Design, synthesis and preliminary bioactivity studies of imidazolidine-2,4-dione derivatives as Bcl-2 inhibitors

Article abstract of DOI:10.1016/j.bmc.2015.10.023

Anti-apoptotic B-cell lymphoma-2 (Bcl-2) proteins are promising targets for cancer therapy. In the present study, a series of imidazolidine-2,4-dione derivatives were designed and synthesized to test their inhibitory activities against anti-apoptotic Bcl-

Full text of DOI:10.1016/j.bmc.2015.10.023

Accepted Manuscript
Design, synthesis and preliminary bioactivitystudiesofimidazolidine-2,4-dione
derivatives as Bcl-2 inhibitors
Gang Wang, Yutao Wang, Lei Wang, Leiqiang Han, Xuben Hou, Huansheng
Fu, Hao Fang
PII:
S0968-0896(15)30101-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2015.10.023
BMC 12623
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 August 2015
14 October 2015
16 October 2015
Please cite this article as: Wang, G., Wang, Y., Wang, L., Han, L., Hou, X., Fu, H., Fang, H., Design, synthesis and
preliminary bioactivity studies of imidazolidine-2,4-dione derivatives as Bcl-2 inhibitors, Bioorganic & Medicinal
Chemistry (2015), doi: http://dx.doi.org/10.1016/j.bmc.2015.10.023
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Design, synthesis and preliminary bioactivity studies of imidazolidine-2,4-dione
derivatives as Bcl-2 inhibitors
Gang Wang, Yutao Wang, Lei Wang, Leiqiang Han, Xuben Hou, Huansheng Fu, Hao
Fang*
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry
of Education), School of Pharmacy, Shandong University, Jinan, Shandong 250012,
PR China
*Address correspondence to this author:
Prof. Hao Fang*, Department of Medicinal Chemistry, School of Pharmacy,
Shandong University, 44 West Wenhua Road, Jinan, PR China.
E-mail: haofangcn@sdu.edu.cn
Tel. 86-531-88382731
Fax. 86-531-88382548
1

Abstract
Anti-apoptotic B-cell lymphoma-2 (Bcl-2) proteins are promising targets for
cancer therapy. In the present study, a series of imidazolidine-2,4-dione derivatives
were designed and synthesized to test their inhibitory activities against anti-apoptotic
Bcl-2 proteins. Among them, compound 8k had better growth inhibitory effects on
K562 and PC-3 cell lines compared to lead compound WL-276.
Keywords: Bcl-2; imidazolidine-2,4-dione; inhibitors; antitumor
1. Introduction
Malignant tumors are major diseases threatening human life. Anti-apoptotic
B-cell lymphoma 2 (Bcl-2) proteins, including Bcl-2, Bcl-xL, Mcl-1, Bfl-1/A1, Bcl-B
and Bcl-w, are contributors of tumor initiation, progression, and resistance to current
anti-tumor treatments.1-4 Development of Bcl-2 inhibitors to promote cell apoptosis
has become one of the important strategies of tumor treatment.5 So far, many small
molecule Bcl-2 inhibitors have been reported as antitumor agents in clinical trials,
such as ABT-199, Gossypol, Obatoclax and etc.6 (Figure 1)
Rhodanine (2-thioxo-4-thiazolidinone) has been extensively studied and used as
a potential scaffold in drug design to develop potent Bcl-2 inhibitors.7-10 Among them,
BH3I-1 (Figure 1) was firstly found to bind the Bcl-2 proteins and induce apoptosis.10
Later, Xing’s group reported a novel rhodanine Bcl-2 inhibitor, WL-276, which
showed good antiproliferative activity against PC-3 cell line11. Our previous studies
focused on the structural modification of WL-276 and found that target compounds
with aromatic amino acid side chain and electron withdrawing group in
benzenesulfonamide would be favor to enhance binding affinities.12 In our on-going
studies, the new heterocycle, imidazolidine-2,4-dione, which was bioisostere of
2

rhodanine and had been widely used in drug discovery13-17, was used as scaffold to
replace rhodanine in WL-276 and the structural modification were performed on the
different substitutions or side chains. This study will report the synthesis, binding
affinities to Bcl-2 proteins and antiproliferative activities against tumor cell lines of
imidazolidine-2,4-dione derivatives.
Figure 1. Representative small molecule inhibitors of anti-apoptotic Bcl-2 proteins.
2. Chemistry
Scheme 1 showed the routes of synthesizing imidazolidine-2,4-dione derivatives.
The intermediates 4a-4b were generated from substituted acids 1a-1b and amine 2
according to reported procedures18. Then, 4a-4b were cyclized to give the key
intermediates 5a-5b by employing triphosgene. Suzuki coupling reaction between
5a-5b and different arylboronic acids formed biaryl intermediates 6a-6d. The
conversion from methyl esters 5a-5b or 6a-6d to acids 7a-7f was conducted in the
presence of hydrochloride. The acids 7a-7f were reacted with substituted
benzenesulfonamides and other amines to yield target compounds 8a-8u using
coupling reagent HATU.
3

Scheme 1. Reagents and conditions: (a) EDCI, HOBt, TEA, DCM, r.t. , 3 h; (b) HCl gas, DCM,
0~5 °C, 0.5 h; (c) Triphosgene, Py, DCM, reflux, 12 h; (d) (Ph3P)4Pd, substituted phenylboronic
acid, Na2CO3, toluene, 80 °C, 2 h; (e) HCl, dioxane, reflux, 16 h; (f) HATU, DIEA, substituted
benzsulfamides and other amines, DCM, 20~25 °C, 2 h.
3. Results and discussion
To gain the initial structure-activity relationships of the synthesized
imidazolidine-2,4-dione derivatives, all target compounds were evaluated for their
binding affinities to Bcl-2 based on competitive binding experiments19 (Table 1). The
results showed that carboxylic acid derivatives (R2 = OH) and amide derivatives (R2 =
side chain of amino acids), such as compounds 7a-7e and 8p-8u, had binding
constants (Ki) of higher than 50 μM in the preliminary evaluation. It indicated that
carboxylic acid and amide derivatives were unfavorable for the target compounds’
affinities to Bcl-2 protein. When the substituted benzenesulfonamide fragments were
introduced in R2 position, compounds 8a-8o exhibited various inhibitions depending
on different structures in R1 and R2. For example, hydrogen and bromo substitution in
R1 gave very poor potency. While introducing aromatic ring in R1 position, most of
the derivatives possessed good inhibition against Bcl-2 (8e-8l). However, low binding
4

