Discovery and development of substituted tyrosine derivatives as Bcl-2/Mcl-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.08.030

Anti-apoptotic Bcl-2 family proteins are vital for cancer cells to escape apoptosis, which make them attractive targets for cancer therapy. Recently, a lead compound 1 was found to modestly inhibit the binding of BH3 peptide to Bcl-2 protein with a K_i value of 5.2 μM. Based on this, a series of substituted tyrosine derivatives were developed and tested for their binding affinities to Bcl-2 protein. Results indicated that these compounds exhibited potent binding affinities to Bcl-2 and Mcl-1 protein but not to Bcl-X_L protein. Promisingly, compound 6i inhibited the binding of BH3 peptide to Bcl-2 and Mcl-1 protein with a K_i value of 450 and 190 nM respectively, and showed obvious anti-proliferative activities against tested cancer cells.

Full text of DOI:10.1016/j.bmc.2018.08.030

Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Discovery and development of substituted tyrosine derivatives as
Bcl-2/Mcl-1 inhibitors
a
a
a
a
b
a,⁎
Renshuai Liu , Lulu Liu , Tingting Liu , Xinying Yang , Yichao Wan , Hao Fang
a
Department of Medicinal Chemistry, Key Laboratory of Chemical Biology (Ministry of Education), School of Pharmacy, Shandong University, Ji’nan, Shandong 250012,
PR China
b
Key Laboratory of Theoretical Organic Chemistry and Functional Molecule, Ministry of Education, College of Chemistry and Chemical Engineering, Hunan University of
Science and Technology, Xiangtan 411201, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Apoptosis
Bcl-2
Mcl-1
Tyrosine
Anti-tumor
Anti-apoptotic Bcl-2 family proteins are vital for cancer cells to escape apoptosis, which make them attractive
targets for cancer therapy. Recently, a lead compound 1 was found to modestly inhibit the binding of BH3
peptide to Bcl-2 protein with a K
developed and tested for their binding affinities to Bcl-2 protein. Results indicated that these compounds ex-
hibited potent binding affinities to Bcl-2 and Mcl-1 protein but not to Bcl-X protein. Promisingly, compound 6i
inhibited the binding of BH3 peptide to Bcl-2 and Mcl-1 protein with a K value of 450 and 190 nM respectively,
and showed obvious anti-proliferative activities against tested cancer cells.
i
value of 5.2 µM. Based on this, a series of substituted tyrosine derivatives were
L
i
1. Introduction
of the BH3-only proteins to bind anti-apoptotic Bcl-2 proteins are called
BH3 mimetics’. It is well known that developing small molecules cap-
‘
Apoptosis is vital for normal tissue homeostasis by removal of da-
able of effectively interfering the protein–protein interactions between
pro- and anti-apoptotic Bcl-2 proteins is challenging. However, progress
has been so far made.6 For example, Venetoclax/ABT-199 (Fig. 1)
has been approved to treat patients with 17p deletion chronic lym-
phocytic leukemia (CLL) on April 11, 2016.8 This shows great promise
to develop novel small molecule Bcl-2 inhibitors to cancer treatment.
maged or unwanted cells. It is generally considered that evasion of
apoptosis is a critical attribute of cancer cells. Among all kinds of
proteins involved in apoptosis, the B-cell lymphoma 2 (Bcl-2) family
proteins are one of the most well-characterized ones. These proteins are
pivotal regulators of the intrinsic apoptosis pathway by the way of
governing mitochondrial outer membrane (MOM) integrity. There are
three groups of structurally related Bcl-2 family proteins: the multi-
,7
In our previous work, thiazolidinones, thiadiazoles and pyrrolidines
have been developed as Bcl-2 inhibitors.9
–15
Interestingly, an inter-
domain anti-apoptotic proteins (Bcl-2, Bcl-X
L
, Mcl-1, Bcl-W, A1/BFL-1),
mediate (1, Fig. 1) to synthesize these compounds was found to mod-
estly inhibit the binding of BH3 peptide to Bcl-2 protein with a K value
of 5.2 µM. In our on-going studies, we developed a series of substituted
tyrosine derivatives based on this compound. This paper shows the
synthesis and biological evaluation of these tyrosine derivatives.
the pro-apoptotic BH3-only proteins (BIM, BID, PUMA, NOXA, BAD,
BMF, BIK, HRK), and the multi-domain pro-apoptotic proteins (Bak and
Bax). In response to apoptotic stimuli (e.g., DNA damage and growth
factor withdrawal), the BH3-only proteins are upregulated by tran-
scriptional and/or posttranscriptional pathways. This will unbalance
anti- and pro-apoptotic Bcl-2 family proteins, and then lead to activa-
tion of BAK and BAX. Activated BAX/BAK oligomerize and permeabi-
lize the mitochondrial outer membrane. Subsequently, mitochondrial
proteins (e.g., cytochrome c) are released to cytosol, which triggers
caspase cascades leading to cell death.1–4
i
2. Chemistry
1
3,16
Tyrosine derivatives were synthesized as outlined in Scheme 1.
As starting material, L-tyrosine 2 was protected by Cu2+, reacted with
benzyl bromides, and then protected by Boc group to yield key inter-
mediates 3a–3e. Then intermediates 3a–3e were treated with different
benzenesulfonamides to give target compounds 4a–4m. The Boc groups
of 4a–4m were removed to generate target compounds 5a–5f. Com-
pounds 5a–5f did coupling reactions with carboxylic acids to obtain
However, cancer cells can upregulate anti-apoptotic Bcl-2 proteins
in various different ways to escape apoptosis. In this case, small mo-
lecule inhibitors targeting these anti-apoptotic proteins have been de-
veloped to restore apoptosis. Molecules which mimic the BH3 domain
5
⁎
Corresponding author at: Department of Medicinal Chemistry, School of Pharmacy, Shandong University, 44 West Wenhua Road, Jinan, PR China.
E-mail address: haofangcn@sdu.edu.cn (H. Fang).
https://doi.org/10.1016/j.bmc.2018.08.030
Received 22 June 2018; Received in revised form 22 August 2018; Accepted 24 August 2018
0968-0896/ © 2018 Elsevier Ltd. All rights reserved.
Please cite this article as: Liu, R., Bioorganic & Medicinal Chemistry (2018), https://doi.org/10.1016/j.bmc.2018.08.030

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 1. Structures of ABT-199 and 1.
4 2 3 2
Scheme 1. Reagents and reaction conditions: (a) (i) 1 M CuSO , 2 M NaOH;(ii) benzyl bromides, MeOH; (b) EDTA •2Na; (c)(Boc) O, Et N, 1,4-Dioxane/H O; (d) (i)
isobutyl chloroformate, NMM, THF; (ii) benzenesulfonamides, NaH, THF; (e) RNH
DMF.
2
, DMF; (f) ethyl acetate saturated with HCl; (g) carboxylic acids, TBTU, NMM,
target compounds 6a–6i.
Table 1
The structures and Bcl-2 binding affinities of target compounds.
3. Results and discussion
A total of twenty-nine target compounds were synthesized. Then
these compounds were investigated for their binding affinities to Bcl-2
protein using the fluorescence polarization (FP) assays. Results were
i
tabulated in Table 1 as K values. As we can see from Table 1, fourteen
target compounds exhibited improved binding affinities to Bcl-2 protein
compared to their predecessor 1, and some of them were even better
than Gossypol.
Compd.
R1
R2
R3
Ki
a
(µM)
4
4
4
4
a
b
c
Ph
H
4-Me-Ph-
4-Cl-Ph-
Ph-O-
H
CN
CN
Ph
Ph
4-Me-Ph-
4-Me-Ph-
Ph-O-
Ph
H
CN
CN
Ph
Ph
Ph
Cl
Cl
Cl
Cl
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
Boc
H
0.62 ± 0.06
N.A. b
0.39 ± 0.03
0.58
0.70 ± 0.04
1.5 ± 0.06
0.80 ± 0.08
N.A.
N.A.
N.A.
N.A.
0.85 ± 0.07
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
0.56 ± 0.06
0.58 ± 0.01
1.1 ± 0.16
0.57 ± 0.08
0.45 ± 0.07
N.A.
0.61 ± 0.03
N.A.
0.45 ± 0.01
5.2 ± 0.45
0.88 ± 0.15
In this work, modifications on compound 1 were made mainly in
three parts – biphenyl part (R
1
), sulfonamide moiety (R
2
) and the Boc
) was vital for
d
group (R ). Among these three parts, the Boc group (R
3
3
4e
Cl
Ph-(CH
Ph-(CH
Cyclopentyl-NH-
Cy-NH-
Ph-(CH ) -NH-
4
4
4
4
f
2
2
)
)
3
-NH-
-NH-
compound’s binding affinity to Bcl-2 protein. The removal of this Boc
group led to the total loss of compound’s activity (e.g. 5a–5f). However,
the binding affinities could be restored when acyl groups were in-
troduced to this position (e.g. 6a–6i). Substitutions on sulfonamide
moiety were beneficial to compound’s potency. Compound 1 with no
g
h
i
3
4j
4k
2
3
Cy-CH
2
-NH-
4
4
5
5
l
Ph-O-(CH
Cy-NH-
Cl
Ph-(CH
Ph-(CH
Cyclopentyl-NH-
Cy-NH-
Ph-(CH
Cl
Cl
Cl
Cl
Ph-(CH
Ph-(CH
2
)
2
-NH-
substitutions on sulfonamide moiety had a K
compounds with substitutions behaved much more better (e.g. 4a
.62 µM, 4l 0.85 µM, 6i 0.45 µM). But this was a different situation
when it came to compounds with cycloalkyl amino groups on sulfo-
namide moiety (e.g. 4h, 4i, 6h). In addition, changes on R had a little
influence on compound's binding affinities compared with those on R
and R (e.g. 4a vs 4c vs 4e, 4f vs 4g, 6e vs 6i).
i
value of 5.2 µM, while
m
a
b
0
2
)
)
3
-NH-
-NH-
H
H
H
H
5c
2
3
5
5
5
6
d
e
f
1
2
2
)
3
-NH-
H
3
a
n-Amyl-CO-
Py-3-CO-
4-CH
Naph-2-CO-
4-CH -Bn-CO-
Naph-2-CO-
4-Ph-Bn-CO-
Naph-2-CO-
To understand how these compounds and Bcl-2 protein interacted
with each other, representative compound 6i were docked to the
binding groove of Bcl-2 protein (PDB code: 4LVT) using Surflex-Dock
software (Fig. 2a). Furthermore, the proposed interactions between 6i
and Bcl-2 protein in the binding groove were demonstrated in Fig. 2b.
