Design, synthesis and preliminary biological studies of pyrrolidine derivatives as Mcl-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.11.014

Anti-apoptotic proteins, such as B-cell lymphoma (Bcl-2) protein, myeloid cell leukemia sequence 1 (Mcl-1) protein, are potential targets for cancer treatment. In the studies, a series of pyrrolidine derivatives were developed as potent Mcl-1 inhibitors. The preliminary biological studies suggested that most of target compounds exhibit good abilities for targeting Mcl-1 protein. Among them, compound 21 (K_i = 0.53 μM) exhibited equal inhibitory activities towards Mcl-1 protein compared to positive control gossypol (K_i = 0.39 μM). This compound also possessed good antiproliferative activities against MDA-MB-231 and PC-3 cancer cells.

Full text of DOI:10.1016/j.bmc.2015.11.014

Bioorganic & Medicinal Chemistry 23 (2015) 7685–7693
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and preliminary biological studies of pyrrolidine
derivatives as Mcl-1 inhibitors
Yichao Wan a,y, Junhua Wang b,y, Feng’e Sun a, Minglu Chen a, Xuben Hou a, Hao Fang a,
⇑
a 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
b Institute of Agro-Food Science and Technology, Shandong Academy of Agricultural Sciences, Jinan, Shandong 250100, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Anti-apoptotic proteins, such as B-cell lymphoma (Bcl-2) protein, myeloid cell leukemia sequence 1
(Mcl-1) protein, are potential targets for cancer treatment. In the studies, a series of pyrrolidine
derivatives were developed as potent Mcl-1 inhibitors. The preliminary biological studies suggested that
most of target compounds exhibit good abilities for targeting Mcl-1 protein. Among them, compound 21
Received 16 October 2015
Revised 9 November 2015
Accepted 13 November 2015
Available online 14 November 2015
(Ki = 0.53
gossypol (Ki = 0.39
MDA-MB-231 and PC-3 cancer cells.
lM) exhibited equal inhibitory activities towards Mcl-1 protein compared to positive control
l
M). This compound also possessed good antiproliferative activities against
Keywords:
Apoptosis
Bcl-2
Ó 2015 Elsevier Ltd. All rights reserved.
Mcl-1
Pyrrolidine
Anti-tumor
1. Introduction
Bik/Nbk, Bim/Bod, Blk, Hrk/DP5, Noxa, Puma/Bbc3, Bnip3, Bmf
and Bcl-GS.5 Abnormal over-expression of anti-apoptotic (pro-sur-
vival) proteins may make tumor cells to acquire the resistance to
anti-tumor agents by evading apoptotic pathways.6
As a program of cell suicide, apoptosis is essential for eliminat-
ing useless or dangerous cells and maintaining proper tissue
development.1 In addition, aberrant apoptosis can result in
many pathological process, such as neurodegenerative diseases,
myelodysplastic syndromes, acquired immunodeficiency syn-
drome, autoimmune diseases and cancers.2 Therefore, modulating
apoptosis by targeting the key regulators in apoptotic pathways
may be an attractive approach to cancer therapy.3
In mitochondria-mediated apoptotic pathway, B-cell lymphoma
(Bcl-2) family proteins are key regulators of apoptosis through act-
ing on the outer mitochondrial membrane (OMM) to adjust its per-
meability and then releasing cell death factors, such as cytochrome
c.4 This family is comprised of anti-apoptotic proteins and pro-
apoptotic proteins. The anti-apoptotic members, such as Bcl-2,
Bcl-XL, Bcl-w, Mcl-1, share three or four Bcl-2 homology (BH)
domains. In the pro-apoptotic subfamily, it can be further divided
into the multidomains proteins and BH3-only proteins. The mul-
tidomains proteins usually include the members of Bax, Bak, Bok/
Mtd, Bcl-XS and Bcl-GL which have two or three BH domains. The
BH3-only proteins can convey death signals to trigger apoptosis
and have only one BH3 domain structure, which include Bad, Bid,
As an important member of anti-apoptotic (pro-survival) pro-
teins subfamily, Mcl-1 protein could lead to the survival and
immortalization of cells when introduced exogenously.7 In 2001,
Zhou et al. found that mice with a human Mcl-1 transgene exhib-
ited high incidence of B-cell lymphoma after an extended period.8
In addition, overexpression of Mcl-1 protein was observed in many
human cancers, such as lung, breast, prostate, pancreatic, ovarian
and cervical cancers,9–13 which is a mechanism for tumor cells to
resist conventional chemotherapy agents.14 Therefore, inhibiting
Mcl-1 protein could be a promising strategy to suppress tumor
cells. So far, many small-molecule Mcl-1 inhibitors have been
designed and synthesized such as S1,15,16 polyphenol derivatives
(Gossypol),17 indole derivatives (GX15-070,18 A-121047719
(Fig. 1).
)
In 2008, professor Xing designed and synthesized a novel Bcl-2
proteins inhibitor WL-276 based on the scaffold of rhodanine.20
And this compound could suppress PC-3 cell growth in a dose-
dependent manner effectively. To investigate SARs of WL-276,
we previous synthesized a series of WL-276 derivatives. The
results showed that modifications of WL-276 effectively enhanced
binding affinities for Mcl-1 over other Bcl-2 proteins and their
water solubility needed to be improved.21 In our recent screening,
pyrrolidine derivatives 1 exhibited a moderate Mcl-1 inhibition
⇑
Corresponding author. Tel.: +86 531 88382731; fax: +86 531 88382548.
E-mail address: haofangcn@sdu.edu.cn (H. Fang).
Y.W. and J.W. contributed equally.
y
http://dx.doi.org/10.1016/j.bmc.2015.11.014
0968-0896/Ó 2015 Elsevier Ltd. All rights reserved.

7686
Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
O
H
different amino acid
side chains
OCH3
N
H3C
CH3
OH
hydroxyl, different substituted
benzenesulfonamides
CN
N
H3C
CHO OH
N
O
HO
HO
OH
CH3
HN
H3C
R2
CHO
R3
O
CH3
O
NH
N
S
OH
H3C
CH3
O
N
NH
Boc
N
Boc
GX15-070
S1
Gossypol
R1
O
O
bromo, different
substituted phenyls
O
1
CH3
O
O
Figure 2. The structures of 1 and the target compounds.
S
O
OH
O
N
H
O
S
N
O
S
O
N
N
O
S
N
N
H3C
CH3
R2
CH3
N
OH
O
O
N
N
O
NH
O
Boc
H3C
OH
O
O
O
N
OH
Boc
CH3
Boc
N
a
c
b
N
HN
O
O
R1
OH
A-1210477
Figure 1. The structures of reported Mcl-1 inhibitors.
WL-276
OH
3
2
1, 7-15
4a-4d
R1
O
O
O
S
R2
O
N
H
e
NH
R3
O
O
O
O
Boc
N
R2
S
R2
d
(Ki = 8.4 lM) and displayed good water solubility, which was
N
OH
H
NH
NH
O
suitable for further research. Therefore, a series of pyrrolidine
derivatives was designed based on the structures 1 (Fig. 2). In this
paper, we will demonstrate the synthesis, binding affinities to
Mcl-1 protein and anti-proliferative activity against some tumor
cell lines of these compounds.
Boc
R3
Boc
R1
16-31
6a-6k
5a-5d
R
R
1= Br, Ph,4-CH3-Ph,4-Cl-Ph
2= different amino acid side chains
R3= H, 4-CH3, 4-Cl, 4-NO2, 3-NO2-4-Cl
Scheme 1. Reagents and conditions: (a) (i) MeOH, CH3COCl; (ii) (Boc)2O, Et3N,
CH2Cl2; (b) (i) PPh3, DEAD, substituted phenols R1-Ph-OH, THF, rt, 12 h; (ii) 1 M
NaOH, THF, rt, overnight; (c) (i) NMM, isobutyl chloroformate, THF; (ii) 1 M NaOH,
THF, rt, overnight; (d) NMM, isobutylchloroformate, NaH, benzenesulfonamide,
ꢀ20 °C to rt, 4 h; (e) (i) ethyl acetate saturated with HCl, rt, overnight; (ii) NMM,
isobutylchloroformate, THF.
2. Chemistry
The synthesis route of pyrrolidine derivatives was showed in
Scheme 1. (2S,4R)-4-hydroxyproline 2 was as the starting material
to get intermediate 3 by amino and carboxyl protection. Then
intermediate 3 was reacted with various substituted phenols by
Mitsunobu reaction to get key intermediates 4a–4d. Different N-
Boc-amino acids 5a–5d coupled with different substituted bensul-
fonamides to yield intermediates 6a–6k by mixed anhydrides
method.22 Finally, the key intermediates 4a–4d was treated with
different amino acid methyl ester hydrochlorides to get target
compounds 1, 7–15. The intermediates 6a–6k was first removed
the protection of Boc group by ethyl acetate saturated with HCl
gas. Sequential reacting with the key intermediates 4a–4d, the tar-
get compounds 16–31 was obtained by mixed anhydrides method.
in the series, and the binding affinity of the compound 21
(Ki = 0.53 lM) showed 15 times more potent than compound 1.
Docking studies were performed so as to study the interaction
between Mcl-1 protein and active compound 21 using Surflex-
Dock (Fig. 3). The docking results showed that biphenyl group of
compound 21 could mimetic the
a-helix of BH3-only protein,
Bim (Fig. 3a). Acyl–sulfonyl group could form two hydrogen bonds
with Arg263 and one hydrogen bond with Asn260. In addition, the
carbonyl of pyrrolidine ring interacted with Thr266 by one hydro-
gen bond. (Fig. 3b).
To explore if pyrrolidine derivatives had inhibitory activities for
other anti-apoptotic Bcl-2 family members, we selected four repre-
sentative compounds to test the inhibitory activities against Bcl-2
and Bcl-XL proteins (Table 2). The results showed that all tested
compounds had good inhibitory activities to the anti-apoptotic
proteins of Bcl-2 family. And these compounds displayed more
potent binding affinity on Mcl-1 protein than other two Bcl-2 pro-
teins except compound 29. In addition, compound 28 exhibited a
profile for selectively inhibiting Mcl-1 protein with 8.2-fold versus
Bcl-2 protein and 15.5-fold versus Bcl-XL protein.
