hz, 2H),
7.04 (d, J = 9.2 Hz, 2H), 5.02 (d, J = 8.0 Hz, 1H), 4.66–4.64 (m, 1H),
3.29–3.24 (m, 1H), 3.12–3.07 (m, 1H), 1.45 (s, 9H).
4.1.2.7. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-((tert-butyldi-
methylsilyl)oxy)phenyl)propanoic acid (4h).
Colorless oily
liquid, yield: 65%. 1H NMR (CDCl3, 300 MHz), d 7.04 (d, J = 8.4 Hz,
2H), 6.77 (d, J = 8.4 Hz, 2H), 4.91 (d, J = 7.8 Hz, 1H), 4.56–4.37 (m,
1H), 3.16–2.91 (m, 2H), 1.42 (s, 9H), 0.97 (s, 9H), 0.18 (s, 6H).
Compounds 4m–4n were synthesized following the procedure
described above.
4.1.3. (S)-3-(4-(Benzyloxy)phenyl)-2-((tert-butoxycarbonyl)
amino)propanoic acid (4i)
4.1.3.4.
[1,10-biphenyl]-4-yl)propanoic acid (4m).
(S)-2-((tert-Butoxycarbonyl)amino)-3-(40-methoxy-
To a solution of
L-tyrosine (12.5 g, 69 mmol) in 2 N NaOH solu-
White powder,
tion (69 mL), 1 N copper sulfate solution (34.5 mL) was added and
heated at 60 °C for 1.5 h. Then 200 mL methanol and benzyl bro-
mide (9.1 mL, 75.9 mmol) were added into the reaction mixture.
After stirred vigorously for 3 h at 60 °C, the reaction mixture was
cooled to room temperature. The precipitate was filtered and
washed by proper amount of water, methanol and ether to get blue
solid. Then the solid was added into the solution of 55.77 g/L
EDTAꢀ2Na solution (368 mL). After stirred overnight at 60 °C, the
reaction mass was cooled to room temperature. The precipitate
was filtered and washed by water to give 24.3 g pale white solid.
Di-tert-butyl dicarbonate (7.9 g, 36 mmol) was slowly added into
a solution of 13.6 g pale white and Et3N (12.5 mL, 90 mmol) in
200 mL mixed solvent (1,4-dioxane–H2O = 2:1) at 0 °C. After stirred
overnight at room temperature, the reaction mixture was concen-
trated under reduced pressure and acidified with HCl solution until
pH 2–3. The mixture was extracted with EtOAc. The combined
EtOAc was washed by brine and dried over anhydrous MgSO4. After
the solvent was concentrated to give white oil, the obtained resi-
due was purified by silica gel chromatography (petroleum ether–
ethyl acetate = 8:1, 0.2% HOAc) to generate 6.5 g compound 4i as
a white powder. Yield: 46%, mp: 112–114 °C. 1H NMR (DMSO-d6,
300 MHz), d 12.52 (s, 1H), 7.45–7.30 (m, 5H), 7.17 (d, J = 8.7 Hz,
2H), 7.08 (d, J = 8.1 Hz, 1H), 6.93 (d, J = 8.7 Hz, 2H), 5.06 (s, 2H),
4.06–3.98 (m, 1H), 2.96–2.90 (m, 1H), 2.78–2.70 (m, 1H), 1.32 (s,
9H).
yield: 64%, mp: 158–160 °C. 1H NMR (CDCl3, 400 MHz), d 7.44–
7.41 (m, 4H), 7.19 (d, J = 8.0 Hz, 2H), 6.91 (d, J = 8.8 Hz, 2H), 4.90
(d, J = 7.6 Hz, 1H), 4.66 (d, J = 6.4 Hz, 1H), 3.78 (s, 3H), 3.18–2.90
(m, 2H), 1.36 (s, 9H).
4.1.3.5. (S)-2-((tert-Butoxycarbonyl)amino)-3-(4-((40-methoxy-
[1,10-biphenyl]-4-yl)methoxy)phenyl)propanoic
acid
(4n).
White powder, yield: 84%, mp: 174–176 °C. 1H NMR
(CDCl3, 400 MHz), d 7.56–7.51 (m, 4H), 7.47 (d, J = 8.4 Hz, 2H),
7.12 (d, J = 8.4 Hz, 2H), 6.98–6.93 (m, 4H), 5.06 (s, 2H), 4.93 (d,
J = 7.6 Hz, 1H), 4.58 (d, J = 6.0 Hz, 1H), 3.15 (s, 3H), 3.16–3.04 (m,
2H), 1.42 (s, 9H).
4.1.4. (2S,4R)-1-tert-Butyl-2-methyl4-hydroxypyrrolidine-1,2-
dicarboxylate (7)
Compound 7 was synthesized following the procedure of com-
pound 2. White powder, yield: 58%, mp: 92–93 °C. 1H NMR (CDCl3,
400 MHz), d 4.49–4.38 (m, 2H), 3.73 (s, 3H), 3.66–3.44 (m, 2H),
2.33–2.19 (m, 2H), 2.10–2.04 (m, 2H), 1.42 (s, 9H).
4.1.5. (2S,4S)-4-([1,10-Biphenyl]-4-yloxy)-1-(tert-butoxycarbonyl)
pyrrolidine-2-carboxylic acid (8a)
In an ice bath, DEAD (0.65 mL, 4 mmol) in 10 mL anhydrous THF
was added slowly into the solution of compound 7 (1.18 g,
4.8 mmol), [1,10-biphenyl]-4-ol (0.68 g, 4 mmol), PPh3 (1.05 g,
4 mmol) and Et3N (0.55 mL, 4 mmol) in 20 mL anhydrous THF for
1 h. After stirred overnight at room temperature, the reaction mix-
ture was concentrated under reduced pressure and extracted with
EtOAc. The EtOAc layer was washed by 1 N NaOH, 1 N citric acid
and brine. Then the organic layer was dried over anhydrous MgSO4
and concentrated to give white crude residue. The residue was
purified by silica gel chromatography (petroleum ether–ethyl acet-
ate = 10:1) to generate 1.1 g white powder. The solution of 1.1 g
white powder in 11 mL THF was treated with 11 mL 1 N NaOH.
Compounds 4j–4k were synthesized following the procedure
described above.
4.1.3.1. (S)-3-(4-((4-Bromobenzyl)oxy)phenyl)-2-((tert-butoxy-
carbonyl)amino)propanoic acid (4j).
White powder, yield:
44%, mp: 116–117 °C. 1H NMR (CDCl3, 400 MHz), d 7.51 (d,
J = 8.4 Hz, 2H), 7.38 (d, J = 8.4 Hz, 2H), 7.11 (d, J = 8.4 Hz, 2H),
7.00 (d, J = 8.4 Hz, 2H), 5.00 (s, 2H), 4.92–4.90 (m, 1H), 4.57–4.55
(m, 1H), 3.15–3.01 (m, 2H), 1.42 (s, 9H).
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Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
7
After stirred for 1.5 h in 60–80 °C, the solvent was removed under
reduced pressure. The residue was acidified with 1 N HCl until pH
2–3 and extracted with EtOAc. The organic layer was dried over
anhydrous MgSO4 and concentrated to give crude residue. Finally,
the residue was purified by recrystallized from methanol/water to
give 0.6 g compound 8a as a white needle solid. Yield: 40%, mp:
158–160 °C. 1H NMR (DMSO-d6, 400 MHz), d 12.52 (s, 1H), 7.61
(d, J = 7.1 Hz, 2H), 7.59 (d, J = 8.4 Hz, 2H), 7.45 (t, J = 7.5 Hz, 2H),
7.33 (t, J = 7.3 Hz, 1H), 6.98–6.94 (m, 2H), 5.07–5.06 (m, 1H),
4.32–4.26 (m, 1H), 3.79–3.71 (m, 1H), 3.47–3.42 (m, 1H), 2.62–
2.58 (m, 1H), 2.24 (d, J = 13.6 Hz, 1H), 1.42 (s, 9H), ESI-MS m/z:
382.5 (MꢁH)ꢁ.
J = 7.5 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H),
4.81 (d, J = 6.6 Hz, 1H), 4.22 (br s, 1H), 3.90 (t, J = 6.6 Hz, 2H),
3.00–2.87 (m, 2H), 1.85–1.76 (m, 2H), 1.39 (s, 9H), 0.99 (t,
J = 7.2 Hz, 3H).
4.1.6.3. (S)-tert-Butyl(1-(4-nitrophenylsulfonamido)-1-oxo-3-
(4-propoxyphenyl)propan-2-yl) carbamate (9d).
low powder, yield: 61%, mp: 246–248 °C. 1H NMR (CDCl3,
400 MHz), 9.49 (s, 1H), 8.38 (d, J = 8.8 Hz, 2H), 8.23 (d,
Pale yel-
d
J = 8.8 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.78 (d, J = 8.4 Hz, 2H),
4.83 (d, J = 6.8 Hz, 1H), 4.20 (d, J = 6.8 Hz, 1H), 3.89 (t, J = 6.5 Hz,
2H), 3.00–2.88 (m, 2H), 1.82–1.77 (m, 2H), 1.40 (s, 9H), 1.05 (t,
J = 8.1 Hz, 3H).
Compounds 8b–8c were synthesized following the procedure
described above.
4.1.6.4. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
4.1.5.1.
biphenyl]-4-yl)oxy)pyrrolidine-2-carboxylic acid (8b).
(2S,4S)-1-(tert-Butoxycarbonyl)-4-((40-methyl-[1,10-
White
1-oxo-3-(4-propoxyphenyl)propan-2-yl)carbamate (9e).
Pale
yellow powder, yield: 32%, mp: 136–137 °C. 1H NMR (CDCl3,
400 MHz), d 9.58 (s, 1H), 8.50 (d, J = 2.1 Hz, 1H), 8.20 (dd,
J1 = 8.4 Hz, J2 = 2.1 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 6.98 (d,
J = 8.4 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 4.87 (d, J = 6.9 Hz, 1H),
4.24–4.17 (m, 1H), 3.90 (t, J = 6.6 Hz, 2H), 3.02–2.87 (m, 2H),
1.86–1.74 (m, 2H), 1.41 (s, 9H), 1.06 (t, J = 7.2 Hz, 3H).
flake solid, yield: 33%, mp: 152–154 °C. 1H NMR (DMSO-d6,
400 MHz), d 12.49 (s, 1H), 7.57 (d, J = 8.6 Hz, 2H), 7.51 (d,
J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.95–6.92 (m, 2H), 5.05–
5.04 (m, 1H), 4.31–4.26 (m, 1H), 3.75–3.70 (m, 1H), 3.46–3.41
(m, 1H), 2.64–2.53 (m, 1H), 2.32 (s, 3H), 2.24 (d, J = 13.6 Hz, 1H),
1.41 (s, 9H), ESI-MS m/z: 396.4 [MꢁH]ꢁ.
4.1.6.5. (S)-tert-Butyl(3-(4-isopropoxyphenyl)-1-oxo-1-(phenyl-
4.1.5.2.
biphenyl]-4-yl)oxy)pyrrolidine-2-carboxylic acid (8c).
(2S,4S)-1-(tert-Butoxycarbonyl)-4-((40-chloro-[1,10-
White
sulfonamido)propan-2-yl)carbamate (9f).
White powder,
yield: 43%, mp: 133–134 °C. 1H NMR (DMSO-d6, 400 MHz), d
12.35 (s, 1H), 7.92 (d, J = 7.8 Hz, 2H), 7.74–7.59 (m, 3H), 7.10 (d,
J = 8.4 Hz, 2H), 7.03 (d, J = 8.1 Hz, 1H), 6.77 (d, J = 8.4 Hz, 2H),
4.60–4.48 (m, 1H), 4.13–4.06 (m, 1H), 2.77–2.72 (m, 1H), 2.55–
2.51 (m, 1H), 1.32 (s, 9H), 1.24 (d, J = 6.0 Hz, 6H).
flake solid, yield: 37%, mp: 162–164 °C. 1H NMR (DMSO-d6,
400 MHz), d 12.55 (s, 1H), 7.65 (d, J = 8.6 Hz, 2H), 7.61 (d,
J = 8.7 Hz, 2H), 7.49 (d, J = 8.6 Hz, 2H), 6.97–6.94 (m, 2H), 5.07–
5.06 (m, 1H), 4.32–4.26 (m, 1H), 3.78–3.71 (m, 1H), 3.46–3.41
(m, 1H), 2.65–2.55 (m, 1H), 2.23 (d, J = 13.2 Hz, 1H), 1.41 (s, 9H),
ESI-MS m/z: 416.5 [MꢁH]ꢁ.
4.1.6.6. (S)-tert-Butyl(3-(4-isobutoxyphenyl)-1-oxo-1-(phenyl-
sulfonamido)propan-2-yl)carbamate (9g).
White powder,
4.1.6. (S)-tert-Butyl(3-(4-bromophenyl)-1-(4-chloro-3-nitrophe-
nylsulfonamido)-1-oxopropan-2-yl)carbamate (9a)
yield: 36%, mp: 122–124 °C. 1H NMR (CDCl3, 400 MHz), d 9.27 (s,
1H), 8.06 (d, J = 7.6 Hz, 2H), 7.68–7.64 (m, 1H), 7.57–7.53 (m,
2H), 6.93 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz, 2H), 4.88 (d,
J = 7.2 Hz, 1H), 4.26 (s, 1H), 3.68 (d, J = 6.4 Hz, 2H), 2.98–2.89 (m,
2H), 2.09–2.01 (m, 1H), 1.39 (s, 9H), 1.02 (d, J = 6.8 Hz, 6H).