Moreover, compounds 4a, 6e and 6i were tested their binding af-
6b
6c
Ph
Ph
Ph
H
CN
CN
Ph
Ph
3
-Bn-CO-
6
6
6
6
d
e
f
2
2
)
)
3
-NH-
-NH-
3
3
g
Cyclopentyl-NH-
Cy-NH-
Ph-(CH
6h
6i
2
)
3
-NH-
4-CH
3
-Bn-CO-
finities to Mcl-1 and Bcl-X
these compounds exhibited no binding affinities to Bcl-X
had potent binding affinities to Mcl-1 protein. It is promising because
Bcl-X protein plays a key role in regulating platelets’ life-span, and the
inhibition of Bcl-X protein results in acute and reversible thrombocy-
topenia. Furthermore, these three compounds were evaluated their
L
protein (Table 2). As depicted in Table 2,
1
L
protein, but
Gossypol
a
L
i
K values were expressed as the mean ± standard deviation of three in-
L
dependent determinations.
1
7
b
No activity.
2

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 2. (a) The docking mode of 6i and Gossypol (grey) in the binding groove of Bcl-2 protein. (b) The proposed interactions between 6i and Bcl-2 protein in the
binding groove.
Table 2
Isoform binding affinities and anti-proliferative activities of representative compounds.
a
a
Compd.
K
i
(μM)
IC50 (μM)
Bcl-X
L
Mcl-1
Jurkat
RS4;11
THP-1
Molt-4
HUEVC
4
6
6
a
e
i
N.A.b
N.A.
N.A.
N.A.
0.45 ± 0.03
0.50 ± 0.04
0.19 ± 0.01
0.53 ± 0.08
23.49 ± 0.28
23.82 ± 0.45
3.37 ± 0.51
1.43 ± 0.25
14.05 ± 2.86
16.80 ± 2.35
3.39 ± 0.87
1.23 ± 0.37
27.25 ± 3.61
23.91 ± 2.00
6.46 ± 0.17
5.80 ± 0.65
44.38 ± 0.92
> 50
8.36 ± 0.30
1.94 ± 0.81
> 50
> 100
> 50
13.41 ± 1.48
Gossypol
a
K
i
and IC50 values were expressed as the mean ± standard deviation of three independent determinations.
b
No activity.
anti-proliferative activities against four cancer cell lines and one normal
human cell line using MTT assay (Table 2). The results showed that
these compounds possessed certain anti-proliferative activities against
tested cancer cell lines, but had less influence on the growth of normal
human cell. Among these three compounds, compound 6i displayed
similar anti-proliferative activities against cancer cells compared to
Gossypol.
were even better than Gossypol. It turns out that these compounds
exhibited potent binding affinities to Bcl-2 and Mcl-1protein, but not to
Bcl-X
cancer cells. Promisingly, compound 6i inhibited the binding of BH3
peptide to Bcl-2 and Mcl-1 protein with a K value of 450 and 190 nM
L
protein. Besides, they these compounds inhibit the growth of
i
respectively, and showed obvious anti-proliferative activities against
tested cancer cells. In future, these results could pave the way for more
potent Bcl-2/Mcl-1 inhibitors.
In addition, compound 6i was evaluated for its ability to induce
apoptosis in the Jurkat cell line using annexin-V and propidium iodide
(
PI) double staining by flow cytometry (Fig. 3). It was found that
compound 6i induced apoptosis in a dose-dependent manner. Treat-
ment of the Jurkat cells by 10 and 20 μM of 6i for 48 h results in 6.2%
and 22.2% of apoptotic cells (early + late) respectively, as compared to
5. Experimental section
5.1. Chemistry: General procedures
2.6% of apoptotic cells in the DMSO control.
Caspase-3 activation is a crucial process involved in the intrinsic
Starting materials used in this work were commercially available.
Unless otherwise specified, solvents and reagents used belong to ana-
lytical reagents. Reactions were all monitored by thin-layer chromato-
graphy (TLC) on 0.25 mm silica gel plates (60GF – 254), and UV light
(254 nM) was used to observed the spots. Products were purified by
silica gel chromatography (200–300 mesh) or/and recrystallization.
The RY-1 electrothermal melting point apparatus was used to measure
melting points. Purities of target compounds were assessed by reverse-
phase HPLC performed on Shimadzu LC-20AT with a Phenomenex
apoptosis pathway, so we determined the ability of compound 6i to
induce caspase-3 activity in the Jurkat cell line (Fig. 4). The obtained
results showed that compound 6i induced activation of caspase-3 ac-
tivity in a dose-dependent manner. Importantly, these results correlate
with the ability of 6i to induce apoposis and inhibit Jurkat cell growth.
4
. Conclusions
1
Synergi C18 column (250 × 4.6 mm, 4 µm) and 1 mL/min flow. H and
13
In conclusion, a series of substituted tyrosine derivatives were de-
C NMR spectra were performed on a 400 MHz Brucker DRX spectro-
veloped based on compound 1. Fourteen of them exhibited improved
binding affinities to Bcl-2 protein compared to 1, and some of them
meter using TMS as an internal standard. Chemical shifts δ were
showed in parts per million (ppm) and coupling constants J were given
3

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
Fig. 3. Analysis of apoptosis induced by 6i in the Jurkat cell line.
5
.1.2. (S)-3-(4-(benzyloxy)phenyl)-2-((tert-butoxycarbonyl)amino)
propanoic acid (3b)
White powder, yield: 46%. H NMR (400 MHz, DMSO-d
H), 7.52–7.27 (m, 5H), 7.16 (d, J = 8.5 Hz, 2H), 7.07 (d, J = 8.3 Hz,
H), 6.92 (d, J = 8.6 Hz, 2H), 5.06 (s, 2H), 4.09–3.89 (m, 1H), 2.94
1
6
) δ 12.58 (s,
1
1
(
(
dd, J = 13.8, 4.5 Hz, 1H), 2.75 (dd, J = 13.7, 10.4 Hz, 1H), 1.41–1.17
m, 9H).
5.1.3. (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4′-methyl-[1,1′-
biphenyl]-4-yl)methoxy)phenyl) propanoic acid (3c)
1
Yellowish-brown powder, yield: 43%.
DMSO‑d ) δ 12.54 (s, 1H), 7.65 (d, J = 8.3 Hz, 2H), 7.56 (d, J = 8.1 Hz,
H), 7.50 (d, J = 8.3 Hz, 2H), 7.27 (d, J = 8.0 Hz, 2H), 7.16 (d,
J = 8.5 Hz, 2H), 7.04 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 5.10
s, 2H), 4.10–3.96 (m, 1H), 2.94 (dd, J = 13.8, 4.5 Hz, 1H), 2.75 (dd,
J = 13.7, 10.3 Hz, 1H), 2.34 (s, 3H), 1.36–1.20 (m, 9H).
H
NMR (400 MHz,
6
2
(
5
.1.4. (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-phenoxybenzyl)oxy)
phenyl)propanoic acid (3d)
White powder, yield: 19%. H NMR (400 MHz, DMSO‑d
H), 7.54–7.33 (m, 4H), 7.20–7.11 (m, 3H), 7.08–6.98 (m, 4H), 6.92 (d,
J = 8.6 Hz, 2H), 5.03 (s, 2H), 4.10–3.97 (m, 1H), 2.94 (dd, J = 13.8,
Fig. 4. Induction of caspase-3 by 6i in the Jurkat cell line. Results shown are the
mean and SEM from two separate experiments.
1
6
) δ 12.58 (s,
1
in hertz (Hz). Multiplicity (s, singlet; d, doublet; t, triplet; m, multiplet).
HRMS were conducted on an Agilent 6510 Quadrupole Time-of-Flight
LC/MS deliver.
4.4 Hz, 1H), 2.76 (dd, J = 13.6, 10.4 Hz, 1H), 1.36–1.24 (m, 9H).
5.1.5. (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4-cyanobenzyl)oxy)
5
.1.1. (S)-3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-2-((tert-
phenyl)propanoic acid (3e)
butoxycarbonyl)amino)propanoic acid (3a)
Colorless oil, yield: 55%. 1H NMR (400 MHz, DMSO‑d
) δ 12.59 (s,
6
To a solution of L-tyrosine (5.83 g, 32.2 mmol) in 2 M NaOH solution
1H), 7.86 (d, J = 8.3 Hz, 2H), 7.63 (d, J = 8.3 Hz, 2H), 7.18 (d,
J = 8.5 Hz, 2H), 7.08 (d, J = 8.3 Hz, 1H), 6.93 (d, J = 8.6 Hz, 2H), 5.19
(s, 2H), 4.09–4.03 (m, 1H), 2.96 (dd, J = 13.8, 4.4 Hz, 1H), 2.76 (dd,
J = 13.7, 10.4 Hz, 1H), 1.42–1.22 (m, 9H).
(
32.2 mL), 1 M copper sulfate solution (16.1 mL) was added, and the
reaction mixture was stirred at 60 °C for 1 h. Then methanol (90 mL) and
-phenylbenzyl bromide (8.75 g, 35.4 mmol) were added. The reaction
4
mixture was stirred vigorously for 3 h at 60 °C. The precipitate was fil-
tered, washed by water, and then added into 55.77 g/L EDTA·2Na so-
lution (168 mL). The resulting mixture was stirred at 60 °C for 12 h.