affinities occurred when aromatic ring was replaced with pyridine ring (8m-8o, Ki >
50 μM), which suggested that phenyl group might be critical for binding affinity in R1
position. In addition, different substituents in aromatic ring of benzenesulfonamide in
R2 position also influenced the binding affinities to Bcl-2. For example, methyl group
in para position (8f, Ki > 50 μM) is unfavorable for enhancing potency compared with
the compound 8e (Ki = 19 μM) without substitution. On the other hand, substitution of
3-NO2-4-Cl gave the most potent target compounds (8g, Ki = 3.7 μM and 8k, Ki = 4.4
μM), which indicated that introducing electron withdrawing groups in
benzenesulfonamide moiety would be favor to enhance the binding affinities with
target protein. The result is in agreement with our previous SAR studies on WL-276
with rhodanine as scaffold.12
Table 1. The binding affinities to Bcl-2 protein of imidazolidine-2,4-dione derivatives
Ki (μM)a
Compd
7a
7b
7c
R1
H
R2
OH
OH
>50
>50
Br
4-Cl-Ph
3-CH3-Ph
2-F-Py-4-
H
OH
>50
OH
>50
7d
7e
OH
>50
Ph-SO2NH
3-NO2-4-Cl-Ph-SO2NH
Ph-SO2NH
3-NO2-4-Cl-Ph-SO2NH
Ph-SO2NH
4-CH3-Ph-SO2NH
>50
8a
8b
8c
H
>50
Br
>50
Br
>50
8d
8e
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
3-CH3-Ph
3-CH3-Ph
19±1.5
>50
8f
3-NO2-4-Cl-Ph-SO2NH 3.7±0.44
8g
8h
8i
3-NO2-Ph-SO2NH
Ph-SO2NH
15±0.81
>50
4-CH3-Ph-SO2NH
13±1.1
8j
3-CH3-Ph 3-NO2-4-Cl-Ph-SO2NH 4.4±0.21
4-CHO-Ph 3-NO2-4-Cl-Ph-SO2NH 13±1.2
8k
8l
5

2-F-Py-4-
2-F-Py-4-
Ph-SO2NH
>50
>50
>50
8m
8n
8o
4-CH3-Ph-SO2NH
2-F-Py-4- 3-NO2-4-Cl-Ph-SO2NH
4-Cl-Ph
>50
>50
8p
8q
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-AcO-Ph-NH
4-OH-Ph-NH
>50
>50
8r
8s
4-Cl-Ph
4-Cl-Ph
>50
8t
>50
8u
WL-276
0.62±0.01
a Three independent fluorescence polarization assays (FPAs) were conducted and results were
expressed as mean ± standard deviations.
To better understanding the interactions of these imidazolidine-2,4-dione
compounds to Bcl-2 protein, we docked compound 8g in the active site of Bcl-2 (PDB
entry: 2O2F) using Surflex Dock. (Figure 2) The results suggested that 8g could bind
well to Bcl-2 protein. In addition, the acyl, nitro and acyl benzenesulfonamide groups
could form several hydrogen bonds with Tyr105, Gly142, Trp141, Asn140 and
Arg143. The above interactions might contribute to the affinity of 8g to Bcl-2.
Figure 2. Proposed binding mode of 8g in Bcl-2.
6

To confirm if the active target compounds could inhibit other antiapoptotic Bcl-2
proteins, five target compounds (8g, 8h, 8j, 8k and 8l) were selected to evaluate their
binding abilities to Bcl-XL and Mcl-1. According to the results in Table 2, the most
active compounds, 8g and 8k, showed similar binding affinities to the three
antiapototic Bcl-2 proteins. They exhibited same activity trends to the three Bcl-2
proteins as the control WL-276 due to their structural similarity. Different from them,
compounds 8h and 8j displayed potent Bcl-2 affinities while had very poor affinities
(Ki > 50 μM) on Bcl-XL and Mcl-1.
Table 2．The binding affinities to three Bcl-2 proteins of representative compounds
Ki (μM)a
Compd
Bcl-XL
6.1±1.6
>50
Bcl-2
3.7±0.44
15±0.81
13±1.1
Mcl-1
2.9±0.47
>50
8g
8h
>50
>50
8j
4.7±0.13
>50
4.4±0.21
13±1.2
2.3±0.08
9.3±2.6
0.25±0.08
8k
8l
WL-276
0.66±0.21
0.62±0.01
a Three independent fluorescence polarization assays (FPAs) were conducted and results were
expressed as mean ± standard deviations.
As Bcl-2 proteins were observed to be overexpressed in many cancer cells, such
as myeloma, prostate and acute leukemias,20 further studies were performed to
examine the activities of imidazolidine-2,4-dione compounds at the cellular level.
These five compounds were evaluated antiproliferative activities by MTT assay using
human K562 (chronic myelogenous leukemia cell), PC-3 (prostatic cancer cell) and
MDA-MB-231 (breast cancer cell). As the results in Table 3, these five compounds
showed obvious inhibition on tumor cell lines’ growth. Among them, compound 8g
and 8k had better antiproliferative activities than the other three compounds.
7

Especially, compound 8k showed better inhibitory activities against K562 and PC-3
cell lines compared with WL-276.
Table 3. Antiproliferative activities of representative compounds
IC50 (μM)a
Compd
K562
PC-3
MDA-MB-231
39.4 ± 2.6
49.0 ± 4.8
46.9 ± 6.6
35.1 ± 3.4
68.4 ± 4.4
44.9 ± 3.2
51.7 ± 2.0
73.3 ± 3.0
56.0 ± 0.4
28.4 ± 5.0
63.8 ± 8.7
39.8 ± 3.9
70.8 ± 4.8
76.4 ± 2.1
63.5 ± 3.2
57.8 ± 3.5
83.4 ± 5.3
35.2 ± 1.2
8g
8h
8j
8k
8l
WL-276
a
Three independent assays were conducted and results were expressed as mean ± standard
deviations.
4. Conclusions
In summary, we developed a series of novel imidazolidine-2,4-dione derivatives
as potential Bcl-2 inhibitors based on our previous structure-activity studies on
WL-276. As a new scaffold for Bcl-2 inhibitiors, imidazolidine-2,4-dione was a good
structural motif in drug discovery and had good structural diversity. In our studies,
some compounds with biphenyl and benzenesulfonamide showed potent Bcl-2
inhibitory activities. Compounds 8g and 8k exhibited most potent binding affinities to
Bcl-2 and Mcl-1. Specifically, compound 8k displayed higher antiproliferative
activities against K562 and PC-3 cell lines compared with the positive control
WL-276, which would be a good starting point for the development of more potent
Bcl-2 inhibitors in the future.
5. Experimental section
5.1. General Chemistry information
Unless otherwise noted, all starting materials, reagents and solvents were
8