The solution of compound 3 (3.4 g, 10 mmol) in 60 mL anhy-
drous DCM was reacted with 4-chloro-3-nitrobenzenesulfonamide
(2.6 g, 11 mmol), HATU (4.56 g, 12 mmol) and ethyldiisopropy-
lamine (2.58 g, 20 mmol) and stirred at room temperature for
6 h. Solvent was removed under reduced pressure. The reaction
mixture was extracted with EtOAc. The combined organic phases
were washed with 1 N citric acid and brine, dried over MgSO4, fil-
tered and concentrated to generate the yellow oil. Finally, it was
purified by silica gel chromatography (petroleum ether–ethyl acet-
ate = 6:1, 0.2% HOAc) to generate 4.1 g compound 9a as a white
powder. Yield: 73%, mp: 144–146 °C. 1H NMR (CDCl3, 400 MHz),
d 9.82 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H), 8.17–8.15 (m, 1H), 7.76 (d,
J = 8.4 Hz, 1H), 7.39 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 8.4 Hz, 2H),
4.92 (br s, 1H), 4.26–4.25 (m, 1H), 3.06–3.01 (m, 1H), 2.91–2.85
(m, 1H), 1.40 (s, 9H).
4.1.6.7. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-3-
(4-isobutoxyphenyl)-1-oxopropan-2-yl)carbamate (9h). White
powder, yield: 51%, mp: 100–102 °C. 1H NMR (CDCl3, 400 MHz), d
9.65 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.17 (dd, J1 = 8.4 Hz,
J2 = 2.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 6.98 (d, J = 8.4 Hz, 2H),
6.79 (d, J = 8.4 Hz, 2H), 4.88 (d, J = 6.8 Hz, 1H), 4.21 (d, J = 6.8 Hz,
1H), 3.69 (d, J = 6.8 Hz, 2H), 2.97–2.88 (m, 2H), 2.08–2.02 (m, 1H),
1.40 (s, 9H), 1.03 (d, J = 6.8 Hz, 6H).
4.1.6.8. (S)-tert-Butyl(3-(4-isobutoxyphenyl)-1-(4-nitrophenyl-
sulfonamido)-1-oxopropan-2-yl)carbamate
(9i).
White
Compounds 9b–9t were synthesized following the procedure
described above.
powder, yield: 41%, mp: 172–174 °C. 1H NMR (CDCl3, 400 MHz),
d 9.56 (s, 1H), 8.38 (d, J = 8.8 Hz, 2H), 8.23 (d, J = 8.8 Hz, 2H), 6.97
(d, J = 8.0 Hz, 2H), 6.78 (d, J = 8.0 Hz, 2H), 4.87 (d, J = 6.8 Hz, 1H),
4.22 (d, J = 6.4 Hz, 1H), 3.68 (d, J = 6.8 Hz, 2H), 2.99–2.87 (m, 2H),
2.12–2.02 (m, 1H), 1.41 (s, 9H), 1.03 (d, J = 6.8 Hz, 6H).
4.1.6.1. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
1-oxo-3-phenylpropan-2-yl)carbamate (9b).
Pale yellow
powder, yield: 98%, mp: 132–134 °C. 1H NMR (CDCl3, 400 MHz),
d 9.56 (s, 1H), 8.49 (d, J = 2.4 Hz, 1H), 8.21–8.18 (m, 1H), 7.75 (d,
J = 8.4 Hz, 1H), 7.30–7.27 (m, 2H), 7.24–7.22 (m, 1H), 7.08–7.07
(m, 2H), 4.87–4.83 (m, 1H), 4.29–4.20 (m, 1H), 3.10–3.05 (m,
1H), 2.99–2.93 (m, 1H), 1.40 (s, 9H).
4.1.6.9. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(4-(hexyloxy)phenyl)-1-oxopropan-2-yl)carbamate
(9j).
Pale yellow powder, yield: 89%, mp: 98–100 °C. 1H NMR
(CDCl3, 400 MHz), d 9.59 (s, 1H), 8.50 (d, J = 2.4 Hz, 1H), 8.20 (dd,
J1 = 8.4 Hz, J2 = 2.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 6.98 (d,
J = 8.0 Hz, 2H), 6.79 (d, J = 8.0 Hz, 2H), 4.86 (d, J = 6.8 Hz, 1H),
4.27–4.14 (m, 1H), 3.93 (d, J = 6.4 Hz, 2H), 3.01–2.89 (m, 2H),
1.80–1.73 (m, 2H), 1.45–1.43 (m, 2H), 1.41 (s, 9H), 1.35–1.25 (m,
4H), 0.92 (t, J = 6.8 Hz, 3H).
4.1.6.2. (S)-tert-Butyl(1-oxo-1-(phenylsulfonamido)-3-(4-pro-
poxyphenyl)propan-2-yl)carbamate (9c).
White powder,
yield: 51%, mp: 108–110 °C. 1H NMR (CDCl3, 300 MHz), d 9.16 (s,
1H), 8.06 (d, J = 7.5 Hz, 2H), 7.69 (t, J = 7.5 Hz, 1H), 7.58 (t,
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

8
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
!4.1.6.10. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
1-oxo-3-(4-phenethoxyphenyl)propan-2-yl)carbamate (9k).
White powder, yield: 73%, mp: 141–143 °C. 1H NMR (CDCl3,
400 MHz), 9.68 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.17 (dd,
J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 7.71 (d, J = 8.8 Hz, 1H), 7.34–7.27 (m,
4H), 7.24–7.22 (m, 1H), 6.96 (d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz,
2H), 4.89 (br s, 1H), 4.22 (d, J = 6.0 Hz, 1H), 4.15 (t, J = 7.2 Hz, 2H),
3.12 (t, J = 7.2 Hz, 2H), 2.99–2.86 (m, 2H), 1.40 (s, 9H).
(t, J = 7.2 Hz, 1H), 7.10 (d, J = 8.0 Hz, 2H), 4.84 (d, J = 6.8 Hz, 1H),
4.27–4.19 (m, 1H), 3.08–3.03 (m, 1H), 2.96–2.90 (m, 1H), 1.34 (s, 9H).
d
4.1.6.18. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(40-methoxy-[1,10-biphenyl]-4-yl)-1-oxopropan-2-yl)carba-
mate (9s).
Pale yellow powder, yield: 48%, mp: 86–88 °C.
1H NMR (CDCl3, 400 MHz), d 9.71 (s, 1H), 8.52 (d, J = 2.0 Hz,
1H), 8.19 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.72 (d, J = 8.4 Hz,
1H), 7.50–7.44 (m, 4H), 7.13 (d, J = 8.0 Hz, 2H), 6.98 (d,
J = 8.8 Hz, 2H), 4.90 (d, J = 6.8 Hz, 1H), 4.29 (d, J = 6.8 Hz, 1H),
3.85 (s, 3H), 3.12–3.07 (m, 1H), 3.01–2.96 (m, 1H), 1.40 (s, 9H).
4.1.6.11. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(4-(naphthalen-2-ylmethoxy)phenyl)-1-oxopropan-2-yl)car-
bamate (9l).
Pale yellow powder, yield: 38%, mp: 104–105 °C.
1H NMR (CDCl3, 400 MHz), d 9.78 (s, 1H), 8.49 (s, 1H), 8.17 (d,
J = 8.4 Hz, 1H), 7.86–7.82 (m, 4H), 7.70 (d, J = 8.4 Hz, 1H), 7.53–
7.47 (m, 3H), 6.98 (d, J = 8.0 Hz, 2H), 6.90 (d, J = 8.0 Hz, 2H), 5.18 (s,
2H), 4.93 (br s, 1H), 4.23 (br s, 1H), 3.00–2.85 (m, 2H), 1.39 (s, 9H).
4.1.6.19. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(4-((40-methoxy-[1,10-biphenyl]-4-yl)methoxy)phenyl)-1-oxo-
propan-2-yl)carbamate (9t).
Pale yellow powder, yield: 43%,
mp: 181–183 °C. 1H NMR (CDCl3, 400 MHz), d 9.60 (s, 1H), 8.50 (s,
1H), 8.19 (d, J = 8.4 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.58–7.52 (m,
4H), 7.48 (d, J = 8.0 Hz, 2H), 7.01–6.97 (m, 4H), 6.90 (d, J = 8.0 Hz,
2H), 5.06 (s, 2H), 4.87 (d, J = 6.8 Hz, 1H), 4.24–4.21 (m, 1H), 3.85
(s, 3H), 3.02–2.89 (m, 2H), 1.40 (s, 9H).
4.1.6.12. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(4-(4-nitrophenoxy)phenyl)-1-oxopropan-2-yl)carbamate
(9m).
Pale yellow powder, yield: 49%, mp: 116–118 °C. 1H
NMR (CDCl3, 400 MHz), d 9.66 (s, 1H), 8.52 (d, J = 2.4 Hz, 1H),
8.22–8.19 (m, 3H), 7.76 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 8.4 Hz,
2H), 7.05–6.99 (m, 4H), 4.91 (d, J = 6.4 Hz, 1H), 4.32–4.28 (m,
1H), 3.15–3.10 (m, 1H), 3.01–2.95 (m, 1H), 1.42 (s, 9H).
4.1.6.20. (S)-tert-Butyl-(3-(4-((tert-butyldimethylsilyl)oxy)phe-
nyl)-1-(4-chloro-3-nitrobenzamido)-1-oxopropan-2-yl)carba-
mate (9u).
Pale yellow oily liquid, yield: 78%. 1H NMR
(CDCl3, 600 MHz), d 9.76 (s, 1H), 8.51 (s, 1H), 8.16 (d, J = 7.8 Hz,
1H), 7.23 (d, J = 8.4 Hz, 1H), 6.92 (d, J = 7.8 Hz, 2H), 6.71 (d,
J = 7.8 Hz, 2H), 4.90 (s, 1H), 4.23 (s, 1H), 2.99–2.97 (m, 1H),
2.90–2.86 (m, 1H), 1.40 (s, 9H), 0.97 (s, 9H), 0.18 (s, 6H).
4.1.6.13. (S)-tert-Butyl(3-(4-(benzyloxy)phenyl)-1-(4-chloro-3-
nitrophenylsulfonamido)-1-oxopropan-2-yl)carbamate
(9n).
Pale yellow powder, yield: 87%, mp: 212–214 °C. 1H
NMR (DMSO-d6, 300 MHz), d 8.32 (d, J = 1.8 Hz, 1H), 8.00 (dd,
J1 = 8.4 Hz, J2 = 1.8 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H), 7.45–7.29 (m,
5H), 6.87 (d, J = 8.4 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 5.85 (d,
J = 7.5 Hz, 1H), 5.02 (s, 2H), 3.85–3.79 (m, 1H), 2.92–2.86 (m, 1H),
2.73–2.66 (m, 1H), 1.31 (s, 9H).
4.1.7. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-3-(4-bromo-
phenyl)-1-(4-chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-
yl)carbamoyl)pyrrolidine-1-carboxylate (1)
Compound 9a (0.68 g, 1.2 mmol) was dissolved in 20 mL EtOAc
saturated with HCl gas and stirred overnight at room temperature.
Then the generated hydrochloride precipitate was collected and
used for the further step. N-methylmorpholine (0.22 g, 2.2 mmol)
and isobutyl chloroformate (0.16 g, 1.1 mmol) were added into a
solution of compound 8a (0.38 g, 1 mmol) in 10 mL anhydrous
THF at ꢁ20 °C. After stirred for 1 h, the hydrochloride precipitate
above was added and stirred overnight at room temperature. Then
THF was evaporated under reduced pressure and extracted by
EtOAc. The EtOAc layer was washed with 1 N citric acid, brine
and dried over anhydrous MgSO4. The concentrated residue was
purified by column chromatography (petroleum ether–ethyl acet-
ate = 3:1–2:1, 0.2% HOAc) to obtain the compound 1 as a white
powder. Yield: 41%, mp: 97–99 °C. 1H NMR (CDCl3, 600 MHz), d
10.34 (s, 1H), 8.52 (s, 1H), 8.18 (d, J = 7.7 Hz, 1H), 7.71 (d,
J = 8.1 Hz, 1H), 7.55–7.53 (m, 4H), 7.43 (t, J = 7.3 Hz, 2H), 7.33 (t,
J = 7.2 Hz, 1H), 7.28 (d, J = 7.0 Hz, 2H), 6.93 (d, J = 7.02 Hz, 2H),
6.86 (d, J = 8.6 Hz, 2H), 6.68 (d, J = 5.9 Hz, 1H), 4.90 (s, 1H), 4.73
(s, 1H), 4.41 (d, J = 10.4 Hz, 1H), 3.77 (d, J = 11.8 Hz, 1H), 3.68 (d,
J = 11.3 Hz, 1H), 3.27 (d, J = 11.0 Hz, 1H), 2.90 (d, J = 8.6 Hz, 1H),
2.58 (d, J = 13.9 Hz, 1H), 2.50–2.46 (m, 1H), 1.39 (s, 9H). 13C NMR
4.1.6.14.