In further studies, three human tumor cell lines, MDA-MB-231
(breast cancer cell), PC-3 (prostatic cancer cell) and K562 (chronic
myelogenous leukemia cell), were chosen to evaluate the antipro-
liferative activities of active compounds 21, 28, 29 and 30 by MTT
assay. Their IC50 values were listed in Table 3. The reported Mcl-1
inhibitor, Gossypol, was evaluated as the positive control in the
3. Results and discussion
We synthesized a series of pyrrolidine derivatives according to
the structures of compound 1. Their competitive binding affinities
for Mcl-1 protein were obtained by fluorescence polarization
assays (FPAs). The results of Ki value were listed in Table 1. Accord-
ing to the preliminary data, some active compounds (16–31) usu-
ally had biphenyl groups on R1, aromatic amino acid side chains on
R2, and different substituted benzenesulfonamides on R3. Espe-
cially, compounds containing 3-NO2-4-Cl substituent on R3 effec-
tively increased potency against Mcl-1 protein. For example,
compound 18 (Ki = 0.94
lM) exhibited almost 9-fold more potent
than compound 1 (Ki = 8.4
l
M). And these compounds with phenyl
or 4-bromophenyl on R2 showed better activities compared to 3-
indolyl and p-hydroxy phenyl substitutions, such as compounds
18, 21, 25, 31. The compound 21 was the most potent compound

Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
7687
Table 1
The binding affinities of pyrrolidine derivatives to Mcl-1 protein
O
R2
R3
O
NH
O
Boc
N
R1
Compd
R1
R2
R3
Mcl-1 Kia
(lM)
1
7
8
Ph
Br
Br
Ph-CH2–
Ph-CH2–
H
OH
OH
OH
8.4 0.13
>10
>10
9
Ph
H
OH
>10
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
Gossypol
Br
Ph
Br
Ph
Br
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
Ph
OH
OH
OH
OH
OH
OH
Ph-SO2NH–
4-CH3–Ph-SO2NH–
3-NO2-4-Cl–Ph-SO2NH–
Ph-SO2NH–
4-CH3–Ph-SO2NH–
3-NO2-4-Cl–Ph-SO2NH–
4-NO2–Ph-SO2NH–
4-Cl–Ph-SO2NH–
Ph-SO2NH–
>10
>10
>10
>10
>10
>10
6.4 1.9
3.3 0.53
0.94 0.15
1.2 0.12
0.99 0.12
0.53 0.09
2.2 0.72
0.74 0.11
3.2 0.60
1.2 0.32
1.6 0.32
1.9 0.26
0.56 0.18
0.63 0.20
0.58 0.07
2.2 0.62
0.39 0.04
Isopropyl
Isopropyl
Isobutyl
Isobutyl
Ph-CH2–
Ph-CH2–
Ph-CH2–
4-Br-Ph-CH2–
4-Br-Ph-CH2–
4-Br-Ph-CH2–
4-Br-Ph-CH2–
4-Br-Ph-CH2–
1H-Indole-3-CH2–
1H-Indole-3-CH2–
Ph-CH2–
Ph-CH2–
Ph-CH2–
3-NO2-4-Cl–Ph-SO2NH–
Ph-SO2NH–
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-CH3-Ph
4-Cl-Ph
Ph
4-CH3–Ph-SO2NH–
3-NO2-4-Cl–Ph-SO2NH–
3-NO2-4-Cl–Ph-SO2NH–
3-NO2-4-Cl–Ph-SO2NH–
3-NO2-4-Cl–Ph-SO2NH–
4-Br-Ph-CH2–
4-Br-Ph-CH2–
4-OH-Ph-CH2–
a
Each value is the result of three separate experiments.
a
b
Figure 3. (a) Docked compound 21 with Mcl-1 protein complexed with Bim protein. (b) The docking mode of compound 21 in the hydrophobic groove of Mcl-1 protein.
Table 3
Table 2
Antiproliferative activities of representative compounds
The binding affinities of representative pyrrolidine derivatives to three Bcl-2 proteins
a
Compd
IC50
(l
M)
Compd
Bcl-XL
Kia
M)
Bcl-2
Kia
Mcl-1
Kia
M)
(
l
(
l
M)
(
l
MDA-MB-231
PC-3
K562
21
28
29
30
2.2 0.06
8.7 2.5
1.2 0.36
2.1 0.17
0.77 0.40
0.93 0.06
4.6 0.15
0.55 0.05
0.83 0.04
0.56 0.06
0.53 0.09
0.56 0.18
0.63 0.20
0.58 0.07
0.39 0.04
21
28
29
30
13.6 0.61
15.4 1.7
14.9 2.4
15.8 1.9
9.42 0.43
10.7 1.1
23.0 1.9
22.4 1.8
22.1 1.5
21.9 0.60
5.91 0.77
13.5 0.42
13.8 0.55
11.2 0.65
5.78 0.36
Gossypol
Gossypol
a
a
Each value was reproduced in three independent assays and expressed with
standard deviations.
Each value was reproduced in three independent assays and expressed with
standard deviations.

7688
Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
same condition. The result showed that all the four compounds
had IC50 values of over 20 M for K562 cell line and similar
5.1.2. (2S,4S)-1-(tert-Butoxycarbonyl)-4-(4-bromophenoxy)
pyrrolidine-2-carboxylic acid (4a)
l
inhibition on MDA-MB-231 and PC-3 cell lines. In addition,
compound 21 possesses similar growth inhibition towards
MDA-MB-231 compared to Gossypol.
The reaction mixture of compound 3 (9.8 g, 40 mmol), 4-bro-
mophenol (8.30 g, 48 mmol) and triphenylphosphine (15.7 g,
60 mmol) in 100 mL anhydrous THF was placed at 0 °C. Then
diethyl azodicarboxylate (DEAD, 9.40 g, 60 mmol) in 20 mL anhy-
drous was added to the above solution dropwise. After the reaction
was warm to room temperature and stirred for 12 h, THF was
removed and extracted by EtOAc. The EtOAc phase was washed
by 0.5 M NaOH, 1 M citric acid and brine successively. After dried
over anhydrous MgSO4, the solution was concentrated to yield
colorless oil liquid. Then the oil was dissolved in 100 mL
mixed-solvent (THF/1 M NaOH = 1:1) and stirred overnight at
room temperature. When the organic solvent was removed, the
residue was acidified with 1 M HCl until pH 2–3. The formed pre-
cipitation was collected and recrystallized from MeOH/H2O to get
compound 4a as a white lamellar crystal. Yield: 62%, mp: 105–
107 °C. 1H NMR (DMSO-d6, 600 MHz), d 1.35 (s, 9H), 2.14–2.20
(m, 1H), 2.52–2.56 (m, 1H), 3.39–3.42 (m, 1H), 3.64–3.70 (m,
1H), 4.22–4.26 (m, 1H), 5.02 (s, 1H), 6.84 (d, J = 7.8 Hz, 2H), 7.44
(d, J = 7.8 Hz, 2H), 12.51 (s, 1H), ESI-MS m/z: 385.1 (MꢀH)ꢀ.
Compounds 4b–4d were synthesized following the procedure
described above.
4. Conclusions
Based on the recent screening, we developed a novel Mcl-1
inhibitor, compound 1, with a new scaffold of pyrrolidine. Different
pyrrolidine derivatives were designed and synthesized by
introducing different amino acid side chains, biphenyl groups
and benzenesulfonamides. According to the data of protein binding
assay and anti-proliferative assay, compound 21 displayed the
promising inhibitory activity against Mcl-1 protein and tumor cell
lines. These results suggest that further optimization of pyrrolidine
derivatives could lead to new anti-tumor agents.
5. Experiment section
5.1. General chemistry information
All the starting materials used were commercially available
and used directly. The products were purified by recrystallization
or silica gel chromatography (200–300 mesh). The completion of
reactions were monitored by conventional thin-layer chromatog-
raphy analysis on 0.25 mm silica gel plates (60GF-254) and
observed under UV light (254 nM or 365 nM) or iodine vapor.
Melting points were tested on an electrothermal melting point
apparatus without correction. Proton nuclear magnetic resonance
(1H NMR) spectra were measured with a Bruker DRX spectrometer
5.1.2.1.
(2S,4S)-4-(4-Phenylphenoxy)-1-(tert-butoxycarbonyl)
pyrrolidine-2-carboxylic acid (4b). White lamellar crystal, yield:
41%, mp: 158–160 °C. 1H NMR (DMSO-d6, 400 MHz), d 1.42 (s, 9H),
2.22 (d, J = 13.2 Hz, 1H), 2.55–2.63 (m, 1H), 3.43–3.46 (m, 1H),
3.71–3.78 (m, 1H), 4.26–4.32 (m, 1H), 5.05 (s, 1H), 6.95–6.97 (m,
2H), 7.31 (t, J = 7.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 2H), 7.59 (d,
J = 8.4 Hz, 2H), 7.61 (d, J = 7.1 Hz, 2H), 12.47 (s, 1H), ESI-MS m/z:
382.5 (MꢀH)ꢀ.
(600 MHz) and
a Bruker Avance spectrometer (300 MHz or
5.1.2.2. (2S,4S)-1-(tert-Butoxycarbonyl)-4-(4-(40-methyl)phenyl-
phenoxy)pyrrolidine-2-carboxylic acid (4c). White lamellar
crystal, yield: 40%, mp: 152–154 °C. 1H NMR (DMSO-d6,
400 MHz), d 1.41 (s, 9H), 2.24 (d, J = 13.6 Hz, 1H), 2.32 (s, 3H),
2.53–2.64 (m, 1H), 3.41–3.46 (m, 1H), 3.70–3.75 (m, 1H), 4.26–
4.31 (m, 1H), 5.04–5.06 (m, 1H), 6.92–6.95 (m, 2H), 7.24 (d,
J = 8.0 Hz, 2H), 7.51 (d, J = 8.0 Hz, 2H), 7.57 (d, J = 8.6 Hz, 2H),
12.49 (s, 1H), ESI-MS m/z: 396.4 [MꢀH]ꢀ.
400 MHz). Chemical shifts are expressed in parts per million
(ppm) using tetramethylsilane (TMS) as an internal standard, and
coupling constants (J values) are given in hertz (Hz). The splitting
patterns were described as s (singlet), d (doublet), dd (doublet dou-
blet), t (triplet), q (quartet), m (multiplet), and br s (broad singlet).
High-resolution mass spectral (HRMS) data were displayed as m/z
on an Agilent 6510 Quadrupole Time-of-Flight LC/MS deliver. The
following yields were for optimized products.