obtained from commercial suppliers and used without further purification. All
reactions were monitored by thin-layer chromatography on 0.25 mm silica gel plates
(60 GF-254) and visualized with UV light (254 nM or 365 nM) or chromogenic
agents. ESI-MS was determined on an Aglient-1100 series LC/MSD trap
spectrometer. Proton nuclear magnetic resonance (1H NMR) spectra were measured
with a Brucker Avance spectrometer (300 MHz or 400 MHz). The chemical shifts
were given in parts per million with tetramethylsilane (TMS) as internal standard and
coupling constants (J values) were given in hertz (Hz). The splitting patterns were
described as s (singlet), d (doublet), dd (doublet doublet), t (triplet), q (quartet), m
(multiplet), and brs (broadsinglet). Most products were purified by column
chromatography (silica gel 100-200 mesh). Melting points were measured on an
electrothermal melting point apparatus without correction. ESI-MS was obtained on
an Aglient-1100 series LC/MSD trap spectrometer. HRMS spectrums were conducted
on an Agilent 6510 Quadrupole Time-of-Flight LC/MS deliver.
5.1.1. (S)-methyl 2-((S)-4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-phenylpropanoate
(5a)
To the solution of 4a (1.81 g, 5 mmol) in pyridine (7.5 mL) and DCM (25 mL),
was added the solution of triphosgene (0.9 g, 3 mmol) in DCM (10 mL) at 5-10 °C
and stirred at this temperature for 1.5 h. Reaction mixture was stirred at reflux for 12
h, and then quenched with hydrochloride solution (1N, 7.5 mL) below 10 °C. Organic
phase was separated, washed with water and brine, dried over sodium sulfate,
concentrated under vacuum to yield yellow oil. This oil was purified by silica gel
column (ethyl acetate: petroleum ether, 1:10, v/v) to afford white solid 5a (1.42 g,
81.0%). 1H-NMR (600 MHz, DMSO-d6): δ 8.24 (s, 1H), 7.24-7.29 (m, 4H), 7.20-7.22
(m, 2H), 7.13 (d, J = 7.8 Hz, 2H), 7.08 (d, J = 7.2 Hz, 2H), 4.8 (dd, J = 10.8 Hz, 4.8
9

Hz, 1H), 4.30-4.33 (m, 1H), 3.61 (s, 3H), 3.28 (dd, J = 13.8 Hz, 4.8 Hz, 1H),
3.07-3.11 (m, 1H), 2.71 (dd, J = 13.8 Hz, 4.8 Hz, 1H), 2.41-2.45 (m, 1H). ESI-MS:
m/z 353.3 (M+H)+.
5.1.1.1. (S)-methyl 2-((S)-4-(4-bromobenzyl)-2,5-dioxoimidazolidin-1-yl)-3-phenyl
propanoate (5b)
Compounds 5b were synthesized following the procedure of 5a. White solid,
1
yield: 82.0%, mp: 148-150 °C. H-NMR (400 MHz, CDCl3): δ 7.44 (d, J = 8.0 Hz,
2H), 7.20-7.33 (m, 5H), 7.00 (d, J = 8.0 Hz, 2H), 5.21 (S, 1H), 4.98 (dd, J = 6.8 Hz,
10.4 Hz, 1H), 4.05 (dd, J = 2.8 Hz, 10.0 Hz, 1H), 3.81 (S, 3H), 3.52 (S, 1H), 3.50 (d,
J = 4.4 Hz, 1H), 3.02 (dd, J = 3.6 Hz, 14.0 Hz, 1H), 2.21 (dd, J = 10.0 Hz, 14.0 Hz,
1H). ESI-MS m/z: 430.7 (M+H)+.
5.1.2.
(S)-methyl
2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimid
azolidin-1-yl)-3-phenylpropanoate (6a)
The mixture of 5b (2.9 g, 6.7 mmol), 4-chlorophenylboronic acid (1.36 g, 10.1
mmol), tetrakis(triphenylphosphine)palladium (0.7 g, 0.6 mmol) and sodium
carbonate (2.8 g, 26.8 mmol) in toluene (29 mL) and water (2.9 mL) was degassed
and heated at 80 °C for 2 h. Filter, the filtrate was diluted with EA (50 mL), washed
with water, dried over sodium sulfate, concentrated under vacuum to yield yellow oil.
The oil was purified by silica gel column (ethyl acetate: petroleum ether, 1:10, v/v) to
1
afford off-white solid 6a (2.7 g, 87.2%). mp: 150-152 °C. H-NMR (400 MHz,
DMSO-d6) : δ 8.22 (S, 1H), 7.06-7.70 (m, 13H), 4.88 (dd, J = 4.8 Hz, 11.2 Hz, 1H),
4.32-4.38 (m, 1H), 3.62 (S, 3H), 3.29-3.31 (m, 1H), 3.10-3.16 (m, 1H), 2.76-2.80 (m,
1H), 2.42-2.47 (m, 1H). ESI-MS m/z: 463.0 (M+H)+.
Compounds 6b-6d were synthesized following the procedure described above.
5.1.2.1.
(S)-methyl
2-((S)-4-((3'-methylbiphenyl-4-yl)methyl)-2,5-dioxo
10

imidazolidin-1-yl)-3-phenylpropanoate (6b)
1
Off-white solid, yield: 90.5%, mp: 137-140°C. H-NMR (400 MHz, DMSO-d6):
δ 8.30 (S, 1H), 7.15-7.56 (m, 13H), 4.88 (dd, J = 5.6 Hz, 10.8 Hz, 1H), 4.37 (m, 1H),
3.61 (S, 3H), 3.29-3.33 (m, 1H), 3.10 (dd, J = 11.2 Hz, 13.6 Hz, 1H), 2.79 (dd, J = 4.8
Hz, 14.0 Hz, 1H), 2.47-2.50 (m, 1H), 2.37 (S, 3H). ESI-MS m/z: 443.2 (M+H)+
5.1.2.2.
(S)-methyl
2-((S)-4-(4-(2-fluoropyridin-4-yl)benzyl)-2,5-dioxo
imidazolidin-1-yl)-3-phenylpropanoate (6c)
1
Off-white solid, yield: 80.4%, mp: 142-144 °C. H-NMR (400 MHz, CDCl3): δ
8.24 (d, J = 5.2 Hz, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.20-7.35 (m, 9H), 5.52 (S, 1H),
4.98 (dd, J = 6.4 Hz, 10.8 Hz, 1H), 4.12 (dd, J = 3.6 Hz, 10.0 Hz, 1H), 3.79 (S, 3H),
3.46-3.50 (m, 2H), 3.09 (dd, J = 8.0 Hz, 14.0 Hz, 1H), 2.30 (dd, J = 10.0 Hz, 14.0 Hz,
1H). ESI-MS m/z: 447.8 (M+H)+.
5.1.2.3.
(S)-methyl
2-((S)-4-((4'-formylbiphenyl-4-yl)methyl)-2,5-dioxo
imidazolidin- 1-yl)-3-phenylpropanoate (6d)
1
Off-white solid, yield: 23.8%, mp: 152-155 °C. H-NMR (400 MHz, CDCl3): δ
10.0 (S, 1H), 7.94 (d, J = 8.4 Hz, 2H), 7.70 (d, J = 8.4 Hz, 2H), 7.57 (d, J = 8.0 Hz,
2H), 7.20~7.32 (m, 7H), 4.98 (dd, J = 6.4 Hz, 10.4 Hz, 1H), 4.12 (dd, J = 3.6 Hz, 9.6
Hz,, 1H), 3.78 (S, 3H), 3.46-3.49 (m, 2H), 3.10 (dd, J = 4.0 Hz, 14.0 Hz, 1H), 2.28
(dd, J = 10.4 Hz, 14.0 Hz, 1H). ESI-MS m/z: 457.1 (M+H)+.
5.1.3. (S)-2-((S)-4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-phenylpropanoic acid (7a)
The mixture of 5a (1.76 g, 5.0 mmol) in dioxane (30 mL) and 3N hydrochloride
(60 mL) was refluxed for 16 h, then cooled to room temperature. Filter, the cake was
washed with methanol to afford white solid (1.63 g, 96.4%). Mp 204-205 °C.
1H-NMR (600 MHz, DMSO-d6): δ 13.09 (s, 1H), 8.17 (s, 1H), 7.23-7.29 (m, 4H),
11