(S)-tert-Butyl(3-(4-(benzyloxy)phenyl)-1-oxo-1-
(9o). White
(phenylsulfonamido)propan-2-yl)carbamate
powder, yield: 37%, mp: 140–142 °C. 1H NMR (CDCl3, 400 MHz),
d 9.25 (s, 1H), 8.05 (d, J = 7.6 Hz, 2H), 7.67–7.63 (m, 1H), 7.55–
7.51 (m, 2H), 7.43–7.31 (m, 5H), 6.95 (d, J = 8.4 Hz, 2H), 6.84 (d,
J = 8.4 Hz, 2H), 5.02 (s, 2H), 4.87 (d, J = 7.6 Hz, 1H), 4.26 (br s,
1H), 2.99–2.87 (m, 2H), 1.39 (s, 9H).
4.1.6.15. (S)-tert-Butyl(3-(4-((4-bromobenzyl)oxy)phenyl)-1-
(4-chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)car-
bamate (9p).
White powder, yield: 45%, mp: 146–148 °C.
1H NMR (CDCl3, 400 MHz), d 9.65 (s, 1H), 8.50 (d, J = 2.4 Hz,
1H), 8.20 (dd, J1 = 8.8 Hz, J2 = 2.4 Hz, 1H), 7.75 (d, J = 8.8 Hz,
1H), 7.52 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.8 Hz, 2H), 7.01 (d,
J = 8.0 Hz, 2H), 6.86 (d, J = 8.8 Hz, 2H), 4.99 (s, 2H), 4.83 (d,
J = 7.2 Hz, 1H), 4.21–4.19 (m, 1H), 3.02–2.89 (m, 2H), 1.40 (s,
9H).
4.1.6.16. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(4-((4-nitrobenzyl)oxy)phenyl)-1-oxopropan-2-yl)carbamate
(9q).
(CDCl3, 400 MHz), d 9.53 (s, 1H), 8.49 (d, J = 2.0 Hz, 1H), 8.26–
8.20 (m, 3H), 7.76 (d, J = 8.4 Hz, 1H), 7.62 (d, J = 8.8 Hz, 2H), 7.31
(d, J = 8.0 Hz, 2H), 7.01 (d, J = 8.0 Hz, 2H), 5.15 (s, 2H), 4.83 (d,
J = 6.8 Hz, 1H), 4.23–4.18 (m, 1H), 3.04–2.91 (m, 2H), 1.40 (s, 9H).
(CDCl3, 100 MHz), d 171.96, 170.04, 155.99, 147.54, 140.18,
138.69, 135.40, 133.42, 132.80, 132.55, 132.05, 131.08, 128.85,
128.69, 127.11, 126.77, 126.13, 121.95, 115.96, 82.80, 60.06,
Yellow powder, yield: 87%, mp: 142–144 °C. 1H NMR
53.36, 53.22, 36.30, 34.60, 28.09. HRMS (AP-ESI) m/z Calcd for C37
H
-
36BrClN4O9S [MꢁH]ꢁ 825.0997, found: 825.1006.
Compounds 10–46 were synthesized following the procedure
described above.
4.1.6.17.
(S)-tert-Butyl(3-([1,10-biphenyl]-4-yl)-1-(4-chloro-3-
nitrophenylsulfonamido)-1-oxopropan-2-yl)carbamate (9r). Pale
4.1.7.1. (2S,4S)-tert-Butyl-2-(((S)-3-(4-bromophenyl)-1-(4-chloro-
3-nitrophenylsulfonamido)-1-oxopropan-2-yl)carbamoyl)-4-
(4-(40-methyl)phenylphenoxy)pyrrolidine-1-carboxylate (10). Yel-
low powder, yield: 31%, mp: 111–113 °C. 1H NMR (CDCl3,
300 MHz), d 8.53 (s, 1H), 8.20 (d, J = 7.8 Hz, 1H), 7.72 (d, J = 8.1 Hz,
yellow powder, yield: 91%, mp: 104–106 °C. 1H NMR (CDCl3,
400 MHz),
d 9.61 (s, 1H), 8.46 (d, J = 2.0 Hz, 1H), 8.13 (dd,
J1 = 8.4 Hz, J2 = 2.4 Hz, 1H), 7.65 (d, J = 8.4 Hz, 1H), 7.49 (d,
J = 7.2 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.39 (t, J = 7.2 Hz, 2H), 7.30
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
9
1H), 7.53 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 7.8 Hz, 2H), 7.31 (m, 2H), 7.23
ESI) m/z Calcd for
726.2846.
C
40H45N3O8S [MꢁH]ꢁ 726.2849, found:
(m, 2H), 6.94 (d, J = 7.5 Hz, 2H), 6.84 (d, J = 8.4 Hz, 2H), 6.61 (m, 1H),
4.90 (s, 1H), 4.73–4.70 (m, 1H), 4.40 (d, J = 9.0 Hz, 1H), 3.77 (d,
J = 12.3 Hz, 1H), 3.68 (dd, J = 12.0 Hz, 3.0 Hz, 1H), 3.31 (d,
J = 13.2 Hz, 1H), 2.90 (dd, J = 13.8 Hz, 5.1 Hz, 1H), 2.60–2.47 (m, 2H),
2.39 (s, 3H), 1.39 (s, 9H).13C NMR (CDCl3, 100 MHz), d 171.75,
169.87, 155.77, 147.54, 138.70, 137.30, 136.89, 133.41, 132.79,
132.54, 132.04, 131.09, 130.91, 129.56, 128.85, 128.46, 126.60,
126.13, 121.94, 115.97, 82.82, 65.57, 60.06, 53.37, 53.22, 36.29,
30.59, 28.08, 21.06. HRMS (AP-ESI) m/z Calcd for C38H38BrClN4O9S
[MꢁH]ꢁ 839.1153, found: 839.1159.
4.1.7.5.
(2S,4S)-tert-Butyl4-((40-methyl-[1,10-biphenyl]-4-yl)
oxy)-2-(((S)-1-(4-nitrophenylsulfonamido)-1-oxo-3-(4-propox-
yphenyl)propan-2-yl)carbamoyl)pyrrolidine-1-carboxylate
(14).
Pale yellow powder, yield: 38%, mp: 88–90 °C. 1H NMR
(CDCl3, 400 MHz), d 10.11 (s, 1H), 8.37 (d, J = 8.6 Hz, 2H), 8.24 (d,
J = 8.6 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H),
7.24 (d, J = 8.0 Hz, 2H), 7.00 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.0 Hz,
2H), 6.72 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 7.3 Hz, 1H), 4.91 (s, 1H),
4.63 (br s, 1H), 4.40 (d, J = 9.6 Hz, 1H), 3.85–3.78 (m, 2H), 3.73–
3.67 (m, 2H), 3.35 (d, J = 11.6 Hz, 1H), 2.87–2.82 (m,1H), 2.53–
2.49 (m, 2H), 2.38 (s, 3H), 1.80–1.75 (m, 2H), 1.36 (s, 9H), 1.04 (t,
J = 7.4 Hz, 3H). 13C NMR (CDCl3, 100 MHz), d 175.45, 171.68,
170.57, 158.73, 155.82, 150.63, 144.39, 137.37, 136.83, 135.27,
130.43, 130.00, 129.54, 128.39, 126.58, 125.88, 123.88, 115.93,
114.92, 82.55, 69.39, 60.17, 53.55, 53.27, 35.99, 34.73, 28.10,
22.57, 21.04, 20.53, 10.53. HRMS (AP-ESI) m/z Calcd for C41H46N4-
4.1.7.2. ((2S,4S)-tert-Butyl-2-(((S)-3-(4-bromophenyl)-1-(4-
chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)car-
bamoyl)-4-(4-(40-chloro)phenylphenoxy)pyrrolidine-1-car-
boxylate (11).
Yellow powder, yield: 26%, mp: 125–
127 °C. 1H NMR (CDCl3, 600 MHz), d 10.26 (s, 1H), 8.53 (s,
1H), 8.20 (d, J = 7.2 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.51 (d,
J = 7.8 Hz, 2H), 7.47 (d, J = 7.8 Hz, 2H), 7.40 (d, J = 7.8 Hz, 2H),
7.30 (d, J = 7.2 Hz, 2H), 6.94 (d, J = 7.8 Hz, 2H), 6.85 (d,
J = 8.4 Hz, 2H), 6.64 (d, J = 7.2 Hz, 1H), 4.90 (s, 1H), 4.73 (s,
1H), 4.40 (d, J = 10.2 Hz, 1H), 3.77 (d, J = 11.4 Hz, 1H), 3.69 (d,
J = 11.4 Hz, 1H), 3.30 (d, J = 10.8 Hz, 1H), 2.90 (d, J = 13.8 Hz,
1H), 2.57 (d, J = 14.4 Hz, 1H), 2.50–2.47 (m, 1H), 1.40 (s,
O
10S [MꢁH]ꢁ 785.2856, found: 785.2860.
4.1.7.6. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-1-
(4-nitrophenylsulfonamido)-1-oxo-3-(4-propoxyphenyl)pro-
pan-2-yl)carbamoyl)pyrrolidine-1-carboxylate
(15).
Pale
9H).13
C
NMR (CDCl3, 100 MHz),
d
172.25, 169.51, 156.21,
yellow powder, yield: 23%, mp: 90–92 °C. 1H NMR (CDCl3,
400 MHz), d 10.12 (s, 1H), 8.36 (d, J = 8.6 Hz, 2H), 8.24 (d,
J = 8.6 Hz, 2H), 7.55–7.52 (m, 4H), 7.44 (t, J = 7.4 Hz, 2H), 7.34–
7.30 (m, 1H), 6.99 (d, J = 8.4 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H),
6.72–6.65 (m, 3H), 4.92 (s, 1H), 4.67–4.62 (m, 1H), 4.41 (dd,
J1 = 2.5 Hz, J2 = 9.9 Hz, 1H), 3.84–3.79 (m, 3H), 3.71–3.67 (m, 1H),
3.35 (d, J = 11.5 Hz, 1H), 2.87–2.83 (m, 1H), 2.57–2.49 (m, 2H),
1.78–1.76 (m, 2H), 1.36 (s, 9H), 1.04 (t, J = 7.4 Hz, 3H). 13C NMR
147.54, 138.64, 134.11, 133.41, 133.20, 133.08, 132.83,
132.74, 132.55, 132.04, 131.05, 128.98, 128.56, 127.99,
126.10, 121.95, 116.03, 82.84, 60.04, 53.35, 53.22, 36.32,
34.54, 28.31, 28.08. HRMS (AP-ESI) m/z Calcd for C37H35BrCl2-
N4O9S [MꢁH]ꢁ 859.0607, found: 859.0610.
4.1.7.3.
(2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenyl-
sulfonamido)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-4-(4-
(CDCl3, 100 MHz), d 171.64, 170.58, 158.74, 156.04, 150.64,
(40-methyl)phenylphenoxy)pyrrolidine-1-carboxylate
144.39, 140.25, 135.29, 130.43, 130.00, 128.82, 128.61, 127.07,
126.74, 125.87, 123.89, 115.93, 114.93, 82.59, 69.39, 60.18, 53.54,
53.27, 35.96, 34.75, 29.69, 28.10, 22.56, 10.53. HRMS (AP-ESI) m/
z Calcd for C40H44N4O10S [MꢁH]ꢁ 771.2700, found: 771.2708.
(12).
(CDCl3, 600 MHz),
Yellow powder, yield: 38%, mp: 90–92 °C. 1H NMR
d
10.21 (s, 1H), 8.46 (s, 1H), 8.23 (d,
J = 7.8 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.51 (d, J = 8.4 Hz, 2H),
7.43 (d, J = 7.8 Hz, 2H), 7.23 (m, 3H), 7.17 (t, J = 7.2 Hz, 2H), 7.04
(d, J = 7.2 Hz, 2H), 6.84 (d, J = 8.3 Hz, 2H), 6.64 (s, 1H), 4.90 (s,
1H), 4.70–4.74 (m, 1H), 4.39 (d, J = 8.4 Hz, 1H), 3.76 (d,
J = 12.6 Hz, 1H), 3.67 (dd, J = 12.6 Hz, 3.0 Hz, 1H), 3.37 (dd,
J = 13.8 Hz, 4.8 Hz, 1H), 2.93 (dd, J = 14.4 Hz, 5.4 Hz, 1H), 2.55
(d, J = 13.8 Hz, 1H), 2.48–2.51 (m, 1H), 2.39 (s, 3H), 1.35 (s,
4.1.7.7. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-1-oxo-3-(4-propoxyphenyl)propan-2-yl)carbamoyl)-
4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-carboxy-
late (16).
Pale yellow powder, yield: 44%, mp: 96–98 °C. 1H
NMR (CDCl3, 400 MHz), d 10.16 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H),
8.25 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.53 (d, J = 8.8 Hz,
2H), 7.44 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H), 6.95 (d,
J = 8.4 Hz, 2H), 6.88 (d, J = 8.4 Hz, 2H), 6.69–6.62 (m, 3H), 4.91 (s,
1H), 4.71–4.66 (m, 1H), 4.40–4.37 (m, 1H), 3.86–3.79 (m, 3H),
3.70–3.66 (m, 1H), 3.39–3.30 (m, 1H), 2.86 (dd, J1 = 5.2 Hz,
J2 = 14.0 Hz, 1H), 2.57–2.45 (m, 2H), 2.39 (s, 3H), 1.82–1.73 (m,
2H), 1.34 (s, 9H), 1.04 (d, J = 7.6 Hz, 3H). 13C NMR (CDCl3,
9H).13
C NMR (CDCl3, 100 MHz), d 171.59, 170.12, 155.81,
147.52, 138.80, 137.39, 136.83, 135.25, 134.46, 132.93, 132.69,
132.48, 130.90, 129.54, 129.34, 129.00, 128.40, 127.78, 126.59,
126.04, 115.89, 82.60, 65.56, 60.15, 53.29, 36.74, 34.74, 30.59,
28.14, 21.05. HRMS (AP-ESI) m/z Calcd for
C38H39ClN4O9S
[MꢁH]ꢁ 761.2048, found: 761.2052.
