Structure-Based Optimization of 3-Phenyl-N-(2-(3-phenylureido)ethyl)thiophene-2-sulfonamide Derivatives as Selective Mcl-1 Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.1c00690

Selective Mcl-1 inhibitors may overcome the drug resistance caused by current anti-apoptotic Bcl-2 protein inhibitors in tumors with Mcl-1 overexpression. Based on previously discovered compounds with a 3-phenylthiophene-2-sulfonamide core moiety, in this work, we have obtained new compounds with improved binding affinity and/or selectivity under the guidance of structure-based design. The most potent compounds achieved sub-micromolar binding affinities to Mcl-1 (K_i～ 0.4 μM) and good cytotoxicity (IC₅₀< 10 μM) on several tumor cells.¹⁵N-heteronuclear single-quantum coherence NMR spectra suggested that these compounds bound to the BH3-binding groove on Mcl-1. Several cellular assays revealed that FWJ-D4 as well as its precursor FWJ-D5 effectively induced caspase-dependent apoptosis, and their target engagement at Mcl-1 was confirmed by co-immunoprecipitation experiments. Treatment with FWJ-D5 at 50 mg/kg every 2 days on an RS4;11 xenograft mouse model for 22 days led to 75% reduction in tumor volume without body weight loss.

Full text of DOI:10.1021/acs.jmedchem.1c00690

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Article
Structure-Based Optimization of 3‑Phenyl‑N‑(2-(3-
phenylureido)ethyl)thiophene-2-sulfonamide Derivatives as
Selective Mcl‑1 Inhibitors
Yan Li,⊥ Wenjie Fan,⊥ Qineng Gong,⊥ Jie Tian, Mi Zhou, Qing Li, Laura B. Uwituze, Zhichao Zhang,*
Ran Hong,* and Renxiao Wang*
Cite This: J. Med. Chem. 2021, 64, 10260−10285
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ABSTRACT: Selective Mcl-1 inhibitors may overcome the drug resistance caused by current anti-apoptotic Bcl-2 protein inhibitors
in tumors with Mcl-1 overexpression. Based on previously discovered compounds with a 3-phenylthiophene-2-sulfonamide core
moiety, in this work, we have obtained new compounds with improved binding affinity and/or selectivity under the guidance of
structure-based design. The most potent compounds achieved sub-micromolar binding affinities to Mcl-1 (Ki ∼ 0.4 μM) and good
cytotoxicity (IC50 < 10 μM) on several tumor cells. 15N-heteronuclear single-quantum coherence NMR spectra suggested that these
compounds bound to the BH3-binding groove on Mcl-1. Several cellular assays revealed that FWJ-D4 as well as its precursor FWJ-
D5 effectively induced caspase-dependent apoptosis, and their target engagement at Mcl-1 was confirmed by co-
immunoprecipitation experiments. Treatment with FWJ-D5 at 50 mg/kg every 2 days on an RS4;11 xenograft mouse model for
22 days led to 75% reduction in tumor volume without body weight loss.
between Bcl-2 family proteins has become an appealing
approach to the development of new anti-cancer drugs since
the beginning of this century.10,11
INTRODUCTION
■
Apoptosis, also known as programmed cell death, is an essential
process for eliminating aging or abnormal cells in the living
system. Dysregulation of apoptosis is critical for various
malignant tumor diseases and drug resistance in anti-tumor
therapies.1−4 Bcl-2 family proteins are key regulators of the
mitochondrial apoptotic pathway, which includes anti-apoptotic
members containing BH1−BH4 domains (such as Bcl-xL, Bcl-2,
Mcl-1, Bcl-w, and A1), pro-apoptotic members with BH1−BH3
domains (such as Bax and Bak), and BH3-only proteins (such as
Bim and Bad).5 At the molecular level, the BH3 domain on the
pro-apoptotic proteins binds to a groove on the surface of the
anti-apoptotic proteins.6 Various studies have shown that
overexpression of the anti-apoptotic proteins is resistant to
chemotherapies in a variety of tumor cells.7−9 Blocking the
protein−protein interactions (PPIs) formed by the anti-
apoptotic proteins by small-molecule inhibitors can release the
apoptotic signal and recover the apoptosis process. Thus,
discovery of small-molecule inhibitors targeting the PPIs
Although PPIs are generally challenging as drug targets, a
range of small-molecule inhibitors of anti-apoptotic Bcl-2
proteins have been reported in the literature.12 A batch of
such small-molecule inhibitors have entered clinical trials.13−20
Among them, the ABT series of compounds developed by
Abbott, such as ABT-263,15 and ABT-737,16 is widely known. In
fact, ABT-199 (i.e., Venetoclax)13 has been approved for
treating chronic lymphocytic leukemia or small lymphocytic
lymphoma. Most of these inhibitors,, such as the ABT
Received: April 15, 2021
Published: July 6, 2021
https://doi.org/10.1021/acs.jmedchem.1c00690
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 10260−10285
10260

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Article
Figure 1. Structures and binding affinity data of some small-molecule Mcl-1 inhibitors reported in the literature.
compounds, are known to target Bcl-2 or Bcl-xL. However, it has
been discovered that selective Bcl-2 or Bcl-xL inhibitors
potentially face drug resistance problems in tumor cells
overexpressing Mcl-1 or other less-noted anti-apoptotic
proteins.21,22 For example, ABT-737 inhibits Bcl-xL, Bcl-2, and
Bcl-w, but it lacks efficacy on some types of tumor cells with Mcl-
1 overexpression.22 Therefore, developing selective inhibitors
targeting other anti-apoptotic Bcl-2 proteins (such as Mcl-1 and
Bcl-w) can be a useful supplement to the current efforts on
targeting the Bcl-2 family proteins.23
The Mcl-1 protein has quite different biological functions as
compared to other anti-apoptotic Bcl-2 proteins (such as Bcl-2
and Bcl-xL).24,25 In fact, the crystal structure of Mcl-1 reveals that
it has a smaller and less flexible binding pocket as compared to
Bcl-2 or Bcl-xL.26 All these prompt that Mcl-1 has its
independent characteristics from other anti-apoptotic Bcl-2
proteins. A number of selective small-molecule Mcl-1 inhibitors
have been reported in the past 10 years or so.27,28 Some of them
are summarized as representatives in Figure 1.29−38 Never-
theless, many of the Mcl-1 inhibitors reported in the literature
are tested only in the in vitro binding assay and cytotoxicity assay.
Only one of them, that is, AMG176, is in the first-phase clinical
trial for relapsed or refractory multiple myeloma.20 Therefore,
development of new classes of Mcl-1 inhibitors is still a
compelling demand.
through virtual screening of over 56,000 commercial com-
pounds. Later, we optimized it into a class of compounds with a
3-phenylthiophene-2-sulfonamide core moiety.38 Our binding
assay results indicated that among the three major anti-apoptotic
Bcl-2 proteins, this class of compounds showed selectivity
toward Mcl-1 and Bcl-2 but not Bcl-xL. Cytotoxicity assay results
indicated that such compounds inhibited the proliferation of
several types of tumor cells, such as A549 (human nonsmall cell
lung cancer cell), H1299 (human lung adenocarcinoma cells),
HL-7702 (human liver cells), BEL-7404 (human liver cancer
cells), and Hep3B (human liver cancer cells). Several most active
compounds, for example, YCW-E5, YCW-E10, and YCW-E11,
were observed to induce mitochondrial-pathway apoptosis at
the micromolar level.
In this work, we further optimized this class of compounds
under the guidance of structural analysis and molecular
modeling. Among the newly synthesized compounds, we
obtained some compounds with improved binding affinity as
well as selectivity toward Mcl-1. We also obtained some
compounds with improved binding affinity as pan inhibitors of
anti-apoptotic Bcl-2 proteins. 15N-heteronuclear single-quan-
tum coherence (HSQC) NMR spectra of two selected
compounds suggested that they bound to the BH3-domain
binding pocket on Mcl-1. Cellular mechanism-of-action studies
indicated that these compounds induced caspase-independent
apoptosis via disruption of the PPIs involving Mcl-1 (such as
Bim/Mcl-1 interactions). Moreover, the most potent com-
In our previous work, we obtained a hit compound containing
a phenyltetrazole and a hydrazinecarbothioamide moiety
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pounds exhibited a significant dose-dependent tumor-inhibition
effect on an RS4;11 xenograft mouse model without obvious
weight loss. Overall, these new compounds are more promising
than the lead compound as potential candidates for developing
new anti-cancer drugs.
butyl (2-((4-bromothiophene)-2-sulfonamido)ethyl)carbamate
and tert-butyl (2-((5-bromothiophene)-2-sulfonamido)ethyl)-
carbamate were key reagents for deciding the positions of
sulfonamide and phenyl groups on the thiophene ring. TJ-A1−
TJ-A10 and TJ-B1−TJ-B3 were tested in a FP-based binding
assay (Table 1), which was applied successfully in our previous
works.39,40 Binding assay results indicate that both the
compounds containing the 5-phenyl-N-(2-(3-phenylureido)-
ethyl)thiophene-2-sulfonamide moiety (i.e., TJ-A1−TJ-A3)
and the compounds containing the 4-phenyl-N-(2-(3-
phenylureido)ethyl) thiophene-2-sulfonamide moiety (i.e., TJ-
B1−TJ-B3) bind to Mcl-1 with inhibition constants ranging
between 0.3 and 1.0 μM. In terms of binding affinity to Mcl-1,
these new compounds are comparable to or slightly better than
the chosen lead compounds (e.g., YCW-E5, Ki = 0.84 μM).