After cooling down, the precipitate was filtered, washed by water,
5.1.6. (S)-2-((tert-butoxycarbonyl)amino)-3-(4-((4′-chloro-[1,1′-
biphenyl]-4-yl)methoxy)phenyl) propanoic acid (3f)
1
White powder, yield: 21%. H NMR (400 MHz, DMSO-d
6
) δ 12.56 (s,
and then diluted with 1,4-dioxane/H
dicarbonate (9.13 g, 41.9 mmol) and Et
2
O (2:1, 150 mL). Di-tert-butyl
N (14.5 mL, 104.6 mmol) was
1H), 7.77–7.63 (m, 4H), 7.59–7.46 (m, 4H), 7.17 (d, J = 8.5 Hz, 2H),
7.05 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 5.12 (s, 2H),
4.10–3.96 (m, 1H), 2.95 (dd, J = 13.8, 4.5 Hz, 1H), 2.76 (dd, J = 13.7,
10.3 Hz, 1H), 1.32 (s, 9H).
3
added at 0 °C. After stirred overnight at room temperature, the reaction
mixture was acidified to pH 2–3 by 6 M HCl solution and extracted with
EtOAc. The combined EtOAc was washed by brine and dried over
MgSO
4
. Then the solvent was evaporated in vacuum. Purification with
5.1.7. Tert-butyl (S)-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-oxo-1-
(phenylsulfonamido)propan- 2-yl) carbamate (1)
To a solution of NaH (0.17 g, 4.2 mmol) in anhydrous THF (10 mL)
at 0 °C was added benzenesulfonamide (0.29 g, 1.84 mmol). The mix-
ture was stirred at 0 °C for 30 min and then at room temperature for
another 4 h to give corresponding salt.
silica gel column chromatography gave 7.56 g (52%) of 3a as yellowish-
1
brown powder. H NMR (400 MHz, DMSO-d
.73–7.60 (m, 4H), 7.56–7.42 (m, 4H), 7.37 (t, J = 7.3 Hz, 1H), 7.17 (d,
J = 8.5 Hz, 2H), 7.07 (d, J = 8.3 Hz, 1H), 6.94 (d, J = 8.6 Hz, 2H), 5.12
s, 2H), 4.11–3.92 (m, 1H), 2.94 (dd, J = 13.8, 4.4 Hz, 1H), 2.76 (dd,
6
) δ 12.57 (s, 1H),
7
(
J = 13.7, 10.3 Hz, 1H), 1.41–1.21 (m, 9H).
Compounds 3b–3e were synthesized following the procedure de-
scribed above.
N-methyl morpholine (0.2 mL, 1.84 mmol) was added at −20 °C to
a solution of 3a (0.75 g, 1.68 mmol) in anhydrous THF (10 mL). After
15 min, isobutyl chloroformate (0.24 mL, 1.84 mmol) was added. The
4

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
resulting mixture was stirred at −20 °C for 45 min and then added
dropwise to the prepared solution of benzenesulfonamide salt. The
mixture was stirred at room temperature for overnight. EtOAc was
added and the mixture was washed with 5% citric acid solution and
5.1.11. Tert-butyl
((4′-chloro-[1,1′-biphenyl]-4-yl)
carbamate (4d)
(S)-(1-((4-chloro-3-nitrophenyl)sulfonamido)-3-(4-
methoxy)phenyl)-1-oxopropan-2-yl)
White powder, yield: 53%, mp: 219–220 °C. HPLC purity: 94.0%,
1
brine, then dried over MgSO
Purification with silica gel column chromatography gave 0.59 g (60%)
of as white powder. mp: 157–158 °C. HPLC purity: 99.9%,
4
. The solvent was evaporated in vacuum.
t
R
= 10.6 min, MeOH/water = 87/13 (0.1% HCOOH).
H NMR
(400 MHz, CDCl ) δ 9.54 (s, 1H), 8.50 (d, J = 2.1 Hz, 1H), 8.18 (dd,
3
1
J = 8.5, 2.1 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H),
7.54–7.45 (m, 4H), 7.41 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H),
6.89 (d, J = 8.5 Hz, 2H), 5.07 (s, 2H), 4.93–4.80 (m, 1H), 4.28–4.14
(m, 1H), 3.00 (dd, J = 14.3, 6.5 Hz, 1H), 2.92 (dd, J = 14.2, 7.3 Hz,
1
t
(
(
R
= 6.8 min, MeOH/water = 83/17 (0.1% HCOOH).
400 MHz, CDCl ) δ 9.43 (s, 1H), 8.05 (d, J = 7.7 Hz, 2H), 7.69–7.57
m, 5H), 7.56–7.50 (m, 2H), 7.51–7.40 (m, 4H), 7.39–7.32 (m, 1H),
H
NMR
3
6
1
7
1
1
5
.94 (d, J = 8.5 Hz, 2H), 6.84 (d, J = 8.6 Hz, 2H), 5.05 (s, 2H), 4.94 (s,
1H), 1.41 (s, 9H). 13C NMR (100 MHz, DMSO‑d
6
) δ 172.58, 157.50,
H), 4.31 (s, 1H), 2.97 (dd, J = 14.2, 6.2 Hz, 1H), 2.89 (dd, J = 14.2,
155.87, 147.53, 139.49, 139.09, 138.75, 137.28, 133.39, 132.86,
132.83, 131.15, 130.73, 129.60, 129.36, 128.89, 128.72, 127.17,
125.59, 114.85, 78.92, 69.22, 57.10, 35.50, 28.42. HRMS (AP-ESI) m/z
1
3
.1 Hz, 1H), 1.38 (s, 9H). C NMR (100 MHz, CDCl ) δ 169.67, 158.02,
3
55.92, 141.05, 140.74, 138.40, 135.87, 134.03, 130.35, 128.95,
28.84, 128.51, 127.99, 127.44, 127.42, 127.15, 115.21, 81.38, 69.78,
−
2 3 8
Calcd for C33H31Cl N O S [M−H] 698.1136, found: 698.1118.
34 2 6
5.97, 36.19, 28.20. HRMS (AP-ESI) m/z Calcd for C33H N O S
−
[
M−H] 585.2065, found: 585.2049.
Compounds 4a–4h were synthesized following the procedure de-
scribed above.
5.1.12. Tert-butyl (S)-(1-((4-chloro-3-nitrophenyl)sulfonamido)-1-oxo-3-
(4-((4-phenoxybenzyl)oxy) phenyl)propan-2-yl)carbamate (4e)
White powder, yield: 77%, mp: 91–93 °C. HPLC purity: 88.1%,
1
5.1.8. Tert-butyl (S)-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((4-
t
R
= 7.5 min, MeOH/water = 87/13 (0.1% HCOOH).
H
NMR
chloro-3-nitrophenyl) sulfonamide)-1-oxopropan-2-yl)carbamate (4a)
Yellowish powder, yield: 87%, mp: 104–106 °C. HPLC purity:
(400 MHz, CDCl
3
) δ 9.61 (s, 1H), 8.53–8.47 (m, 1H), 8.18 (dd, J = 8.5,
1.9 Hz, 1H), 7.73 (d, J = 8.5 Hz, 1H), 7.42–7.35 (m, 2H), 7.35–7.31 (m,
1H), 7.11 (t, J = 7.4 Hz, 1H), 7.08–6.96 (m, 5H), 6.91–6.81 (m, 2H),
4.99 (s, 2H), 4.88 (d, J = 5.9 Hz, 1H), 4.22 (d, J = 6.3 Hz, 1H), 2.99
(dd, J = 14.2, 6.4 Hz, 1H), 2.91 (dd, J = 14.2, 7.4 Hz, 1H), 1.40 (s, 9H).
9
1.3%, t
400 MHz, CDCl
J = 8.5, 1.9 Hz, 1H), 7.70 (d, J = 8.5 Hz, 1H), 7.64–7.54 (m, 4H),
.54–7.39 (m, 4H), 7.38–7.29 (m, 1H), 6.97 (d, J = 8.5 Hz, 2H), 6.87
d, J = 8.6 Hz, 2H), 5.05 (s, 2H), 4.98 (s, 1H), 4.26 (s, 1H), 2.97 (dd,
R
= 8.3 min, MeOH/water = 87/13 (0.1% HCOOH). 1H NMR
(
3
) δ 9.80 (s, 1H), 8.49 (d, J = 2.1 Hz, 1H), 8.16 (dd,
1
3
7
3
C NMR (100 MHz, CDCl ) δ 170.12, 158.14, 157.27, 156.97, 147.63,
(
138.36, 133.04, 132.72, 131.44, 130.27, 129.82, 129.27, 127.07,
13
J = 14.2, 6.4 Hz, 1H), 2.88 (dd, J = 14.2, 7.5 Hz, 1H), 1.39 (s, 9H).
C
125.88, 123.50, 119.08, 118.82, 115.20, 81.92, 69.64, 56.20, 36.14,
28.17. HRMS (AP-ESI) m/z Calcd for C H32ClN O S [M−H]
33 3 9
680.1475, found: 680.1450.
−
NMR (100 MHz, CDCl
3
) δ 170.02, 158.17, 156.19, 147.64, 141.05,
1
1
2
6
40.71, 138.36, 135.79, 133.04, 132.70, 130.28, 128.82, 127.97,
27.44, 127.39, 127.11, 125.89, 115.27, 81.98, 69.80, 56.23, 36.05,
8.16. HRMS (AP-ESI) m/z Calcd for
64.1526, found: 664.1500.
−
33 3 8
C H32ClN O S [M−H]
5.1.13. Tert-butyl (S)-(3-(4-(benzyloxy)phenyl)-1-((3-nitro-4-((3-
phenylpropyl)amino)phenyl) sulfonamido)-1-oxopropan-2-yl)carbamate (4f)
5
.1.9. Tert-butyl (S)-(1-((4-chloro-3-nitrophenyl)sulfonamido)-3-(4-((4′-
Yellow powder, yield: 63%, mp: 144–146 °C. HPLC purity: 92.1%,
1
chloro-[1,1′-biphenyl]-4-yl) methoxy)phenyl)-1-oxopropan-2-yl)carbamate
4b)
White powder, yield: 53%, mp: 219–220 °C. HPLC purity: 99.2%,
t
R
= 12.6 min, MeOH/water = 80/20 (0.1% HCOOH).