4.1.7.4. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-1-
100 MHz), d 171.41, 170.71, 158.79, 155.80, 147.47, 138.86,
oxo-1-(phenylsulfonamido)-3-(4-propoxyphenyl)propan-2-yl)-
137.38, 136.83, 135.26, 132.99, 132.68, 132.51, 130.39, 129.55,
128.42, 126.60, 126.10, 125.74, 115.90, 114.86, 82.61, 69.34,
60.16, 53.30, 35.95, 34.71, 29.71, 28.06, 22.58, 21.06, 10.56. HRMS
(AP-ESI) m/z Calcd for C41H45ClN4O10S [MꢁH]ꢁ 819.2467, found:
819.2474.
carbamoyl)pyrrolidine-1-carboxylate (13).
Pale yellow
powder, yield: 35%, mp: 94–96 °C. 1H NMR (CDCl3, 400 MHz), d
9.88 (s, 1H), 8.06 (d, J = 7.6 Hz, 2H), 7.64–7.62 (m, 1H), 7.54–7.52
(m, 6H), 7.44–7.40 (m, 2H), 7.33–7.30 (m, 1H), 6.93–6.89 (m,
4H), 6.67 (d, J = 8.2 Hz, 2H), 6.61 (br s, 1H), 4.91 (s, 1H), 4.70 (br
s, 1H), 4.43 (d, J = 9.0 Hz, 1H), 3.84–3.68 (m, 4H), 3.38 (d,
J = 13.0 Hz, 1H), 2.82–2.78 (m, 1H), 2.61 (d, J = 14.4 Hz, 1H), 2.49–
2.44 (m, 1H), 1.79–1.74 (m, 2H), 1.35 (s, 9H), 1.03 (t, J = 7.4 Hz,
3H). 13C NMR (CDCl3, 100 MHz), d 171.48, 170.28, 158.56, 156.15,
140.35, 138.94, 135.16, 133.61, 130.48, 128.79, 128.67,
128.58,126.99, 126.76, 126.16, 115.94, 114.85, 82.30, 69.33,
60.16, 53.25, 36.01, 34.72, 29.70, 28.11, 22.58, 10.56. HRMS (AP-
4.1.7.8. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-1-oxo-3-(4-propoxyphenyl)propan-2-yl)carbamoyl)-
4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-carboxy-
late (17).
Pale yellow powder, yield: 58%, mp: 104–106 °C. 1H
NMR (CDCl3, 400 MHz), d 10.14 (s, 1H), 8.50 (d, J = 2.0 Hz, 1H), 8.25
(d, J = 8.8 Hz, 1H), 7.74 (d, J = 8.8 Hz, 1H), 7.51 (d, J = 8.8 Hz, 2H),
7.47 (d, J = 8.4 Hz, 2H), 7.40 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 8.4 Hz,
2H), 6.89 (d, J = 8.8 Hz, 2H), 6.69–6.63 (m, 3H), 4.92 (s, 1H), 4.71–
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

10
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.66 (m, 1H), 4.40–4.38 (m, 1H), 3.86–3.78 (m, 3H), 3.72–3.69 (m,
1H), 3.36–3.30 (m, 1H), 2.85 (dd, J1 = 5.2 Hz, J2 = 14.0 Hz, 1H), 2.58–
2.45 (m, 2H), 1.82–1.73 (m, 2H), 1.35 (s, 9H), 1.04 (d, J = 7.6 Hz,
3H). 13C NMR (CDCl3, 100 MHz), d 171.40, 170.59, 158.78, 156.23,
155.92, 147.47, 138.83, 138.72, 133.96, 133.12, 132.98, 132.70,
132.50, 130.38, 128.96, 128.51, 127.98, 126.07, 125.71, 115.95,
114.84, 82.63, 69.35, 60.10, 53.29, 35.99, 34.59, 29.70, 28.05,
J = 2.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H),
7.51–7.45 (m, 4H), 7.40 (d, J = 8.4 Hz, 2H), 7.00–6.87 (m, 4H),
6.69–6.63 (m, 3H), 4.93 (s, 1H), 4.70–4.66 (m, 1H), 4.41–4.38
(m, 1H), 3.85–3.69 (m, 2H), 3.66–3.57 (m, 2H), 3.36–3.34 (m,
1H), 2.84 (dd, J1 = 5.2 Hz, J2 = 14.0 Hz, 1H), 2.59–2.46 (m, 2H),
2.09–1.99 (m, 1H), 1.34 (s, 9H), 1.02 (d, J = 6.8 Hz, 6H). 13C NMR
(CDCl3, 100 MHz), d 171.34, 170.60, 158.88, 156.23, 155.92,
147.47, 138.87, 138.72, 133.95, 133.12, 133.00, 132.70, 132.50,
130.38, 128.96, 128.53, 127.99, 126.07, 125.65, 115.94, 114.83,
82.64, 74.22, 60.09, 53.31, 35.99, 34.58, 29.71, 28.29, 28.05,
22.58, 10.55. HRMS (AP-ESI) m/z Calcd for
C40H42Cl2N4O10S
[MꢁH]ꢁ 839.1920, found: 839.1918.
4.1.7.9. (2S,4S)-tert-Butyl-4-([1,10-biphenyl]-4-yloxy)-2-(((S)-3-
(4-((tert-butyldimethylsilyl)oxy)phenyl)-1-(4-chloro-3-nitro-
phenylsulfonamido)-1-oxopropan-2-yl)carbamoyl)pyrrolidine-
19.27, 19.23. HRMS (AP-ESI) m/z Calcd for C41H44Cl2N4O10
S
[MꢁH]ꢁ 853.2077, found: 853.2076.
1-carboxylate (18).
Pale yellow powder, yield: 34%, mp: 102–
4.1.7.13. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-isobutoxyphenyl)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-
104 °C. 1H NMR (CDCl3, 600 MHz), d 10.2 (s, 1H), 8.52 (s, 1H), 8.21
(d, J = 7.8 Hz, 1H), 7.71 (d, J = 7.8 Hz, 1H), 7.55–7.53 (m, 4H), 7.42 (t,
J = 7.2 Hz, 2H), 7.32 (t, J = 7.2 Hz, 1H), 6.88–6.92 (m, 4H), 6.69 (d,
J = 6.6 Hz, 1H), 6.63 (d, J = 7.8 Hz, 2H), 4.93 (s, 1H), 4.70 (s, 1H),
4.39 (d, J = 9.6 Hz, 1H), 3.79 (d, J = 12.0 Hz, 1H), 3.70 (d,
J = 11.4 Hz, 1H), 3.32 (d, J = 12.0 Hz, 1H), 2.84 (d, J = 9.0 Hz, 1H),
2.57–2.55 (d, J = 13.8 Hz, 1H), 2.51–2.49 (m, 1H), 1.37 (s, 9H),
0.96 (s, 9H), 0.15–0.14 (m, 6H). 13C NMR (DMSO-d6, 100 MHz), d
171.48, 170.68, 156.42, 153.63, 153.22, 147.00, 139.63, 139.03,
132.80, 132.29, 130.50, 130.16, 128.92, 128.72, 127.67, 126.65,
126.08, 125.00, 119.22, 115.70, 78.95, 73.76, 58.70, 54.21, 51.24,
35.84, 27.86, 27.53, 25.40, 17.74, ꢁ4.73. HRMS (AP-ESI) m/z Calcd
for C43H51ClN4O10SSi [MꢁH]ꢁ 877.2705, Found: 877.2708.
1-carboxylate (22).
Pale yellow powder, yield: 42%, mp:
100–102 °C. 1H NMR (CDCl3, 400 MHz), d 10.14 (s, 1H), 8.51
(d, J = 2.0 Hz, 1H), 8.26 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz,
1H), 7.53 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.24 (d,
J = 8.0 Hz, 2H), 6.95 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.4 Hz, 2H),
6.69 (d, J = 8.8 Hz, 2H), 6.63 (d, J = 7.6 Hz, 1H), 4.91 (s, 1H),
4.71–4.64 (m, 1H), 4.40–4.37 (m, 1H), 3.82 (d, J = 12.0 Hz, 1H),
3.71–3.66 (m, 1H), 3.64–3.57 (m, 2H), 3.39–3.34 (m, 1H), 2.84
(dd, J1 = 5.2 Hz, J1 = 14.0 Hz, 1H), 2.57–2.45 (m, 2H), 2.39 (s,
3H), 2.09–2.01 (m, 1H), 1.33 (s, 9H), 1.02 (d, J = 6.8 Hz, 6H).
13C NMR (CDCl3, 100 MHz), d 171.33, 170.75, 158.89, 155.93,
155.79, 147.46, 138.85, 137.37, 136.84, 135.25, 133.02,
132.69, 132.51, 130.39, 129.55, 128.44, 126.61, 126.11,
125.66, 115.88, 114.85, 82.62, 74.20, 60.15, 53.32, 35.93,
34.70, 29.72, 28.29, 28.06, 21.08, 19.29, 19.25. HRMS (AP-ESI)
4.1.7.10. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-3-
(4-isopropoxyphenyl)-1-oxo-1-(phenylsulfonamido)propan-2-
yl)carbamoyl)pyrrolidine-1-carboxylate (19).
Pale yellow
powder, yield: 30%, mp: 93–95 °C. 1H NMR (CDCl3, 400 MHz), d
9.86 (s, 1H), 8.07 (d, J = 7.5 Hz, 2H), 7.66 (t, J = 7.3 Hz, 1H), 7.55–
7.53 (m, 6H), 7.44–7.41 (m, 2H), 7.34–7.30 (m, 1H), 6.91–6.89
(m, 4H), 6.64–6.62 (m, 3H), 4.91 (s, 1H), 4.72 (br s, 1H), 4.46 (m,
2H), 3.78–3.68 (m, 2H), 3.38 (d, J = 12.4 Hz, 1H), 2.81–2.77 (m,
1H), 2.61 (d, J = 14.2 Hz, 1H), 2.50–2.47 (m, 1H), 1.35 (s, 9H), 1.29
(d, J = 4.1 Hz, 6H). 13C NMR (CDCl3, 100 MHz), d 171.21, 170.28,
157.36, 156.16, 155.76, 140.37, 138.94, 135.15, 133.61, 130.53,
128.79, 128.67, 128.58, 126.98, 126.76, 126.09, 115.94, 82.30,
69.66, 60.16, 53.23, 36.02, 34.75, 29.65, 28.11, 22.06. HRMS (AP-
m/z Calcd for
833.2626.
C
42H47ClN4O10
S
[MꢁH]ꢁ 833.2623, found:
4.1.7.14. (2S,4S)-tert-Butyl-2-(((S)-3-(4-isobutoxyphenyl)-1-(4-
nitrophenylsulfonamido)-1-oxopropan-2-yl)carbamoyl)-4-((40-
methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-carboxylate
(23).
Pale yellow powder, yield: 53%, mp: 104–105 °C. 1H
NMR (CDCl3, 400 MHz), d 10.12 (s, 1H), 8.36 (d, J = 8.4 Hz, 2H),
8.24 (d, J = 8.4 Hz, 2H), 7.54 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz,
2H), 7.24 (d, J = 8.0 Hz, 2H), 6.99 (d, J = 8.0 Hz, 2H), 6.89 (d,
J = 8.4 Hz, 2H), 6.71–6.62 (m, 3H), 4.92 (s, 1H), 4.65 (br s, 1H),
4.40 (d, J = 10.0 Hz, 1H), 3.82 (d, J = 12.0 Hz, 1H), 3.71–3.58 (m,
3H), 3.39–3.29 (m, 1H), 2.86 (dd, J1 = 5.2 Hz, J2 = 14.0 Hz, 1H),
2.58–2.45 (m, 2H), 2.39 (s, 3H), 2.10–2.00 (m, 1H), 1.35 (s, 9H),
1.02 (d, J = 6.8 Hz, 6H). 13C NMR (CDCl3, 100 MHz), d 171.58,
170.62, 158.84, 155.81, 150.61, 144.38, 137.35, 136.85, 130.93,
130.45, 130.02, 129.55, 128.86, 128.42, 126.59, 125.76, 123.89,
115.89, 114.90, 82.58, 74.26, 65.58, 60.17, 53.30, 35.96, 34.70,
ESI) m/z Calcd for
726.2844.
C
40H45N3O8S [MꢁH]ꢁ 726.2849, found:
4.1.7.11. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-3-
(4-isobutoxyphenyl)-1-oxo-1-(phenylsulfonamido)propan-2-
yl)carbamoyl)pyrrolidine-1-carboxylate (20).