7.18-7.21 (m, 2H), 7.13 (d, J = 6.6 Hz, 2H), 7.09 (d, J = 6.6 Hz, 2H), 4.72-4.75 (dd, J
= 11.4 Hz, 5.4 Hz, 1H), 4.26-4.28 (m, 1H), 3.18-3.31 (dd, J = 13.8 Hz, 5.4 Hz, 1H),
3.11-3.15 (m, 1H), 2.71-2.74 (dd, J = 13.8 Hz, 5.4 Hz, 1H), 2.35-2.40 (m, 1H). HRMS
(AP-ESI) Calcd. for C19H18N2O4: 337.1194 (M-H)-, Found: 337.1206.
Compounds 7b-7f were synthesized following the procedure described above.
5.1.3.1.
(S)-2-((S)-4-(4-bromobenzyl)-2,5-dioxoimidazolidin-1-yl)-3-phenyl
propanoic acid (7b)
1
White solid, mp: 214-216 °C. H-NMR (300 MHz, DMSO-d6): δ 13.07 (s, 1H),
8.15 (s, 1H), 7.39 (d, J = 8.1 Hz, 2H), 7.17-7.29 (m, 3H), 7.10 (d, J = 6.6 Hz, 2H),
7.02 (d, J = 8.4 Hz, 2H), 4.70 (dd, J = 11.1 Hz, 5.1 Hz, 1H), 4.26-4.30 (m, 1H),
3.27-3.30 (m, 1H), 3.12-3.20 (m, 1H), 2.67 (dd, J = 16.8 Hz, 5.4 Hz, 1H), 2.37-2.44
(m, 1H). HRMS (AP-ESI) Calcd. for C19H17BrN2O4: 415.0299 (M-H)-, Found:
415.0302.
5.1.3.2. (S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-yl)-
3-phenylpropanoic acid (7c)
Off-white solid, yield: 82.5%, mp: 186-188 °C. 1H-NMR (400 MHz, DMSO-d6):
δ 8.22 (S, 1H), 7.67 (d, J = 8.8 Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.8 Hz,
2H), 7.25-7.29 (m, 2H), 7.14-7.21 (m, 5H), 4.76 (dd, J = 5.2 Hz, 11.2 Hz, 1H),
4.31-4.34 (m, 1H), 3.29-3.33 (m, 1H), 3.14-3.20 (m, 1H), 2.77 (dd, J = 5.2 Hz, 14.0
Hz, 1H), 2.45 (dd, J = 5.2 Hz, 14.0 Hz, 1H). HRMS (AP-ESI) m/z Calcd for
C25H22ClN2O4 (M+H)+: 449.1263, Found: 449.1265.
5.1.3.3. (S)-2-((S)-4-((3'-methylbiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-yl)-
12

3-phenylpropanoic acid (7d)
1
Off-white solid, yield: 59.0%, mp: >250°C. H-NMR (400 MHz, DMSO-d6): δ
7.90 (S, 1H), 7.52 (d, J = 8.0 Hz, 2H), 7.41-7.44 (m, 2H), 7.34 (t, J = 7.6 Hz, 1H),
7.08-7.24 (m, 8H), 4.33 (dd, J =4.4 Hz, 12.0 Hz, 1H), 4.14 (dd, J = 5.6 Hz, 6.8 Hz,
1H), 3.24-3.40 (m, 3H), 2.85 (dd, J = 4.4 Hz, 14.0 Hz, 1H), 2.38 (S, 3H). HRMS
(AP-ESI) m/z Calcd for C26H25N2O4 (M+H)+: 429.1809, Found: 429.1808.
5.1.3.4. (S)-2-((S)-4-(4-(2-fluoropyridin-4-yl)benzyl)-2,5-dioxoimidazolidin-1-yl)-
3-phenylpropanoic acid (7e)
1
Off-white solid, yield: 87.3%, mp: >250 °C. H-NMR (400 MHz, DMSO-d6): δ
8.30 (d, J = 5.2 Hz, 1H), 8.00 (d, J = 25.2 Hz, 1H), 7.76 (dd, J = 6.8 Hz, 8.0 Hz, 2H),
7.69 (d, J = 5.2 Hz, 1H), 7.51 (s, 1H), 7.31 (dd, J = 8.0 Hz, 14.8 Hz, 2H), 7.21 (t, J =
7.2 Hz, 1H), 7.05-7.14 (m, 3H), 6.95 (d, J = 7.2 Hz, 1H), 4.24-4.38 (m, 1H),
4.20-4.23 (m, 1H), 3.22-3.31 (m, 2H), 2.85~3.03 (m, 1H), 2.55~2.77 (m, 1H). HRMS
(AP-ESI) m/z Calcd for C24H21FN3O4 (M+H)+: 434.1511, Found: 434.1509.
5.1.3.5. (S)-2-((S)-4-((4'-formylbiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-yl)-
3-phenylpropanoic acid (7f)
1
Off-white solid, yield: 61.9%, mp: 192-193 °C. H-NMR (400 MHz, MeOD): δ
7.19-7.29 (m, 5H), 4.93 (t, J = 8.4 Hz, 1H), 3.80 (q, J = 18.0Hz, 2H), 3.46 (d, J =
8.4Hz, 2H). HRMS (AP-ESI) m/z Calcd for C12H13N2O4 (M+H)+: 249.0870, Found:
249.0871.
5.1.4. (S)-2-((S)-4-benzyl-2,5-dioxoimidazolidin-1-yl)-3-phenyl-N-(phenylsulfonyl)
propanamide (8a)
To the mixture of 7a (0.38 g, 1.11 mmol) in DCM (15 mL), was added DIEA
(0.29 g, 2.22 mmol) and HATU (0.5 g, 1.31 mmol) in order below 5 °C. Then another
mixture of benzenesulfonamide (0.11 g, 0.67 mmol) in DCM (3 mL) was added
13