H NMR
(
(400 MHz, CDCl
3
) δ 8.80 (d, J = 2.2 Hz, 1H), 8.47 (t, J = 5.2 Hz, 1H),
8.00 (d, J = 8.7 Hz, 1H), 7.44–7.34 (m, 4H), 7.34–7.27 (m, 3H), 7.23
(d, J = 7.3 Hz, 1H), 7.19 (d, J = 7.1 Hz, 2H), 6.97 (d, J = 8.5 Hz, 2H),
6.88–6.76 (m, 3H), 5.00 (s, 2H), 4.94 (s, 1H), 4.28 (s, 1H), 3.35 (dd,
J = 12.6, 6.8 Hz, 2H), 2.97 (dd, J = 14.2, 6.2 Hz, 1H), 2.89 (dd,
J = 14.2, 7.3 Hz, 1H), 2.77 (t, J = 7.4 Hz, 2H), 2.13–2.01 (m, 2H), 1.38
1
t
R
= 6.4 min, MeOH/water = 83/17 (0.1% HCOOH).
H
NMR
(
400 MHz, CDCl ) δ 9.54 (s, 1H), 8.50 (d, J = 2.1 Hz, 1H), 8.18 (dd,
3
J = 8.5, 2.1 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.57 (d, J = 8.2 Hz, 2H),
7
6
.54–7.45 (m, 4H), 7.41 (d, J = 8.5 Hz, 2H), 7.00 (d, J = 8.2 Hz, 2H),
.89 (d, J = 8.5 Hz, 2H), 5.07 (s, 2H), 4.93–4.80 (m, 1H), 4.28–4.14
(s, 9H). 13C NMR (100 MHz, CDCl
) δ 169.89, 158.06, 155.89, 147.99,
3
(
m, 1H), 3.00 (dd, J = 14.3, 6.5 Hz, 1H), 2.92 (dd, J = 14.2, 7.3 Hz,
140.31, 136.85, 135.16, 130.68, 130.33, 129.25, 128.70, 128.62,
128.38, 128.04, 127.51, 127.38, 126.43, 124.05, 115.16, 113.92,
1
3
1
1
1
1
6
H), 1.41 (s, 9H). C NMR (100 MHz, DMSO‑d ) δ 172.58, 157.50,
55.87, 147.53, 139.49, 139.09, 138.75, 137.28, 133.39, 132.86,
32.83, 131.15, 130.73, 129.60, 129.36, 128.89, 128.72, 127.17,
81.45, 69.99, 56.02, 42.49, 36.25, 32.96, 30.05, 28.18. HRMS (AP-ESI)
−
40 4 8
m/z Calcd for C36H N O S [M−H] 687.2494, found: 687.2478.
25.59, 114.85, 78.92, 69.22, 57.10, 35.50, 28.42. HRMS (AP-ESI) m/z
−
3 8
Calcd for C27H28ClN O S [M−H] 588.1213, found: 588.1198.
5.1.14. Tert-butyl (S)-(3-(4-((4-cyanobenzyl)oxy)phenyl)-1-((3-nitro-4-((3-
5
(
.1.10. Tert-butyl
(4′-methyl-[1,1′-biphenyl]-4-yl)
carbamate (4c)
Yellowish powder, yield: 88%, mp: 118–119 °C. HPLC purity:
(S)-(1-((4-chloro-3-nitrophenyl)sulfonamido)-3-(4-
phenylpropyl)amino)phenyl) sulfonamido)-1-oxopropan-2-yl)carbamate (4g)
methoxy)phenyl)-1-oxopropan-2-yl)
Yellow powder, yield: 86%, mp: 161–162 °C. HPLC purity: 95.2%,
1
t
R
= 5.3 min, MeOH/water = 83/17 (0.1% HCOOH). H NMR (400 MHz,
CDCl ) δ 9.31 (s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.48 (t, J = 5.2 Hz, 1H),
3
1
9
8.7%, t
400 MHz, CDCl
J = 8.5, 2.1 Hz, 1H), 7.72 (d, J = 8.5 Hz, 1H), 7.60 (d, J = 8.2 Hz, 2H),
R
= 10.1 min, MeOH/water = 87/13 (0.1% HCOOH). H NMR
8.02 (d, J = 8.7 Hz, 1H), 7.66 (d, J = 8.3 Hz, 2H), 7.52 (d, J = 8.3 Hz,
2H), 7.31 (t, J = 7.3 Hz, 2H), 7.25–7.21 (m, 1H), 7.21–7.16 (m, 2H), 7.01
(d, J = 8.5 Hz, 2H), 6.85 (d, J = 9.4 Hz, 1H), 6.82 (d, J = 8.6 Hz, 2H),
5.07 (s, 2H), 4.94 (s, 1H), 4.27 (s, 1H), 3.37 (dd, J = 12.6, 6.9 Hz, 2H),
2.98 (dd, J = 14.1, 6.3 Hz, 1H), 2.91 (dd, J = 14.1, 7.2 Hz, 1H), 2.78 (t,
J = 7.4 Hz, 2H), 2.16–2.00 (m, 2H), 1.38 (s, 9H). 13C NMR (100 MHz,
(
3
) δ 9.55 (s, 1H), 8.50 (d, J = 2.1 Hz, 1H), 8.18 (dd,
7
6
.48 (dd, J = 8.0, 5.4 Hz, 4H), 7.24 (s, 1H), 7.00 (d, J = 8.2 Hz, 2H),
.90 (d, J = 8.6 Hz, 2H), 5.07 (s, 2H), 4.84 (d, J = 6.9 Hz, 1H), 4.20 (d,
J = 6.6 Hz, 1H), 3.00 (dd, J = 14.3, 6.4 Hz, 1H), 2.92 (dd, J = 14.3,
1
3
7
1
1
1
.4 Hz, 1H), 2.40 (s, 3H), 1.40 (s, 9H). C NMR (100 MHz, CDCl
3
) δ
3
CDCl ) δ 169.74, 157.49, 155.79, 148.01, 142.37, 140.26, 135.23, 132.43,
130.71, 130.48, 129.18, 128.71, 128.36, 128.01, 127.59, 126.46, 124.03,
70.03, 158.19, 147.63, 140.98, 138.34, 137.81, 137.25, 135.46,
33.04, 132.71, 130.27, 129.55, 127.97, 127.18, 126.94, 125.90,
118.70, 115.13, 113.89, 111.73, 81.50, 68.90, 56.02, 42.49, 36.26, 32.95,
−
15.25, 81.98, 69.83, 56.21, 36.10, 28.17, 21.13. HRMS (AP-ESI) m/z
39 5 8
30.05, 28.16. HRMS (AP-ESI) m/z Calcd for C37H N O S [M−H]
−
Calcd for C34
H
34ClN
3
O
8
S [M−H] 678.1682, found: 678.1669.
712.2447, found: 712.2423.
5

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
5
.1.15. Tert-butyl (S)-(3-(4-((4-cyanobenzyl)oxy)phenyl)-1-((4-
(400 MHz, CDCl
3
) δ 9.12 (s, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.57 (t,
(cyclopentylamino)-3-nitrophenyl) sulfonamido)-1-oxopropan-2-yl)carbamate
J = 5.2 Hz, 1H), 8.02 (dd, J = 9.2, 2.0 Hz, 1H), 7.60 (d, J = 8.2 Hz,
2H), 7.54–7.44 (m, 4H), 7.25 (d, J = 7.3 Hz, 2H), 7.01 (d, J = 8.5 Hz,
2H), 6.96–6.83 (m, 3H), 5.05 (s, 2H), 4.81 (d, J = 7.1 Hz, 1H), 4.24 (s,
1H), 3.26–3.15 (m, 2H), 3.05–2.88 (m, 2H), 2.40 (s, 3H), 1.89–1.74 (m,
4H), 1.74–1.64 (m, 2H), 1.40 (s, 9H), 1.35–1.16 (m, 3H), 1.11–0.98 (m,
(4h)
Yellow powder, yield: 82%, mp: 177–178 °C. HPLC purity: 97.8%,
1
t
(
R
= 6.7 min, MeOH/water = 80/20 (0.1% HCOOH).
400 MHz, CDCl
H
NMR
3
) δ 9.32 (s, 1H), 8.78 (d, J = 2.2 Hz, 1H), 8.51 (d,
J = 6.3 Hz, 1H), 8.03 (d, J = 8.7 Hz, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.53
d, J = 8.2 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.97 (d, J = 9.3 Hz, 1H),
2H). 13C NMR (100 MHz, CDCl
3
) δ 169.83, 158.11, 148.26, 140.95,
(
137.86, 137.21, 135.54, 135.09, 130.61, 130.34, 129.54, 129.30,
128.00, 127.40, 127.16, 126.95, 123.81, 115.19, 114.03, 81.52, 69.82,
56.04, 49.83, 37.26, 36.35, 31.05, 28.19, 26.21, 25.73, 21.12. HRMS
(AP-ESI) m/z Calcd for C41H N O S [M−H] 755.3120, found:
48 4 8
755.3098.