Note that the lead compounds exhibit selectivity to Mcl-1 and
Bcl-2 without obvious binding to Bcl-xL, but these new
compounds bind to all three major anti-apoptotic Bcl-2 proteins
(i.e., Bcl-2, Bcl-xL, and Mcl-1) with inhibition constants around
the micromolar level. Therefore, these new compounds may
serve better as pan inhibitors of anti-apoptotic Bcl-2 proteins.
When R3 is replaced with a nitro group, the resulting compounds
(i.e., TJ-A4−TJ-A10) generally lose their binding affinities to all
three anti-apoptotic Bcl-2 proteins. It is thus clear that keeping
the two hydroxyl groups as R3 is essential for maintaining the
desired binding affinity for this class of compounds. In addition,
CCK8 cell viability assay results indicate that compounds TJ-
A1−TJ-A3 exhibit obvious cytotoxicity (IC50 < 20 μM) on the
two leukemia cell lines (i.e., HL-60 and RS4;11) compared to
HeLa (Table 1). However, they exhibit obvious cytotoxicity on
HEK293T cells as well. In contrast, compounds TJ-B1 and TJ-
B2 also exhibit obvious cytotoxicity on HL-60 and RS4;11 cells
(IC50 < 10 μM) but far less cytotoxicity on HEK293T (IC50 > 20
μM). In particular, TJ-B2 has the IC50 value of 1.3 and 3.9 μM
on HL-60 and RS4;11 cells, respectively, without obvious
toxicity on HEK293T at the maximal applied concentration of
50 μM.
RESULTS AND DISCUSSION
■
Structural Optimization and Structure−Activity Rela-
tionship. In this study, three most active compounds developed
in our previous work,38 that is, YCW-E5, YCW-E10, and YCW-
E11, were chosen as the starting point for structural
optimization. In fact, these three lead compounds share the
same structural scaffold with minor difference in substituent
groups (Figure 2a). In this work, we have synthesized five sets of
In order to recover the selectivity toward Mcl-1, we then kept
the 3-phenyl-N-(2-(3-phenylureido)ethyl)thiophene-2-sulfona-
mide core moiety on the lead compound and tried to replace the
dihydroxyphenyl moiety (i.e., α-ring in Figure 2a) with other
aromatic groups. Such derivative compounds were obtained
with the methods outlined in Scheme 1, producing compounds
TJ-C1−TJ-C34 (Table 2). Binding assay results indicate that
when this dihydroxyphenyl moiety is changed to a benzyl, 4-
aminophenyl, or 4-nitrophenyl moiety, most of the compounds
totally lose their binding affinity to the three anti-apoptotic Bcl-2
proteins. Only a few compounds (such as TJ-C5, TJ-C6, TJ-C8,
TJ-C9, TJ-C14, TJ-C16, and TJ-C18) maintain the binding
affinity to Mcl-1 with Ki values between 1.0 and 13 μM, but the
structure−activity relationship is not clear. Moreover, these
several compounds do not have the desired cytotoxicity profile
on HL-60, RS4;11, and HEK293T cells. Therefore, our efforts of
changing the dihydroxyphenyl moiety on the lead compounds
did not lead to improved bioactivities.
To explain the structure−activity relationship of the newly
synthesized compounds, we applied an MD simulation of 100 ns
long to obtain the binding mode of the lead compound YCW-
E10 to Mcl-1 (Figure 2b). Based on this complex structural
model, several critical hydration sites inside the binding pocket
before ligand binding were predicted with the WATsite
method.41 As shown in Figure 2b, site #1 can form a hydrogen
bond with the backbone amide group on residue V249. The high
Figure 2. (A) Chemical structures of the lead compounds YCW-E5 [X
= S, R1 = 3-CF3, R2 = 5-CF3, R3 = OH, Ki(Bcl-2) = 5.6 1.6 μM;
Ki(Mcl-1) = 0.84 0.32 μM; no binding to Bcl-xL], YCW-E10 [X = O,
R1 = 3-CF3, R2 = 5-CF3, R3 = OH, Ki(Bcl-2) = 17 0.6 μM; Ki(Mcl-1)
= 2.1 0.58 μM; no binding to Bcl-xL], and YCW-E11 [X = O, R1 = 3-
Cl, R2 = 5-Cl, R3 = OH, Ki(Bcl-2) = 4.6 1.4 μM; Ki(Mcl-1) = 1.4
0.54 μM; no binding to Bcl-xL]. (B) Illustration of the binding modes of
YCW-E10 (colored in cyan) and FWJ-D4 (colored in green) to Mcl-1,
which were predicted by molecular dynamics (MD) simulation. The
binding pocket is displayed with a translucent surface, with the ligand
and key residue colored in cyan and gray, respectively. Several critical
hydration sites predicted by WATsite are displayed as spheres in pink.
The desolvation energies (in kcal/mol) associated with these
hydrations sites are 3.94 (#1), 3.68 (#2), 2.66 (#3), 2.06 (#4), and
0.96 (#5). The Mcl-1 protein structure used in MD simulation was
from the Mcl-1/BRD inhibitor complex (PDB code: 4WMR).
derivative compounds based on these lead compounds. Below,
we will describe these new compounds and discuss their
activities in a stepwise manner, so the readers can understand the
basic logic in our molecular design.
Our first attempt of structural optimization was to alter the
relative position of the sulfonamide and the phenyl group on the
thiophene moiety while keeping the other parts unchanged. As a
result, compounds TJ-A1−TJ-A10 and TJ-B1−TJ-B3 were
synthesized with the methods outlined in Scheme 1. Here, tert-
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a
Scheme 1. Synthesis of Compounds TJ-A1−TJ-A10, TJ-B1−TJ-B3, TJ-C1−TJ-C34, and FWJ-D1−FWJ-D20
a
Reagents and conditions: (a) NaH, THF, reflux; (b) Pd/C, N2H4·H2O, EtOH, N2; (c) (1) H2SO4, NaNO2, 0 °C; (2) KI; (d) (1) nBuLi, THF;
(2) B(OMe)3, −78 °C; (3). HCl, CH2Cl2; (e) DCM, triethylamine (TEA); (f) Pd(OAc)2, PPh3, K3PO4, THF, reflux; (g) CF3COOH, CH2Cl2,
RT; (h) aryl isothiocyanate, THF, RT.
Table 1. Chemical Structures, Binding Data, and Cytotoxicity of Compounds TJ-A1−TJ-A10 and TJ-B1−TJ-B3
a
b
binding data (Ki, μM)
cytotoxicity (IC50, μM)
compound
X
R1
R2
R3
Bcl-xL
Bcl-2
Mcl-1
HeLa
HL-60
RS4;11
HEK293T
TJ-A1
TJ-A2
TJ-A3
TJ-A4
TJ-A5
TJ-A6
TJ-A7
TJ-A8
TJ-A9
S
3-CF3
3-CF3
3-Cl
4-Cl
3-OMe
3-Cl
5-CF3
5-CF3
5-Cl
H
OH
OH
OH
0.93 0.27 2.6 0.60 0.37 0.17
>20
>20
>20
>20
>20
18.3 0.06
10.5 0.28
10.4 0.72
8.1 1.07
6.7 0.66
2.3 0.57
9.1 0.61
0.34 0.10
5.4 0.21
1.9 0.26
0.6 0.23
1.3 0.57
>20
4.3 0.51
5.7 0.18
6.9 1.24
2.8 0.13
0.077 0.0057
2.46 0.21
10.3 0.84
4.9 0.10
4.5 0.25
6.7 1.10
1.3 0.09
2.1 0.79
3.6 0.76
3.0 0.59
1.4 0.13
2.4 0.11
4.5 0.77
3.9 0.81
>50
4.8 0.05
4.9 0.17
6.2 0.32
3.3 0.22
0.0043 0.00019
7.31 0.83
12.3 1.96
17.1 0.95
12.4 0.25
>20
17.8 1.90
>20
O
O
O
O
O
O
O
O
O
S
O
O
S
O
O
S
1.5 0.49
1.7 0.17
N.A.
3.9 0.85 0.42 0.10
3.6 0.80 0.94 0.37
NO2
NO2
NO2
NO2
NO2
NO2
NO2
OH
OH
OH
OH
OH
N.A.
N.A.
5.9 2.3
N.A.
H
N.A.
5-Cl
5-Me
H
N.A.
N.A.
N.A.
>20
3-Me
weak
N.A.
N.A.
12.0 0.19
>20
>50
4-NO2
4-OMe
3-CF3
3-CF3
3-CF3
3-Cl
3-CF3
3-CF3
3-Cl
N.A.
N.A.
N.A.
16.6 0.86
18.5 0.87
>50
H
N.A.
N.A.
N.A.
>20
>50
>20
>20
>50
>50
>50
>50
TJ-A10
TJ-B1
TJ-B2
5-CF3
5-CF3
5-CF3
5-Cl
5-CF3
5-CF3
5-Cl
5-CF3
N.A.
N.A.
N.A.
1.0 0.39
1.7 0.12
1.7 0.50
N.A.
N.A.
N.A.
4.6 2.1
5.2 0.2
8.4 4.9
5.6 1.6
17.0 0.6
4.6 1.4
N.A.
0.32 0.12
0.51 0.08
0.77 0.10
0.84 0.32
2.1 0.58
1.4 0.54
N.A.