below 10 °C. Reaction mixture was quenched with 1N HCl after stirring for 2 h at
20-25 °C. Organic phase was separated and water phase was extracted with DCM
once. Organic phases were combined, washed with water and brine, dried over
sodium sulfate, concentrated under vacuum to yield yellow oil. This oil was purified
by silica gel column (ethyl acetate: petroleum ether, 1:20, v/v) to afford white powder
1
(0.23 g, 44.0%). Mp: 196-197 °C. H-NMR (600 MHz, DMSO-d6): δ 12.39 (s, 1H),
8.16 (s, 1H), 7.90 (d, J = 7.8 Hz, 2H), 7.71-7.80 (m, 1H), 7.62-7.64 (m, 2H),
7.26-7.28 (m, 4H), 7.18-7.25 (m, 2H), 7.10 (d, J = 6.6 Hz, 2H), 7.07 (d, J = 7.8 Hz,
2H), 4.76 (s, 1H), 4.07-4.09 (m, 1H), 3.23-3.26 (dd, J = 13.8 Hz, 5.4 Hz, 1H),
2.95-2.99 (m, 1H), 2.59-2.61 (m, 1H), 2.12-2.16 (m, 1H). HRMS (AP-ESI) Calcd. for
C25H23N3O5S: 476.1286 (M-H)-, Found: 476.1309.
Compounds 8b-8o were synthesized following the procedure described above.
5.1.4.1.
(S)-2-((S)-4-benzyl-2,5-dioxoimidazolidin-1-yl)-N-((4-chloro-3-nitro
phenyl)sulfonyl)-3-phenylpropanamide (8b)
White solid, yield: 39.4%，mp: 214-215 °C。1H-NMR (300 MHz, DMSO-d6): δ
8.47 (s, 1H), 8.17 (brs, 1H), 8.12 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 8.03(d, J = 8.4 Hz, 1H),
7.17-7.28 (m, 6H), 7.07-7.11 (m, 4H), 4.77 (dd, J = 10.5 Hz, 5.4 Hz, 1H), 4.07-4.11
(m, 1H), 3.19-3.25 (dd, J = 13.8 Hz, 5.1 Hz, 1H), 2.82-2.90 (m, 1H), 2.59-2.65 (dd, J
= 13.8 Hz, 5.1 Hz, 1H), 2.12-2.16 (m, 1H). HRMS (AP-ESI) Calcd. for
C25H21ClN4O7S: 555.0447 (M-H)-, Found: 555.0761.
5.1.4.2.
(S)-2-((S)-4-(4-bromobenzyl)-2,5-dioxoimidazolidin-1-yl)-3-phenyl-N-
(phenyl sulfonyl)propan–amide (8c)
1
White solid, yield: 32.3%, mp: 209-210 °C. H-NMR (300 MHz, DMSO-d6): δ
12.41 (s, 1H), 8.15 (s, 1H), 7.90-7.93 (m, 2H), 7.71-7.76 (m, 1H), 7.61-7.66 (m, 2H),
7.41 (d, J = 11.4 Hz, 2H), 7.17-7.28 (m, 3H), 7.09 (d, J = 6.6 Hz, 2H), 7.01 (d, J = 8.4
14

Hz, 2H), 4.74 (dd, J = 11.1 Hz, 4.8 Hz, 1H), 4.06-4.11 (m, 1H), 3.22 (dd, J = 13.8 Hz,
4.8 Hz, 1H), 2.98-3.06 (m, 1H), 2.55 (dd, J = 14.1 Hz, 5.1 Hz, 1H), 2.09-2.16 (m, 1H).
HRMS (AP-ESI) Calcd. for C25H22BrN3O5: 554.0391 (M-H)-, Found: 554.0409.
5.1.4.3. (S)-2-((S)-4-(4-bromobenzyl)-2,5-dioxoimidazolidin-1-yl)-N-((4-chloro-3-
nitrophenyl)sulfonyl)-3-phenylpropanamide (8d)
1
White solid, yield: 31.4%, mp: 199-201 °C. H-NMR (300 MHz, DMSO-d6): δ
8.47 (d, J = 2.1 Hz, 1H), 8.16 (s, 1H), 8.12 (d, J = 2.1 Hz, 1H), 8.03-8.06 (m, 1H),
7.42-7.44 (m, 2H), 7.16-7.28 (m, 3H), 7.07-7.09 (m, 2H), 7.02-7.05 (m, 2H), 4.77 (dd,
J = 10.8 Hz, 5.1 Hz, 1H), 4.08-4.12 (m, 1H), 3.20 (dd, J = 13.8 Hz, 5.1 Hz, 1H),
2.87-2.96 (m, 1H), 2.56 (dd, J = 13.8 Hz, 5.1 Hz, 1H), 2.11-2.21 (m, 1H). HRMS
(AP-ESI) Calcd. for C25H20BrClN4O7S: 632.9852 (M-H)-, Found: 632.9873.
5.1.4.4.
(S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-
yl)-3-phenyl-N-(phenylsulfonyl)propanamide (8e)
Off-white solid, yield: 61.3%, mp: >250 °C. 1H-NMR (400 MHz, MeOD): δ 7.99
(d, J = 7.2 Hz, 2H), 7.41-7.58 (m, 9H), 7.16-7.26 (m, 7H), 4.78 (dd, J = 5.2 Hz, 11.6
Hz, 1H), 4.16 (dd, J = 4.8 Hz, 8.8 Hz, 1H), 3.44 (dd, J = 4.8 Hz, 14.0 Hz, 1H),
3.28-3.34 (m, 1H), 2.87 (dd, J = 4.8 Hz, 13.6 Hz, 1H), 2.37 (dd, J = 8.8 Hz, 13.6 Hz,
1H). HRMS (AP-ESI) m/z Calcd for C31H27ClN3O5S (M+H)+: 588.1354, Found:
588.1355.
5.1.4.5.
(S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-
yl)-3-phenyl-N-tosylpropanamide (8f)
1
Off-white solid, yield: 27.0%, mp: >250 °C. H-NMR (400 MHz, PYR-d5): δ
9.61 (s, 1H), 8.32 (d, J = 8.0 Hz, 1H), 7.36-7.51 (m, 9H), 7.22-7.26 (m, 4H),
7.13-7.15 (m, 1H), 7.07 (d, J = 8.0 Hz, 2H), 5.47 (dd, J = 6.0 Hz, 10.8 Hz, 1H), 4.30
(dd, J = 2.0 Hz, 10.0 Hz, 1H), 3.90-3.96 (m, 2H), 3.28 (dd, J = 3.2 Hz, 14.0 Hz, 1H),
15