6
4
7
1
1
1
8
.83 (d, J = 8.6 Hz, 2H), 5.07 (s, 2H), 4.95 (s, 1H), 4.29 (s, 1H),
.08–3.97 (m, 1H), 2.99 (dd, J = 14.1, 6.3 Hz, 1H), 2.92 (dd, J = 14.1,
.1 Hz, 1H), 2.24–2.06 (m, 2H), 1.88–1.78 (m, 2H), 1.78–1.70 (m, 2H),
−
1
3
.70–1.60 (m, 2H), 1.39 (s, 9H). C NMR (100 MHz, CDCl
3
) δ 169.74,
57.47, 155.77, 147.62, 142.39, 134.99, 132.43, 130.61, 130.50,
29.27, 128.00, 127.59, 123.70, 118.72, 115.10, 114.72, 111.71,
5.1.19. Tert-butyl
(S)-(3-(4-((4′-methyl-[1,1′-biphenyl]-4-yl)methoxy)
1.46, 68.88, 55.98, 54.53, 36.29, 33.45, 28.17, 24.00. HRMS (AP-ESI)
phenyl)-1-((3-nitro-4-((2-
oxopropan-2-yl)carbamate (4l)
phenoxyethyl)amino)phenyl)sulfonamido)-1-
−
m/z Calcd for C33
37 5 8
H N O S [M−H] 662.2290, found: 662.2260.
Yellow powder, yield: 70%, mp: 140–142 °C. HPLC purity: 92.8%,
5
.1.16. Tert-butyl
(S)-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((4-
1
t
(
R
= 8.7 min, MeOH/water = 87/13 (0.1% HCOOH).
400 MHz, CDCl ) δ 9.19 (s, 1H), 8.83 (d, J = 2.3 Hz, 1H), 8.79 (t,
J = 5.3 Hz, 1H), 8.07 (dd, J = 9.2, 2.0 Hz, 1H), 7.59 (d, J = 8.3 Hz,
H
NMR
(cyclohexylamino)-3- nitrophenyl)sulfonamido)-1-oxopropan-2-yl)carbamate
3
(4i)
Cyclohexanamine (0.57 mL, 5 mmol) was added to a solution of 4b
2
7
H), 7.53–7.43 (m, 4H), 7.34–7.27 (m, 2H), 7.26–7.19 (m, 2H),
.09–6.96 (m, 4H), 6.96–6.82 (m, 4H), 5.06 (s, 2H), 4.82 (d,
(
1
666 mg, 1 mmol) in DMF (10 mL). Then the mixture was stirred at
20 °C for 8 h. EtOAc was added and the mixture was washed with 5%
citric acid solution and brine, then dried over MgSO . The solvent was
J = 7.2 Hz, 1H), 4.33–4.16 (m, 3H), 3.78 (dd, J = 10.5, 5.3 Hz, 2H),
4
13
2
.99–2.86 (m, 2H), 2.40 (s, 3H), 1.39 (s, 9H). C NMR (100 MHz,
evaporated in vacuum. Purification with silica gel column chromato-
graphy gave 510 mg (70%) of 4i as yellow powder. mp: 171–173 °C.
CDCl
3
) δ 169.88, 158.09, 158.04, 147.99, 140.93, 137.82, 137.23,
1
1
8
35.55, 135.21, 131.10, 130.34, 129.68, 129.55, 129.17, 128.01,
27.44, 127.15, 126.94, 124.57, 121.71, 115.19, 114.68, 113.98,
HPLC purity: 95.9%,
t
R
= 9.8 min, MeOH/water = 87/13 (0.1%
NMR (400 MHz, CDCl 9.09 (s, 1H), 8.81 (d,
J = 2.3 Hz, 1H), 8.52 (d, J = 7.5 Hz, 1H), 8.01 (dd, J = 9.2, 1.9 Hz,
1
HCOOH).
H
3
) δ
1.55, 69.82, 65.53, 56.07, 42.63, 36.31, 28.18, 21.13. HRMS (AP-ESI)
−
44 4 9
m/z Calcd for C42H N O S [M−H] 779.2756, found: 779.2733.
1
7
5
3
H), 7.60 (t, J = 8.2 Hz, 4H), 7.53–7.40 (m, 4H), 7.39–7.32 (m, 1H),
.01 (d, J = 8.4 Hz, 2H), 6.94 (d, J = 9.4 Hz, 1H), 6.91–6.84 (m, 2H),
.06 (s, 2H), 4.82 (d, J = 7.4 Hz, 1H), 4.24 (s, 1H), 3.58 (s, 1H),
5
1
.1.20. Tert-butyl (S)-(1-((4-(cyclohexylamino)-3-nitrophenyl)sulfonamido)-
-oxo-3-(4-((4-phenoxy benzyl)oxy)phenyl)propan-2-yl)carbamate (4m)
.06–2.88 (m, 2H), 2.12–2.00 (m, 2H), 1.88–1.75 (m, 2H), 1.71–1.64
m, 1H), 1.52–1.25 (m, 14H). 1 C NMR (100 MHz, CDCl
3
Yellow powder, yield: 61%, mp: 160–162 °C. HPLC purity: 88.8%,
(
3
) δ 169.86,
1
t
R
= 9.6 min, MeOH/water = 87/13 (0.1% HCOOH).
400 MHz, CDCl
H
NMR
1
1
1
58.08, 147.20, 141.00, 140.76, 135.88, 134.99, 130.48, 130.36,
29.53, 128.81, 128.01, 127.41, 127.36, 127.12, 123.58, 115.17,
14.26, 81.45, 69.78, 56.08, 51.60, 36.39, 32.45, 28.18, 25.35, 24.36.
(
3
) δ 9.13 (s, 1H), 8.81 (d, J = 2.2 Hz, 1H), 8.52 (d,
J = 7.4 Hz, 1H), 8.01 (d, J = 9.2 Hz, 1H), 7.45–7.30 (m, 4H), 7.11 (t,
J = 7.4 Hz, 1H), 7.05–6.96 (m, 5H), 6.94 (d, J = 9.4 Hz, 1H), 6.86 (d,
J = 8.4 Hz, 2H), 4.98 (s, 2H), 4.84 (d, J = 7.3 Hz, 1H), 4.24 (s, 1H),
−
HRMS (AP-ESI) m/z Calcd for C39
27.2790.
Compounds 4j-4m were synthesized following the procedure de-
scribed above.
44 4 8
H N O S [M−H] 727.2807, found:
7
3
1
1
1
1
3
.58 (s, 1H), 3.04–2.86 (m, 2H), 2.13–1.98 (m, 2H), 1.88–1.76 (m, 2H),
.74–1.65 (m, 1H), 1.52–1.30 (m, 14H). 13C NMR (100 MHz, CDCl
69.81, 158.07, 157.22, 157.01, 147.20, 135.00, 131.54, 130.48,
30.34, 129.80, 129.52, 129.41, 129.29, 127.44, 123.57, 123.46,
19.05, 118.82, 115.15, 114.25, 81.46, 69.63, 56.02, 51.60, 36.31,
) δ
3
5.1.17. Tert-butyl (S)-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((3-
nitro-4-((3-phenylpropyl) amino)phenyl)sulfonamido)-1-oxopropan-2-yl)
carbamate (4j)
44 4 9
2.45, 28.18, 25.36, 24.36. HRMS (AP-ESI) m/z Calcd for C39H N O S
−
[
M−H] 743.2756, found: 743.2743.
Yellow powder, yield: 55%, mp: 83–85 °C. HPLC purity: 92.2%,
1
t
R
= 9.0 min, MeOH/water = 87/13 (0.1% HCOOH).
400 MHz, CDCl
J = 5.1 Hz, 1H), 8.00 (d, J = 8.2 Hz, 1H), 7.58 (t, J = 7.6 Hz, 4H),
H
NMR
(
3
) δ 9.42 (s, 1H), 8.80 (d, J = 2.2 Hz, 1H), 8.46 (t,
5.1.21. (S)-3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-2-amino-N-((4-
chloro-3-nitrophenyl)sulfonyl) propanamide hydrochloride (5a)
7
7
6
4
.49–7.39 (m, 4H), 7.38–7.32 (m, 1H), 7.29 (t, J = 7.3 Hz, 2H),
.24–7.20 (m, 1H), 7.17 (d, J = 7.0 Hz, 2H), 6.99 (d, J = 8.5 Hz, 2H),
.85 (d, J = 8.7 Hz, 2H), 6.83–6.79 (m, 1H), 5.03 (s, 2H), 4.97 (s, 1H),
.31 (s, 1H), 3.39–3.29 (m, 2H), 2.98 (dd, J = 14.1, 6.2 Hz, 1H), 2.89
4a (1.5 g, 2.25 mmol) was dissolved in 30 mL EtOAc saturated with
HCl gas and stirred overnight at room temperature. The precipitate was
filtered and washed with EtOAc to afford 1.3 g (96%) of 5a as yellowish
powder. mp: 196–198 °C. HPLC purity: 98.9%, t
water = 80/20 (0.1% HCOOH). 1H NMR (400 MHz, DMSO‑d
6
R
= 5.7 min, MeOH/
(
dd, J = 14.1, 7.3 Hz, 1H), 2.76 (t, J = 7.4 Hz, 2H), 2.13–2.00 (m, 2H),
) δ 8.53
1
3
1
1
1
1
3
.38 (s, 9H). C NMR (100 MHz, CDCl
3
) δ 169.88, 158.06, 155.80,
(d, J = 2.1 Hz, 1H), 8.27 (s, 3H), 8.15 (dd, J = 8.5, 2.1 Hz, 1H), 8.04 (d,
J = 8.5 Hz, 1H), 7.69 (t, J = 7.6 Hz, 4H), 7.54 (d, J = 8.2 Hz, 2H), 7.48
(t, J = 7.6 Hz, 2H), 7.38 (t, J = 7.3 Hz, 1H), 7.01 (d, J = 8.6 Hz, 2H),
6.85 (d, J = 8.6 Hz, 2H), 5.11 (s, 2H), 4.04–3.97 (m, 1H), 3.05 (dd,
47.96, 140.98, 140.71, 140.27, 135.85, 135.14, 130.70, 130.33,
29.19, 128.80, 128.68, 128.34, 127.98, 127.39, 127.34, 127.09,
26.40, 124.11, 115.17, 113.87, 81.41, 69.76, 56.06, 42.47, 36.32,
1
3
2.94, 30.03, 28.16. HRMS (AP-ESI) m/z Calcd for C42
H
44
4
N O
8
S
J = 14.2, 5.7 Hz, 1H), 2.98 (dd, J = 14.2, 6.6 Hz, 1H). C NMR
(100 MHz, DMSO‑d ) δ 169.83, 157.96, 147.49, 140.82, 140.26,
40.19, 136.74, 133.14, 133.02, 131.16, 130.40, 129.44, 128.72,
127.99, 127.24, 127.14, 126.74, 125.61, 115.04, 69.26, 54.88, 35.59.