5.1.2.3. (2S,4S)-1-(tert-Butoxycarbonyl)-4-(4-(40-chloro)phenyl-
phenoxy)pyrrolidine-2-carboxylic acid (4d). White lamellar crys-
tal, yield: 43%, mp: 162–164 °C. 1H NMR (DMSO-d6, 600 MHz), d
1.41 (s, 9H), 2.20–2.22 (m, 1H), 2.59–2.62 (m, 1H), 3.41–3.45 (m,
1H), 3.71–3.77 (m, 1H), 4.29 (dd, J = 21 Hz, 9.6 Hz, 1H), 5.06–5.09
(m, 1H), 6.94–6.97 (m, 2H), 7.48 (d, J = 7.8 Hz, 2H), 7.61 (d,
J = 7.8 Hz, 2H), 7.65 (d, J = 8.4 Hz, 2H), 12.54 (s, 1H), ESI-MS m/z:
416.5 [MꢀH]ꢀ.
5.1.1. (2S,4R)-1-tert-Butyl 2-methyl 4-hydroxypyrrolidine-1,2-
dicarboxylate (3)
Acetyl chloride (14 mL, 198 mmol) was slowly added into the
anhydrous methanol (100 mL) in an ice bath. After stirred for
30 min, compound 2 (7.87 g, 60.0 mmol) was added to the solu-
tion. Then the reaction was refluxed for 4 h in an oil bath. After
the methanol was removed under reduced pressure, about 10 mL
acetone was added into the residue and filtered to give 8.67 g of
(2S,4R)-methyl 4-hydroxypyrrolidine-2-carboxylate hydrochloride
as a white powder. Yield: 88%, mp: 160–164 °C. Di-tert-butyl dicar-
bonate (11.42 g, 52.3 mmol) in 50 mL dichloromethane was slowly
added to the solution of (2S,4R)-methyl 4-hydroxypyrrolidine-2-
carboxylate hydrochloride (8.64 g, 47.6 mmol), triethylamine
(13.5 mL, 96.0 mmol) in 150 mL dichloromethane. After stirred
overnight at room temperature, the reaction mixture was concen-
trated under reduced pressure and dissolved with EtOAc. The
EtOAc layer was washed two times by 1 M citric acid, saturated
sodium bicarbonate and brine, respectively. Finally, the organic
layer was dried over anhydrous MgSO4 and concentrated to obtain
compound 3 as a white powder. Yield: 88%, mp: 93–94 °C. 1H NMR
(CDCl3, 300 MHz), d 1.41 (s, 9H), 2.06–2.09 (m, 1H), 2.27–2.34 (m,
1H), 3.43–3.64 (m, 2H), 3.73 (s, 3H), 4.37–4.48 (m, 2H), ESI-MS m/z:
246.3 (M+H)+.
5.1.3. (S)-tert-Butyl(1-oxo-3-phenyl-1-(phenylsulfonamido)
propan-2-yl)carbamate (6a)
N-Methylmorpholine (0.45 g, 4.4 mmol) and isobutyl chlorofor-
mate (0.60 g, 4.4 mmol) was added into a solution of N-Boc-L-
phenylalanine (1.06 g, 4 mmol) in 20 mL anhydrous THF at
ꢀ20 °C. After stirred for 1 h, a solution of benzenesulfonamide
(0.94 g, 6 mmol) and NaH (0.48 g, 12 mmol) in 15 mL anhydrous
THF was added and stirred overnight at room temperature. Then
the solvent was removed under reduced pressure and the right
amount of EtOAc was added. The solution was washed by 1 M citric
acid, brine and dried over anhydrous MgSO4 successively. After
removal of EtOAc, the crude product was further purified by col-
umn chromatography (petroleum ether/ethyl acetate = 5:1) to
yield pure compound 6a as a white powder. Yield: 68%, mp:
133–135 °C. 1H NMR (CDCl3, 300 MHz), d 1.38 (s, 9H), 2.93 (dd,

Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
7689
J = 14.1 Hz, 6.9 Hz, 1H), 3.05 (dd, J = 14.1 Hz, 6.3 Hz, 1H), 4.31 (s,
1H), 4.90 (s, 1H), 7.02–7.04 (m, 2H), 7.04–7.23 (m, 3H), 7.52–7.57
(m, 2H), 7.57–7.66 (m, 1H), 8.05 (d, J = 7.5 Hz, 2H), 9.34 (s, 1H),
ESI-MS m/z: 403.1 (MꢀH)ꢀ.
5.1.3.9. (S)-tert-Butyl(1-(4-chloro-3-nitrophenylsulfonamido)-
3-(1H-indol-3-yl)-1-oxopropan-2-yl)carbamate (6j). Yellow
powder, yield: 85%, mp: 90–92 °C. 1H NMR (CDCl3, 300 MHz), d
1.40 (s, 9H), 3.18–3.20 (d, J = 6.6 Hz, 2H), 4.38–4.40 (m, 1H), 5.00
(d, J = 6.9 Hz, 1H), 6.97 (s, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.22 (t,
J = 7.2 Hz, 1H), 7.37 (d, J = 8.4 Hz, 1H), 7.49 (d, J = 8.1 Hz, 1H),
7.68 (d, J = 8.4 Hz, 1H), 8.12 (d, J = 2.1 Hz, 1H), 8.15 (d, J = 2.1 Hz,
1H), 8.42 (d, J = 2.1 Hz, 1H), 9.43 (br s, 1H), ESI-MS m/z: 521.3
(MꢀH)ꢀ.
Compounds 6b–6j were synthesized following the procedure
described above.
5.1.3.1. (S)-tert-Butyl-(1-(4-methylphenylsulfonamido)-1-oxo-
3-phenylpropan-2-yl)carbamate (6b). White powder, the pure
compound 6b was not obtained by column chromatography and
it was used into the next step directly.
5.1.3.10. (S)-tert-Butyl(3-(4-((tert-butyldimethylsilyl)oxy)phe-
nyl)-1-(4-chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-
yl)carbamate (6k). tert-Butyl dimethylsilyl chloride (1.13 g,
7.5 mmol) and imidazole (2.04 g, 30 mmol) was added into a solu-
tion of N-Boc-L-tyrosine (1.40 g, 5 mmol) in 30 mL N,N-dimethyl-
formamide (DMF) at 0 °C. After stirred for 1 h at 0 °C, the mixture
was allowed to stir overnight at room temperature. Then the reac-
tion mixture was transferred to a separating funnel by adding to
much water and extracted with EtOAc two times. The combined
organic layer was washed by 1 M citric acid, brine, dried over anhy-
drous MgSO4 and concentrated to yield colorless oil. Finally, the
resulting oil was treated with the procedure of the synthesis of
compound 6a to obtain yellow oil compound 6k. Yield: 78%, 1H
NMR (CDCl3, 600 MHz), d 0.18 (s, 6H), 0.97 (s, 9H), 1.40 (s, 9H),
2.86–2.90 (m, 1H), 2.97–2.99 (m, 1H), 4.23 (s, 1H), 4.90 (s, 1H),
6.71 (d, J = 7.8 Hz, 2H), 6.92 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 8.4 Hz,
1H), 8.16 (d, J = 7.8 Hz, 1H), 8.51 (s, 1H), 9.76 (s, 1H), ESI-MS m/z:
612.5 (MꢀH)ꢀ.
5.1.3.2. (S)-tert-Butyl-(1-(4-chloro-3-nitrophenylsulfonamido)-
1-oxo-3-phenylpropan-2-yl)carbamate (6c). Yellow powder,
yield: 80%, mp: 147–149 °C. 1H NMR (CDCl3, 300 MHz), d 1.40
(s, 9H), 2.96 (dd, J = 14.1 Hz, 7.8 Hz, 1H), 3.07 (dd, J = 14.1 Hz,
6.3 Hz, 1H), 4.20–4.27 (m, 1H), 4.81 (d, J = 6.6 Hz, 1H), 7.07–7.10
(m, 2H), 7.26–7.28 (m, 3H), 7.73 (d, J = 8.4 Hz, 1H), 8.19
(dd, J = 8.4 Hz, 2.1 Hz, 1H), 8.48 (d, J = 2.1 Hz, 1H), 9.50 (s, 1H),
ESI-MS m/z: 482.1 (MꢀH)ꢀ.
5.1.3.3. (S)-tert-Butyl(3-(4-bromophenyl)-1-oxo-1-(phenylsul-
fonamido)propan-2-yl)carbamate (6d). White powder, yield:
87%, mp: 119–121 °C. 1H NMR (DMSO-d6, 300 MHz), d 1.26 (s,
9H), 2.57–2.61 (m, 1H), 2.79–2.85 (m, 1H), 4.06–4.13 (m, 1H),
7.07 (br s, 1H), 7.16 (d, J = 8.1 Hz, 2H), 7.42 (d, J = 8.1 Hz, 2H),
7.58–7.66 (m, 2H), 7.70 (t, J = 7.2 Hz, 1H), 7.88–7.95 (m, 2H),
12.43 (s, 1H), ESI-MS m/z: 483.3 (MꢀH)ꢀ.
5.1.3.4. (S)-tert-Butyl(3-(4-bromophenyl)-1-(4-methylphenyl-
sulfonamido)-1-oxopropan-2-yl)carbamate (6e). White pow-
der, yield: 85%, mp: 114–115 °C. 1H NMR (DMSO-d6, 300 MHz), d
1.31 (s, 9H), 2.35 (s, 3H), 2.68–2.75 (m, 1H), 2.89–2.95 (m, 1H),
3.84–3.86 (m, 1H), 6.04 (br s, 1H), 6.92 (d, J = 8.1 Hz, 2H), 7.21–
7.35 (m, 4H), 7.65 (d, J = 8.1 Hz, 2H), 12.34 (s, 1H), ESI-MS m/z:
497.4 (MꢀH)ꢀ.