White pow-
der, yield: 20%, mp: 86–88 °C. 1H NMR (CDCl3, 400 MHz), d 9.82
(s, 1H), 8.07 (d, J = 7.7 Hz, 2H), 7.67–7.63 (m, 1H), 7.60–7.54 (m,
6H), 7.44 (t, J = 7.4 Hz, 2H), 7.34–7.32 (m, 1H), 6.93–6.90 (m, 4H),
6.67 (d, J = 8.2 Hz, 2H), 6.60 (d, J = 8.2 Hz, 1H), 4.92 (s, 1H), 4.70
(br s, 1H), 4.43 (d, J = 9.4 Hz, 1H), 3.80–3.66 (m, 2H), 3.65–3.58
(m, 2H), 3.41 (d, J = 12.2 Hz, 1H), 3.02 (br s,1H), 2.80–2.76 (m,
1H), 2.61 (d, J = 13.2 Hz, 1H), 2.50–2.48 (m, 1H), 1.34 (s, 9H), 1.02
(d, J = 6.7 Hz, 6H). 13C NMR (CDCl3, 100 MHz), d 171.18, 170.22,
158.67, 156.16, 155.75, 140.35, 138.96, 135.15, 133.61, 130.48,
128.79, 128.67, 128.59, 126.99, 126.77, 126.10, 115.93, 114.86,
82.28, 74.25, 60.16, 53.27, 36.02, 34.73, 28.28, 28.11, 19.27,
29.71, 28.29, 28.10, 21.06, 19.24. HRMS (AP-ESI) m/z Calcd for C42
H
-
48N4O10S [MꢁH]ꢁ 799.3013, found: 799.3015.
4.1.7.15. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-(hexyloxy)phenyl)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (24).
Pale yellow powder, yield: 47%, mp: 84–
10.14 (s, 1H), 8.50 (d,
86 °C. 1H NMR (CDCl3, 400 MHz),
d
J = 2.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H),
7.51–7.44 (m, 4H), 7.40 (d, J = 8.4 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H),
6.88 (d, J = 8.4 Hz, 2H), 6.69 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 7.6 Hz,
1H), 4.92 (s, 1H), 4.68–4.66 (m, 1H), 4.41–4.38 (m, 1H), 3.89–
3.78 (m, 3H), 3.71–3.68 (m, 1H), 3.35–3.31 (m, 1H), 2.85 (dd,
J1 = 5.2 Hz, J2 = 14.0 Hz, 1H), 2.58–2.46 (m, 2H), 1.77–1.70 (m,
2H), 1.49–1.41 (m, 2H), 1.35 (s, 9H), 1.35–1.24 (m, 4H), 0.92 (t,
J = 6.8 Hz, 3H). 13C NMR (CDCl3, 100 MHz), d 171.39, 170.60,
158.79, 156.22, 155.92, 147.47, 138.83, 138.72, 133.96, 133.13,
19.25. HRMS (AP-ESI) m/z Calcd for
740.3006, found: 740.3025.
C
41H47N3O8S [MꢁH]ꢁ
4.1.7.12. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenyl-
sulfonamido)-3-(4-isobutoxyphenyl)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (21).
Pale yellow powder, yield: 51%, mp: 102–
104 °C. 1H NMR (CDCl3, 400 MHz), d 10.14 (s, 1H), 8.50 (d,
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
11
132.98, 132.70, 132.50, 130.37, 128.96, 128.51, 127.98, 126.07,
125.69, 115.95, 114.83, 82.62, 67.89, 60.10, 53.41, 53.28, 35.98,
34.60, 31.59, 29.25, 28.06, 25.78, 22.60, 14.03. HRMS (AP-ESI) m/
z Calcd for C43H48Cl2N4O10S [MꢁH]ꢁ 881.2390, found: 881.2388.
110 °C. 1H NMR (CDCl3, 400 MHz), d 10.20 (s, 1H), 8.52 (d,
J = 1.6 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.85–7.79 (m, 4H), 7.73 (d,
J = 8.0 Hz, 1H), 7.53–7.48 (m, 7H), 7.45–7.39 (m, 2H), 7.34–7.30
(m, 1H), 6.99 (d, J = 8.4 Hz, 2H), 6.88–6.82 (m, 4H), 6.65 (d,
J = 7.2 Hz, 1H), 5.12 (s, 2H), 4.88–4.81 (m, 1H), 4.69–4.65 (m, 1H),
4.41–4.37 (m, 1H), 3.78–3.73 (m, 1H), 3.68–3.64 (m, 1H), 3.33–
3.30 (m, 1H), 2.91–2.86 (m, 1H), 2.56–2.48 (m, 2H), 1.38 (s, 9H).
13C NMR (CDCl3, 100 MHz), d 171.55, 170.60, 158.37, 155.97,
147.45, 140.19, 138.77, 133.25, 133.06, 132.69, 132.50, 130.46,
128.82, 128.60, 128.40, 127.93, 127.73, 127.05, 126.73, 126.47,
126.27, 126.15, 126.09, 125.15, 115.90, 115.27, 82.61, 69.95,
60.12, 53.49, 53.26, 35.97, 34.63, 28.33, 28.10, 20.46. HRMS (AP-
ESI) m/z Calcd for C48H45ClN4O10S [MꢁH]ꢁ 903.2472, found:
903.2470.
4.1.7.16. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-(hexyloxy)phenyl)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (25).
Pale yellow powder, yield: 40%, mp: 90–
10.15 (s, 1H), 8.51 (d,
92 °C. 1H NMR (CDCl3, 400 MHz),
d
J = 2.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H),
7.53 (d, J = 8.4 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz,
2H), 6.95 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.69 (d,
J = 8.4 Hz, 2H), 6.63 (d, J = 7.6 Hz, 1H), 4.91 (s, 1H), 4.67 (br s,
1H), 4.40–4.37 (m, 1H), 3.86–3.78 (m, 3H), 3.70–3.66 (m, 1H),
3.36–3.32 (m, 1H), 2.86 (dd, J1 = 5.2 Hz, J2 = 14.0 Hz, 1H), 2.57–
2.45 (m, 2H), 2.39 (s, 3H), 1.77–1.70 (m, 2H), 1.49–1.40 (m, 2H),
1.35 (s, 9H), 1.33–1.26 (m, 4H), 0.92 (t, J = 6.8 Hz, 3H). 13C NMR
4.1.7.20. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-(naphthalen-2-ylmethoxy)phenyl)-1-oxo-
propan-2-yl)carbamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)
(CDCl3, 100 MHz),
d
171.41, 170.73, 158.81, 155.94, 155.79,
pyrrolidine-1-carboxylate (29).
White powder, yield: 44%,
147.46, 138.84, 137.37, 136.83, 135.25, 133.00, 132.69, 132.51,
130.39, 129.55, 128.42, 126.60, 126.12, 125.70, 115.89, 114.84,
82.60, 67.88, 60.16, 53.30, 35.95, 34.70, 31.61, 29.26, 28.07,
mp: 174–176 °C. 1H NMR (CDCl3, 400 MHz), d 10.20 (s, 1H), 8.52
(s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.85–7.80 (m, 4H), 7.73 (d,
J = 8.4 Hz, 1H), 7.52–7.41 (m, 7H), 7.22 (d, J = 8.0 Hz, 2H), 6.99 (d,
J = 8.4 Hz, 2H), 6.84 (d, J = 8.0 Hz, 4H), 6.65 (d, J = 6.4 Hz, 1H),
5.12 (s, 2H), 4.87 (br s, 1H), 4.66 (br s, 1H), 4.40 (d, J = 8.0 Hz,
1H), 3.79–3.65 (m, 2H), 3.33–3.30 (m, 1H), 2.91–2.87 (m, 1H),
2.56–2.47 (m, 2H), 2.37 (s, 3H), 1.37 (s, 9H). 13C NMR (CDCl3,
25.79, 22.62, 21.08, 14.05. HRMS (AP-ESI) m/z Calcd for C44H51
-
ClN4O10S [MꢁH]ꢁ 861.2936, found: 861.2936.
4.1.7.17. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-1-oxo-3-(4-phenethoxyphenyl)propan-2-yl)car-
bamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
100 MHz),
d 171.47, 170.57, 158.34, 155.72, 147.41, 138.74,
137.28, 136.78, 135.19, 134.07, 133.22, 133.04, 132.94, 132.66,
132.47, 130.43, 129.51, 128.36, 127.90, 127.70, 126.54, 126.25,
126.07, 125.13, 115.89, 115.23, 82.55, 69.91, 60.10, 53.41, 53.24,
carboxylate (26).
Pale yellow powder, yield: 68%, mp: 98–
100 °C. 1H NMR (CDCl3, 400 MHz), d 10.19 (s, 1H), 8.50 (s, 1H),
8.22 (d, J = 8.4 Hz, 1H), 7.71 (d, J = 8.4 Hz, 1H), 7.52 (d, J = 8.4 Hz,
2H), 7.43 (d, J = 8.0 Hz, 2H), 7.33–7.26 (m, 3H), 7.25–7.19 (m,
4H), 6.94 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.71–6.63 (m,
3H), 4.89 (s, 1H), 4.68 (br s, 1H), 4.39 (d, J = 10.0 Hz, 1H), 4.07–
4.02 (m, 2H), 3.79–3.65 (m, 2H), 3.34–3.30 (m, 1H), 3.07–3.00
(m, 2H), 2.86–2.81 (m, 1H), 2.56–2.43 (m, 2H), 2.38 (s, 3H), 1.28
(s, 9H). 13C NMR (CDCl3, 100 MHz), d 171.45, 170.66, 158.44,
155.92, 155.78, 147.46, 138.82, 138.20, 137.37, 136.85, 135.24,
132.95, 132.70, 132.52, 130.42, 129.57, 129.03, 128.50, 128.43,
126.61, 126.54, 126.12, 126.05, 115.91, 114.89, 82.59, 68.53,
60.14, 53.30, 35.98, 35.80, 34.68, 28.35, 28.02, 21.09. HRMS (AP-
ESI) m/z Calcd for C46H47ClN4O10S [MꢁH]ꢁ 881.2623, found:
881.2623.
35.89, 34.61, 28.07, 21.02. HRMS (AP-ESI) m/z Calcd for C49H47
-
ClN4O10S [MꢁH]ꢁ 917.2623, found: 917.2623.
4.1.7.21. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-(naphthalen-2-ylmethoxy)phenyl)-1-oxo-
propan-2-yl)carbamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)
pyrrolidine-1-carboxylate (30).
Pale yellow powder, yield:
50%, mp: 120–122 °C. 1H NMR (CDCl3, 400 MHz), d 10.23 (s, 1H),
8.51 (s, 1H), 8.24 (d, J = 8.0 Hz, 1H), 7.85–7.80 (m, 4H), 7.72 (d,
J = 8.0 Hz, 1H), 7.50–7.34 (m, 9H), 6.98 (d, J = 8.4 Hz, 2H), 6.86–
6.83 (m, 4H), 6.66 (br s, 1H), 5.11 (s, 2H), 4.87–4.81 (m, 1H),
4.69–4.64 (m, 1H), 4.41–4.38 (m, 1H), 3.79–3.65 (m, 2H), 3.31–
3.25 (m, 1H), 2.91–2.86 (m, 1H), 2.57–2.44 (m, 2H), 1.38 (s, 9H).
13C NMR (CDCl3, 100 MHz), d 171.55, 158.35, 156.18, 155.90,
147.44, 138.75, 138.65, 134.08, 133.24, 133.06, 132.71, 132.50,
130.45, 128.94, 128.46, 128.40, 128.24, 127.94, 127.87, 127.73,
126.46, 126.32, 126.26, 126.17, 126.05, 125.13, 115.96, 115.23,
82.60, 81.85, 69.95, 60.08, 53.51, 53.24, 36.06, 34.54, 28.33,
28.10. HRMS (AP-ESI) m/z Calcd for C48H44Cl2N4O10S [MꢁH]ꢁ
937.2077, found: 937.2077.
4.1.7.18. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-1-oxo-3-(4-phenethoxyphenyl)propan-2-yl)car-
bamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (27).
White powder, yield: 77%, mp: 103–104 °C.
1H NMR (CDCl3, 400 MHz), d 10.15 (s, 1H), 8.50 (s, 1H), 8.23 (d,
J = 8.4 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.49–7.43 (m, 4H), 7.39 (d,
J = 8.4 Hz, 2H), 7.33–7.29 (m, 3H), 7.23–7.19 (m, 2H), 6.94 (d,
J = 8.8 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.72–6.62 (m, 3H), 4.90 (br
s, 1H), 4.69–4.64 (m, 1H), 4.40–4.37 (m, 1H), 4.08–4.02 (m, 2H),
3.78–3.58 (m, 2H), 3.34–3.30 (m, 1H), 3.07–2.99 (m, 2H), 2.85–
2.81 (m, 1H), 2.57–2.45 (m, 2H), 1.29 (s, 9H). 13C NMR (CDCl3,
4.1.7.22. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-(4-nitrophenoxy)phenyl)-1-oxopropan-2-yl)-
carbamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-
1-carboxylate (31).
Pale yellow powder, yield: 24%, mp: 118–
100 MHz),
d
171.43, 170.54, 158.43, 156.21, 155.91, 147.45,
120 °C. 1H NMR (CDCl3, 400 MHz), d 10.30 (s, 1H), 8.51 (s, 1H),
8.21–8.06 (m, 3H), 7.72 (d, J = 8.0 Hz, 1H), 7.52–7.36 (m, 6H),
7.16 (d, J = 8.4 Hz, 2H), 6.96–6.82 (m, 7H), 4.92 (s, 1H), 4.76 (br s,
1H), 4.44 (d, J = 10.0 Hz, 1H), 3.78–3.61 (m, 2H), 3.28–3.13 (m,
1H), 3.05–3.00 (m, 1H), 2.60–2.45 (m, 2H), 1.44 (s, 9H). 13C NMR
138.78, 138.71, 138.17, 133.95, 133.13, 132.97, 132.73, 132.52,
130.41, 129.02, 128.98, 128.51, 127.99, 126.56, 126.08, 115.95,
114.86, 82.61, 68.53, 60.08, 53.29, 36.02, 35.79, 34.56, 28.34,
28.01. HRMS (AP-ESI) m/z Calcd for C45H44Cl2N4O10S [MꢁH]ꢁ
901.2077, found: 901.2076.