>20
>50
>20
TJ-B3
YCW-E5
YCW-E10
YCW-E11
YCW-E16
7.5 0.41
8.9 1.06
10.7 1.58
6.8 0.08
>20
OH
OMe
3-CF3
ABT-199
A1210477
N.A.
6.4 0.74
>20
<0.001
N.A.
<0.001
N.A.
N.A.
<0.001
>20
>50
a
The mean values and standard deviations of the Ki values were derived based on the outcomes of three parallel measurements. N.A.: no obvious
sign of binding was observed; weak: dose-dependent FP signals were observed, but the change in the FP signals (up to a 100 μM concentration)
b
was not significant enough to determine the Ki value accurately. The mean values and standard deviations of the IC50 values were derived based on
the outcomes of three parallel measurements.
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Table 2. Chemical Structures, Binding Data, and Cytotoxicity of Compounds TJ-C1−TJ-C34
a
b
Ki (μM)
Bcl-xL Bcl-2
cytotoxicity (IC50, μM)
compound
X
R1
R2
R3
Mcl-1
N.A.
N.A.
N.A.
N.A.
11 3.9
4.3 0.76
N.A.
9.0 1.7
10.0 3.4
N.A.
weak
N.A.
N.A.
1.6 0.66
N.A.
13.0 6.5
N.A.
4.6 0.36
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
Hela
HL-60
RS4;11
>50
HEK293T
TJ-C1
TJ-C2
TJ-C3
TJ-C4
TJ-C5
TJ-C6
TJ-C7
TJ-C8
S
O
S
O
O
S
O
S
3-CF3
3-CF3
3-Cl
3-Me
3-OMe
4-Cl
3-NO2
4-NO2
3-NO2
3-Cl
4-OMe
4-Cl
3-CF3
3-Me
3-CF3
3-OMe
3-Cl
4-OMe
4-NO2
3-NO2
3-CF3
3-Cl
4-OMe
3-Me
3-CF3
3-Cl
5-CF3
5-CF3
H
5-Me
H
H
H
H
H
5-Cl
H
H
5-CF3
5-Me
5-CF3
H
H
H
H
H
5-CF3
5-Cl
H
5-Me
5-CF3
H
5-CF3
5-Cl
H
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
Bn
4-NH2-Ph
4-NH2-Ph
4-NH2-Ph
4-NO2-Ph
4-NO2-Ph
4-NO2-Ph
4-NO2-Ph
3-OH-Bn
3-OH-Bn
3-OH-Bn
3-OH-Bn
3-OH-Bn
3-OH-Bn
3-OH-Bn
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
N.A.
4.7 0.06
13.5 0.34
8.4 0.25
>50
2.1 0.14
11.4 0.32
>50
2.2 0.18
1.7 0.39
>50
8.5 0.76
>20
10.2 0.58
>20
>50
>50
>50
>20
>50
>20
>50
>20
7.0 0.17
>20
7.1 0.86
12.8 0.24
>20
12.1 1.15
13.3 0.86
8.9 1.17
18.3 0.53
41% @ 20 μM
>50
>20
>20
>20
6.0 0.59
>20
4.99 0.05
>20
>20
13.8 0.81
>20
>20
>20
>20
>20
17.4 0.53
50% @ 20 μM
>50
10.5 1.01
9.0 0.56
>20
14.1 0.57
20.0 1.23
8.6 0.18
2.8 0.22
>50
11.3 0.78
11.6 1.34
>20
20.0 0.12
13.6 0.16
11.9 1.53
>20
TJ-C9
S
>50
11.7 0.81
>50
>50
>50
>50
7.2 1.50
>50
TJ-C10
TJ-C11
TJ-C12
TJ-C13
TJ-C14
TJ-C15
TJ-C16
TJ-C17
TJ-C18
TJ-C19
TJ-C20
TJ-C21
TJ-C22
TJ-C23
TJ-C24
TJ-C25
TJ-C26
TJ-C27
TJ-C28
TJ-C29
TJ-C30
TJ-C31
TJ-C32
TJ-C33
TJ-C34
O
O
O
O
O
S
S
S
O
S
S
O
O
O
O
S
>50
>20
>20
>20
9.9 0.49
>50
4.6 0.04
>20
28% @ 20 μM
11.1 0.99
17.3 0.42
>50
6.3 0.90
>50
>50
>50
>50
>50
10.8 0.83
11.4 0.64
4.0 0.83
2.8 0.45
>20
10.1 1.42
9.8 0.54
3.0 0.33
>50
>20
>20
9.4 0.13
5.8 0.22
12.0 0.58
5.8 0.22
10.1 1.06
14.1 0.61
1.5 0.75
4.1 0.07
6.3 0.34
9.3 0.66
9.4 0.84
12.4 0.01
>20
>50
14.9 1.04
10.7 0.59
>20
S
3-OH-Bn
1.8 0.36
7.1 0.89
>20
11.6 1.19
8.9 0.22
2.6 1.08
3.1 0.44
>50
O
O
S
O
S
O
S
O
3-CF3
3-Cl
trans-4-OH-Cy
trans-4-OH-Cy
trans-4-OH-Cy
trans-4-OH-Cy
cis-4-OH-Cy
cis-4-OH-Cy
cis-4-OH-Cy
cis-4-OH-Cy
>20
4-NO2
4-NO2
3-CF3
3-CF3
4-NO2
4-NO2
5.4 0.55
10.5 1.38
10.9 0.50
14.1 1.37
>20
H
5-CF3
5-CF3
H
H
N.A.
>20
>20
a
The mean values and standard deviations of the Ki values were derived based on the outcomes of three parallel measurements. N.A.: no obvious
sign of binding was observed; weak: dose-dependent FP signals were observed, but the change in the FP signals (up to a 100 μM concentration)
b
was not significant enough to determine the Ki value accurately. The mean values and standard deviations of the IC50 values were derived based on
the outcomes of three parallel measurements. Cell viability percentage at 20 μM was provided for the case that could not be normally fitted.
positive energy of 3.94 kcal/mol associated with this site
indicates its poor stability inside the binding pocket. According
to our predicted binding mode, the terminal hydroxyl group on
YCW-E10 forms a hydrogen bond with V249 after displacing
the unstable water molecule at this site. This may explain why
the newly synthesized compounds with hydrogen bond donors
at this location can maintain good binding affinity to Mcl-1. In
addition, there are two high-energy sites at the right part of the
binding pocket, that is, site #2 and site #4. Interestingly,
locations of these two sites overlap with the 3- and 5-subsitutent
positions on the γ-phenyl ring on YCW-E10 (Figure 2a). In fact,
the compounds with 3,5-dichloro or 3,5-ditrifluoromethyl
substituents on this phenyl ring generally show high binding
affinity to Mcl-1. Besides, this binding mode also reveals that the
α-phenyl ring on YCW-E10 fills up a relatively narrow cavity
formed by residues M231, V249, and M250. It thus explains why
the attempt of replacing the α-phenyl ring essentially failed.
It appears that this predicted binding mode is able to interpret
the basic structure−activity relationship observed among the
newly obtained compounds summarized in Tables 1 and 2.
Encouraged by the outcome of this analysis, we turned our
attention to the other two hydration sites with positive energies
near the β-phenyl ring on the lead compound, that is, site #3
(2.66 kcal/mol) and site #5 (0.96 kcal/mol). These two sites are
surrounded mainly by hydrophobic residues, such as A227,
F228, and M231. Therefore, introduction of small-size,
hydrophobic groups onto the β-phenyl ring may further improve
binding affinity to Mcl-1 protein.
Accordingly, a total of 20 compounds with 4-methyl or 5-
isopropyl substitutions on the β-phenyl ring (i.e., FWJ-D1−
D20) were synthesized with the methods outlined in Scheme 1
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(Table 3). Note that this set of compounds shares exactly the
same structural scaffold as the lead compounds. Binding assay
results indicate that most of these new compounds reserve the
selectivity toward Mcl-1 as the lead compounds. Here, some
derivative compounds with the 4-Me or 5-i-Pr group on the β-
phenyl ring achieve improved binding affinity to Mcl-1. For
example, the Mcl-1 binding affinity of FWJ-D4 or FWJ-D6 is
improved by 4−6 times as compared to YCW-E10. Considering
that the methyl or isopropyl group is rather small in size, this
amount of improvement is promising. The predicted binding
mode of FWJ-D4 to Mcl-1 protein is shown in Figure 2b. One
can see here that the additional methyl group introduced on the
β-phenyl ring displaces the high-energy water site #3, which
provides an explanation for the improvement in binding affinity.
In addition, FWJ-D4 and FWJ-D6 now bind to Mcl-1
specifically, while YCW-E10 is still a dual inhibitor of Mcl-1
and Bcl-2. Here, another observation is that if the two hydroxyl
groups on the α-phenyl ring are protected as methoxy groups,
Mcl-1 binding affinity of the resulting compounds (i.e., FWJ-D3,
FWJ-D5, FWJ-D7, FWJ-D10, and FWJ-D15) will decrease
significantly or even lose completely. This again verifies the
critical role of the two hydroxyl groups. In terms of cytotoxicity
on cell lines, most of this set of compounds shows good
cytotoxicity (IC50 < 10 μM) on two leukemia cell lines (i.e., HL-
60 and RS4;11) and obvious cytotoxicity (typically IC50 < 12
μM) on HEK293T cells as well. Nevertheless, FWJ-D4 and
FWJ-D9 show much less toxicity (IC50 > 20 μM) on HEK293T
while maintaining IC50 values of 3−7 μM on the two leukemia
cell lines. Selective cytotoxicity toward leukemia cells is another
notable improvement achieved in our optimization.