5.1.4. (S)-2-((2S,4S)-4-(4-Phenylphenoxy)-1-(tert-butoxycarbonyl)
pyrrolidine-2-carboxamido)-3-phenylpropanoic acid (1)
The intermediate 4b (0.38 g, 1 mmol) was dissolved in 15 mL
anhydrous THF at ꢀ20 °C. 10 min later, NMM (0.22 g, 2.2 mmol)
and isobutylchloroformate (0.16 g, 1.1 mmol) were added succes-
sively. After stirred for 1 h, phenylalanine methyl ester hydrochlo-
ride (0.20 g, 1.1 mmol) was added and the reaction was warmed
slowly to rt and stirred overnight. The solvent was removed under
low pressure. The resulting residue was extracted with EtOAc and
washed with 1 M citric acid, saturated NaHCO3 and brine, respec-
tively. After dried over MgSO4 and concentrated, the obtained oil
was dissolved in 10 mL mixed-solvent (THF/1 M NaOH = 1:1) and
stirred for 4 h at room temperature. When the reaction was com-
pleted, the solvent was removed and the residue was acidified with
1 M HCl until pH 2–3. The generated white precipitation was col-
lected and recrystallized from MeOH/H2O to yield compound 1
as a white crystal. Yield: 88%, mp: 202–204 °C. 1H NMR (DMSO-
d6, 300 MHz), d 1.29 (s, 9H), 1.99–2.20 (m, 1H), 2.61 (br s, 1H),
2.91–2.98 (m, 1H), 3.04–3.10 (m, 1H), 3.37–3.42 (m, 1H), 3.82–
3.87 (m, 1H), 4.18–4.28 (m, 1H), 4.46 (br s, 1H), 5.00 (br s, 1H),
6.96 (d, J = 8.7 Hz, 2H), 7.16–7.24 (m, 5H), 7.28–7.33 (m, 1H),
7.43 (t, J = 7.8 Hz, 2H), 7.57–7.62 (m, 4H), 7.78–7.84 (m, 1H),
12.76 (br s, 1H). 13C NMR (CDCl3, 75 MHz), d 176.57, 174.63,
172.11, 156.34, 140.56, 135.69, 134.73, 130.92, 129.43, 128.74,
128.60, 128.37, 127.17, 126.84, 126.76, 115.90, 81.66, 75.67,
60.04, 52.97, 37.86, 28.09, 20.71, 19.18. HRMS (AP-ESI) m/z calcd
for C31H34N2O6 [MꢀH]ꢀ: 529.2339, found: 529.2340.
5.1.3.5. (S)-tert-Butyl(3-(4-bromophenyl)-1-(4-chloro-3-nitro-
phenylsulfonamido)-1-oxopropan-2-yl)carbamate
(6f).
Yellow powder, yield: 72%, mp: 116–118 °C. 1H-NMR (DMSO-d6,
300 MHz), d 1.31 (s, 9H), 2.68–2.75 (m, 1H), 2.91–2.98 (m, 1H),
3.82–3.89 (m, 1H), 6.01 (d, J = 7.8 Hz, 1H), 6.94 (d, J = 8.1 Hz, 2H),
7.28 (d, J = 8.1 Hz, 2H), 7.82 (d, J = 8.1 Hz, 1H), 7.98 (d, J = 8.4 Hz,
1H), 8.32 (s, 1H), ESI-MS m/z: 562.2 (MꢀH)ꢀ.
5.1.3.6. (S)-tert-Butyl(3-(4-bromophenyl)-1-(4-nitrophenylsul-
fonamido)-1-oxopropan-2-yl)carbamate (6g). Yellow powder,
the pure compound 7g was also not obtained by column chro-
matography and it was used into the next step directly.
5.1.3.7. (S)-tert-Butyl(3-(4-bromophenyl)-1-(4-chlorophenylsul-
fonamido)-1-oxopropan-2-yl)carbamate (6h). White powder,
yield: 75%, mp: 121–123 °C. 1H NMR (DMSO-d6, 300 MHz), d 1.31
(s, 9H), 2.70–2.77 (m, 1H), 2.91–2.97 (m, 1H), 3.80–3.86 (m, 1H),
5.88 (d, J = 7.5 Hz, 1H), 6.89 (d, J = 8.1 Hz, 2H), 7.27 (d, J = 8.4 Hz,
2H), 7.45 (d, J = 6.9 Hz, 2H), 7.74 (d, J = 8.7 Hz, 2H), ESI-MS m/z:
517.3 (MꢀH)ꢀ.
Compounds 7–15 were synthesized following the procedure
described above.
5.1.3.8. (S)-tert-Butyl(3-(1H-indol-3-yl)-1-oxo-1-(phenylsulfon-
amido)propan-2-yl)carbamate (6i). Yellow powder, yield: 80%,
mp: 95–97 °C. 1H NMR (CDCl3, 300 MHz), d 1.39 (s, 9H), 3.19–
3.21 (m, 2H), 4.38 (br s, 1H), 4.89 (br s, 1H), 6.96 (s, 1H), 7.13 (t,
J = 7.2 Hz, 1H), 7.22–7.24 (m, 1H), 7.38 (d, J = 8.1 Hz, 1H), 7.52–
7.57 (m, 3H), 7.66 (t, J = 7.5 Hz, 1H), 8.03 (d, J = 7.5 Hz, 2H), 8.08
(br s, 1H), 8.94 (br s, 1H), ESI-MS m/z: 442.5 (MꢀH)ꢀ.
5.1.4.1. (S)-2-((2S,4S)-4-(4-Bromophenoxy)-1-(tert-butoxycar-
bonyl)pyrrolidine-2-carboxamido)-3-phenylpropanoic
acid
(7). White crystal, yield: 87%, mp: 185–187 °C. 1H NMR
(DMSO-d6, 300 MHz), d 1.24 (s, 9H), 1.93–1.98 (m, 1H), 2.57 (s,
1H), 2.89–2.96 (m, 1H), 3.02–3.09 (m, 1H), 3.37–3.38 (m, 1H),
3.77–3.83 (m, 1H), 4.16 (br s, 1H), 4.44 (br s, 1H), 4.93 (s, 1H),
6.82 (d, J = 8.7 Hz, 2H), 7.14–7.26 (m, 5H), 7.40–7.46 (m, 2H),

7690
Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
7.80–7.83 (m, 1H), 12.71 (br s, 1H). 13C NMR (CDCl3, 75 MHz), d
174.08, 171.89, 155.88, 135.62, 132.54, 129.37, 128.58, 127.17,
117.42, 113.92, 81.75, 75.84, 59.95, 53.10, 52.84, 37.73, 28.09.
HRMS (AP-ESI) m/z calcd for C25H29BrN2O6 [MꢀH]ꢀ: 531.1131,
found: 531.1133.
2.24–2.25 (m, 1H), 2.35–2.59 (m, 1H), 2.60–2.90 (m, 1H), 3.80 (s,
2H), 4.41–4.55 (m, 2H), 4.89 (s, 1H), 6.87 (d, J = 8.4 Hz, 2H), 7.29–
7.32 (m, 1H), 7.39–7.43 (m, 2H), 7.49–7.54 (m, 4H). 13C NMR (CDCl3,
75 MHz), d 175.01, 172.22, 156.55, 140.57, 134.68, 128.74, 128.30,
126.84, 126.76, 115.95, 81.67, 76.05, 60.36, 57.31, 53.27, 31.35,
29.69, 28.20, 19.06, 17.91. HRMS (AP-ESI) m/z calcd for C27H34N2O6
[MꢀH]ꢀ: 481.2339, found: 481.2361.
5.1.4.2. 2-((2S,4S)-4-(4-Bromophenoxy)-1-(tert-butoxycarbonyl)
pyrrolidine-2-carboxamido)acetic acid (8). White crystal, yield:
32%, mp: 72–74 °C. 1H NMR (CDCl3, 400 MHz), d 1.45 (s, 9H),
2.45–2.69 (m, 2H), 3.75 (s, 2H), 4.00–4.13 (m, 2H), 4.46 (br s,
1H), 4.84 (s, 1H), 6.67 (d, J = 8.8 Hz, 2H), 7.11 (br s, 1H), 7.73 (d,
J = 8.8 Hz, 2H). 13C NMR (CDCl3, 100 MHz), d 172.72, 171.98,
155.64, 154.99, 132.52, 117.42, 113.89, 81.91, 75.33, 59.96, 52.77,
41.46, 35.82, 28.25.HRMS (AP-ESI) m/z calcd for C18H23BrN2O6
[MꢀH]ꢀ: 441.0661, found: 441.0677.
5.1.4.8. (S)-2-((2S,4S)-4-(4-Bromophenoxy)-1-(tert-butoxycarbon-
yl)pyrrolidine-2-carboxamido)-4-methylpentanoic acid (14).
White crystal, yield: 85%, mp: 152–154 °C. 1H NMR (CDCl3,
400 MHz), d 0.90–0.93 (m, 6H), 1.46 (s, 9H), 1.55–1.60 (m, 1H),
1.65–1.73 (m, 2H), 2.24–2.46 (m, 1H), 2.54–2.79 (m, 1H), 3.74 (s,
2H), 4.45 (br s, 1H), 4.58 (br s, 1H), 4.80 (s, 1H), 6.68 (d, J = 8.8 Hz,
2H), 7.36 (d, J = 8.8 Hz, 2H). 13C NMR (CDCl3, 75 MHz), d 178.79,
175.02, 158.87, 135.39, 132.49, 120.33, 117.43, 116.83, 84.73,
79.01, 63.01, 55.95, 53.80, 44.52, 31.07, 27.56, 25.59, 24.89. HRMS
(AP-ESI) m/z calcd for C22H31BrN2O6 [MꢀH]ꢀ: 497.1287, found:
497.1314.
5.1.4.3. 2-((2S,4S)-4-(4-Phenylphenoxy)-1-(tert-butoxycarbonyl)
pyrrolidine-2-carboxamido)acetic acid (9). White crystal, yield:
95%, mp: 195–197 °C. 1H NMR (CDCl3, 400 MHz), d 1.46 (s, 9H),
2.48–2.71 (m, 2H), 3.76–3.82 (m, 2H), 4.00–4.20 (m, 2H), 4.46 (br
s, 1H), 4.91 (s, 1H), 6.84 (d, J = 8.0 Hz, 2H), 7.12 (d, J = 4.0 Hz, 1H),
7.27–7.31 (m, 1H), 7.37 (t, J = 7.2 Hz, 2H), 7.46–7.50 (m, 4H). 13C
NMR (CDCl3, 100 MHz), d 172.94, 172.20, 156.05, 155.34, 140.56,
134.73, 128.73, 128.37, 126.83, 126.76, 115.86, 81.84, 75.14,
60.11, 52.86, 41.50, 28.26.HRMS (AP-ESI) m/z calcd for
5.1.4.9. (S)-2-((2S,4S)-4-(4-Phenylphenoxy)-1-(tert-butoxycar-
bonyl)pyrrolidine-2-carboxamido)-4-methylpentanoic
(15).
acid
White crystal, yield: 95%, mp: 182–184 °C. 1H NMR
(CDCl3, 400 MHz), d 0.91–0.96 (m, 6H), 1.47 (s, 9H), 1.59–1.63
(m, 1H), 1.69–1.75 (m, 2H), 2.33–2.56 (m, 1H), 2.60–2.86 (m,
1H), 3.78 (s, 2H), 4.61 (br s, 1H), 4.63 (br s, 1H), 4.89 (s, 1H),
6.86 (d, J = 8.4 Hz, 2H), 7.29–7.33 (m, 1H), 7.39 (t, J = 7.6 Hz, 2H),
C
24H28N2O6 [MꢀH]ꢀ: 439.1869, found: 439.1876.