(CDCl3, 100 MHz),
d 172.39, 169.99, 162.74, 156.11, 154.33,
147.51, 142.87, 138.60, 138.55, 133.24, 132.91, 132.77, 132.60,
131.32, 131.21, 128.99, 128.51, 128.39, 127.91, 126.07, 125.95,
120.72, 117.29, 117.14, 115.98, 82.71, 60.08, 53.83, 53.33, 36.39,
4.1.7.19. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-1-
(4-chloro-3-nitrophenylsulfonamido)-3-(4-(naphthalen-2-
ylmethoxy)phenyl)-1-oxopropan-2-yl)carbamoyl)pyrrolidine-
34.54, 28.32, 28.19. HRMS (AP-ESI) m/z Calcd for C43H39Cl2N5O12
S
1-carboxylate (28).
Pale yellow powder, yield: 28%, mp: 108–
[MꢁH]ꢁ 918.1615, found: 918.1615.
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

12
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
4.1.7.23. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-3-
(4-(benzyloxy)phenyl)-1-(4-chloro-3-nitrophenylsulfonamido)-
1-oxopropan-2-yl)carbamoyl)pyrrolidine-1-carboxylate
130.46, 129.54, 128.59, 128.39, 128.04, 127.42, 126.59, 126.40,
126.09, 115.94, 115.21, 82.57, 69.86, 60.15, 53.47, 53.28, 35.98,
34.70, 28.35, 28.10, 21.05. HRMS (AP-ESI) m/z Calcd for C45H45
-
(32).
Pale yellow powder, yield: 33%, mp: 102–104 °C. 1H
ClN4O10S [MꢁH]ꢁ 867.2467, found: 867.2469.
NMR (CDCl3, 400 MHz), d 10.25 (s, 1H), 8.51 (s, 1H), 8.24 (d,
J = 8.0 Hz, 1H), 7.72 (d, J = 8.0 Hz, 1H), 7.54–7.52 (m, 4H), 7.43–
7.30 (m, 8H), 6.97 (d, J = 8.4 Hz, 2H), 6.87 (d, J = 8.4 Hz, 2H), 6.78
(d, J = 8.2 Hz, 2H), 6.68 (d, J = 7.7 Hz, 1H), 4.95 (d, J = 15.6 Hz, 3H),
4.70 (br s, 1H), 4.41–4.38 (m, 1H), 3.80–3.66 (m, 2H), 3.34–3.29
(m,1H), 2.90–2.85 (m, 1H), 2.58–2.49 (m, 2H), 1.37 (s, 9H). 13C
NMR (CDCl3, 100 MHz), d 175.22, 171.85, 158.72, 155.99, 147.49,
132.97, 132.72, 132.52, 132.17, 132.14, 132.11, 132.08, 130.44,
128.84, 128.68, 128.62, 128.56, 128.07, 127.43, 127.09, 126.76,
115.98, 115.32, 82.91, 69.91, 60.22, 53.55, 35.88, 28.14. HRMS
(AP-ESI) m/z Calcd for C44H43ClN4O10S [MꢁH]ꢁ 853.2310, found:
853.2308.
4.1.7.27. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-3-
(4-((4-bromobenzyl)oxy)phenyl)-1-(4-chloro-3-nitrophenylsul-
fonamido)-1-oxopropan-2-yl)carbamoyl)pyrrolidine-1-car-
boxylate (36).
Pale yellow powder, yield: 54%, mp: 150–
152 °C. 1H NMR (CDCl3, 400 MHz), d 10.20 (s, 1H), 8.50 (s, 1H),
8.24 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.53–7.47 (m, 6H),
7.43–7.40 (m, 2H), 7.34–7.31 (m, 1H), 7.25 (d, J = 8.4 Hz, 2H),
6.98 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.75 (d, J = 8.4 Hz,
2H), 6.66 (d, J = 6.8 Hz, 1H), 4.91–4.87 (m, 3H), 4.70–4.68 (m,
1H), 4.41–4.38 (m, 1H), 3.78–3.67 (m, 2H), 3.31–3.27 (m, 1H),
2.91–2.86 (m, 1H), 2.57–2.46 (m, 2H), 1.36 (s, 9H). 13C NMR (CDCl3,
100 MHz),
d 171.55, 170.49, 158.05, 155.97, 147.44, 140.16,
4.1.7.24. (2S,4S)-tert-Butyl-4-([1,10-biphenyl]-4-yloxy)-2-(((S)-3-
(4-(benzyloxy)phenyl)-1-oxo-1-(phenylsulfonamido)propan-2-
138.75, 135.65, 135.21, 132.99, 132.71, 132.52, 131.70, 130.48,
128.97, 128.84, 128.59, 127.08, 126.72, 126.06, 121.92, 115.90,
115.13, 82.54, 69.02, 60.11, 53.48, 53.27, 36.00, 34.62, 28.08. HRMS
(AP-ESI) m/z Calcd for C44H42BrClN4O10S [MꢁH]ꢁ 931.1415, found:
931.1414.
yl)carbamoyl)pyrrolidine-1-carboxylate (33).
White pow-
der, yield: 49%, mp: 100–102 °C. 1H NMR (CDCl3, 400 MHz), d
9.90 (s, 1H), 8.05 (d, J = 7.0 Hz, 2H), 7.64–7.60 (m, 1H), 7.57–7.49
(m, 6H), 7.41–7.29 (m, 8H), 6.94 (d, J = 8.4 Hz, 2H), 6.89 (d,
J = 7.7 Hz, 2H), 6.74 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 6.9 Hz, 1H),
4.94–4.90 (m, 3H), 4.74–4.72 (m, 1H), 4.44 (d, J = 9.4 Hz, 1H),
3.73 (d, J = 12.5 Hz, 2H), 3.35–3.32 (m, 1H), 2.84–2.82 (m,1H),
2.61–2.43 (m, 2H), 1.37 (s, 9H). 13C NMR (CDCl3, 100 MHz), d
171.26, 170.07, 158.19, 156.14, 155.73, 140.31, 138.88, 136.76,
135.12, 133.62, 130.55, 128.79, 128.67, 128.59, 128.04, 127.40,
126.98, 126.76, 115.93, 115.15, 82.22, 69.89, 60.13, 53.24, 36.03,
34.64, 28.14. HRMS (AP-ESI) m/z Calcd for C44H45N3O8S [MꢁH]ꢁ
774.2849, found: 774.2848.
4.1.7.28.
(2S,4S)-tert-Butyl-2-(((S)-3-(4-((4-bromobenzyl)oxy)
phenyl)-1-(4-chloro-3-nitrophenylsulfonamido)-1-oxopropan-
2-yl)carbamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrro-
lidine-1-carboxylate (37).
White powder, yield: 36%, mp:
174–176 °C. 1H NMR (CDCl3, 400 MHz), d 10.23 (s, 1H), 8.50 (d,
J = 6.4 Hz, 1H), 8.23 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 7.6 Hz, 1H),
7.50–7.41 (m, 6H), 7.25–7.21 (m, 4H), 6.97–6.93 (m, 2H), 6.84–
6.82 (m, 2H), 6.75–6.73 (m, 2H), 6.64–6.60 (m, 1H), 4.89–4.87
(m, 3H), 4.68 (br s, 1H), 4.40–4.35 (m, 1H), 3.78–3.66 (m, 2H),
3.30–3.26 (m, 1H), 2.89–2.86 (m, 1H), 2.56–2.45 (m, 2H), 2.39 (s,
3H), 1.36 (s, 9H). 13C NMR (CDCl3, 100 MHz), d 171.64, 170.54,
158.08, 155.78, 147.48, 138.81, 137.31, 136.87, 135.70, 135.23,
132.97, 132.71, 132.53, 131.71, 130.50, 129.57, 128.99, 128.38,
126.72, 126.57, 126.08, 121.93, 115.96, 115.17, 82.55, 69.05,
60.15, 53.59, 53.30, 36.02, 34.69, 28.11, 21.08. HRMS (AP-ESI) m/
z Calcd for C45H44BrClN4O10S [MꢁH]ꢁ 945.1572, found: 945.1576.
4.1.7.25. (2S,4S)-tert-Butyl-2-(((S)-3-(4-(benzyloxy)phenyl)-1-(3-
chloro-4-nitrophenylsulfonamido)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (34).
Pale yellow powder, yield: 38%, mp: 88–
90 °C. 1H NMR (CDCl3, 400 MHz), d 10.21 (s, 1H), 8.51 (s, 1H),
8.23 (d, J = 7.6 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.49–7.45 (m, 4H),
7.38–7.36 (m, 7H), 6.97 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.2 Hz, 2H),
6.79 (d, J = 8.2 Hz, 2H), 6.67 (d, J = 6.9 Hz, 1H), 4.95 (s, 2H), 4.90
(s, 1H), 4.70–4.65 (m, 1H), 4.41 (d, J = 9.7 Hz, 1H), 3.78–3.67 (m,
2H), 3.31 (d, J = 13.6 Hz,1H), 2.90–2.85 (m, 1H), 2.58–2.48 (m,
2H), 1.37 (s, 9H). 13C NMR (CDCl3, 100 MHz), d 171.83, 170.29,
158.40, 156.24, 147.52, 138.83, 138.71, 136.65, 133.14, 132.94,
132.70, 132.52, 130.45, 128.96, 128.61, 128.49, 128.07, 127.98,
127.42, 126.44, 126.07, 116.03, 115.23, 82.64, 69.89, 60.12, 53.62,
53.27, 36.04, 34.63, 28.11. HRMS (AP-ESI) m/z Calcd for C44H42Cl2-
N4O10S [MꢁH]ꢁ 887.1920, found: 887.1918.
4.1.7.29.
(2S,4S)-tert-Butyl-2-(((S)-3-(4-((4-bromobenzyl)oxy)
phenyl)-1-(4-chloro-3-nitrophenylsulfonamido)-1-oxopropan-
2-yl)carbamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrro-
lidine-1-carboxylate (38).
Pale yellow powder, yield: 46%,
mp: 106–108 °C. 1H NMR (CDCl3, 400 MHz), d 10.18 (s, 1H), 8.50
(d, J = 1.6 Hz, 1H), 8.25 (d, J = 8.0 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H),
7.50–7.43 (m, 6H), 7.39 (d, J = 8.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H),
6.98 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.4 Hz, 2H), 6.76 (d, J = 8.4 Hz,
2H), 6.65 (d, J = 7.2 Hz, 1H), 4.90 (s, 3H), 4.69–4.65 (m, 1H), 4.41–
4.38 (m, 1H), 3.78–3.67 (m, 2H), 3.32–3.28 (m, 1H), 2.90–2.86
(m, 1H), 2.57–2.48 (m, 2H), 1.36 (s, 9H). 13C NMR (CDCl3,
4.1.7.26. (2S,4S)-tert-Butyl-2-(((S)-3-(4-(benzyloxy)phenyl)-1-(4-
chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
100 MHz),
d 171.52, 170.39, 158.06, 156.18, 155.90, 147.45,
138.75, 138.64, 135.64, 133.93, 133.13, 133.00, 132.73, 132.52,
131.72, 130.49, 128.97, 128.48, 127.95, 126.64, 126.04, 121.96,
115.96, 115.12, 82.57, 69.03, 60.06, 53.50, 53.28, 36.01, 34.50,
28.09. HRMS (AP-ESI) m/z Calcd for C44H41BrCl2N4O10S [MꢁH]ꢁ
965.1026, found: 965.1001.
carboxylate (35).
Pale yellow powder, yield: 28%, mp: 100–
102 °C. 1H NMR (CDCl3, 400 MHz), d 10.18 (s, 1H), 8.52 (d,
J = 2.0 Hz, 1H), 8.24 (d, J = 7.6 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H),
7.52 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.0 Hz, 2H), 7.38–7.32 (m, 5H),
7.23 (d, J = 8.0 Hz, 2H), 6.98 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz,
2H), 6.79 (d, J = 8.4 Hz, 2H), 6.64 (d, J = 7.6 Hz, 1H), 4.98 (s, 2H),
4.90 (s, 1H), 4.71–4.63 (m, 1H), 4.40–4.37 (m, 1H), 3.80–3.73 (m,
1H), 3.71–3.65 (m, 1H), 3.35–3.31 (m, 1H), 2.89 (dd, J1 = 5.2 Hz,
J1 = 14.0 Hz, 1H), 2.56–2.44 (m, 2H), 2.38 (s, 3H), 1.36 (s, 9H). 13C
NMR (CDCl3, 100 MHz), d 171.53, 170.58, 158.39, 155.79, 147.48,
138.84, 137.35, 136.81, 136.67, 135.25, 132.91, 132.66, 132.50,
4.1.7.30. (2S,4S)-tert-Butyl4-([1,10-biphenyl]-4-yloxy)-2-(((S)-1-
(4-chloro-3-nitrophenylsulfonamido)-3-(4-((4-nitrobenzyl)oxy)
phenyl)-1-oxopropan-2-yl)carbamoyl)pyrrolidine-1-carboxy-
late (39).