−
[
M−H] 763.2807, found: 763.2789.
6
1
5.1.18. Tert-butyl (S)-(1-((4-((cyclohexylmethyl)amino)-3-nitrophenyl)
−
sulfonamido)-3-(4-((4′-methyl- [1,1′-biphenyl]-4-yl)methoxy)phenyl)-1-
oxopropan-2-yl)carbamate (4k)
3 6
HRMS (AP-ESI) m/z Calcd for C28H24ClN O S [M−H] 564.1002,
found: 564.0982.
Yellow powder, yield: 41%, mp: 181–183 °C. HPLC purity: 98.0%,
Compounds 5b-5f were synthesized following the procedure de-
scribed above.
1
t
R
= 6.4 min, MeOH/water = 92/8 (0.1% HCOOH).
H
NMR
6

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
1
5.1.22. (S)-2-amino-3-(4-(benzyloxy)phenyl)-N-((3-nitro-4-((3-
t
R
= 5.5 min, MeOH/water = 83/17 (0.1% HCOOH).
H
NMR
phenylpropyl)amino)phenyl) sulfonyl)propanamide hydrochloride (5b)
(400 MHz, DMSO‑d
6
) δ 8.76 (t, J = 5.7 Hz, 1H), 8.58 (d, J = 2.3 Hz,
Yellow powder, yield: 94%, mp: 216–218 °C. HPLC purity: 99.6%,
1H), 8.44 (s, 3H), 7.86 (dd, J = 9.2, 2.2 Hz, 1H), 7.73–7.63 (m, 4H),
7.56–7.45 (m, 4H), 7.41–7.34 (m, 1H), 7.30–7.14 (m, 6H), 6.95 (d,
J = 8.6 Hz, 2H), 6.80 (d, J = 8.7 Hz, 2H), 5.07 (s, 2H), 4.09 (s, 1H),
3.49 (dd, J = 13.4, 6.7 Hz, 2H), 3.10–2.94 (m, 2H), 2.72–2.62 (m, 2H),
1
t
(
1
7
R
= 7.6 min, MeOH/water = 74/26 (0.1% HCOOH).
400 MHz, DMSO‑d
H
NMR
6
) δ 8.73 (t, J = 5.8 Hz, 1H), 8.57 (d, J = 2.3 Hz,
H), 8.42 (s, 3H), 7.86 (dd, J = 9.2, 2.2 Hz, 1H), 7.48–7.36 (m, 4H),
.36–7.30 (m, 1H), 7.30–7.13 (m, 6H), 6.94 (d, J = 8.6 Hz, 2H), 6.77
2.00–1.87 (m, 2H). 13C NMR (100 MHz, DMSO‑d
) δ 167.22, 156.91,
6
(
3
d, J = 8.7 Hz, 2H), 5.01 (s, 2H), 4.07 (s, 1H), 3.54–3.40 (m, 2H),
.11–2.94 (m, 2H), 2.75–2.60 (m, 2H), 2.04–1.84 (m, 2H). C NMR
146.85, 140.60, 139.16, 139.13, 135.59, 133.39, 130.10, 129.08,
128.36, 127.73, 127.66, 127.63, 126.93, 126.14, 126.06, 125.27,
1
3
(
100 MHz, DMSO‑d
34.47, 131.15, 130.19, 128.90, 128.81, 128.71, 128.32, 128.12,
26.35, 126.31, 124.08, 115.64, 114.97, 69.61, 54.26, 42.75, 35.49,
32 4 6
2.81, 30.18. HRMS (AP-ESI) m/z Calcd for C31H N O S [M−H]
6
) δ 168.32, 158.01, 147.91, 141.68, 137.47,
125.21, 122.89, 114.60, 113.90, 68.18, 53.15, 41.68, 34.40, 31.73,
29.10. HRMS (AP-ESI) m/z Calcd for C37H N O S [M−H] 663.2283,
36 4 6
found: 663.2261.
−
1
1
3
5
−
87.1970, found: 587.1955.
5.1.27. (S)-N-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((4-chloro-3-
nitrophenyl)sulfonamido)-1- oxopropan-2-yl)hexanamide (6a)
5
.1.23. (S)-2-amino-3-(4-((4-cyanobenzyl)oxy)phenyl)-N-((3-nitro-4-
TBTU (320 mg, 1.0 mmol), NMM (0.28 mL, 2.49 mmol) and 5a
(500 mg, 0.83 mmol) was added to a solution of hexanoic acid (0.1 mL,
0.83 mmol) in anhydrous DMF (15 mL) at 0 °C. The reaction mixture
was stirred at room temperature for overnight. EtOAc was added and
the mixture was washed with 5% citric acid solution and brine, then
((3-phenylpropyl)amino) phenyl)sulfonyl)propanamide hydrochloride (5c)
Yellow powder, yield: 81%, mp: 222–224 °C. HPLC purity: 99.2%,
1
t
(
R
= 7.7 min, MeOH/water = 70/30 (0.1% HCOOH).
400 MHz, DMSO‑d
H
NMR
6
) δ 8.73 (t, J = 5.7 Hz, 1H), 8.56 (d, J = 2.3 Hz,
1
7
5
H), 8.44 (s, 3H), 7.93–7.78 (m, 3H), 7.62 (d, J = 8.3 Hz, 2H),
dried over MgSO
with silica gel column chromatography gave 160 mg (29%) of 6a as
yellowish powder. mp: 148–149 °C. HPLC purity: 98.9%, t = 8.9 min,
MeOH/water = 87/13 (0.1% HCOOH). 1H NMR (400 MHz, DMSO‑d
)
6
4
. The solvent was evaporated in vacuum. Purification
.36–7.11 (m, 6H), 6.98 (d, J = 8.6 Hz, 2H), 6.79 (d, J = 8.7 Hz, 2H),
.14 (s, 2H), 4.09 (s, 1H), 3.49 (dd, J = 13.4, 6.7 Hz, 2H), 3.14–2.91
R
(
m, 2H), 2.78–2.60 (m, 2H), 2.04–1.83 (m, 2H). 13C NMR (100 MHz,
DMSO‑d
6
) δ 168.30, 157.62, 147.92, 143.31, 141.67, 134.47, 132.87,
31.23, 130.19, 128.81, 128.75, 128.70, 128.49, 126.71, 126.36,
24.06, 119.20, 115.64, 115.01, 110.97, 68.66, 54.21, 42.74, 35.48,
δ 8.52 (d, J = 2.2 Hz, 1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 8.08 (d,
J = 7.4 Hz, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.72–7.63 (m, 4H), 7.56–7.43
(m, 4H), 7.41–7.33 (m, 1H), 7.09 (d, J = 8.6 Hz, 2H), 6.87 (d,
J = 8.7 Hz, 2H), 5.09 (s, 2H), 4.47–4.35 (m, 1H), 2.86 (dd, J = 13.7,
5.1 Hz, 1H), 2.62 (dd, J = 13.7, 9.5 Hz, 1H), 1.98 (t, J = 7.3 Hz, 2H),
1
1
3
6
−
31 5 6
2.80, 30.17. HRMS (AP-ESI) m/z Calcd for C32H N O S [M−H]
12.1922, found: 612.1901.
1
.32 (dd, J = 15.0, 7.5 Hz, 2H), 1.17 (dd, J = 14.7, 7.4 Hz, 2H),
13
5.1.24. (S)-2-amino-3-(4-((4-cyanobenzyl)oxy)phenyl)-N-((4-
1.09–0.96 (m, 2H), 0.78 (t, J = 7.3 Hz, 3H). C NMR (100 MHz,
DMSO) δ 171.18, 165.46, 157.52, 147.83, 141.67, 140.28, 140.13,
136.77, 134.81, 134.22, 131.99, 130.66, 130.16, 130.09, 129.66,
129.39, 128.78, 128.71, 128.46, 127.94, 127.16, 127.11, 126.31,
(
(
cyclopentylamino)-3-nitrophenyl) sulfonyl)propanamide hydrochloride
5d)
Yellow powder, yield: 82%, mp: 224–226 °C. HPLC purity: 94.2%,
1
t
(
R
= 5.9 min, MeOH/water = 70/30 (0.1% HCOOH).
400 MHz, DMSO‑d
H
NMR
124.48, 124.34, 123.04, 115.66, 114.87, 69.26, 56.03, 42.69, 35.82,
32.81, 30.10. HRMS (AP-ESI) m/z Calcd for C34H34ClN O S [M−H]
3 7
662.1733, found: 662.1718.
−
6
) δ 8.56 (d, J = 2.3 Hz, 1H), 8.44 (s, 3H), 8.37 (d,
J = 6.8 Hz, 1H), 7.97––7.80 (m, 3H), 7.64 (d, J = 8.3 Hz, 2H), 7.30 (d,
J = 9.4 Hz, 1H), 6.99 (d, J = 8.6 Hz, 2H), 6.80 (d, J = 8.6 Hz, 2H), 5.16
Compounds 6b-6i were synthesized following the procedure de-
(
s, 2H), 4.22–4.02 (m, 2H), 3.12–2.89 (m, 2H), 2.17–1.96 (m, 2H),
scribed above.
1
3
1
1
1
6
6
.85–1.67 (m, 2H), 1.67–1.47 (m, 4H). C NMR (100 MHz, DMSO‑d ) δ
68.32, 157.64, 147.49, 143.33, 134.55, 132.89, 131.23, 130.23,
28.66, 128.50, 126.73, 124.44, 119.20, 116.33, 115.03, 110.98,
5.1.28. (S)-N-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((4-chloro-3-
nitrophenyl)sulfonamido)-1- oxopropan-2-yl)nicotinamide (6b)
8.70, 54.58, 54.23, 35.47, 33.02, 24.05. HRMS (AP-ESI) m/z Calcd for
White powder, yield: 61%, mp: 102–104 °C. HPLC purity: 98.0%,
−
1
C
28
H
29
N
5
O
6
S [M−H] 562.1766, found: 562.1753.
t
(
R
= 8.4 min, MeOH/water = 80/20 (0.1% HCOOH).