5.1.4.4. (S)-2-((2S,4S)-4-(4-Bromophenoxy)-1-(tert-butoxycarbon-
yl)pyrrolidine-2-carboxamido)-2-phenylacetic acid (10).
White crystal, yield: 50%, mp: 96–98 °C. 1H NMR (CDCl3,
400 MHz), d 1.47 (s, 9H), 2.18–2.42 (m, 1H), 2.48–2.66 (m, 1H),
3.66 (s, 2H), 4.45 (br s, 1H), 4.74 (s, 1H), 5.57 (d, J = 6.8 Hz, 1H),
6.20–6.35 (m, 2H), 7.18 (d, J = 7.0 Hz, 2H), 7.29–7.33 (m, 3H), 7.38
7.49–7.54 (m, 4H). 13C NMR (CDCl3, 75 MHz),
d 176.39,
176.23, 156.35, 155.25, 140.55, 134.74, 128.74, 128.32, 126.85,
126.76, 115.86, 81.68, 75.81, 60.17, 53.16, 50.86, 41.70,
30.57, 28.17, 24.67, 22.68, 22.03, 20.69, 19.17. HRMS
(AP-ESI) m/z calcd for C28H36N2O6 [MꢀH]ꢀ: 495.2495, found:
495.2505.
(d, J = 7.2 Hz, 2H). 13C NMR (CDCl3, 75 MHz),
d
172.90,
171.79, 155.53, 155.37, 136.43, 132.30, 128.97, 128.50, 127.52,
117.46, 113.78, 82.00, 75.66, 60.16, 56.40, 52.91, 29.68, 28.19. HRMS
(AP-ESI) m/z calcd for C24H27BrN2O6 [MꢀH]ꢀ: 517.0974, found:
517.0982.
5.1.5. (2S,4S)-tert-Butyl 4-(4-phenylphenoxy)-2-(((S)-1-oxo-3-
phenyl-1-(phenylsulfonamido)propan-2-yl)carbamoyl)
pyrrolidine-1-carboxylate (16)
The intermediate 6a (0.49 g, 1.2 mmol) was dissolved in EtOAc
saturated with HCl and stirred at room temperature overnight.
Then the generated hydrochloride precipitate was collected and
dried for the next step. The other intermediate 4b (0.38 g, 1 mmol)
was dissolved in 10 mL anhydrous THF at ꢀ20 °C. 10 min later,
N-methylmorpholine (0.22 g, 2.2 mmol) and isobutyl chlorofor-
mate (0.16 g, 1.1 mmol) was added successively. After 1 h, the
hydrochloride precipitate previously dried was added and the
reaction mixture was warm slowly to rt and stirred until no start-
ing materials was existed. Then the solvent was evaporated under
reduced pressure and was extracted with EtOAc, washed with 1 M
citric acid, saturated NaHCO3, brine and dried over MgSO4. The
concentrated residue was purified by column chromatography
(petroleum ether/ethyl acetate = 2:1) to obtain the compound 16
as white powder. Yield: 40%, mp: 94–96 °C. 1H NMR (CDCl3,
600 MHz), d 1.38 (s, 9H), 2.48–2.51 (m, 1H), 2.63 (d, J = 7.1 Hz,
1H), 2.91 (d, J = 10.1 Hz, 1H), 3.40 (d, J = 12.6 Hz, 1H), 3.71 (d,
J = 11.5 Hz, 1H), 3.76 (d, J = 11.2 Hz, 1H), 4.46 (d, J = 10.3 Hz, 1H),
4.83 (s, 1H), 4.94 (s, 1H), 6.71 (d, J = 7.3 Hz, 1H), 6.91 (d,
J = 7.7 Hz, 2H), 7.01 (d, J = 7.5 Hz, 2H), 7.14 (m, 2H), 7.22 (m, 1H),
7.34 (t, J = 7.0 Hz, 1H), 7.45 (t, J = 6.8 Hz, 2H), 7.56–7.57 (m, 6H),
7.65–7.66 (m, 1H), 8.06 (d, J = 6.8 Hz, 2H), 10.00 (s, 1H). 13C NMR
5.1.4.5. (S)-2-((2S,4S)-4-(4-Phenylphenoxy)-1-(tert-butoxycarbo-
nyl)pyrrolidine-2-carboxamido)-2-phenylacetic acid (11).
White crystal, yield: 39%, mp: 211–213 °C. 1H NMR (CDCl3,
400 MHz), d 1.48 (s, 9H), 2.23–2.42 (m, 1H), 2.55–2.70 (m, 1H),
3.71–3.78 (m, 2H), 4.48 (br s, 1H), 4.85 (s, 1H), 5.60 (d, J = 6.8 Hz,
1H), 6.45–6.53 (m, 2H), 7.25 (s, 1H), 7.29–7.32 (m, 5H), 7.39–7.42
(m, 4H), 7.48 (d, J = 7.6 Hz, 2H). 13C NMR (CDCl3, 75 MHz), d
173.56, 172.03, 156.00, 155.34, 140.65, 134.60, 129.02, 128.73,
128.55, 128.20, 127.55, 126.81, 126.78, 115.87, 81.82, 75.56, 60.37,
56.49, 53.05, 29.70, 28.21. HRMS (AP-ESI) m/z calcd for
C
30H32N2O6 [MꢀH]ꢀ: 515.2182, found: 515.2206.
5.1.4.6. (S)-2-((2S,4S)-4-(4-Bromophenoxy)-1-(tert-butoxycarbon-
yl)pyrrolidine-2-carboxamido)-3-methylbutanoic acid (12).
White crystal, yield: 33%, mp: 80–82 °C. 1H NMR (CDCl3,
400 MHz), d 0.93 (dd, J = 11.2 Hz, 6.8 Hz, 6H), 1.46 (s, 9H), 2.21–
2.23 (m, 1H), 2.33–2.51 (m, 1H), 2.56–2.89 (m, 1H), 3.72–3.86 (m,
2H), 4.49 (d, J = 5.6 Hz, 2H), 4.80 (s, 1H), 6.68 (d, J = 8.8 Hz, 2H),
7.35 (d, J = 8.8 Hz, 2H). 13C NMR (CDCl3, 75 MHz), d 174.82, 172.15,
156.13, 155.22, 132.48, 117.49, 113.87, 81.75, 76.29, 60.21, 57.25,
53.15, 31.29, 28.18, 19.03, 17.80. HRMS (AP-ESI) m/z calcd for
(CDCl3, 100 MHz),
d 171.58, 169.88, 156.16, 140.37, 138.87,
C
21H29BrN2O6 [MꢀH]ꢀ: 483.1131, found: 483.1138.
135.10, 134.75, 133.65, 129.45, 128.93, 128.80, 128.70, 128.63,
128.57, 127.50, 126.99, 126.77, 115.91, 82.30, 60.13, 53.24, 36.80,
34.66, 28.17. HRMS (AP-ESI) m/z calcd for C37H39N3O7S [MꢀH]ꢀ
668.2430, found: 668.2436.
Compounds 17–31 were synthesized following the procedure
described above.
5.1.4.7. (S)-2-((2S,4S)-4-(4-Phenylphenoxy)-1-(tert-butoxycarbon-
yl)pyrrolidine-2-carboxamido)-3-methylbutanoic acid (13).
White crystal, yield: 90%, mp: 185–186 °C. 1H NMR (CDCl3,
400 MHz),
d 0.96 (dd, J = 12.8 Hz, 6.8 Hz, 6H), 1.46 (s, 9H),

Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
7691
5.1.5.1. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-1-(4-meth-
ylphenylsulfonamido)-1-oxo-3-phenylpropan-2-yl)carbamoyl)py-
rrolidine-1-carboxylate (17). White powder, yield: 31%, mp: 89–
91 °C. 1H NMR (CDCl3, 600 MHz), d 1.37 (s, 9H), 2.46 (s, 3H),
2.49–2.50 (m, 1H), 2.61 (d, J = 14.1 Hz, 1H), 2.90 (d, J = 13.0 Hz, 1H),
3.41 (d, J = 13.2 Hz, 1H), 3.70–3.71 (m, 1H), 3.75 (d, J = 10.7 Hz,
1H), 4.44 (d, J = 10.1 Hz, 1H), 4.79 (br s, 1H), 4.92 (s, 1H), 6.66 (d,
J = 5.9 Hz, 1H), 6.90 (d, J = 5.7 Hz, 2H), 7.04 (d, J = 6.4 Hz, 2H), 7.14–
7.15 (m, 2H), 7.21–7.23 (m, 1H), 7.33–7.34 (m, 3H), 7.42–7.44 (m,
2H), 7.55–7.56 (m, 4H), 7.93 (d, J = 5.2 Hz, 2H), 9.87 (s, 1H). 13C
NMR (CDCl3, 100 MHz), d 171.44, 169.95, 156.18, 144.63, 140.38,
135.92, 135.08, 134.80, 129.48, 129.32, 128.89, 128.80, 128.71,
128.56, 127.47, 126.99, 126.77, 115.91, 82.29, 60.12, 53.11, 36.81,
34.66, 28.17, 21.66. HRMS (AP-ESI) m/z calcd for C38H41N3O7S
[MꢀH]ꢀ 682.2587, found: 682.2600.
(AP-ESI) m/z calcd for C38H40BrN3O7S [MꢀH]ꢀ 760.1692, found:
760.1695.
5.1.5.5. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-3-(4-bro-
mophenyl)-1-(4-chloro-3-nitrophenylsulfonamido)-1-oxopropan-
2-yl)carbamoyl)pyrrolidine-1-carboxylate
(21).
Yellow
powder, yield: 41%, mp: 97–99 °C. 1H NMR (CDCl3, 600 MHz), d
1.39 (s, 9H), 2.46–2.50 (m, 1H), 2.58 (d, J = 13.9 Hz, 1H), 2.90 (d,
J = 8.6 Hz, 1H), 3.27 (d, J = 11.0 Hz, 1H), 3.68 (d, J = 11.3 Hz, 1H),
3.77 (d, J = 11.8 Hz, 1H), 4.41 (d, J = 10.4 Hz, 1H), 4.73 (s, 1H), 4.90
(s, 1H), 6.68 (d, J = 5.9 Hz, 1H), 6.86 (d, J = 8.6 Hz, 2H), 6.93 (d,
J = 7.02 Hz, 2H), 7.28 (d, J = 7.0 Hz, 2H), 7.33 (t, J = 7.2 Hz, 1H), 7.43
(t, J = 7.3 Hz, 2H), 7.53–7.55 (m, 4H), 7.71 (d, J = 8.1 Hz, 1H), 8.18 (d,
J = 7.7 Hz, 1H), 8.52 (s, 1H), 10.34 (s, 1H). 13C NMR (CDCl3,
100 MHz), d 171.83, 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, 53.36, 53.23, 36.30,
34.60, 28.09. HRMS (AP-ESI) m/z calcd for C37H36BrClN4O9S [MꢀH]ꢀ
825.0997, found: 825.1006.