Two-dimensional HSQC spectroscopy was employed in our
work to characterize the binding of these active compounds to
15N-labeled Mcl-1 protein. Three of our compounds, including
YCW-E10, FWJ-D2, and FWJ-D4, were selected for this test. In
addition, a known selective Mcl-1 inhibitor, A-1210477,31 was
also considered as the positive control. The HSQC spectrum of
the free Mcl-1, which is publicly available from the Biological
Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/,
access number 19654), was used as the reference for assignment
of chemical shifts. After superimposing the 15N-HSQC spectra
of the free Mcl-1 protein and the Mcl-1 complex, the chemical
shift of each residue was calculated. By comparing the 15N-
HSQC spectra of the Mcl-1 protein in complex with A-1210477
and those with our thiophene-2-sulfonamide compounds, a
number of residues with similar chemical shifts can be found
(Figure 3a−d). Here, the residues with significant chemical
shifts after adding the compounds include H224, F228, M231,
L235, M250, and R263. These residues generally locate around
the BH3 binding groove on Mcl-1. Although HSQC spectros-
copy does not provide structural information at the atomic level,
the results shown in Figure 3 are still strong proofs of our
hypothesis, that is, the thiophene-2-sulfonamide derivative
compounds bind at the BH3 binding groove on Mcl-1 as
expected. It also supports the predicted binding mode shown in
Figure 2b.
Despite the encouraging results obtained in bioassays, we
have observed that many compounds obtained in our work
encounter water solubility problem in sample preparation. We
suspect that it is one of the reasons that prevent this class of
compounds that achieves an even higher level of potency.
Among the newly synthesized compounds, we managed to
obtain the crystal structure of FWJ-D7 (Figure 4), which was
deposited into the CSD database on May 20, 2019 (access
Figure 3. Superimposed 15N-HSQC spectra of the free Mcl-1 (in black)
and Mcl-1 in complex with compounds (A) YCW-E10, (B) FWJ-D2,
(C) FWJ-D4, and (D) A1210477. The six residues with obvious
chemical shifts near the BH3 peptide binding pocket are labeled
explicitly. The enlarged view of the focused residues is provided in the
upper right corner of each figure.
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compounds in our work. Moreover, all such weak Mcl-1
inhibitors still have obvious cytotoxicity (typically IC50 < 10
μM) on the two leukemia cell lines (i.e., HL-60 and RS4;11) and
a reasonable selectivity toward HEK293T cells (IC50 > 20 μM).
In contrast, the corresponding compounds with the two critical
hydroxyl groups protected in the methoxy form do not exhibit
any obvious cytotoxicity on all four cell lines considered in our
work. We therefore conclude that this set of compounds still has
potential if their structures are properly optimized. This
hypothesis, however, was not testified in this work because we
had obtained other more promising candidates for further
investigation.
Characterization of the Pro-Apoptotic Effects of the
Thiophene-2-Sulfonamide Compounds. Three com-
pounds obtained in our work, that is, FWJ-D4, FWJ-D5, and
YCW-E16, were selected and examined in several types of assays
to characterize their apoptosis-inducing effects on the RS4;11
cells. Here, FWJ-D4 was the most potent one in the binding
assay and cell viability assay. FWJ-D5 is the precursor of FWJ-
D4 with the hydroxyl groups protected by methoxy groups. The
binding affinity of FWJ-D5 to Mcl-1 is about 10-fold weaker
than that of FWJ-D4. However, it has similar toxicities to FWJ-
D4 on HL-60 and RS4;11 cell lines. The lead compound YCW-
E16 was chosen for making a comparison. ABT-199 was used as
the positive reference in all assays.
Several commonly used indicators for characterizing
apoptosis include changes in mitochondrial membrane potential
(MOMP), transfer of phosphatidyl-serine (PS), and changes of
plasma membrane permeability. These indicators could be
quantified by using the corresponding dyeing methods. In our
study, flow cytometry analysis with MitoDamage staining was
employed to detect the three indicators of apoptosis mentioned
above. After treatment by the selected compounds at five doses,
that is, 2.5, 5, 10, 20, and 40 μM for 24 h, RS4;11 cells were
stained with 7-AAD, annexin V, and MitoSense Red and then
analyzed by flow cytometry. Annexin V and 7-AAD were used to
detect the state of PS and the plasma membrane permeability of
the cells, respectively. The combination of 7-AAD and annexin V
was used to differentiate cells in early apoptosis [annexin V (+)
and 7-ADD (−)] and late apoptosis [annexin V (+) and 7-ADD
(+)], corresponding to the bottom and upper right regions in
Figure 5. As for the cells treated with dimethyl sulfoxide
(DMSO), less than 5% of them were observed to be in the early
or late stage of apoptosis. After the tested compounds were
added, the ratio of apoptotic cells increased in an obvious dose-
dependent manner (as shown in Figure S1 in the Supporting
Information, Part II). For example, when the cells were treated
by the given compound at 20 μM, the ratio of apoptotic cells
reached 97, 88, and 98% for YCW-E16, FWJ-D4, and FWJ-D5,
respectively (Figure 5). MitoSense Red was used to detect the
changes in MOMP, where cells with intact MOMP exhibited
high Red2 fluorescence and cells with impaired MOMP
exhibited low Red2 fluorescence. As shown in Figure 6, Red2
fluorescence gradually shifted to the left as the concentration of
the given compound increased, which was consistent with the
results obtained from 7-AAD + annexin V staining. These results
indicate that these tested compounds are able to induce cell
apoptosis.
Figure 4. (A) Crystal packing of FWJ-D7 (CSD number: 1917331);
(B) two-dimensional illustration of the hydrogen bonding network
observed in this crystal structure.
number: 1917331). This structure indicates that there is a
complex hydrogen bond network formed between FWJ-D7
molecules due to crystal packing (Figure 4). Such a hydrogen
bond network certainly enhances the crystal lattice energy,
which in turn results in the poor water solubility.
In an attempt to reduce this effect, we designed another set of
compounds by replacing the ethylenediamine moiety with a
piperazine ring (Table 4). The aim was to remove the inter-
molecular hydrogen bonds existing in the crystal structure. The
piperazine ring was chosen mainly due to the convenience of
synthesis. As a result, compounds FWJ-E1−FWJ-E18 were
synthesized with the methods outlined in Scheme 2. Although
water solubility of this set of compounds was somewhat
improved according to our qualitative judgement, basically, all
of them do not have the desired affinity to any of the three anti-
apoptotic Bcl-2 proteins. About half of these compounds, such
as FWJ-E1 and FWJ-E9, retain weak affinity to Mcl-1 (i.e.,
inhibition ratio >50% in the FP binding assay at a dose of 100
μM). Interestingly, all such weak Mcl-1 inhibitors have two
naked hydroxyl groups on the α-phenyl ring, which is consistent
with the structure−activity relationship observed in other sets of
To further verify the pro-apoptotic mechanism of these
compounds, they were tested in a western blot assay for
detecting caspase activation. Here, poly-ADP-ribose polymerase
protein (PARP) is a major substrate of activated caspases.
Cleavage of PARP is one of the indicators of caspase-dependent
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Table 4. Chemical Structures, Binding Data, and Cytotoxicity of Compounds FWJ-E1−FWJ-E18
a
inhibition at 100 μM
cytotoxicity (IC50, μM)
Bcl-xL Bcl-2 Mcl-1
compound
X
R1
R2
R3
R4
(%)
(%)
(%)
HeLa
HL-60
RS4;11
HEK293T
FWJ-E1
FWJ-E2
FWJ-E3
FWJ-E4
FWJ-E5
FWJ-E6
FWJ-E7
FWJ-E8
FWJ-E9
FWJ-E10
FWJ-E11
FWJ-E12
FWJ-E13
FWJ-E14
FWJ-E15
FWJ-E16
FWJ-E17
FWJ-E18
S
S
S
O
O
O
O
O
S
S
S
O
O
S
S
S
O
O
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
4-NO2
3-NO2
3-NO2
3-CF3
3-CF3
3-CF3
3-CF3
3-CF3
4-Cl
H
H
H
H
H
H
H
H
5-CF3
5-CF3
5-CF3
5-CF3
5-CF3
H
H
H
H
H
OH
H
H
5-NO2
H
H
5-NO2
H
H
H
5-NO2
H
H
H
H
H
5-NO2
H
28
18
27
29
20
44
34
19
12
14
29
−8
24
3
31
16
16
33
17
19
30
18
20
12
25
7
23
2
18
18
33
15
81
33
26
63
22
37
74
31
72
52
66
47
44
20
43
41
95
28
49% @ 20 μM
>50
>50
>20
>50
>50
>20
>50
>20
>20
>50
20% @ 20 μM
>50
>50
>50
>50
14.8 2.08
>20
5.3 0.25
>50
>50
4.9 0.33
>50
>50
9.0 0.89
>50
>50
14.3 1.17
>50
>50
9.8 1.00
>50
7.3 0.28
9.5 0.30
>50
10% @ 20 μM
>50
7.5 0.48
>50
>20
>20
>50
>20
>20
>20
>20
>20
>20
>20
42% @ 20 μM
29% @ 20 μM
>20
>50
>20
>50
15.5 1.29
>50
OMe
OMe
OH
OMe
OMe
OH
OMe
OH
OH
OMe
OH
OMe
OH
OMe
OMe
OH
4.5 0.27
>50
2.2 0.22
8.4 0.42
38% @ 20 μM
15% @ 20 μM
>20
5.5 0.39
>50
>50
4-Cl
4-Cl
4-F
4-F
33
37
32
33
>50
6.8 0.28
14.9 3.10
8.1 1.56
>20
OMe
H
a
The mean values and standard deviations of the IC50 values were derived based on the outcomes of three parallel measurements. Cell viability
percentage at 20 μM was provided for the case that could not be normally fitted.
apoptosis. In our work, YCW-E16 and FWJ-D5 exhibited
significant pro-apoptotic signals in flow cytometry analysis, and
thus, they were tested in the caspase activation assay. Again,
DMSO and ABT-199 were used as the negative and positive
controls, respectively. Our results showed that the full-length
PARP (116 kDa) degraded and the cleaved PARP (89 kDa)
increased after treatment with these two compounds (Figure 7).