400 MHz, DMSO‑d
H
NMR
6
) δ 8.96 (d, J = 7.4 Hz, 2H), 8.75 (s, 1H), 8.57 (d,
5
(
(
.1.25. (S)-3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-2-amino-N-((4-
cyclohexylamino)-3-nitro phenyl)sulfonyl)propanamide hydrochloride
5e)
J = 2.1 Hz, 1H), 8.18 (dd, J = 8.5, 2.1 Hz, 1H), 8.13 (d, J = 7.9 Hz,
1H), 8.06 (d, J = 8.5 Hz, 1H), 7.72–7.61 (m, 4H), 7.58–7.42 (m, 5H),
7.37 (t, J = 7.3 Hz, 1H), 7.22 (d, J = 8.5 Hz, 2H), 6.91 (d, J = 8.6 Hz,
Yellow powder, yield: 85%, mp: 204–206 °C. HPLC purity: 99.4%,
2H), 5.09 (s, 2H), 4.70–4.57 (m, 1H), 3.04 (dd, J = 13.7, 4.6 Hz, 1H),
1
13
t
(
R
= 6.0 min, MeOH/water = 83/17 (0.1% HCOOH).
400 MHz, DMSO‑d
.87 (dd, J = 9.3, 2.2 Hz, 1H), 7.69 (t, J = 8.1 Hz, 4H), 7.58–7.43 (m,
H
NMR
6
2.85 (dd, J = 13.5, 10.3 Hz, 1H). C NMR (100 MHz, DMSO‑d ) δ
6
) δ 8.58 (d, J = 2.3 Hz, 1H), 8.47–8.24 (m, 4H),
170.76, 164.48, 156.47, 151.32, 147.69, 146.61, 139.21, 139.06,
138.56, 135.71, 134.84, 132.42, 131.70, 130.04, 129.64, 128.50,
128.34, 127.67, 126.89, 126.11, 126.06, 124.46, 123.10, 113.84,
7
4
2
1
1
H), 7.39 (d, J = 7.4 Hz, 1H), 7.37–7.30 (m, 1H), 6.95 (d, J = 8.6 Hz,
H), 6.80 (d, J = 8.7 Hz, 2H), 5.08 (s, 2H), 4.07 (s, 1H), 3.81–3.67 (m,
H), 3.12–2.93 (m, 2H), 1.99–1.87 (m, 2H), 1.76–1.62 (m, 2H),
4 7
68.16, 55.16, 34.51. HRMS (AP-ESI) m/z Calcd for C34H27ClN O S
−
[M−H] 669.1216, found: 669.1200.
1
3
.62–1.51 (m, 1H), 1.49–1.30 (m, 4H), 1.28–1.18 (m, 1H). C NMR
(
100 MHz, DMSO‑d
6
) δ 171.18, 165.46, 157.52, 147.83, 141.67,
5.1.29. (S)-3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-N-((4-chloro-3-
nitrophenyl)sulfonyl)-2- (2-(p-tolyl)acetamido)propanamide (6c)
1
1
1
4
40.28, 140.13, 136.77, 134.81, 134.22, 131.99, 130.66, 130.16,
30.09, 129.66, 129.39, 128.78, 128.71, 128.46, 127.94, 127.16,
27.11, 126.31, 124.48, 124.34, 123.04, 115.66, 114.87, 69.26, 56.03,
Yellowish powder, yield: 79%, mp: 180–181 °C. HPLC purity:
1
91.9%, t
(400 MHz, DMSO‑d
1H), 8.13 (dd, J = 8.5, 2.2 Hz, 1H), 8.01 (d, J = 8.5 Hz, 1H), 7.73–7.63
m, 4H), 7.54 (d, J = 8.2 Hz, 2H), 7.47 (t, J = 7.6 Hz, 2H), 7.37 (t,
R
= 9.4 min, MeOH/water = 87/13 (0.1% HCOOH). H NMR
2.69, 35.82, 32.81, 30.10. HRMS (AP-ESI) m/z Calcd for C34
H
36
4
N O
6
S
6
) δ 8.54 (d, J = 2.2 Hz, 1H), 8.37 (d, J = 7.4 Hz,
−
[
M−H] 627.2283, found: 627.2272.
(
5
4
.1.26. (S)-3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-2-amino-N-((3-nitro-
-((3-phenylpropyl) amino)phenyl)sulfonyl)propanamide hydrochloride (5f)
Yellow powder, yield: 65%, mp: 217–219 °C. HPLC purity: 97.2%,
J = 7.3 Hz, 1H), 7.05 (d, J = 8.6 Hz, 2H), 7.01 (d, J = 7.9 Hz, 2H), 6.93
(d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.6 Hz, 2H), 5.10 (s, 2H), 4.46–4.38
(m, 1H), 3.32–3.25 (m, 2H), 2.88 (dd, J = 13.7, 5.0 Hz, 1H), 2.65 (dd,
7

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
J = 13.7, 9.3 Hz, 1H), 2.23 (s, 3H). 13C NMR (100 MHz, DMSO‑d
) δ
t
= 11.3 min, MeOH/water = 80/20 (0.1% HCOOH).
1
H NMR
6
R
1
1
1
1
3
6
71.18, 165.46, 157.52, 147.83, 141.67, 140.28, 140.13, 136.77,
34.81, 134.22, 131.99, 130.66, 130.16, 130.09, 129.66, 129.39,
28.78, 128.71, 128.46, 127.94, 127.16, 127.11, 126.31, 124.48,
24.34, 123.04, 115.66, 114.87, 69.26, 56.03, 42.69, 35.82, 32.81,
0.10. HRMS (AP-ESI) m/z Calcd for
(400 MHz, DMSO‑d
6
) δ 12.45 (s, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.42 (d,
J = 8.0 Hz, 1H), 8.35 (d, J = 6.7 Hz, 1H), 7.89 (dd, J = 9.2, 2.2 Hz,
1H), 7.84 (d, J = 8.3 Hz, 2H), 7.65–7.55 (m, 4H), 7.49 (d, J = 8.2 Hz,
2H), 7.45–7.38 (m, 2H), 7.37–7.31 (m, 1H), 7.27 (d, J = 9.5 Hz, 1H),
7.12 (d, J = 8.2 Hz, 2H), 7.06 (d, J = 8.6 Hz, 2H), 6.82 (d, J = 8.7 Hz,
2H), 5.11 (s, 2H), 4.53–4.42 (m, 1H), 4.09 (dd, J = 12.4, 6.1 Hz, 1H),
−
37 3 7
C H32ClN O S [M−H]
96.1577, found: 696.1548.
3.47–3.36 (m, 2H), 2.89 (dd, J = 13.6, 4.6 Hz, 1H), 2.64 (dd, J = 13.6,
5
.1.30. (S)-N-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((4-chloro-3-
9.7 Hz, 1H), 2.14–1.97 (m, 2H), 1.77–1.64 (m, 2H), 1.64–1.48 (m, 4H).
13
nitrophenyl)sulfonamido)-1- oxopropan-2-yl)-2-naphthamide (6d)
6
C NMR (100 MHz, DMSO‑d ) δ 171.18, 170.53, 157.15, 147.43,
143.38, 140.41, 138.62, 135.76, 134.30, 132.84, 130.80, 130.18,
129.92, 129.58, 129.35, 128.41, 127.74, 126.93, 126.78, 124.61,
Yellowish powder, yield: 60%, mp: 182–184 °C. HPLC purity:
1
9
1.6%, t
R
= 10.8 min, MeOH/water = 87/13 (0.1% HCOOH). H NMR
(
1
400 MHz, DMSO‑d
H), 8.38 (s, 1H), 8.18 (dd, J = 8.5, 2.2 Hz, 1H), 8.06 (d, J = 8.5 Hz,
6
) δ 8.86 (d, J = 7.2 Hz, 1H), 8.57 (d, J = 2.2 Hz,
119.23, 116.37, 114.76, 110.89, 68.63, 54.89, 54.53, 41.90, 36.36,
32.97, 24.03. HRMS (AP-ESI) m/z Calcd for C42H N O S [M−H]
39 5 7
−
1
7
6
H), 8.04–7.92 (m, 3H), 7.87–7.80 (m, 1H), 7.68–7.56 (m, 6H),
.54–7.42 (m, 4H), 7.36 (t, J = 7.3 Hz, 1H), 7.24 (d, J = 8.6 Hz, 2H),
.91 (d, J = 8.7 Hz, 2H), 5.08 (s, 2H), 4.70–4.61 (m, 1H), 3.04 (dd,
756.2497, found: 756.2466.
5.1.34. (S)-N-(3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-1-((4-
(cyclohexylamino)-3-nitrophenyl)
1
3
J = 13.6, 4.8 Hz, 1H), 2.97–2.85 (m, 1H).
C
NMR (100 MHz,
sulfonamido)-1-oxopropan-2-yl)-2-
DMSO‑d
6
) δ 171.18, 165.46, 157.52, 147.83, 141.67, 140.28, 140.13,
naphthamide (6h)
1
1
1
3
7
36.77, 134.81, 134.22, 131.99, 130.66, 130.16, 130.09, 129.66,
29.39, 128.78, 128.71, 128.46, 127.94, 127.16, 127.11, 126.31,
24.48, 124.34, 123.04, 115.66, 114.87, 69.26, 56.03, 42.69, 35.82,
2.81, 30.10. HRMS (AP-ESI) m/z Calcd for C39
18.1420, found: 718.1406.
Yellow powder, yield: 63%, mp: 118–120 °C. HPLC purity: 95.3%,
1
t
R
= 12.7 min, MeOH/water = 87/13 (0.1% HCOOH).