5.1.5.2. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-1-(4-chlo-
ro-3-nitrophenylsulfonamido)-1-oxo-3-phenylpropan-2-yl)-
carbamoyl)pyrrolidine-1-carboxylate (18). Yellow powder,
yield: 35%, mp: 106–109 °C. 1H NMR (CDCl3, 600 MHz), d 1.35
(s, 9H), 2.50–2.51 (m, 1H), 2.56 (d, J = 14.2 Hz, 1H), 2.92–2.94
(m, 1H), 3.37–3.38 (m, 1H), 3.68–3.40 (m, 1H), 3.77 (d,
J = 11.8 Hz, 1H), 4.40 (d, J = 8.7 Hz, 1H), 4.73 (s, 1H), 4.91 (s,
1H), 6.65 (d, J = 7.4 Hz, 1H), 6.86 (d, J = 8.2 Hz, 2H), 7.04 (d,
J = 7.4 Hz, 2H), 7.17 (t, J = 7.5 Hz, 2H), 7.23 (d, J = 7.3 Hz, 1H),
7.33 (t, J = 7.4 Hz, 1H), 7.43 (t, J = 7.5 Hz, 2H), 7.53–7.54 (m, 4H),
7.74 (d, J = 8.6 Hz, 1H), 8.23 (d, J = 8.4 Hz, 1H), 8.48 (s, 1H),
10.21 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 171.67, 170.42,
156.04, 147.52, 140.27, 138.81, 134.48, 132.93, 132.68, 132.49,
129.32, 128.99, 128.83, 128.61, 127.77, 127.06, 126.76, 126.03,
115.88, 82.60, 60.14, 53.28, 36.81, 34.70, 28.14, 18.95. HRMS
(AP-ESI) m/z calcd for C37H37ClN4O9S [MꢀH]ꢀ 747.1892, found:
747.1901.
5.1.5.6. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-3-(4-bro-
mophenyl)-1-(4-nitrophenylsulfonamido)-1-oxopropan-2-yl)-
carbamoyl)pyrrolidine-1-carboxylate (22). Yellow powder,
yield: 30%, mp: 128–130 °C. 1H NMR (CDCl3, 600 MHz), d 1.35 (s,
9H), 2.48–2.51 (m, 1H), 2.57 (d, J = 13.8 Hz, 1H), 2.90 (dd,
J = 13.8 Hz, 4.2 Hz, 1H), 3.31 (d, J = 11.4 Hz, 1H), 3.69 (d, J = 9 Hz,
1H), 3.79 (d, J = 12.6 Hz, 1H), 4.41 (d, J = 9.6 Hz, 1H), 4.71 (s, 1H),
4.91 (s, 1H), 6.64 (d, J = 7.8 Hz, 1H), 6.87 (d, J = 8.4 Hz, 2H), 6.97
(d, J = 7.8 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.34 (t, J = 7.2 Hz, 1H),
7.43 (t, J = 7.2 Hz, 2H), 7.54–7.56 (m, 4H), 8.24 (d, J = 8.4 Hz, 2H),
8.37 (d, J = 8.4 Hz, 2H), 10.20 (s, 1H). 13C NMR (CDCl3, 100 MHz),
d 172.18, 169.8, 156.00, 150.69, 144.22, 140.16, 135.41, 133.45,
132.05, 131.12, 130.01, 128.85, 128.68, 127.13, 126.76, 123.94,
121.90, 115.96, 82.77, 60.09, 53.36, 53.21, 36.11, 34.36, 28.12.
HRMS (AP-ESI) m/z calcd for C37H37BrN4O9S [MꢀH]ꢀ 791.1386,
found: 791.1387.
5.1.5.3. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-3-(4-bro-
mophenyl)-1-oxo-1-(phenylsulfonamido)propan-2-yl)carbamoyl)
pyrrolidine-1-carboxylate (19). White powder, yield: 51%, mp: 96–
98 °C. 1H NMR (CDCl3, 600 MHz), d 1.40 (s, 9H), 2.45–2.46 (m, 1H), 2.62
(d, J = 13.4 Hz, 1H), 2.84 (d, J = 10.7 Hz, 1H), 3.32 (d, J = 11.4 Hz, 1H),
3.71–3.74 (m, 2H), 4.44 (d, J = 9.5 Hz, 1H), 4.80 (s, 1H), 4.91
(s, 1H), 6.62 (d, J = 8.7 Hz, 1H), 6.86 (d, J = 8.0 Hz, 2H), 6.88 (d,
J = 8.5 Hz, 2H), 7.21 (d, J = 6.9 Hz, 2H), 7.32 (t, J = 7.3 Hz, 1H),
7.42 (d, J = 7.4 Hz, 2H), 7.53–7.54 (m, 6H), 7.65 (t, J = 7.0 Hz, 1H), 8.04
(d, J = 6.1 Hz, 2H), 9.96 (s, 1H). 13C NMR (CDCl3, 100 MHz), d
171.99, 169.54, 156.13, 140.27, 138.79, 135.18, 133.73,
131.90, 131.17, 128.81, 128.75, 128.63, 127.03, 126.77, 121.59,
115.94, 81.93, 60.05, 53.36, 52.84, 36.34, 34.55, 28.12. HRMS
(AP-ESI) m/z calcd for C37H38BrN3O7S [MꢀH]ꢀ 746.1536, found:
746.1546.
5.1.5.7. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-3-(4-bro-
mophenyl)-1-(4-chlorophenylsulfonamido)-1-oxopropan-2-yl)-
carbamoyl)pyrrolidine-1-carboxylate
(23). White
powder,
yield: 26%, mp: 125–127 °C. 1H NMR ((CD3)2CO, 600 MHz), d 1.35
(s, 9H), 2.19–2.27 (m, 1H), 2.53–2.54 (m, 1H), 2.91–2.92 (m, 1H),
3.03 (dd, J = 13.2 Hz, 4.8 Hz, 1H), 3.51–3.56 (m, 1H), 3.75–3.76 (m,
1H), 4.21–4.26 (m, 1H), 4.60–4.70 (m, 1H), 5.00 (br s, 1H), 6.74–
6.88 (m, 2H), 6.98 (d, J = 7.8 Hz, 2H), 7.20–7.23 (m, 3H), 7.33 (t,
J = 7.8 Hz, 2H), 7.49–7.52 (m, 4H), 7.58 (d, J = 7.8 Hz, 2H), 7.89 (d,
J = 7.8 Hz, 2H). 13C NMR (CDCl3, 100 MHz), d 171.84, 169.69,
156.06, 140.44, 140.23, 137.17, 133.64, 131.97, 131.14, 130.15,
129.05, 128.83, 128.66, 127.08, 126.77, 115.95, 82.58, 60.07, 53.36,
52.92, 28.11.HRMS (AP-ESI) m/z calcd for C37H37BrClN3O7S [MꢀH]ꢀ
780.1146, found: 780.1149.
5.1.5.4. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-3-(4-bro-
mophenyl)-1-(4-methylphenylsulfonamido)-1-oxopropan-2-yl)-
carbamoyl)pyrrolidine-1-carboxylate (20). White powder, yield:
37%, mp: 95–97 °C. 1H NMR (CDCl3, 600 MHz), d 1.38 (s, 9H), 2.44
(s, 3H), 2.46–2.47 (m, 1H), 2.59 (d, J = 13.7 Hz, 1H), 2.82 (d,
J = 7.6 Hz, 1H), 3.32 (d, J = 15.4 Hz, 1H), 3.69–3.74 (m, 2H), 4.41
(d, J = 10.1 Hz, 1H), 4.75 (s, 1H), 4.88 (s, 1H), 6.56 (d, J = 7.9 Hz,
1H), 6.86–6.88 (m, 4H), 7.20 (d, J = 7.4 Hz, 2H), 7.30–7.32 (m,
3H), 7.41 (t, J = 7.6 Hz, 2H), 7.51–7.52 (m, 4H), 7.89 (d, J = 7.0 Hz,
2H), 9.85 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 171.69, 169.69,
156.14, 144.77, 140.28, 135.82, 135.15, 133.74, 131.84, 131.19,
129.36, 128.81, 128.66, 128.61, 127.02, 126.77, 121.54, 115.93,
82.40, 60.04, 53.36, 52.83, 36.36, 34.37, 28.12, 21.66. HRMS
5.1.5.8.
(2S,4S)-tert-Butyl-2-(((S)-3-(1H-indol-3-yl)-1-oxo-1-
(phenylsulfonamido)propan-2-yl)carbamoyl)-4-(4-phenylphe-
noxy)pyrrolidine-1-carboxylate (24). Yellow powder, yield:
28%, mp: 115–117 °C. 1H NMR (CDCl3, 600 MHz), d 1.11 (s, 9H),
2.53–2.55 (m, 1H), 2.61 (d, J = 14.3 Hz, 1H), 3.15 (dd, J = 14.8 Hz,
4.1 Hz, 1H), 3.56 (dd, J = 14.8 Hz, 3.4 Hz, 1H), 3.62 (dd, J = 11.9 Hz,
3.7 Hz, 1H), 3.76 (d, J = 12.1 Hz, 1H), 4.41 (d, J = 9.5 Hz, 1H), 4.87–
4.88 (m, 1H), 4.97 (s, 1H), 6.81 (s, 1H), 6.85–6.86 (m, 2H), 7.00
(d, J = 8.4 Hz, 2H), 7.17 (t, J = 7.4 Hz, 1H), 7.33–7.36 (m, 2H), 7.44
(t, J = 7.6 Hz, 2H), 7.53–7.59 (m, 6H), 7.65 (d, J = 7.1 Hz, 2H), 8.05
(d, J = 7.7 Hz, 1H), 8.07 (s, 1H), 9.92 (s, 1H). 13C NMR (CDCl3,
100 MHz),
d 171.49, 170.91, 156.29, 155.77, 140.41, 139.08,

7692
Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
136.07, 135.05, 133.55, 128.80, 128.62, 127.54, 126.97, 126.78,
124.31, 122.39, 120.03, 118.22, 115.72, 111.69, 108.08, 82.13,
60.25, 54.78, 52.88, 34.83, 27.71, 26.67. HRMS (AP-ESI) m/z calcd
for C39H40N4O7S [MꢀH]ꢀ 707.2539, found: 707.2546.