After the compound concentration was increased from 2.5 to 40
μM, the cleaved PARP, cleaved caspase-3, and cleaved caspase-9
were increased generally in a dose-dependent manner. This
observation again confirms the results of our flow cytometry
analysis that these compounds are able to induce the caspase-
dependent apoptosis.
To verify target engagement at the cellular level, we
performed the co-immunoprecipitation (co-IP) assay on
MCF-7 cells that expressed high levels of both Bcl-2 and Mcl-
1. ABT-199 (a selective Bcl-2 inhibitor) and A-1210477 (a
selective Mcl-1 inhibitor) were used as controls in this assay. As
can be seen in Figure 8, both compounds produced the
biological effects as expected. Here, FWJ-D4 and FWJ-D5
treatment at 10 μM led to the disruption of Mcl-1/Bim
interactions by 70 and 30%, respectively, where they had no
obvious effect on the Bcl-2/Bim complex. Notably, YCW-E16
treatment at 10 μM disrupted both Mcl-1/Bim and Bcl-2/Bim
interactions by 30 and 20%, respectively. All these observations
were in full agreement with the results obtained in our binding
assay, that is, FWJ-D4 and FWJ-D5 are selective Mcl-1
inhibitors, while YCW-E16 is a dual inhibitor of Bcl-2 and
Mcl-1.
xenograft mouse model of human acute lymphoblastic leukemia
cells (RS4;11) because it was among the most potent
compounds identified in our binding assay and cellular assays.
At the same time, it was expected to have a low toxicity due to
protection of the dihydroxyl groups. When the tumor on the
xenograft mice grew to a volume of 250 mm3, the mice were
grouped and treated intraperitoneally with FWJ-D5 in 12.5, 25,
and 50 mg/kg every 2 days for 22 days. Tumor volumes were
measured every 3 days. Among the high-dose group (i.e., 50 mg/
kg), tumor volume was reduced by up to 75% (Figure 9a).
Treatment of FWJ-D5 at 12.5 and 25 mg/kg also suppressed the
tumor growth significantly, with an inhibition rate over 60%.
Notably, FWJ-D5 was well tolerated by the mice without
obvious body weight loss as compared to the blank group
(Figure 9b).
FWJ-D5 was also evaluated in terms of the tumor mitotic
index (Ki-67) using histological and immunohistochemical
techniques. When the RS4;11 xenograft mice were treated by
FWJ-D5 for 19 days, tumor samples were collected and
analyzed. Tumor tissues from the blank group (i.e., DMSO
only) were stained strongly with Ki-67, indicating a large
fraction of highly proliferative cells (Figure 10a). In comparison,
positive cells under Ki-67 staining decreased in a dose-
dependent manner under the treatment of FWJ-D5 (Figure
10c−e). When the xenograft mice were treated by FWJ-D5 in 50
mg/kg, positive cells under Ki-67 staining generally disappeared,
achieving an effect comparable to that of ABT-199 (Figure
10b). The results of this assay proved that FWJ-D5 was able to
attenuate the proliferation of RS4;11 tumor cells.
In Vivo Efficacy of FWJ-D5 on the Xenograft Mouse
Model. Compound FWJ-D5 was chosen to be tested on a
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Scheme 2. Synthesis of Compounds FWJ-E1, FWJ-E2, FWJ-E4, FWJ-E5, FWJ-E7−FWJ-E9, FWJ-E11−FWJ-E15, FWJ-E17, and
a
FWJ-E18
a
Reagents and conditions: (a) TEA, DCM, 0 °C; (b) Pd(OAc)2, K3PO4, Ph2Pcy, THF; (c) TFA, DCM; (d) aryl isothiocyanate, DCM; (e) BBr3,
DCM.
body weight loss. In conclusion, this class of compounds can
serve as promising candidates for developing new anti-cancer
drugs by targeting the anti-apoptotic Bcl-2 family proteins.
CONCLUSIONS
■
In this work, we have selected the compounds obtained in our
previous work, which contain a 3-phenylthiophene-2-sulfona-
mide core moiety, for structural optimization with the aim of
improving their binding affinity and selectivity toward Mcl-1.
15N-HSQC spectra of a chosen lead compound (i.e., YCW-E10)
suggested that it bound to the BH3-binding groove on Mcl-1. Its
binding mode to Mcl-1 was predicted by MD simulation and
then used to guide structure-based optimization. A total of 85
new compounds were synthesized and all tested in a FP-based
binding assay and cytotoxicity assays. As compared to the lead
compound, some new compounds, such as FWJ-D4, FWJ-D6,
and FWJ-D9, achieved 4−6-fold higher affinity and excellent
selectivity toward Mcl-1 in the binding assay. Moreover, such
compounds exhibited a micromolar level of cytotoxicity on HL-
60 and RS4;11 cells and much less cytotoxicity on HEK293T
cells. Interestingly, we also obtained some compounds, such as
TJ-A1−TJ-A3 and TJ-B1−TJ-B3, with a microlevel of binding
affinity to all three targets (i.e., Mcl-1, Bcl-2, and Bcl-xL). Their
potential as pan inhibitors of anti-apoptotic Bcl-2 family proteins
provides other possibilities for further investigation. In this work,
we selected FWJ-D4 and its precursor FWJ-D5 for in-depth
study. Several apoptotic assays indicated that they effectively
induced caspase-dependent apoptosis. An additional co-
immunoprecipitation assay proved target engagement at Mcl-1
at the cellular level for both compounds. FWJ-D5 was further
tested on an RS4;11 xenograft mouse model, where it exhibited a
tumor inhibition effect in a clear dose-dependent manner.
Treatment of xenograft mice with FWJ-D5 at the dose of 12.5−
50 mg/kg led to 60−75% reduction in tumor volume without
EXPERIMENTAL SECTION
■
Synthesis. The overall synthetic route of all new compounds
essentially followed the protocol reported in our previous work.38 All
reagents and starting materials used in our synthesis were purchased
from Lancaster, Acros, and Shanghai Chemical Reagent Corp. They
were used without further purification unless specified.
The general procedure for the synthesis of compounds TJ-A1−TJ-
A10, TJ-B1−TJ-B3, TJ-C1−TJ-C34, and FWJ-D1−FWJ-D20 (see
Scheme 1) is described as follows:
Phenol 1 (25.9 mmol) and 2-nitrofluorobenzene 2 (23.5 mmol)
were added into a 100 mL flask with 60% NaH (39.9 mmol) and
tetrahydrofuran (THF) (38 mL). The mixture was heated at reflux for 2
h with stirring to give 3. 3 (23.3 mmol) was reduced to its amino
product 4 by reacting with 10% Pd/C (640 mg), EtOH (80 mL), and
hydrazine (13 mL) under nitrogen protection. The reaction of 4 (22.8
mmol) with H2SO4 (68.4 mmol), NaNO2 (45.6 mmol), and KI (45.6
mmol) in an ice-water bath gave iodine product 5. Under argon
protection, 5 (19.1 mmol) was reacted with n-butyllithium (38.2
mmol) and trimethyl borate (47.8 mmol) to obtain product 6.
Intermediate 9 was obtained via the reaction of bromo-substituted 2-
sulfonamidothiophene 7 (7.64 mmol) with single Boc-protected
ethylenediamine 8 (11.46 mmol).
Compound 6 (2.56 mmol), 9 (2.13 mmol), palladium acetate (0.213
mmol), triphenylphosphine (0.425 mmol), and potassium phosphate
(6.38 mmol) were added into a 100 mL flask. After full deoxidation,
THF (25 mL) and water (5 mL) were added to dissolve the reactants.
The solution was then heated under reflux for 4 h. The residue was
concentrated and extracted by water (15 mL) and ethyl acetate (30
mL) three times. It was purified by chromatography (EtOAc/
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Figure 5. Results of flow cytometric analysis on Annexin V- and 7-AAD-stained RS4;11 cells for DMSO [(A), 1‰], ABT-199 [(B), 1 μM], and three
selected compounds, that is, YCW-E16 [(C), 20 μM], FWJ-D4 [(D), 20 μM], and FWJ-D5 [(E), 20 μM]. Cells were treated by compounds for 24 h.
Here, DMSO and ABT-199 were used as the negative control and the positive control, respectively.
Figure 6. Results of flow cytometric analysis with MitoSense Red-stained RS4;11 cells for three selected compounds: (A) YCW-E16; (B) FWJ-D4;
(C) FWJ-D5. Cells were treated by compounds at doses of 2.5 μM (orange), 5 μM (green), 10 μM (dark green), 20 μM (cyan), and 40 μM (purple)
for 24 h. Here, DMSO (red) and ABT-199 (1 μM, blue) were used as the negative control and the positive control, respectively.
petroleum ether 1:10−1:5) to obtain 10, which was further reacted with
DCM and TFA to give 11 by removing the Boc-protecting group.