Pale yellow powder, yield: 42%, mp: 108–110 °C. 1H
NMR (CDCl3, 400 MHz), d 10.23 (s, 1H), 8.51 (d, J = 2.0 Hz, 1H),
8.26–8.14 (m, 3H), 7.75 (d, J = 8.4 Hz, 1H), 7.55–7.51 (m, 6H),
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
13
7.43–7.40 (m, 2H), 7.35–7.31 (m, 1H), 7.00 (d, J = 8.4 Hz, 2H), 6.85
(d, J = 8.4 Hz, 2H), 6.77 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 7.2 Hz, 1H),
5.03 (s, 2H), 4.92–4.90 (m, 1H), 4.71–4.61 (m, 1H), 4.42 (d,
J = 9.6 Hz, 1H), 3.80–3.67 (m, 2H), 3.31–3.26 (m, 1H), 2.94 (dd,
J1 = 4.8 Hz, J2 = 14.0 Hz, 1H), 2.58–2.49 (m, 2H), 1.37 (s, 9H). 13C
NMR (CDCl3, 100 MHz), d 171.73, 170.48, 157.66, 156.01, 147.60,
147.48, 144.08, 140.13, 138.78, 135.23, 133.01, 132.73, 132.56,
130.60, 128.87, 128.57, 127.51, 127.13, 126.69, 126.03, 123.79,
115.97, 115.12, 82.49, 68.48, 60.14, 53.74, 53.31, 36.09, 34.63,
28.12. HRMS (AP-ESI) m/z Calcd for C44H42ClN5O12S [MꢁH]ꢁ
898.2161, found: 898.2161.
4.1.7.34. (2S,4S)-tert-Butyl-2-(((S)-3-([1,10-biphenyl]-4-yl)-1-(4-
chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (43).
Pale yellow powder, yield: 62%, mp: 148–
149 °C. 1H NMR (CDCl3, 400 MHz), d 10.30 (s, 1H), 8.54 (s, 1H),
8.24 (d, J = 8.0 Hz, 1H), 7.73 (d, J = 8.4 Hz, 1H), 7.51–7.47 (m, 3H),
7.45–7.38 (m, 8H), 7.36–7.32 (m, 2H), 7.15 (d, J = 8.0 Hz, 2H),
6.85 (d, J = 8.4 Hz, 2H), 6.73 (d, J = 7.2 Hz, 1H), 4.91–4.90 (m, 1H),
4.77–4.73 (m, 1H), 4.43–4.40 (m, 1H), 3.78–3.67 (m, 2H), 3.39–
3.34 (m, 1H), 3.03–2.98 (m, 1H), 2.59–2.46 (m, 2H), 1.20 (s, 9H).
13C NMR (CDCl3, 100 MHz), d 171.85, 170.29, 156.25, 147.50,
140.44, 139.91, 138.78, 138.68, 133.89, 133.43, 133.09, 132.73,
132.53, 129.80, 128.93, 128.84, 128.46, 127.96, 127.57, 127.42,
126.83, 126.10, 116.01, 82.60, 60.06, 58.43, 53.66, 53.28, 36.51,
34.54, 28.00, 18.40. HRMS (AP-ESI) m/z Calcd for C43H40Cl2N4O9S
[MꢁH]ꢁ 857.1815, found: 857.1814.
4.1.7.31. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-((4-nitrobenzyl)oxy)phenyl)-1-oxopropan-2-yl)
carbamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-
1-carboxylate (40).
Pale yellow powder, yield: 29%, mp: 108–
110 °C. 1H NMR (CDCl3, 400 MHz), d 10.20 (s, 1H), 8.50 (d,
J = 2.4 Hz, 1H), 8.26 (d, J = 8.0 Hz, 1H), 8.21 (d, J = 8.4 Hz, 2H),
7.75 (d, J = 8.8 Hz, 1H), 7.53–7.49 (m, 4H), 7.42 (d, J = 8.4 Hz, 2H),
7.23 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.83 (d, J = 8.4 Hz,
2H), 6.77 (d, J = 8.4 Hz, 2H), 6.65 (d, J = 6.4 Hz, 1H), 5.03 (s, 2H),
4.89 (s, 1H), 4.69–4.66 (m, 1H), 4.41–4.38 (m, 1H), 3.80–3.66 (m,
2H), 3.30–3.26 (m, 1H), 2.94–2.90 (m, 1H), 2.56–2.45 (m, 2H),
2.39 (s, 3H), 1.37 (s, 9H). 13C NMR (CDCl3, 100 MHz), d 171.83,
170.57, 157.70, 155.78, 147.63, 147.53, 144.07, 138.91, 136.96,
135.29, 132.97, 132.69, 132.54, 130.59, 129.59, 128.35, 127.51,
126.54, 126.01, 123.79, 116.04, 115.20, 82.54, 68.52, 60.20, 53.82,
4.1.7.35. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(40-methoxy-[1,10-biphenyl]-4-yl)-1-oxopropan-2-
yl)carbamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrro-
lidine-1-carboxylate (44).
Pale yellow powder, yield: 62%,
mp: 114–116 °C. 1H NMR (CDCl3, 400 MHz), d 10.26 (s, 1H), 8.55
(d, J = 2.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H),
7.51 (d, J = 8.4 Hz, 2H), 7.46–7.37 (m, 6H), 7.24 (d, J = 8.0 Hz, 2H),
7.12 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.86 (d, J = 8.4 Hz,
2H), 6.68 (d, J = 7.6 Hz, 1H), 4.90 (br s, 1H), 4.76–4.71 (m, 1H),
4.41 (d, J = 10.0 Hz, 1H), 3.85 (s, 3H), 3.80–3.77 (m, 1H), 3.69–
3.65 (m, 1H), 3.42–3.37 (m, 1H), 2.99–2.95 (m, 1H), 2.57–2.43
(m, 2H), 2.39 (s, 3H), 1.26 (s, 9H). 13C NMR (CDCl3, 100 MHz), d
171.67, 170.50, 159.34, 155.79, 147.47, 140.11, 138.78, 137.32,
136.81, 135.21, 132.90, 132.73, 132.53, 129.77, 129.52, 128.39,
127.87, 126.98, 126.58, 126.15, 115.94, 114.24, 82.62, 60.11,
55.32, 53.28, 36.36, 34.64, 27.98, 21.06. HRMS (AP-ESI) m/z Calcd
for C45H45ClN4O10S [MꢁH]ꢁ 867.2467, found: 867.2468.
53.31, 36.01, 34.76, 28.14, 21.05. HRMS (AP-ESI) m/z Calcd for C45
H
-
44ClN5O12S [MꢁH]ꢁ 912.2317, found: 912.2317.
4.1.7.32. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-((4-nitrobenzyl)oxy)phenyl)-1-oxopropan-2-
yl)carbamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrro-
lidine-1-carboxylate (41).
Pale yellow powder, yield: 27%,
mp: 114–115 °C. 1H NMR (CDCl3, 400 MHz), d 10.17 (s, 1H), 8.49
(d, J = 1.6 Hz, 1H), 8.26–8.21 (m, 3H), 7.75 (d, J = 8.4 Hz, 1H), 7.56
(d, J = 8.4 Hz, 2H), 7.50 (d, J = 8.4 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H),
7.39 (d, J = 8.4 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 8.0 Hz,
2H), 6.78 (d, J = 8.4 Hz, 2H), 6.67 (d, J = 7.6 Hz, 1H), 5.05 (s, 2H),
4.91 (s, 1H), 4.69–4.65 (m, 1H), 4.41–4.39 (m, 1H), 3.79–3.68 (m,
2H), 3.31–3.27 (m, 1H), 2.92–2.89 (m, 1H), 2.58–2.48 (m, 2H),
1.37 (s, 9H). 13C NMR (CDCl3, 100 MHz), d 171.70, 170.58, 157.66,
156.20, 147.61, 147.48, 144.05, 138.77, 138.64, 133.01, 132.75,
132.55, 130.64, 128.99, 128.46, 127.94, 127.50, 126.01, 123.81,
115.98, 115.08, 82.50, 68.48, 60.04, 53.69, 53.35, 36.10, 34.46,
28.11. HRMS (AP-ESI) m/z Calcd for C44H41Cl2N5O12S [MꢁH]ꢁ
932.1771, found: 932.1773.
4.1.7.36. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(40-methoxy-[1,10-biphenyl]-4-yl)-1-oxopropan-2-
yl)carbamoyl)-4-((40-chloro-[1,10-biphenyl]-4-yl)oxy)pyrro-
lidine-1-carboxylate (45).
Pale yellow powder, yield: 56%,
mp: 118–120 °C. 1H NMR (CDCl3, 400 MHz), d 10.27 (s, 1H), 8.54
(s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.75 (d, J = 8.4 Hz, 1H), 7.52–7.37
(m, 10H), 7.11 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.4 Hz, 2H), 6.85 (d,
J = 8.4 Hz, 2H), 6.71 (d, J = 7.2 Hz, 1H), 4.90 (s, 1H), 4.76–4.69 (m,
1H), 4.42 (d, J = 10.0 Hz, 1H), 3.84 (s, 3H), 3.79–3.76 (m, 1H),
3.72–3.66 (m, 1H), 3.39–3.34 (m, 1H), 3.00 (dd, J1 = 4.8 Hz,
J2 = 14.0 Hz, 1H), 2.58–2.48 (m, 2H), 1.27 (s, 9H). 13C NMR (CDCl3,
100 MHz),
d 171.71, 170.37, 159.34, 156.21, 155.96, 147.46,
140.07, 138.74, 138.65, 133.90, 133.08, 132.90, 132.75, 132.53,
132.33, 129.75, 128.93, 128.47, 127.95, 127.84, 126.92, 126.11,
116.00, 114.25, 82.64, 60.05, 55.33, 53.48, 53.28, 36.42, 34.50,
27.98. HRMS (AP-ESI) m/z Calcd for C44H42Cl2N4O10S [MꢁH]ꢁ
887.1920, found: 887.1920.
4.1.7.33. (2S,4S)-tert-Butyl-2-(((S)-3-([1,10-biphenyl]-4-yl)-1-(4-
chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)car-
bamoyl)-4-((40-methyl-[1,10-biphenyl]-4-yl)oxy)pyrrolidine-1-
carboxylate (42).
Pale yellow powder, yield: 51%, mp: 114–
116 °C. 1H NMR (CDCl3, 400 MHz), d 10.25 (s, 1H), 8.55 (d,
J = 2.0 Hz, 1H), 8.25 (d, J = 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H),
7.52–7.49 (m, 4H), 7.44–7.39 (m, 6H), 7.36–7.32 (m, 1H), 7.24 (d,
J = 8.0 Hz, 2H), 7.16 (d, J = 8.0 Hz, 2H), 6.86 (d, J = 8.4 Hz, 2H), 6.69
(d, J = 7.6 Hz, 1H), 4.89 (s, 1H), 4.76–4.73 (br s, 1H), 4.41–4.38 (m,
1H), 3.80 (d, J = 12.0 Hz, 1H), 3.68–3.65 (m, 1H), 3.42–3.38 (m,
1H), 3.02 (dd, J1 = 4.8 Hz, J2 = 14.0 Hz, 1H), 2.58–2.45 (m, 2H), 2.39
(s, 3H), 1.26 (s, 9H). 13C NMR (CDCl3, 100 MHz), d 171.82, 170.42,
155.83, 147.51, 140.48, 139.95, 138.81, 137.34, 136.82, 135.23,
133.41, 132.85, 132.73, 132.54, 129.85, 129.54, 128.83, 128.40,
127.56, 127.47, 126.86, 126.59, 126.16, 115.97, 82.60, 60.12,
53.56, 53.31, 36.47, 34.65, 28.01, 21.07. HRMS (AP-ESI) m/z Calcd
for C44H43ClN4O9S [MꢁH]ꢁ 837.2361, found: 837.2363.
4.1.7.37. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-((40-methoxy-[1,10-biphenyl]-4-yl)methoxy)
phenyl)-1-oxopropan-2-yl)carbamoyl)-4-((40-methyl-[1,10-
biphenyl]-4-yl)oxy)pyrrolidine-1-carboxylate
(46).
Pale
yellow powder, yield: 44%, mp: 106–108 °C. 1H NMR (CDCl3,
400 MHz), d 10.22 (s, 1H), 8.52 (s, 1H), 8.24 (d, J = 8.4 Hz, 1H), 7.73
(d, J = 8.4 Hz, 1H), 7.56–7.49 (m, 6H), 7.43 (d, J = 7.2 Hz, 4H), 7.23
(d, J = 8.0 Hz, 2H), 6.99–6.97 (m, 4H), 6.86–6.79 (m, 4H), 6.67 (d,
J = 7.6 Hz, 1H), 4.98 (s, 2H), 4.90 (br s, 1H), 4.70–4.66 (m, 1H),
4.41–4.38 (m, 1H), 3.86 (s, 3H), 3.82–3.75 (m, 1H), 3.69–3.64 (m,
1H), 3.35–3.30 (m, 1H), 2.90 (dd, J1 = 4.8 Hz, J2 = 14.0 Hz, 1H),
2.57–2.46 (m, 2H), 2.38 (s, 3H), 1.38 (s, 9H). 13C NMR (CDCl3,
100 MHz),
d 171.50, 170.62, 159.27, 158.38, 155.76, 147.45,
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

14
Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
140.64, 138.78, 137.32, 136.81, 135.22, 134.92, 133.19, 132.94,
132.69, 132.51, 130.46, 129.54, 128.39, 128.09, 127.96, 126.87,
126.58, 126.39, 126.10, 115.91, 115.19, 114.25, 82.59, 69.64, 60.13,
55.35, 53.45, 53.27, 35.98, 34.66, 28.11, 21.06. HRMS (AP-ESI) m/z
Calcd for C52H51ClN4O11S [MꢁH]ꢁ 973.2885, found: 973.2884.