(s, 1H), 6.84 (d, J = 8.3 Hz, 2H), 7.04 (d, J = 7.2 Hz, 2H), 7.17 (t,
J = 7.2 Hz, 2H), 7.23 (m, 3H), 7.43 (d, J = 7.8 Hz, 2H), 7.51 (d,
J = 8.4 Hz, 2H), 7.73 (d, J = 8.4 Hz, 1H), 8.23 (d, J = 7.8 Hz, 1H),
8.46 (s, 1H), 10.21 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 171.59,
170.43, 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
5.1.5.9. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-1-(4-chlo-
ro-3-nitrophenylsulfonamido)-3-(1H-indol-3-yl)-1-oxopropan-
2-yl)carbamoyl)pyrrolidine-1-carboxylate (25). Yellow pow-
der, yield: 33%, mp: 123–125 °C. 1H NMR (CDCl3, 600 MHz), d
1.09 (s, 9H), 2.53–2.60 (m, 2H), 3.14 (dd, J = 15.0 Hz, 4.2 Hz, 1H),
3.52 (d, J = 15.0 Hz, 1H), 3.62 (d, J = 8.4 Hz, 1H), 3.78 (d, J = 6.0 Hz,
1H), 4.37 (d, J = 8.4 Hz, 1H), 4.84 (s, 1H), 4.96 (s, 1H), 6.74 (s, 1H),
6.83–6.88 (m, 2H), 6.95 (d, J = 7.8 Hz, 1H), 7.17 (t, J = 7.2 Hz, 1H),
7.26–7.36 (m, 2H), 7.42 (t, J = 7.2 Hz, 2H), 7.54–7.56 (m, 4H), 7.62
(d, J = 7.8 Hz, 1H), 7.73 (d, J = 7.8 Hz, 1H), 8.12 (br s, 1H), 8.24 (d,
J = 8.4 Hz, 1H), 8.33 (s, 1H), 10.19 (s, 1H). 13C NMR (CDCl3,
C
38H39ClN4O9S [MꢀH]ꢀ 761.2048, found: 761.2052.
5.1.5.13.
(2S,4S)-tert-butyl-2-(((S)-3-(4-bromophenyl)-1-(4-
chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)carbam-
oyl)-4-(4-(40-methyl)phenylphenoxy)pyrrolidine-1-carboxylate
(29). Yellow powder, yield: 31%, mp: 111–113 °C. 1H NMR (CDCl3,
300 MHz), d 1.39 (s, 9H), 2.39 (s, 3H), 2.47–2.60 (m, 2H), 2.90 (dd,
J = 13.8 Hz, 5.1 Hz, 1H), 3.31 (d, J = 13.2 Hz, 1H), 3.68 (dd,
J = 12.0 Hz, 3.0 Hz, 1H), 3.77 (d, J = 12.3 Hz, 1H), 4.40 (d,
J = 9.0 Hz, 1H), 4.70–4.73 (m, 1H), 4.90 (s, 1H), 6.61 (m, 1H), 6.84
(d, J = 8.4 Hz, 2H), 6.94 (d, J = 7.5 Hz, 2H), 7.23 (m, 2H), 7.31 (m,
2H), 7.44 (d, J = 7.8 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H), 7.72 (d,
J = 8.1 Hz, 1H), 8.20 (d, J = 7.8 Hz, 1H), 8.53 (s, 1H). 13C NMR (CDCl3,
100 MHz),
d 171.48, 156.16, 155.94, 147.61, 140.33, 139.03,
136.13, 135.20, 133.20, 132.35, 128.82, 128.67, 127.36, 127.03,
126.77, 125.69, 123.84, 122.65, 120.19, 118.13, 115.70, 111.81,
108.23, 82.39, 60.28, 54.87, 52.97, 34.82, 27.65, 26.61. HRMS
(AP-ESI) m/z calcd for C39H38ClN5O9S [MꢀH]ꢀ 786.2001, found:
786.1996.
100 MHz),
d 172.14, 169.69, 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 [MꢀH]ꢀ 839.1153, found: 839.1159.
5.1.5.10. (2S,4S)-tert-Butyl-4-(4-(40-methyl)phenylphenoxy)-2-
(((S)-1-oxo-3-phenyl-1-(phenylsulfonamido)propan-2-yl)carba-
moyl)pyrrolidine-1-carboxylate (26). White powder, yield:
35%, mp: 86–88 °C. 1H NMR (CDCl3, 600 MHz), d 1.37 (s, 9H),
2.41 (s, 3H), 2.47–2.51 (m, 1H), 2.61 (d, J = 14.4 Hz, 1H), 2.90 (dd,
J = 13.7 Hz, 5.0 Hz, 1H), 3.43 (dd, J = 13.7 Hz, 3.2 Hz, 1H), 3.70–
3.72 (m, 1H), 3.76 (d, J = 11.8 Hz, 1H), 4.44 (d, J = 9.9 Hz, 1H),
4.79 (s, 1H), 4.92 (s, 1H), 6.63 (d, J = 8.2 Hz, 1H), 6.89 (d,
J = 8.1 Hz, 2H), 7.03 (d, J = 7.14 Hz, 2H), 7.16 (t, J = 7.4 Hz, 2H),
7.22–7.23 (m, 1H), 7.26 (d, J = 7.9 Hz, 2H), 7.46 (d, J = 7.7 Hz, 2H),
7.53–7.57 (m, 4H), 7.67 (t, J = 7.3 Hz, 1H), 8.07 (d, J = 7.6 Hz, 2H),
9.91 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 171.99, 170.03, 155.95,
138.89, 137.50, 136.74, 135.11, 134.76, 133.64, 129.52, 129.45,
128.93, 128.70, 128.63, 128.36, 127.49, 126.61, 115.91, 82.30,
65.68, 60.14, 53.25, 36.81, 34.68, 29.70, 28.17, 21.06. HRMS (AP-
ESI) m/z calcd for C38H41N3O7S [MꢀH]ꢀ 682.2587, found: 682.2587.
5.1.5.14.
(2S,4S)-tert-Butyl-2-(((S)-3-(4-bromophenyl)-1-(4-
chloro-3-nitrophenylsulfonamido)-1-oxopropan-2-yl)carba-
moyl)-4-(4-(40-chloro)phenylphenoxy)pyrrolidine-1-carboxy-
late (30). Yellow powder, yield: 26%. Mp: 125–127 °C. 1H NMR
(CDCl3, 600 MHz), d 1.40 (s, 9H), 2.47–2.50 (m, 1H), 2.57 (d,
J = 14.4 Hz, 1H), 2.90 (d, J = 13.8 Hz, 1H), 3.30 (d, J = 10.8 Hz, 1H),
3.69 (d, J = 11.4 Hz, 1H), 3.77 (d, J = 11.4 Hz, 1H), 4.40 (d,
J = 10.2 Hz, 1H), 4.73 (s, 1H), 4.90 (s, 1H), 6.64 (d, J = 7.2 Hz, 1H),
6.85 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 7.8 Hz, 2H), 7.30 (d, J = 7.2 Hz,
2H), 7.40 (d, J = 7.8 Hz, 2H), 7.47 (d, J = 7.8 Hz, 2H), 7.51 (d,
J = 7.8 Hz, 2H), 7.73 (d, J = 8.4 Hz, 1H), 8.20 (d, J = 7.2 Hz, 1H),
8.53 (s, 1H), 10.26 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 171.80,
170.02, 156.21, 147.54, 138.64, 134.11, 133.42, 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 C37H35BrCl2N4O9S
[MꢀH]ꢀ 859.0607, found: 859.0610.
5.1.5.11. (2S,4S)-tert-Butyl-4-(4-(40-methyl)phenylphenoxy)-2-
(((S)-1-(4-methylphenylsulfonamido)-1-oxo-3-phenylpropan-
2-yl)carbamoyl)pyrrolidine-1-carboxylate (27). White pow-
der, yield: 34%, mp: 96–98 °C. 1H NMR (CDCl3, 600 MHz), d 1.35
(s, 9H), 2.38 (s, 3H), 2.44 (s, 3H), 2.47–2.48 (m, 1H), 2.58 (d,
J = 14.8 Hz, 1H), 2.89 (dd, J = 13.8 Hz, 4.3 Hz, 1H), 3.39 (d,
J = 11.2 Hz, 1H), 3.67 (d, J = 14.4 Hz, 1H), 3.72 (d, J = 10.9 Hz, 1H),
4.41 (d, J = 9.1 Hz, 1H), 4.75 (s, 1H), 4.89 (s, 1H), 6.81 (d,
J = 7.7 Hz, 1H), 6.86 (d, J = 8.0 Hz, 2H), 7.02 (d, J = 7.3 Hz, 2H),
7.14 (t, J = 7.7 Hz, 2H), 7.19–7.24 (m, 3H), 7.31 (d, J = 7.6 Hz, 2H),
7.43 (d, J = 7.9 Hz, 2H), 7.51 (d, J = 7.9 Hz, 2H), 7.92 (d, J = 7.7 Hz,
2H), 9.84 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 171.38, 170.03,
155.97, 144.61, 137.52, 136.73, 135.94, 135.06, 134.82, 129.51,
129.49, 129.32, 128.88, 128.69, 128.34, 127.45, 126.61, 115.91,
82.25, 60.13, 53.23, 36.84, 34.68, 28.17, 21.65, 21.06, 19.20. HRMS
(AP-ESI) m/z calcd for C39H43N3O7S [MꢀH]ꢀ 696.2743, found:
696.2749.