2.54 (dd, J = 10.8 Hz, 13.2 Hz, 1H), 2.07 (s, 3H). HRMS (AP-ESI) m/z Calcd for
C32H29ClN3O5S (M+H)+: 602.1511, Found: 602.1497.
5.1.4.6. (S)-N-(4-chloro-3-nitrophenylsulfonyl)-2-((S)-4-((4'-chlorobiphenyl-4-yl)
methyl)-2,5-dioxoimidazolidin-1-yl)-3-phenylpropanamide (8g)
Off-white solid, yield: 19.2%, mp: >250 °C. 1H-NMR (400 MHz, MeOD): δ 8.45
(d, J = 2.0 Hz, 1H), 8.12 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 7.74 (d, J = 8.4 Hz, 1H), 7.57
(d, J = 8.4 Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.42 (d, J = 8.4 Hz, 2H), 7.16-7.24 (m,
7H), 4.73 (dd, J = 4.8 Hz, 11.6 Hz, 1H), 4.17 (dd, J = 4.8 Hz, 8.8 Hz, 1H), 3.45 (dd, J
= 4.8 Hz, 14.0 Hz, 1H), 3.25-3.29 (m, 1H), 2.89 (dd, J = 4.4 Hz, 14.0 Hz, 1H), 2.40
(dd, J = 8.8 Hz, 14.0 Hz, 1H). HRMS (AP-ESI) m/z Calcd for C31H25Cl2N4O7S
(M+H)+: 667.0816, Found: 667.0815.
5.1.4.7.
(S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-
yl)-N-(3-nitrophenylsulfonyl)-3-phenylpropanamide (8h)
1
Off-white solid, yield: 28.5%, mp: 246-248 °C. H-NMR (400 MHz, MeOD): δ
8.78 (t, J = 2.0 Hz, 1H), 8.30-8.38 (m, 2H), 7.72 (t, J = 8.0 Hz, 1H), 7.58 (d, J = 8.8
Hz, 2H), 7.51 (d, J = 8.4 Hz, 2H), 7.43 (d, J = 8.4 Hz, 2H), 7.16-7.26 (m, 7H), 4.73
(dd, J = 4.8 Hz, 12.0 Hz, 1H), 4.16 (dd, J = 4.8 Hz, 9.2 Hz, 1H), 3.46 (dd, J = 5.2 Hz,
14.0 Hz, 1H), 3.30-3.32 (m, 1H), 2.90 (dd, J = 4.8 Hz, 14.0 Hz, 1H), 2.39 (dd, J = 9.2
Hz, 14.0 Hz, 1H). HRMS (AP-ESI) m/z Calcd for C31H26ClN4O7S (M+H)+: 633.1205,
Found: 633.1209.
5.1.4.8.
(S)-2-((S)-4-((3'-methylbiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-
yl)-3-phenyl-N-(phenylsulfonyl)propanamide (8i)
Off-white solid, yield: 17.3%, mp: >250 °C. 1H-NMR (400 MHz, MeOD), δ 7.99
(d, J = 8.0 Hz, 2H), 7.56 (d, J = 7.2 Hz, 1H), 7.48-7.52 (m, 4H), 7.24-7.38 (m, 5H),
7.14~7.19 (m, 6H), 4.81 (dd, J = 5.2 Hz, 11.6 Hz, 1H), 4.16 (dd, J = 4.4 Hz, 8.8 Hz,
16

1H ), 3.41 (dd, J = 5.2 Hz, 14.0 Hz, 1H), 3.26 (dd, J = 11.6 Hz, 14.0 Hz 1H), 2.85 (dd,
J = 4.4 Hz, 14.0 Hz, 1H), 2.39 (s, 3H), 2.34 (dd, J = 8.8 Hz, 14.0 Hz, 1H). HRMS
(AP-ESI) m/z Calcd for C32H30N3O5S (M+H)+: 568.1908, Found: 568.1901.
5.1.4.9.
(S)-2-((S)-4-((3'-methylbiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-
yl)-3-phenyl-N-tosylpropanamide (8j)
Off-white solid, yield: 16.9%, mp: >250 °C. 1H-NMR (400 MHz, MeOD): δ 7.84
(d, J = 8.0 Hz, 2H), 7.50 (d, J = 8.0 Hz, 2H), 7.14-7.41 (m, 13H), 4.74 (dd, J =4.8 Hz,
12.0 Hz, 1H), 4.14 (dd, J = 4.4 Hz, 9.2 Hz, 1H), 3.49 (dd, J = 4.8 Hz, 14.0 Hz, 1H),
3.40 (d, J = 12.0 Hz, 1H), 2.90 (dd, J = 4.0 Hz, 13.6 Hz, 1H), 2.40 (d, J = 7.2 Hz, 6H),
2.34 (dd, J = 9.2 Hz, 13.6 Hz, 1H). HRMS (AP-ESI) m/z Calcd for C33H32N3O5S
(M+H)+: 582.2057, Found: 582.2059.
5.1.4.10. (S)-N-(4-chloro-3-nitrophenylsulfonyl)-2-((S)-4-((3'-methylbiphenyl-4-yl)
methyl)-2,5-dioxoimidazolidin-1-yl)-3-phenylpropanamide (8k)
Off-white solid, yield: 26.7%, mp: >250 °C. 1H-NMR (400 MHz, MeOD): δ 8.45
(d, J = 2.0 Hz, 1H), 8.12 (dd, J = 2.4 Hz, 8.4 Hz, 1H ), 7.75 (d, J = 8.4 Hz, 1H ), 7.51
(d, J = 8.0 Hz, 2H), 7.14-7.40 (m, 11H), 4.72 (dd, J = 5.2 Hz, 12.0 Hz, 1H), 4.17 (dd,
J = 4.4 Hz, 8.8 Hz, 1H), 3.45 (dd, J = 4.8 Hz, 14.0 Hz, 1H), 3.25-3.29 (m, 1H), 2.90
(dd, J = 4.4Hz, 14.0 Hz, 1H), 2.41 (s, 3H), 2.37 (dd, J = 9.2 Hz, 14.0 Hz, 1H). HRMS
(AP-ESI) m/z Calcd for C32H28ClN4O7S (M+H)+: 647.1362, Found: 647.1364.
5.1.4.11. (S)-N-(4-chloro-3-nitrophenylsulfonyl)-2-((S)-4-((4'-formylbiphenyl-4-yl)
methyl)-2,5-dioxoimidazolidin-1-yl)-3-phenylpropanamide (8l)
1
Off-white solid, yield: 17.8%, mp: 165-166 °C. H-NMR (400 MHz, MeOD): δ
10.04 (S, 1H), 8.45 (d, J = 2.0 Hz, 1H), 8.13 (dd, J = 2.0 Hz, 8.4 Hz, 1H), 8.00 (d, J =
8.4 Hz, 2H), 7.84 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 8.4 Hz, 1H), 7.63 (d, J = 8.4 Hz,
2H), 7.16-7.27 (m, 7H), 4.72 (dd, J = 4.8 Hz, 11.6 Hz, 1H), 4.20 (dd, J = 4.8 Hz, 8.4
17