H NMR
(400 MHz, DMSO‑d
J = 2.2 Hz, 1H), 8.39 (s, 1H), 8.35 (d, J = 7.7 Hz, 1H), 8.00 (d,
J = 8.7 Hz, 1H), 7.96 (d, J = 8.6 Hz, 2H), 7.92–7.80 (m, 2H), 7.69–7.55
6
) δ 12.51 (s, 1H), 8.78 (d, J = 7.7 Hz, 1H), 8.59 (d,
−
H
30ClN
3
O
7
S [M−H]
(
m, 6H), 7.53–7.42 (m, 4H), 7.40–7.34 (m, 1H), 7.31 (d, J = 9.5 Hz,
5
.1.31. (S)-3-(4-(benzyloxy)phenyl)-N-((3-nitro-4-((3-phenylpropyl)
1H), 7.23 (d, J = 8.5 Hz, 2H), 6.89 (d, J = 8.6 Hz, 2H), 5.06 (s, 2H),
4.75–4.63 (m, 1H), 3.71 (s, 1H), 3.00 (dd, J = 13.6, 4.7 Hz, 1H),
2.93–2.81 (m, 1H), 1.98–1.85 (m, 2H), 1.77–1.63 (m, 2H), 1.61–1.52
amino)phenyl)sulfonyl)-2-(2- (p-tolyl)acetamido)propanamide (6e)
Yellow powder, yield: 83%, mp: 152–154 °C. HPLC purity: 96.9%,
1
13
t
R
= 5.8 min, MeOH/water = 87/13 (0.1% HCOOH).
400 MHz, CDCl
J = 5.2 Hz, 1H), 7.92 (dd, J = 9.2, 2.2 Hz, 1H), 7.43–7.34 (m, 4H),
H
NMR
(m, 1H), 1.47–1.33 (m, 4H), 1.27–1.19 (m, 1H). C NMR (100 MHz,
(
3
) δ 10.39 (s, 1H), 8.79 (d, J = 2.3 Hz, 1H), 8.46 (t,
6
DMSO‑d ) δ 171.18, 165.46, 157.52, 147.83, 141.67, 140.28, 140.13,
136.77, 134.81, 134.22, 131.99, 130.66, 130.16, 130.09, 129.66,
129.39, 128.78, 128.71, 128.46, 127.94, 127.16, 127.11, 126.31,
7
7
6
1
2
3
1
1
1
7
.34–7.31 (m, 1H), 7.29 (d, J = 7.5 Hz, 2H), 7.24–7.21 (m, 1H),
.21–7.15 (m, 2H), 7.10 (d, J = 7.8 Hz, 2H), 6.98 (d, J = 8.0 Hz, 2H),
.81 (d, J = 9.3 Hz, 1H), 6.74 (d, J = 8.8 Hz, 2H), 6.69 (d, J = 8.8 Hz,
H), 5.95 (d, J = 7.8 Hz, 1H), 4.97 (s, 2H), 4.85–4.71 (m, 1H), 3.51 (s,
H), 3.39–3.29 (m, 2H), 2.86 (dd, J = 14.2, 6.1 Hz, 1H), 2.81–2.69 (m,
124.48, 124.34, 123.04, 115.66, 114.87, 69.26, 56.03, 42.69, 35.82,
−
42 4 7
32.81, 30.10. HRMS (AP-ESI) m/z Calcd for C45H N O S [M−H]
781.2702, found: 781.2688.
1
3
H), 2.33 (s, 3H), 2.14–1.98 (m, 2H). C NMR (100 MHz, CDCl
3
) δ
5.1.35. (S)-3-(4-([1,1′-biphenyl]-4-ylmethoxy)phenyl)-N-((3-nitro-4-((3-
phenylpropyl)amino)phenyl) sulfonyl)-2-(2-(p-tolyl)acetamido)propanamide
(6i)
75.88, 175.49, 162.28, 152.59, 146.44, 142.36, 140.39, 138.96,
38.12, 135.45, 134.88, 133.97, 133.89, 133.80, 133.64, 133.55,
33.47, 133.25, 133.03, 132.84, 131.08, 129.03, 120.41, 119.45,
Yellow powder, yield: 44%, mp: 146–148 °C. HPLC purity: 95.5%,
1
4.35, 59.61, 47.46, 46.59, 41.11, 37.57, 34.89, 25.83. HRMS (AP-ESI)
t
R
= 10.2 min, MeOH/water = 87/13 (0.1% HCOOH).
H NMR
−
m/z Calcd for C40
H
40
N
4
O
7
S [M−H] 719.2545, found: 719.2529.
(400 MHz, DMSO‑d
6
) δ 12.38 (s, 1H), 8.70 (t, J = 5.7 Hz, 1H), 8.55 (d,
J = 2.3 Hz, 1H), 8.27 (d, J = 7.8 Hz, 1H), 7.83 (dd, J = 9.2, 2.2 Hz,
1H), 7.72–7.64 (m, 4H), 7.56–7.43 (m, 4H), 7.37 (t, J = 7.3 Hz, 1H),
7.29–7.23 (m, 2H), 7.23–7.13 (m, 4H), 7.05–6.97 (m, 4H), 6.92 (d,
J = 8.0 Hz, 2H), 6.81 (d, J = 8.7 Hz, 2H), 5.07 (s, 2H), 4.49–4.37 (m,
1H), 3.46 (dd, J = 13.3, 6.7 Hz, 2H), 3.32–3.23 (m, 2H), 2.83 (dd,
J = 13.7, 4.9 Hz, 1H), 2.71–2.65 (m, 2H), 2.65–2.58 (m, 1H), 2.22 (s,
5
.1.32. (S)-N-(3-(4-((4-cyanobenzyl)oxy)phenyl)-1-((3-nitro-4-((3-
phenylpropyl)amino)phenyl) sulfonamido)-1-oxopropan-2-yl)-2-naphthamide
6f)
Yellow powder, yield: 67%, mp: 112–114 °C. HPLC purity: 97.7%,
(
1
t
R
= 9.5 min, MeOH/water = 80/20 (0.1% HCOOH).
H
NMR
(
400 MHz, DMSO‑d ) δ 12.52 (s, 1H), 8.80 (d, J = 7.8 Hz, 1H), 8.68 (t,
6
3H), 1.99–1.88 (m, 2H). 13C NMR (100 MHz, DMSO‑d
6
) δ 171.14,
J = 5.7 Hz, 1H), 8.58 (d, J = 2.3 Hz, 1H), 8.39 (s, 1H), 8.04–7.92 (m,
170.75, 157.52, 147.85, 141.68, 140.28, 140.16, 136.83, 135.65,
134.22, 133.37, 130.72, 130.14, 129.42, 129.23, 129.17, 129.06,
128.79, 128.72, 128.68, 128.50, 127.97, 127.21, 127.13, 126.33,
3
4
5
H), 7.88 (dd, J = 9.2, 2.1 Hz, 1H), 7.86–7.78 (m, 3H), 7.66–7.55 (m,
H), 7.29–7.22 (m, 4H), 7.22–7.12 (m, 4H), 6.88 (d, J = 8.6 Hz, 2H),
.13 (s, 2H), 4.75–4.63 (m, 1H), 3.45 (dd, J = 13.3, 6.6 Hz, 2H), 3.01
124.31, 115.66, 114.74, 69.28, 54.87, 42.72, 41.85, 36.38, 32.83,
−
(
dd, J = 13.6, 4.5 Hz, 1H), 2.89 (dd, J = 13.4, 10.4 Hz, 1H), 2.67 (t,
44 4 7
30.14, 21.08. HRMS (AP-ESI) m/z Calcd for C46H N O S [M−H]
1
3
J = 7.6 Hz, 2H), 1.99–1.86 (m, 2H). C NMR (100 MHz, DMSO‑d
6
) δ
795.2858, found: 795.2839.
1
1
1
1
3
7
71.41, 166.90, 157.12, 147.83, 143.42, 141.68, 134.70, 134.24,
32.82, 132.45, 131.25, 130.77, 130.17, 130.14, 129.32, 128.79,
28.71, 128.49, 128.30, 128.24, 128.09, 127.27, 126.32, 124.60,
5.2. In vitro binding assay for Bcl-2 proteins
24.35, 119.21, 115.68, 114.86, 110.88, 68.60, 56.02, 42.68, 35.80,
A labeled Bid-BH3 peptide (5-FAM-QEDIIRNIARHLAQVGDSMDRS-
IPPG) can bind to Bcl-2, Bcl-X and Mcl-1 protein with K values of
30–60 nM. And when it binds to these proteins, high polarization values
milipolarization units, mP) is generated. However, test compounds can
−
2.80, 30.10. HRMS (AP-ESI) m/z Calcd for C43
H
37
N
5
O
7
S [M−H]
L
d
66.2341, found: 766.2312.
(
5
.1.33. (S)-2-(2-([1,1′-biphenyl]-4-yl)acetamido)-3-(4-((4-cyanobenzyl)
compete with this Bid-BH3 peptide to bind to these proteins, which will
lead to the decrease of mP values. The IC50 values of test compounds are
calculated according to mP values and corresponding concentrations.
oxy)phenyl)-N-((4- (cyclopentylamino)-3-nitrophenyl)sulfonyl)propanamide
6g)
Yellow powder, yield: 64%, mp: 143–145 °C. HPLC purity: 97.0%,
(
i
Then the K values are calculated based on concentrations of the Bid-
8

R. Liu et al.
Bioorganic & Medicinal Chemistry xxx (xxxx) xxx–xxx
China (Grant No. 21672127), Key Research and Development Project of
Shandong Province (Grant No. 2017CXGC1401) and the Fundamental
Research Funds of Shandong University (Grant No. 2016JC018).
BH3 peptide and Bcl-2 proteins, the K
d
value and the IC50 values.18,19
5.3. Docking study
Surflex-dock program in Sybyl-X 2.1.1 was used with default values
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Acknowledgments
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This work was supported by National Natural Science Foundation of
9