5.1.5.15. (2S,4S)-tert-Butyl-4-(4-phenylphenoxy)-2-(((S)-1-(4-chlo-
ro-3-nitrophenylsulfonamido)-3-(4-hydroxyphenyl)-1-oxoprop-
an-2-yl)carbamoyl)pyrrolidine-1-carboxylate (31). Yellow pow-
der, yield 88%. Mp: 116–118 °C. 1H NMR (CDCl3, 600 MHz), d 1.38
(s, 9H), 2.48–2.52 (m, 2H), 2.84 (dd, J = 13.8 Hz, 4.2 Hz, 1H), 3.31 (d,
J = 11.4 Hz, 1H), 3.69 (d, J = 10.2 Hz, 1H), 3.81 (d, J = 12.6 Hz,
1H), 4.39 (d, J = 9.6 Hz, 1H), 4.69 (s, 1H), 4.91 (s, 1H), 5.38 (br s,
1H), 6.61 (d, J = 7.8 Hz, 2H), 6.69 (d, J = 6.6 Hz, 1H), 6.88–6.89 (m,
4H), 7.32 (t, J = 7.2 Hz, 1H), 7.42 (t, J = 7.8 Hz, 2H), 7.53–7.55 (m,
4H), 7.72 (d, J = 7.8 Hz, 1H), 8.82 (d, J = 8.4 Hz, 1H), 8.45 (s, 1H),
10.19 (s, 1H). 13C NMR (CDCl3, 100 MHz), d 172.34, 170.56, 156.01,
155.61, 147.46, 140.21, 138.71, 135.24, 132.95, 132.69, 132.56,
130.52, 128.83, 128.61, 127.06, 126.74, 125.98, 125.84, 115.93,
115.87, 82.76, 60.16, 53.73, 53.27, 35.71, 34.77, 28.06. HRMS (AP-
5.1.5.12. (2S,4S)-tert-Butyl-2-(((S)-1-(4-chloro-3-nitrophenylsul-
fonamido)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-4-(4-(40-meth-
ESI) m/z Calcd for
763.1830.
C
37H37ClN4O10
S
[MꢀH]ꢀ 763.1841. Found:
yl)phenylphenoxy)pyrrolidine-1-carboxylate (28).
Yellow
powder, yield: 38%. Mp: 90–92 °C. 1H NMR (CDCl3, 600 MHz), d
1.35 (s, 9H), 2.39 (s, 3H), 2.48–2.51 (m, 1H), 2.55 (d, J = 13.8 Hz,
1H), 2.93 (dd, J = 14.4 Hz, 5.4 Hz, 1H), 3.37 (dd, J = 13.8 Hz, 4.8 Hz,
1H), 3.67 (dd, J = 12.6 Hz, 3.0 Hz, 1H), 3.76 (d, J = 12.6 Hz, 1H),
4.39 (d, J = 8.4 Hz, 1H), 4.70–4.74 (m, 1H), 4.90 (s, 1H), 6.64
5.2. In vitro binding assay for Bcl-2 proteins
Fluorescence polarization assay was used to test the activities to
Bcl-2 family proteins.
A Bid-BH3 peptide was marked with

Y. Wan et al. / Bioorg. Med. Chem. 23 (2015) 7685–7693
7693
5-carboxyfluorescein succinimidyl ester (FAM) as the fluorescence
Acknowledgments
labeled peptide (5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG).
The assay was started from mixing the Mcl-1 protein with test
compounds in PBS (pH 7.3, 140 mM NaCl, 2.7 mM KCl, 10 mM
NHPO4 and 1.8 mM KH2PO4) and preincubated for 30 min at room
Thanks for the binding assay for Bcl-2 family proteins and the
help in docking studies 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.
21172133), Shandong Natural Science Foundation for Distin-
guished Young Scholars (No. JQ201319), Program for New Century
Excellent Talents in University (NCET-12-0337).
temperature in the dark. Then 20
lL 5-FAM-Bid-BH3 peptide solu-
tion in PBS was added and generated a final total volume of 200
lL
and incubated another 20 min. The final concentration of Mcl-1
protein and 5-FAM-Bid-BH3 peptide were 195 nM and 10 nM,
respectively. Finally 60 lL above solution and 60 lL calibration
solution (1 nmol/L fluorescein and 10 mmol/L NaOH) were respec-
tively, added into a black 384-well plate (Three parallel groups).
Then the polarization values (milipolarization units, mP) were
obtained by using the Tecan GENios-Pro Injector Reader (Tecan
Group Ltd). The excitation wavelength was at 485 nm and the
emission wavelength was at 535 nm. The measured data were pro-
cessed with the GraphPad Prism software to get IC50 value and cal-
culate Ki according to the literature method.23
The methods of binding assay for Bcl-2 protein and Bcl-XL pro-
tein were almost the same as the above procedure except that the
final concentrations of Bcl-2 and Bcl-XL were 376 nM and 170 nM,
respectively. In addition, the tested pyrrolidine derivatives were
References and notes
1. Adams, J. M.; Cory, S. Oncogene 2007, 26, 1324.
2. Vinatier, D.; Dufour, P.; Subtil, D. Eur. J. Obstet. Gynecol. Reprod. Biol. 1996, 67,
85.
3. Tan, T. T.; White, E. Adv. Exp. Med. Biol. 2008, 615, 81.
4. Youle, R. J.; Strasser, A. Nat. Rev. Mol. Cell Biol. 2008, 9, 47.
5. Droin, N. M.; Green, D. R. Biochim. Biophys. Acta 2004, 1644, 179.
6. Johnstone, R. W.; Ruefli, A. A.; Lowe, S. W. Cell 2002, 108, 153.
7. Zhou, P.; Qian, L.; Bieszczad, C. K.; Noelle, R.; Binder, M.; Levy, N. B.; Craig, R. W.
Blood 1998, 92, 3226.
8. Zhou, P.; Levy, N. B.; Xie, H.; Qian, L.; Lee, C. Y.; Gascoyne, R. D.; Craig, R. W.
Blood 2001, 97, 3902.
9. Song, L.; Coppola, D.; Livingston, S.; Cress, D.; Haura, E. B. Cancer Biol. Ther.
2005, 4, 267.
10. Ding, Q.; He, X.; Xia, W.; Hsu, J. M.; Chen, C. T.; Li, L. Y.; Lee, D. F.; Yang, J. Y.; Xie,
X.; Liu, J. C.; Hung, M. C. Cancer Res. 2007, 67, 4564.
11. Krajewska, M.; Krajewski, S.; Epstein, J. I.; Shabaik, A.; Sauvageot, J.; Song, K.;
Kitada, S.; Reed, J. C. Am. J. Pathol. 1996, 148, 1567.
prepared at seven concentrations (1 nM, 10 nM, 100 nM, 1
10 M, 50 M, 100 M) in dimethylsulfoxide (DMSO) solvent.
lM,
l
l
l
12. Miyamoto, Y.; Hosotani, R.; Wada, M.; Lee, J. U.; Koshiba, T.; Fujimoto, K.; Tsuji,
S.; Nakajima, S.; Doi, R.; Kato, M.; Shimada, Y.; Imamura, M. Oncology 1999, 56,
73.
5.3. Cell culture and antiproliferative assay
13. Brotin, E.; Meryet-Figuiere, M.; Simonin, K.; Duval, R. E.; Villedieu, M.; Leroy-
Dudal, J.; Saison-Behmoaras, E.; Gauduchon, P.; Denoyelle, C.; Poulain, L. Int. J.
Cancer 2010, 126, 885.
Three human tumor cell lines (MDA-MB-231, PC-3 and K562)
were incubated at the condition of 5% CO2 at 37 °C with the
RPMI1640 medium supplemented with 10% fetal bovine serum.
When cells were at logarithmic growth phase, cancer cells were
seeded in 96-well plates at 5000 cells per well and incubated for
12 h. Then a range of concentrations of tested compounds were
added into 96-well plates and incubated for 48 h before adding
14. Wertz, I. E.; Kusam, S.; Lam, C.; Okamoto, T.; Sandoval, W.; Anderson, D. J.;
Helgason, E.; Ernst, J. A.; Eby, M.; Liu, J.; Belmont, L. D.; Kaminker, J. S.;
O’Rourke, K. M.; Pujara, K.; Kohli, P. B.; Johnson, A. R.; Chiu, M. L.; Lill, J. R.;
Jackson, P. K.; Fairbrother, W. J.; Seshagiri, S.; Ludlam, M. J.; Leong, K. G.;
Dueber, E. C.; Maecker, H.; Huang, D. C.; Dixit, V. M. Nature 2011, 471, 110.
15. Zhang, Z.; Wu, G.; Xie, F.; Song, T.; Chang, X. J. Med. Chem. 2011, 54, 1101.
16. Song, T.; Chen, Q.; Li, X.; Chai, G.; Zhang, Z. J. Med. Chem. 2013, 56, 9366.
17. Qiu, J.; Levin, L. R.; Buck, J.; Reidenberg, M. M. Exp. Biol. Med. (Maywood) 2002,
227, 398.
10
and 150
using
l
L MTT (5 mg/mL) per well. After 4 h, the medium was removed
L DMSO/well was added. The absorbance was measured
microtiter-plate reader (Thermo Varioskan Flash) at
l
18. Becattini, B.; Kitada, S.; Leone, M.; Monosov, E.; Chandler, S.; Zhai, D.; Kipps, T.
J.; Reed, J. C.; Pellecchia, M. Chem. Biol. 2004, 11, 389.
a
570 nm after mixing for 5 min. The IC50 values were calculated
according to the inhibitory ratios.
19. Bruncko, M.; Wang, L.; Sheppard, G. S.; Phillips, D. C.; Tahir, S. K.; Xue, J.;
Erickson, S.; Fidanze, S.; Fry, E.; Hasvold, L.; Jenkins, G. J.; Jin, S.; Judge, R. A.;
Kovar, P. J.; Madar, D.; Nimmer, P.; Park, C.; Petros, A. M.; Rosenberg, S. H.;
Smith, M. L.; Song, X.; Sun, C.; Tao, Z.-F.; Wang, X.; Xiao, Y.; Zhang, H.; Tse, C.;
Leverson, J. D.; Elmore, S. W.; Souers, A. J. J. Med. Chem. 2015, 58, 2180.
20. Wang, L.; Sloper, D. T.; Addo, S. N.; Tian, D.; Slaton, J. W.; Xing, C. Cancer Res.
2008, 68, 4377.
5.4. Docking study
The docking study was performed based on the Surflex-Dock
program in Sybyl 7.3. The parameters were set at default values
except that the structure of compound 21 was added Gasteiger–
Hückel charges. The Mcl-1 protein (PDB code: 2PQK) was down-
loaded from the Protein Data Bank (PDB).
21. Wan, Y.; Wu, S.; Xiao, G.; Liu, T.; Hou, X.; Chen, C.; Guan, P.; Yang, X.; Fang, H.
Bioorg. Med. Chem. 1994, 2015, 23.
22. Wu, S. L.; Chen, M. L.; Wang, Y. T.; Hou, X. B.; Yang, X. Y.; Su, L.; Fang, H. Drug
Disc. Ther. 2012, 6, 62.
23. Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.; Li, P.; Roller, P.
P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A. Anal. Biochem. 2004, 332, 261.