Hz, 1H), 3.45 (dd, J = 4.8 Hz, 14.0 Hz, 1H), 3.25~3.29 (m, 1H), 2.93 (dd, J = 4.8 Hz,
14.0 Hz, 1H), 2.46 (dd, J = 8.8 Hz, 13.6 Hz, 1H). HRMS (AP-ESI) m/z Calcd for
C32H26ClN4O8S (M+H)+: 661.1154, Found: 661.1166.
5.1.4.12.
(S)-2-((S)-4-(4-(2-fluoropyridin-4-yl)benzyl)-2,5-dioxoimidazolidin-1-
yl)-3-phenyl-N-(phenylsulfonyl)propanamide (8m)
1
Off-white solid, yield: 30.1%, mp: >250 °C. H-NMR (400 MHz, DMSO-d6): δ
8.29 (d, J = 5.2 Hz, 1H), 7.95-8.05 (m, 2H), 7.69-7.77 (m, 5H), 6.95-7.50 (m, 10H),
4.27-4.39 (m, 1H), 4.17 (dd, J = 1.6 Hz, 10.0 Hz, 1H), 3.16-3.31 (m, 2H), 2.98 (dd, J
= 4.8 Hz, 14.0 Hz, 1H), 2.56-2.61 (m, 1H). HRMS (AP-ESI) m/z Calcd for
C30H26FN4O5S (M+H)+: 573.1602, Found: 573.1605.
5.1.4.13.
(S)-2-((S)-4-(4-(2-fluoropyridin-4-yl)benzyl)-2,5-dioxoimidazolidin-1
-yl)-3-phenyl-N-tosylpropanamide (8n)
Off-white solid, yield: 29.4%, mp: >250 °C. 1H-NMR (400 MHz, MeOD), δ 8.24
(d, J = 5.2Hz, 1H), 7.82 (d, J = 8.4 Hz, 2H), 7.67 (d, J = 8.4 Hz, 2H), 7.58-7.60 (m,
1H), 7.07-7.33 (m, 10H), 4.67 (dd, J = 4.8 Hz, 11.6 Hz, 1H), 4.19 (dd, J = 4.8 Hz, 8.0
Hz, 1H), 3.48 (dd, J = 4.8 Hz, 14.4 Hz, 1H), 3.38-3.42 (m, 1H), 3.10 (dd, J = 4.8 Hz,
14.0 Hz, 1H), 2.72 (dd, J = 8.0 Hz, 14.0 Hz, 1H), 2.40 (S, 3H). HRMS (AP-ESI) m/z
Calcd for C31H28FN4O5S (M+H)+: 587.1759, Found: 587.1761.
5.1.4.14. (S)-N-(4-chloro-3-nitrophenylsulfonyl)-2-((S)-4-(4-(2-fluoropyridin-4-yl)
benzyl)-2,5-dioxoimidazolidin-1-yl)-3-phenylpropanamide (8o)
Off-white solid, yield: 37.5%, mp: >250 °C. 1H-NMR (400 MHz, MeOD): δ 8.43
(dd, J = 2.0 Hz, 9.6 Hz, 1H), 8.24 (dd, J = 2.8 Hz, 5.2 Hz, 1H), 8.09-8.13 (m, 1H),
7.76 (d, J = 8.0 Hz, 1H), 7.68 (d, J = 8.0 Hz, 2H), 7.05-7.35 (m, 9H), 4.64-4.74 (m,
1H), 4.19 (dd, J = 4.8 Hz, 8.0 Hz, 1H), 3.46 (dd, J = 4.8 Hz, 14.4 Hz, 1H), 3.11 (dd, J
= 4.4 Hz, 14.0 Hz, 1H), 2.93 (dd, J = 4.8 Hz, 14.0 Hz, 1H), 2.75 (dd, J = 8.0 Hz, 14.0
18

Hz, 1H). HRMS (AP-ESI) m/z Calcd for C30H24ClFN5O7S (M+H)+: 652.1064, Found:
652.1049.
5.1.4.15. (S)-methyl 2-((S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxo
imidazolidin-1-yl)-3-phenylpropanamido)-3-methylbutanoate (8p)
To the mixture of 7c (0.3 g, 0.67 mmol) in DCM (5 mL), was added HOBt (0.11
g, 0.8 mmol) and EDC.HCl (0.14 g, 0.74 mmol) in order below 5 °C. To another
mixture of L-valine methyl ester hydrochloride (0.11 g, 0.67 mmol) in DCM (3 mL),
was added TEA (0.07 g, 0.67 mmol) at room temperature. Then this mixture was
added into the mixture of 7c below 10 °C. Reaction mixture was quenched with water
after stirring for 2 h at 20-25 °C. Organic phase was separated and water phase was
extracted with DCM once. Organic phases were combined, washed with water and
brine, dried with sodium sulfate, concentrated under vacuum to yield colorless oil.
This oil was purified by silica gel column (ethyl acetate: petroleum ether, 1:20, v/v) to
1
afford off-white solid (0.28 g, 74.6%). mp: 70-72 °C. H-NMR (400 MHz, CDCl3): δ
7.48-7.52 (m, 4H), 7.33-7.43 (m, 4H), 7.17-7.27 (m, 5H), 5.47 (S, 1H), 5.03 (t, J = 8.4
Hz, 1H), 4.58 (dd, J = 4.8 Hz, 8.4 Hz, 1H), 4.10-4.13 (m, 1H), 3.74 (S, 3H), 3.49 (d, J
= 8.4 Hz, 2H ), 3.07 (dd, J = 3.6 Hz, 14.0 Hz, 1H), 2.17-2.25 (m, 1H), 2.02-2.09 (m,
1H), 0.97 (d, J = 6.8 Hz, 3H), 0.91 (d, J = 6.8 Hz, 3H). HRMS (AP-ESI) m/z Calcd
for C31H33ClN3O5 (M+H)+: 562.2103, Found: 562.2100.
5.1.4.16.
(S)-2-((S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazo
lidin-1-yl)-3-phenylpropanamido)-3-methylbutanoic acid (8q)
Compound 8q was synthesized from 8p following the procedure of 7a. Off-white
1
solid, yield: 61.6%, mp: 180-182 ℃. H-NMR (400 MHz, MeOD): δ 7.53~7.59 (m,
4H), 7.42 (dd, J = 2.0 Hz, 6.8 Hz, 2H), 7.21-7.32 (m, 7H), 4.95 (dd, J = 6.0 Hz, 11.2
Hz, 1H), 4.28 (d, J = 4.4 Hz, 1H), 4.24 (dd, J = 4.8Hz, 8.4 Hz, 1H), 3.37-3.48 (m, 2H),
19