4.1.8.2. (2S,4S)-N-((S)-3-(4-Bromophenyl)-1-(4-chloro-3-nitro-
phenylsulfonamido)-1-oxopropan-2-yl)-4-((40-chloro-[1,10-
biphenyl]-4-yl)oxy)pyrrolidine-2-carboxamide (50).
powder, yield: 54%, mp: 138–140 °C. 1H NMR (DMSO-d6,
400 MHz), 8.32 (d, J = 2.0 Hz, 1H), 8.01 (dd, J1 = 2.0 Hz,
Yellow
d
J2 = 8.4 Hz, 1H), 7.97 (d, J = 8.0 Hz, 1H), 7.83 (d, J = 8.4 Hz, 1H),
7.65 (d, J = 8.4 Hz, 2H), 7.60 (d, J = 8.4 Hz, 2H), 7.48 (d, J = 8.4 Hz,
2H), 7.05 (d, J = 8.0 Hz, 2H), 6.96 (d, J = 8.4 Hz, 2H), 6.87 (d,
J = 8.0 Hz, 2H), 4.87 (s, 1H), 4.24–4.17 (m, 1H), 3.61–3.58 (m, 1H),
3.31–3.28 (m, 1H), 2.90 (d, J = 4.8 Hz, 2H), 2.82 (d, J = 10.0 Hz,
1H), 2.39–2.32 (m, 1H), 2.02 (s, 1H). 13C NMR (DMSO-d6,
4.1.7.38. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-3-(4-((40-methoxy-[1,10-biphenyl]-4-yl)methoxy)
phenyl)-1-oxopropan-2-yl)carbamoyl)-4-((40-chloro-[1,10-
biphenyl]-4-yl)oxy)pyrrolidine-1-carboxylate
(47).
Pale
yellow powder, yield: 27%, mp: 110–112 °C. 1H NMR (CDCl3,
400 MHz), d 10.22 (s, 1H), 8.52 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H),
7.74 (d, J = 8.4 Hz, 1H), 7.56–7.48 (m, 6H), 7.45–7.36 (m, 6H),
7.00–6.96 (m, 4H), 6.86 (d, J = 8.0 Hz, 2H), 6.81 (d, J = 8.0 Hz, 2H),
6.67 (d, J = 7.6 Hz, 1H), 4.98 (s, 2H), 4.91 (s, 1H), 4.71–4.67 (m,
1H), 4.42 (d, J = 9.6 Hz, 1H), 3.86 (s, 3H), 3.80–3.77 (m, 1H), 3.70–
3.67 (m, 1H), 3.33–3.29 (m, 1H), 2.90 (dd, J1 = 5.2 Hz, J2 = 14.0 Hz,
1H), 2.58–2.49 (m, 2H), 1.38 (s, 9H). 13C NMR (DMSO-d6,
100 MHz),
d 175.16, 172.60, 157.84, 146.95, 146.43, 137.38,
132.74, 132.26, 131.96, 131.77, 131.59, 130.65, 129.24, 128.36,
128.29, 127.09, 124.71, 119.61, 116.25, 78.35, 60.17, 55.28, 52.67,
38.05, 36.78. HRMS (AP-ESI) m/z Calcd for C32H27BrCl2N4O7S
[MꢁH]ꢁ 759.0088, found: 759.0083.
4.1.8.3. (2S,4S)-N-((S)-1-(4-Chloro-3-nitrophenylsulfonamido)-
100 MHz),
d
172.39, 171.20, 159.42, 157.59, 157.26, 147.59,
1-oxo-3-phenylpropan-2-yl)-4-((40-methyl-[1,10-biphenyl]-4-yl)
139.77, 139.51, 139.00, 135.93, 133.43, 132.85, 132.59, 132.05,
132.00, 131.18, 130.68, 129.24, 128.62, 128.37, 128.26, 128.18,
126.60, 125.61, 116.41, 114.84, 114.79, 79.53, 74.32, 69.28, 59.25,
oxy)pyrrolidine-2-carboxamide (51).
Yellow powder, yield:
17%, mp: 156–158 °C. 1H NMR (DMSO-d6, 400 MHz), d 8.31 (d,
J = 1.6 Hz, 1H), 8.28 (d, J = 8.0 Hz, 1H), 7.98–7.96 (m, 1H), 7.77 (d,
J = 8.4 Hz, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.52 (d, J = 8.0 Hz, 2H),
7.24 (d, J = 8.0 Hz, 2H), 7.09–7.04 (m, 3H), 6.99–6.89 (m, 4H),
5.03 (s, 1H), 4.29–4.24 (m, 1H), 4.05 (br s, 1H), 3.47–3.42 (m,
1H), 3.28 (d, J = 12.0 Hz, 1H), 3.03–2.99 (m, 1H), 2.86–2.81 (m,
1H), 2.71–2.65 (m, 1H), 2.32 (s, 3H), 2.06–2.03 (m, 1H). 13C NMR
(DMSO-d6, 100 MHz), d 175.27, 156.73, 146.87, 146.60, 138.28,
137.35, 133.42, 132.65, 131.71, 129.92, 128.04, 126.97, 126.53,
126.35, 124.76, 116.47, 76.82, 59.32, 56.48, 51.57, 38.48, 36.52,
55.64, 54.86, 36.32, 28.45, 28.08. HRMS (AP-ESI) m/z Calcd for C51
H
-
48Cl2N4O11S [MꢁH]ꢁ 993.2339, found: 993.2340.
4.1.8. (2S,4S)-4-([1,10-biphenyl]-4-yloxy)-N-((S)-3-(4-
bromophenyl)-1-(4-chloro-3-nitrophenylsulfonamido)-1-
oxopropan-2-yl)pyrrolidine-2-carboxamide (48)
Compound 1 (0.4 g, 0.48 mmol) was dissolved in 10 mL EtOAc
saturated with HCl gas and stirred overnight at room temperature.
Then some amount of saturated NaHCO3 solution was added and
sequentially extracted with EtOAc. The EtOAc layer was washed
by brine and dried over anhydrous MgSO4. Finally, the solvent
was evaporated under reduced pressure. The concentrated residue
was purified by column chromatography (dichloromethane–
methanol = 20:1) to obtain 0.16 g compound 48 as a yellow pow-
der. Yield: 46%, mp: 134–136 °C. 1H NMR (DMSO-d6, 400 MHz), d
10.20–10.09 (m, 1H), 8.92–8.83 (m, 2H), 8.52 (s, 1H), 8.13 (d,
J = 8.8 Hz, 1H), 7.99 (d, J = 8.4 Hz, 1H), 7.63–7.60 (m, 4H), 7.46–
7.42 (m, 2H), 7.36–7.31 (m, 3H), 7.11 (d, J = 8.0 Hz, 2H), 6.96 (d,
J = 8.8 Hz, 2H), 5.14 (s, 1H), 4.54–4.53 (m, 1H), 4.30–4.28 (m, 1H),
3.55–3.43 (m, 3H), 3.04–2.99 (m, 1H), 2.79–2.71 (m, 1H), 2.14–
2.09 (m, 1H). 13C NMR (DMSO-d6, 100 MHz), d 170.82, 168.17,
156.24, 147.51, 140.13, 136.42, 133.96, 133.39, 132.79, 131.97,
131.38, 130.91, 129.34, 128.35, 127.35, 126.74, 125.47, 120.36,
116.72, 75.48, 58.32, 55.57, 50.55, 36.28, 35.99. HRMS (AP-ESI) m/
z Calcd for C32H28BrClN4O7S [MꢁH]ꢁ 725.0472, found: 725.0471.
Compounds 49–51 were synthesized following the procedure
described above.
21.10. HRMS (AP-ESI) m/z Calcd for
661.1524, found: 661.1521.
C
33H31ClN4O7S [MꢁH]ꢁ
4.2. Binding assay for Bcl-2 family proteins
Fluorescence polarization technique was employed in the bind-
ing assay for Bcl-2 family proteins. In this assay, a fluorescence-
labeled BH3 peptide was used as the tracer. The binding affinities
of tested compounds were characterized quantitatively based on
the changes of FP signals when the addition of the tested com-
pounds at different concentrations.
In the competitive binding experiments for Mcl-1 protein, a
26-reside BH3 peptide derived from Bid protein (QEDIIRNIARH-
LAQVGDSMDRSIPPG) was labeled at the N-terminus by 5-car-
boxyfluorescein succinimidyl ester (FAM) as the fluorescence
labeled tracer (5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG).16 Mcl-
1 protein and these tested compounds were preincubated in
the PBS assay solution for 30 min in dark at room temperature.
Then 20
into the solution to obtain a total volume of 200
bated for another 20 min. Finally, 60 L solutions were added
l
L 5-FAM-Bid-BH3 peptide solution in PBS was added
lL and incu-
4.1.8.1. (2S,4S)-N-((S)-3-(4-bromophenyl)-1-(4-chloro-3-nitro-
l
phenylsulfonamido)-1-oxopropan-2-yl)-4-((40-methyl-[1,10-
into every well of Corning 384-well, black, flat-bottom plates
(Corning Inc.) and three wells per a concentration of every sam-
ple. The polarization values (milipolarization units, mP) were
measured under the condition of an excitation wavelength at
485 nm and an emission wavelength at 535 nm using the Tecan
GENios-Pro Injector Reader (Tecan Group Ltd). In these experi-
ments, all tested pyrrolidine derivatives were prepared in
dimethylsulfoxide (DMSO) at seven concentrations (1 nM,
biphenyl]-4-yl)oxy)pyrrolidine-2-carboxamide (49).
Pale
yellow powder, yield: 53%, mp: 158–160 °C. 1H NMR (DMSO-d6,
400 MHz), d 8.32 (d, J = 2.0 Hz, 1H), 8.19 (d, J = 8.0 Hz, 1H), 8.04
(dd, J1 = 2.0 Hz, J2 = 8.4 Hz, 1H), 7.79 (d, J = 8.4 Hz, 1H), 7.57 (d,
J = 8.8 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.24 (d, J = 8.0 Hz, 2H),
7.17 (d, J = 8.0 Hz, 2H), 6.96–6.90 (m, 4H), 4.98 (s, 1H), 4.26–4.21
(m, 1H), 3.94–3.93 (m, 1H), 3.43–3.39 (m, 1H), 3.16 (d,
J = 12.0 Hz, 1H), 2.98–2.93 (m, 1H), 2.87–2.82 (m, 1H), 2.61–2.54
(m, 1H), 2.32 (s, 3H), 2.06–2.02 (m, 1H). 13C NMR (DMSO-d6,
10 nM, 100 nM, 1 lM, 10 lM, 50 lM, 100 lM). The final concen-
trations of Mcl-1 protein and 5-FAM-Bid-BH3 peptide were
100 MHz),
d 174.94, 156.66, 146.90, 146.50, 137.65, 137.36,
160 nM and 10 nM, respectively.
136.44, 133.49, 132.65, 132.11, 131.72, 130.86, 129.92, 128.06,
127.03, 126.54, 124.77, 119.66, 116.49, 76.81, 59.31, 56.23, 51.62,
37.86, 36.53, 21.10. HRMS (AP-ESI) m/z Calcd for C33H30BrClN4O7S
[MꢁH]ꢁ 739.0629, found: 739.0632.
The binding assay for Bcl-2 protein was almost as well as that
for Mcl-1 protein except that the total concentration of Bcl-2 pro-
tein was 230 nM. However, in the experiment for Bcl-XL protein, a
different 5-FAM-Bim-BH3 peptide (5-FAM-DMRPEIWIAQELRRIG-
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020

Y. Wan et al. / Bioorg. Med. Chem. xxx (2016) xxx–xxx
15
DEFNAYYARR) was used as the fluorescence tracer. The total con-
References and notes
centration of Bcl-XL protein was 205 nM. The competitive inhibi-
tion constant (Ki) of each tested compound was calculated using
the methods developed by Wang et al.17
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4.3. MTT assay
MDA-MB-231 (breast cancer cell), PC-3 (prostatic cancer cell)
and K562 (chronic myelogenous leukemia cells were cultured in
RPMI1640/DEME medium containing 10% FBS and incubated in
humidified incubator (37 °C, 5% CO2). When these cancer cells
were growing at logarithmic growth phase, they were seeded in
the 96-well plates (5000 cells per well) and incubated for 12 h.
Then a series of different concentrated tested compounds were
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Thanks for the binding assay for Bcl-2 family proteins and the
help in molecular docking 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), Shandong Natural Science Foundation for Distin-
guished Young Scholars (No. JQ201319).
A. Supplementary data
Supplementary data (the NMR spectra of all the target com-
pounds) associated with this article can be found, in the online ver-
sion, at http://dx.doi.org/10.1016/j.bmc.2016.10.020.
Please cite this article in press as: Wan, Y.; et al. Bioorg. Med. Chem. (2016), http://dx.doi.org/10.1016/j.bmc.2016.10.020