Compound 11 (∼50 mg), CH2Cl2 (3 mL), and 1.2 equiv of
isocyanate or isothiocyanate were added into a 25 mL flask. The
mixture was stirred at room temperature until the reaction was
complete. The residue was purified by chromatography (EtOAc/
petroleum ether 1:3) to obtain the final compounds described in this
work, including TJ-A1−TJ-A10, TJ-B1−TJ-B3, TJ-C1−TJ-C34, and
FWJ-D1−FWJ-D20.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-5-(2-
(3,4-dihydroxyphenoxy)phenyl)thiophene-2-sulfonamide (TJ-A1).
1
Overall yield: 9.4%, purity: 96.6%. H NMR (400 MHz, CDCl3): δ
8.32 (s, 1H), 7.81 (s, 2H), 7.62 (d, J = 8.0 Hz, 1H), 7.55 (s, 1H), 7.50
(d, J = 4.0 Hz, 1H), 7.35 (d, J = 4.0 Hz, 1H), 7.24 (t, J = 8.4 Hz, 1H),
7.10 (t, J = 7.6 Hz, 1H), 7.01−6.99 (m, 1H), 6.91 (d, J = 8.4 Hz, 1H),
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Figure 7. Activation of caspase-3, caspase-9, and PARP by YCW-E16 and FWJ-D5. RS4;11 cells were treated by the given compounds at multiple
doses for 48 h. Expression levels of caspase-3, caspase-9, and PARP were detected by western blot.
Figure 8. MCF-7 cells were treated with ABT-199 (1 μM), A-1210477
(3 μM), FWJ-D4 (10 μM), FWJ-D5 (10 μM), and YCW-E16 (10
μM). Bcl-2 and Mcl-1 immunoprecipitation assays were performed, and
the immunoprecipitated fractions were analyzed by immunoblotting for
the indicated proteins. The numbers underneath the blots were scanned
and semi-quantified by densitometry, which were normalized to loading
control (β-actin).
6.73 (d, J = 8.4 Hz, 1H), 6.59 (d, J = 2.8 Hz, 1H), 6.34 (dd, J = 8.4, 2.8
Hz, 1H), 6.03 (s, 1H), 5.74−5.72 (m, 1H), 3.72−3.71 (m, 2H), 3.20−
3.19 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.5, 155.1, 149.1,
146.8, 145.4, 142.7, 142.4, 141.2, 132.3, 132.2, 132.0, 131.7, 131.5,
130.7, 128.8, 125.7, 125.3, 124.0, 123.8, 123.5, 123.1, 118.9, 117.6,
116.5, 111.3, 108.2, 44.6, 42.9; ESI-MS: m/z 677.9 [M + H]+, 699.8 [M
+ Na]+; MALDI-HRMS C27H22N3O5F6S3 [M + H]+ calcd, 678.0620;
obs., 678.0623.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-(3,4-
dihydroxyphenoxy)phenyl)thiophene-2-sulfonamide (TJ-A2). Over-
all yield: 13.3%, purity: 96.7%. 1H NMR (400 MHz, acetone-d6): δ 8.57
(s, 1H), 7.98 (s, 2H), 7.73 (dd, J = 8.0, 1.2 Hz, 1H), 7.51 (d, J = 4.0 Hz,
1H), 7.45 (d, J = 4.0 Hz, 1H), 7.37 (s, 1H), 7.19 (dt, J = 7.8, 1.2 Hz,
1H), 7.02 (dt, J = 7.6, 0.8 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 6.73−6.70
(m, 2H), 6.47 (d, J = 2.8 Hz, 1H), 6.30 (dd, J = 8.4, 2.8 Hz, 1H), 6.10
(m, 1H), 3.29−3.25 (m, 2H), 3.06−3.02 (m, 2H); 13C NMR (100
MHz, acetone-d6): δ 156.0, 155.3, 149.6, 147.1, 145.4, 143.5, 143.0,
141.8, 132.9, 132.6, 132.2, 132.1, 131.9, 130.8, 129.1, 128.6, 125.9,
124.2, 123.1, 119.2, 118.5, 118.5, 116.6, 114.9, 111.3, 108.3, 44.4, 40.4;
ESI-MS: m/z 661.9 [M + H]+, 683.9 [M + Na]+; MALDI-HRMS
C27H22N3O6F6S2 [M + H]+ calcd, 662.0849; obs., 662.0850.
Figure 9. Inhibition of tumor growth of FWJ-D5 on an RS4;11
xenograft mouse model. (A) Changes in tumor volume. (B) Changes in
the body weight of the xenograft mouse. Here, xenograft mice were
administrated with the vehicle control (red line) or ABT-199 in 12.5
mg/kg (orange line) and FWJ-D5 in 12.5 mg/kg (green line), 25 mg/
kg (blue line), and 50 mg/kg (purple line) every 2 days for 22 days.
3.40−3.36 (m, 2H), 3.19−3.15 (m, 2H); 13C NMR (100 MHz,
acetone-d6): δ 155.9, 155.2, 149.4, 147.0, 145.2, 143.8, 142.9, 141.6,
135.4, 132.0, 130.8, 129.0, 125.8, 124.1, 124.0, 121.5, 119.0, 116.9,
116.6, 111.3, 108.3, 44.4, 40.3; ESI-MS: m/z 593.9 [M + H]+; MALDI-
HRMS C25H21N3O6Cl2S2Na [M + Na]+ calcd, 616.0141; obs.,
616.0143.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-5-(2-(3,4-
dihydroxyphenoxy)phenyl)thiophene-2-sulfonamide (TJ-A3). Over-
all yield: 14.3%, purity: 95.3%. 1H NMR (400 MHz, acetone-d6): δ 8.39
(s, 1H), 8.21 (s, 1H), 7.95 (s, 1H), 7.87 (dd, J = 8.0, 1.6 Hz,1H), 7.65
(d, J = 4.0 Hz, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.51 (d, J = 1.6 Hz, 1H),
7.33 (td, J = 7.6, 1.6 Hz, 1H), 7.17 (td, J = 7.6, 1.2 Hz, 1H), 6.97 (t, J =
1.6 Hz, 1H), 6.94 (dd, J = 8.4, 0.8 Hz, 1H), 6.87−6.83 (m, 2H), 6.61 (d,
J = 2.8 Hz, 1H), 6.44 (dd, J = 8.4, 2.8 Hz, 1H), 6.14−6.13 (m, 1H),
N-(2-(3-(4-Chlorophenyl)ureido)ethyl)-5-(2-(3,4-dinitrophenoxy)-
phenyl)thiophene-2-sulfonamide (TJ-A4). Overall yield: 11.4%,
1
purity: 98.8%. H NMR (400 MHz, acetone-d6): δ 8.24 (d, J = 9.2
Hz, 1H), 8.15 (s, 1H), 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.70 (d, J = 2.4 Hz,
1H), 7.58−7.55 (m, 3H), 7.48−7.43 (m, 4H), 7.39 (dd, J = 8.0, 0.8 Hz,
1H), 7.22−7.20 (m, 2H), 6.93−6.90 (m, 1H), 6.01−5.98 (m, 1H),
3.34−3.30 (m, 2H), 3.14−3.10 (m, 2H); 13C NMR (100 MHz,
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Figure 10. FWJ-D5 inhibited the proliferation of RS4;11 cells in xenograft mice in a dose-dependent manner. Results of Ki-67 staining in tumor tissues
treated with (A) DMSO, (B) ABT-199 in 12.5 mg/kg, (C) FWJ-D5 in 12.5 mg/kg, (D) FWJ-D5 in 25 mg/kg, and (E) FWJ-D5 in 50 mg/kg.
acetone-d6): δ 162.0, 156.3, 151.0, 146.1, 143.5, 142.8, 140.3, 137.3,
132.4, 131.8, 130.7, 129.3, 129.1, 127.9, 127.5, 126.5, 126.4, 122.9,
121.2, 120.3, 114.2, 44.8, 40.2; ESI-MS: m/z 617.9 [M + H]+; DART-
HRMS C25H19ClN5O8S2 [M − H]− calcd, 616.0369; obs., 616.0350.