2.87 (dd, J = 4.8 Hz, 14.0 Hz, 1H), 2.27 (dd, J =4.8 Hz, 14.0 Hz, 1H), 2.18 (m, 1H),
0.98 (d, J = 6.8 Hz, 3H), 0.93 (d, J = 6.8 Hz, 3H). HRMS (AP-ESI) m/z Calcd for
C30H31ClN3O5 (M+H)+: 548.1947, Found: 548.1947.
5.1.4.17. 4-((S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1
-yl)-3-phenylpropanamido)phenyl acetate (8r)
Compound 8r was synthesized following the procedure of 8p. Off-white solid,
1
yield: 61.6%, mp: >250 ℃. H-NMR (400 MHz, DMSO-d6): δ 9.93 (S, 1H), 8.24 (S,
1H), 7.49-7.67 (m, 8H), 7.21-7.33 (m, 7H), 7.09 (d, J = 8.8 Hz, 2H), 4.89 (dd, J = 5.2
Hz, 10.8 Hz, 1H), 4.24 (dd, J = 5.6 Hz, 6.8 Hz, 1H), 3.45 (dd, J = 5.2 Hz, 13.6 Hz,
1H), 3.27 (dd, J = 11.6 Hz, 13.6 Hz, 1H), 2.79 (dd, J = 4.8 Hz, 14.0 Hz, 1H), 2.42 (dd,
J = 8.0 Hz, 14.0 Hz, 1H), 2.26 (S, 3H). HRMS (AP-ESI) m/z Calcd for C33H29ClN3O5
(M+H)+: 582.1790, Found: 582.1798.
5.1.4.18. (S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazolidin-1-yl)
-N-(4-hydroxyphenyl)-3-phenylpropanamide (8s)
Compound 8s was synthesized from 8r following the procedure of 7a. Off-white
solid, yield: 38.9%, mp: >250 ℃. 1H-NMR (400 MHz, MeOD): δ 7.40-7.59 (m, 8H),
7.20-7.31 (m, 9H), 4.93-4.99 (m, 1H), 4.22-4.28 (m, 1H), 3.40-3.43 (m, 1H),
3.23-3.29 (m, 1H), 2.87-2.95 (m, 1H), 2.44-2.58 (m, 1H). HRMS (AP-ESI) m/z Calcd
for C31H27ClN3O4 (M+H)+: 540.1685, Found: 540.1690.
5.1.4.19. (S)-methyl 2-((S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxo
imidazolidin-1-yl)-3-phenylpropanamido)-3-phenylpropanoate (8t)
Compound 8t was synthesized following the procedure of 8p. Off-white solid,
1
yield: 74.1%, mp: 72-74 ℃. H-NMR (400 MHz, CDCl3), δ 7.47-7.51 (m, 4H),
7.40-7.42 (m, 2H), 7.09-7.31 (m, 12H), 5.55 (S,1H), 4.86-4.96 (m, 2H), 4.03-4.07 (m,
1H), 3.72 (S, 3H), 3.35-3.37 (m, 2H), 3.19 (dd, J = 5.6 Hz, 14.0 Hz, 1H), 3.09 (dd, J =
20

6.8 Hz, 14.0 Hz, 1H), 3.01 (dd, J = 3.6 Hz, 14.0 Hz, 1H), 2.02 (dd, J = 3.2 Hz, 14.0
Hz, 1H). HRMS (AP-ESI) m/z Calcd for C35H33ClN3O5 (M+H)+: 610.2103, Found:
610.2111.
5.1.4.20.
(S)-2-((S)-2-((S)-4-((4'-chlorobiphenyl-4-yl)methyl)-2,5-dioxoimidazo
lidin- 1-yl)-3-phenylpropanamido)-3-phenylpropanoic acid (8u)
Compound 8u was synthesized from 8t following the procedure of 7a. Off-white
solid, yield: 61.5%, mp: 218-220 ℃. 1H-NMR (400 MHz, MeOD): δ 7.55 (dd, J = 8.4
Hz, 12.8 Hz, 4H), 7.42 (d, J = 8.8 Hz, 2H), 7.16-7.28 (m, 12H), 4.82 (dd, J = 7.6 Hz,
9.2 Hz, 1H), 4.52 (t, J = 5.2 Hz, 1H), 4.17 (dd, J = 4.8 Hz, 8.4 Hz, 1H), 3.26-3.29 (m,
2H), 3.20 (dd, J = 4.8 Hz, 13.6 Hz, 1H), 3.02 (dd, J = 7.2 Hz, 14.0 Hz, 1H), 2.86 (dd,
J = 4.4 Hz, 14.0 Hz, 1H), 2.30 (dd, J = 8.4 Hz, 14.0 Hz, 1H). HRMS (AP-ESI) m/z
Calcd for C34H31ClN3O5 (M+H)+: 596.1947, Found: 596.1946.
5.2 Binding assay for Bcl-2 proteins based on Fluorescence polarization
technique
A 26-reside Bid-BH3 peptide (QEDIIRNIARHLAQVGDSMDRSIPPG) was
labeled at the N-terminus by 5-carboxyfluorescein succinimidyl ester (FAM) as the
fluorescence labeled peptide (5-FAM-QEDIIRNIARHLAQVGDSMDRSIPPG). The
5-FAM-Bid-BH3 peptide had a Kd value of 58 nM to bind to Bcl-2 protein.
In the competitive binding experiments, Bcl-2 protein and tested compounds
were preincubated in the PBS assay solution for 30 min at room temperature. Then
the 5-FAM-Bid-BH3 peptide was added into the solution and incubated for 20min.
The volume of total solution was 200 μL. Finally, the solutions were transferred into
384-well, black, flat-bottom plates (Corning Inc.) with 60 μL per well and three wells
per sample. The polarization values (milipolarization units, mP) were measured under
the condition of an excitation wavelength at 485 nm and an emission wavelength at
21

535 nm using the Tecan GENios-Pro Injector Reader (Tecan Group Ltd). In the
binding experiments, the hydantoin derivatives were prepared in DMSO at seven
concentrations, which were, 1 nM, 10 nM, 100 nM, 1μM, 10 μM, 50 μM and 100 μM.
The final concentrations of Bcl-2 protein and 5-FAM-Bid-BH3 peptide were 290nM
and 10nM, respectively.
The procedures of the binding assay for Bcl-XL and Mcl-1were almost same as
that for Bcl-2 except the total concentration of Bcl-XL as 125 nM and the total
concentration of Mcl-1 as 105 nM.
5.3. Molecular docking
Surflex-Dock program in Sybyl 7.3 was used with default values. The structures
of 8g and the Bcl-2 protein were optimized according to reference.21 The top-scored
conformation was selected as the best docking result.
5.4 MTT assay
The antiproliferative activities of 6 representative compounds were evaluated by
the MTT (3-[4, 5-dimethyl-2-thiazolyl]-2,5-diphenyl-2H-tetrazolium bromide) assay.
The three cancer cells were incubated in RPMI1640 medium containing 10% FBS in
humidified incubator (37 °C, 5% CO2). After the cells were seeded at 5000 cells per
well in the 96-well plates and incubated for 12 h, the growing cells were treated with
the indicated compounds at 15 μM, 20 μM, 30 μM, 40 μM and 50 μM for 48 h. 10 μL
MTT (5 mg/mL in PBS) solution was added into every well. The plates were
incubated for further 4 h. Finally, the formed formazan in every well was dissolved in
150 μL DMSO and mixed for 15 min. Optical density was read with a microtiter-plate
reader at 570 nm.
Acknowledgments
We thank Prof. Renxiao Wang from Shanghai Institute of Organic Chemistry in
22

China for the binding assay for Bcl-2 proteins. This work was supported by National
Natural Science Foundation of China (No. 21172133), Shandong Natural Science
Fund for Distinguished Young Scholars (No. JQ201319) and the Program for New
Century Excellent Talents in University (No. NCET-12-0337).
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Graphical abstract
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