N-(2-(3-(3-Methoxyphenyl)ureido)ethyl)-5-(2-(3,4-
dinitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-A5). Overall
yield: 11.4%, purity: 98.1%. 1H NMR (400 MHz, acetone-d6): δ 8.23
(d, J = 8.8 Hz, 1H), 8.00 (s, 1H), 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.70 (d,
J = 2.0 Hz, 1H), 7.58−7.55 (m, 3H),7.48−7.41 (m, 3H), 7.23 (t, J = 2.0
Hz, 1H),7.10 (t, J = 8.0 Hz, 1H), 6.96−6.89 (m, 2H), 6.50 (dd, J = 8.0,
1.6 Hz, 1H), 5.96−5.93 (m, 1H), 3.73 (s, 3H), 3.34−3.30 (m, 2H),
3.14−3.10 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 162.1, 161.1,
156.5, 151.0, 143.6, 142.9, 142.6, 132.4, 131.8, 130.8, 130.2, 129.1,
127.9, 127.5, 126.5, 122.9, 121.3, 114.2, 111.3, 107.8, 104.9, 55.3, 45.0,
40.2; ESI-MS: m/z 612.0 [M − H]−; DART-HRMS C26H22N5O9S2 [M
− H]− calcd, 612.0864; obs., 612.0845.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-5-(2-(3,4-
dinitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-A6). Overall
yield: 10.1% purity: 97.2%. 1H NMR (400 MHz, acetone-d6): δ 8.35 (s,
1H), 8.25 (d, J = 8.8 Hz, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.71 (d, J = 2.0
Hz, 1H), 7.59−7.45 (m, 7H), 7.40 (d, J = 8.0 Hz, 1H), 6.97 (t, J = 2.0
Hz, 1H), 6.87−6.84 (m, 1H), 6.10−6.08 (m, 1H), 3.35−3.30 (m, 2H),
3.15−3.11 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 162.1, 155.8,
151.1, 143.9, 143.7, 143.0, 137.5, 135.5, 132.5, 131.9, 130.8, 129.1,
128.0, 127.6, 126.6, 122.9, 121.6, 121.3, 117.0, 116.9, 114.3, 44.6, 40.3;
ESI-MS: m/z 651.9 [M + H]+; ESI-HRMS C25H18Cl2N5O8S2 [M −
H]− calcd, 649.9979; obs., 649.9952.
127.5, 126.4, 125.7, 122.9, 121.2, 117.9, 117.9, 114.2, 44.4, 40.2; ESI-
MS: m/z 627.0 [M − H]−; ESI-HRMS C25H19N6O10S2 [M − H]−
calcd, 627.0610; obs., 627.0591.
N-(2-(3-(4-Methoxyphenyl)ureido)ethyl)-5-(2-(3,4-
dinitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-A9). Overall
yield: 21.1%, purity: 99.3%. 1H NMR (400 MHz, acetone-d6): δ 8.23
(d, J = 9.2 Hz, 1H), 7.94 (dd, J = 8.0, 1.2 Hz, 1H), 7.79 (s, 1H), 7.70 (d,
J = 2.8 Hz, 1H), 7.58−7.54 (m, 3H), 7.49−7.41 (m, 3H), 7.39−7.34
(m, 2H), 7.01−6.98 (m, 1H), 6.81−6.79 (m, 2H), 5.88−5.85 (m, 1H),
3.73 (s, 3H), 3.31−3.30 (m, 2H), 3.11−3.10 (m, 2H); 13C NMR (100
MHz, acetone-d6): δ 162.1, 157.0, 155.9, 151.0, 143.5, 142.9, 135.9,
134.3, 132.4, 131.8, 130.8, 129.1, 127.9, 127.5, 126.5, 122.9, 121.3,
121.1, 120.9, 114.7, 114.2, 55.6, 45.2, 40.2; ESI-MS: m/z 612.1 [M −
H]−; ESI-HRMS C26H22N5O9S2 [M − H]− calcd, 612.0864; obs.,
612.0846.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-(3,4-
dinitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-A10). Overall
yield: 15.2%, purity: 97.1%. 1H NMR (400 MHz, acetone-d6): δ 8.69 (s,
1H), 8.24 (d, J = 9.2 Hz, 1H), 8.12 (s, 2H), 7.94 (dd, J = 8.0, 1.6 Hz,
1H), 7.71 (d, J = 2.8 Hz, 1H), 7.60−7.54 (m, 3H), 7.52 (s, 1H), 7.48−
7.46 (m, 2H), 7.40 (dd, J = 8.0, 0.8 Hz, 1H), 6.88−6.85 (m, 1H),6.23−
6.22 (m, 1H), 3.39−3.35 (m, 2H), 3.18−3.13 (m, 2H); 13C NMR (100
MHz, acetone-d6): δ 162.1, 155.8, 151.0, 143.6, 143.4, 142.9, 132.5,
132.4, 132.2, 131.8, 130.7, 129.1, 128.0, 127.5, 126.5, 125.8, 123.1,
122.9, 121.2, 118.4, 114.9, 114.2, 44.3, 40.3; ESI-MS: m/z718.1 [M −
H]−; ESI-HRMS C27H18N5O8F6S2 [M − H]− calcd, 718.0506; obs.,
718.0518.
N-(2-(3-(3,5-Dimethylphenyl)ureido)ethyl)-5-(2-(3,4-
dinitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-A7). Overall
yield: 15.3%, purity: 99.5%. 1H NMR (400 MHz, acetone-d6): δ 8.22
(d, J = 9.2 Hz, 1H), 7.93 (dd, J = 8.0, 1.6 Hz, 1H), 7.82 (s, 1H), 7.69 (d,
J = 2.4 Hz, 1H), 7.59−7.54 (m, 3H), 7.48−7.38 (m, 3H), 7.07 (s, 2H),
6.97−6.94 (m, 1H), 6.58 (s, 1H), 5.93−5.91 (m, 1H), 3.33−3.29 (m,
2H), 3.13−3.09 (m, 2H), 2.20 (s, 6H); 13C NMR (100 MHz, acetone-
d6): δ 162.1, 156.6, 151.0, 143.5, 143.3, 142.9, 141.2, 138.8, 132.4,
131.8, 130.8, 129.1, 127.9, 127.5, 126.5, 124.1, 122.9, 121.3, 116.9,
114.2, 45.1, 40.1, 21.5; ESI-MS: m/z 612.0 [M + H]+; ESI-HRMS
C27H25N5O8S2Na [M + Na]+ calcd, 634.1037; obs., 634.1061.
N-(2-(3-(4-Nitrophenyl)ureido)ethyl)-5-(2-(3,4-dinitrophenoxy)-
phenyl)thiophene-2-sulfonamide (TJ-A8). Overall yield: 2.6%, purity:
97.3%. 1H NMR (400 MHz, acetone-d6): δ 8.71 (s, 1H), 8.25 (d, J = 9.2
Hz, 1H), 8.13−8.10 (m, 2H), 7.94 (dd, J = 8.0, 1.6 Hz, 1H), 7.71−7.67
(m, 3H), 7.59−7.54 (m, 3H), 7.47−7.44 (m, 2H), 7.39 (t, J = 8.0 Hz,
1H), 6.88−6.85 (m, 1H), 6.20 (m, 1H), 3.38−3.34 (m, 2H), 3.18−3.13
(m, 2H); 13C NMR (100 MHz, acetone-d6): δ 162.1, 155.6, 151.0,
147.8, 143.6, 142.9, 142.3, 137.3, 132.4, 131.8, 130.7, 129.1, 127.9,
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-4-(2-
(3,4-dihydroxyphenoxy)phenyl)thiophene-2-sulfonamide (TJ-B1).
1
Overall yield: 15.8%, purity: 97.9%. H NMR (400 MHz, acetone-
d6): δ 9.37 (s, 1H), 8.18 (s, 2H), 7.96 (d, J = 1.2 Hz, 1H), 7.94 (s, 1H),
7.88 (d, J = 1.6 Hz, 1H), 7.69 (s, 1H), 7.62 (s, 1H), 7.58 (s, 1H), 7.55
(dd, J = 7.6, 1.6 Hz, 1H), 7.19 (dt, J = 7.8, 1.6 Hz, 1H), 7.03 (dt, J = 7.6,
0.8 Hz, 1H), 6.82−6.80 (m, 2H), 6.68 (d, J = 8.8 Hz, 1H), 6.40 (d, J =
2.8 Hz, 1H), 6.24 (dd, J = 8.8, 2.8 Hz, 1H), 3.72−3.67 (m, 2H), 3.20−
3.15 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.7, 155.7, 150.4,
146.9, 142.7, 142.3, 141.8, 139.0, 133.3, 132.4, 132.1, 131.7, 131.4,
130.7, 130.5, 130.1, 130.1, 128.4, 126.3, 125.7, 124.2, 123.6, 123.0,
119.6, 115.8, 117.7, 116.5, 110.6, 107.7, 44.6, 43.1; ESI-MS: m/z 677.8
[M + H]+, 699.8 [M + Na]+; MALDI-HRMS C27H22N3O5F6S3 [M +
H]+ calcd, 678.0620; obs., 678.0630.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-4-(2-(3,4-
dihydroxyphenoxy)phenyl)thiophene-2-sulfonamide (TJ-B2). Over-
all yield: 11.8%, purity: 98.4%. 1H NMR (400 MHz, acetone-d6): δ 8.59
(s, 1H), 8.00 (s, 2H), 7.94 (d, J = 1.2 Hz, 1H), 7.86 (d, J = 2.0 Hz, 1H),
7.54 (dd, J = 7.6, 1.6 Hz, 1H), 7.39 (s, 1H), 7.19 (dt, J = 7.6, 1.6 Hz,
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1H), 7.03 (dt, J = 7.6, 0.8 Hz, 1H), 6.81 (d, J = 8.4 Hz, 1H), 7.34 (s,
1H), 6.66 (d, J = 8.4 Hz, 1H), 6.40 (d, J = 2.8 Hz, 1H), 6.21 (dd, J = 8.4,
2.8 Hz, 1H), 6.13−6.10 (m, 1H), 3.29−3.25 (m, 2H), 3.03−3.00 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 156.0, 155.6, 150.6, 147.0,
143.4, 142.0, 141.8, 138.9, 133.2, 132.8, 132.5, 132.2, 131.9, 130.7,
130.1, 130.0, 126.5, 125.8, 124.3, 123.1, 119.8, 118.5, 116.5, 114.9,
110.4, 107.6, 44.4, 40.3; ESI-MS: m/z 661.8 [M + H]+, 683.7 [M +
Na]+; MALDI-HRMS C27H22N3O6F6S2 [M + H]+ calcd, 662.0849;
obs., 662.0848.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-4-(2-(3,4-
dihydroxyphenoxy)phenyl)thiophene-2-sulfonamide (TJ-B3). Over-
all yield: 10.1%, purity: 97.2%. 1H NMR (400 MHz, acetone-d6): δ 8.25
(s, 1H), 7.95 (s, 1H), 7.92 (d, J = 1.2 Hz, 1H), 7.84 (d, J = 1.2 Hz, 1H),
7.66 (s, 1H), 7.53 (dd, J = 7.6, 1.2 Hz, 1H), 7.38 (d,J = 1.6, 2H), 7.18
(dt, J = 7.8, 1.2 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.84 (t, J = 1.6 Hz,
1H), 6.81 (d, J = 8.4 Hz, 1H), 6.74−6.71 (m, 1H), 6.66 (d, J = 8.4 Hz,
1H), 6.40 (d, J = 2.8 Hz, 1H), 6.20 (dd, J = 8.4, 2.8 Hz, 1H), 6.01−5.89
(m, 1H), 3.26−3.21 (m, 2H), 3.02−2.98 (m, 2H); 13C NMR (100
MHz, acetone-d6): δ 156.0, 155.6, 150.7, 147.0, 143.8, 142.3, 142.0,
139.0, 135.5, 133.2, 130.8, 130.2, 130.0, 126.6, 124.4, 121.6, 120.0,
117.0, 116.6, 110.5, 107.6, 44.6, 40.3; ESI-MS: m/z 594.0 [M + H]+;
MALDI-HRMS C25H21N3O6Cl2S2Na [M + Na]+ calcd, 616.0141; obs.,
616.0146.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-5-(2-
(benzyloxy)phenyl)thiophene-2-sulfonamide (TJ-C1). Overall yield:
24.4%, purity: 98.4%. 1H NMR (400 MHz, acetone-d6): δ 9.54 (s, 1H),
8.33 (s, 2H), 7.81−7.77 (m, 2H), 7.71 (s, 1H), 7.62 (d, J = 4.0 Hz, 1H),
7.58 (d, J = 4.0 Hz, 1H),7.54 (d, J = 7.2 Hz, 2H), 7.41 (t, J = 7.6 Hz,
2H), 7.34 (t, J = 7.6 Hz, 2H), 7.24 (d, J = 8.4 Hz, 1H), 7.04 (td, J = 7.6,
1.2 Hz, 1H), 6.87 (m, 1H), 5.35 (s, 2H), 3.87−3.82 (m, 2H), 3.34−3.30
(m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.6, 155.5, 146.0,
142.7, 141.1, 137.5, 132.1, 131.9, 131.8, 130.8, 129.4, 129.0, 128.9,
128.7, 125.7, 125.5, 123.5, 123.0, 122.5, 122.2, 117.6, 114.3, 71.4, 44.7,
43.1; ESI-MS: m/z 659.9 [M + H]+, 681.8 [M + Na]+; MALDI-HRMS
C28H24N3O3F6S3 [M + H]+ calcd, 660.0878; obs., 660.0881.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-
(benzyloxy)phenyl)thiophene-2-sulfonamide (TJ-C2). Overall yield:
32.9%, Purity: 99.1%. 1H NMR (400 MHz, acetone-d6): δ 8.72 (s, 1H),
8.13 (s, 2H), 7.79 (dd, J = 8.0, 1.2 Hz, 1H), 7.62 (d, J = 4.0 Hz, 1H),
7.56−7.52 (m, 4H), 7.41 (t, J = 7.6 Hz, 2H), 7.37−7.33 (m, 2H), 7.25
(d, J = 8.4 Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.79−6.76 (m, 1H), 6.26−
6.23 (m, 1H), 5.36 (s, 2H), 3.43−3.38 (m, 2H), 3.20−3.15 (m, 2H);
13C NMR (100 MHz, acetone-d6): δ 155.9, 155.5, 145.9, 143.5, 141.3,
137.5, 132.8, 132.5, 132.2, 131.8, 131.7, 130.8, 129.4, 128.9, 128.9,
128.7, 125.8, 125.5, 123.1, 122.6, 122.2, 118.4, 114.8, 114.3, 71.3, 44.4,
40.4; ESI-MS: m/z 643.9 [M + H]+, 665.8 [M + Na]+; ESI-HRMS
C28H23N3O4F6S2Na [M + Na]+ calcd, 666.0926; obs., 666.0927.
N-(2-(3-(3-Chlorophenyl)thioureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C3). Overall yield: 39.5%,
purity: 98.7%. 1H NMR (400 MHz, acetone-d6): δ 9.09 (s, 1H), 7.82
(dd, J = 7.6, 1.6 Hz, 1H), 7.65 (s, 1H), 7.63 (d, J = 4.0 Hz, 1H), 7.57−
7.54 (m, 3H), 7.49 (m, 1H), 7.42 (t, J = 7.6 Hz, 2H), 7.37−7.31 (m,
4H), 7.27 (t, J = 7.6 Hz, 1H), 7.15 (d, J = 7.6 Hz, 1H), 7.06 (t, J = 7.6
Hz, 1H), 6.90−6.87 (m, 1H), 5.37 (s, 2H), 3.84−3.79 (m, 2H), 3.31−
3.27 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.6, 155.6, 145.9,
141.3, 137.5, 134.6, 131.8, 131.1, 130.8, 129.4, 129.0, 128.9, 128.7,
125.6, 125.4, 124.2, 122.8, 122.6, 122.2, 114.4, 71.4, 44.7, 43.5; ESI-
MS: m/z 557.8 [M + H]+, 579.8 [M + Na]+; ESI-HRMS
C26H24Cl1N3O3S3Na [M + Na]+calcd, 580.0561; obs., 580.0574.
N-(2-(3-(3,5-Dimethylphenyl)ureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C4). Overall yield: 32.4%,
purity: 99.1%. 1H NMR (400 MHz, acetone-d6): δ 7.87 (s, 1H), 7.77
(dd, J = 7.6, 1.6 Hz, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.53−7.51 (m, 3H),
7.39 (t, J = 7.6 Hz, 2H), 7.35−7.31 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H),
7.07 (s, 2H), 7.03 (t, J = 7.6 Hz, 1H), 6.90−6.87 (m, 1H), 6.56 (s, 1H),
5.98−5.95 (m, 1H), 5.31 (s, 2H), 3.38−3.33 (m, 2H), 3.15−3.11 (m,
2H), 2.18 (s, 6H); 13C NMR (100 MHz, acetone-d6): δ 156.7, 155.4,
145.8, 141.3, 141.1, 138.8, 137.5, 131.7, 130.7, 129.4, 129.0, 128.9,
128.7, 125.5, 124.2, 122.6, 122.2, 117.0, 114.3, 71.3, 45.1, 40.2, 21.5;
ESI-MS: m/z 536.0 [M + H]+, 558.0 [M + Na]+; ESI-HRMS
C28H29N3O4S2Na [M + Na]+ calcd, 558.1492; obs., 558.1491.
N-(2-(3-(3-Methoxyphenyl)ureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C5). Overall yield: 33.8%,
purity: 98.0%. 1H NMR (400 MHz, acetone-d6): δ 8.04 (s, 1H), 7.77
(dd, J = 8.0, 1.6 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.54−7.51 (m, 3H),
7.39 (t, J = 7.6 Hz, 2H), 7.34−7.31 (m, 2H), 7.25−7.21 (m, 2H), 7.09
(t, J = 7.6 Hz, 1H), 7.03 (t, J = 7.6 Hz, 1H), 6.92 (dd, J = 8.0, 1.2 Hz,
1H), 6.88−6.85 (m, 1H), 6.50 (dd, J = 8.0, 1.6 Hz, 1H), 6.00−5.97 (m,
1H), 5.32 (s, 2H), 3.71 (s, 3H), 3.39−3.34 (m, 2H), 3.16−3.12 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 161.1, 156.5, 155.4, 145.8,
142.5, 141.3, 137.5, 131.7, 130.7, 130.1, 129.4, 129.0, 128.9, 128.7,
125.5, 122.6, 122.2, 114.3, 111.3, 107.8, 104.9, 71.3, 55.3, 44.9, 40.2;
ESI-MS: m/z 537.9 [M + H]+, 560.0 [M + Na]+; ESI-HRMS
C27H27N3O5S2Na [M + Na]+ calcd, 560.1284; obs., 560.1279.
N-(2-(3-(4-Chlorophenyl)thioureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C6). Overall yield: 32.0%,
purity: 97.5%. 1H NMR (400 MHz, acetone-d6): δ 9.03 (s, 1H), 7.81
(dd, J = 7.6, 1.2 Hz, 1H), 7.61 (d, J = 4.0 Hz, 1H), 7.56−7.53 (m, 3H),
7.46−7.29 (m, 9H), 7.24 (d, J = 8.0 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H),
6.90−6.87 (m, 1H), 5.35 (s, 2H), 3.84−3.79 (m, 2H), 3.31−3.26 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 182.6, 155.4, 145.8, 141.2,
138.4, 137.4, 131.8, 130.8, 130.4, 129.6, 129.4, 129.3, 129.0, 128.9,
128.7, 126.3, 125.5, 122.6, 122.2, 114.3, 71.3, 44.6, 43.6; ESI-MS: m/z
557.9 [M + H]+, 579.8 [M + Na]+; ESI-HRMS C26H24ClN3O3S3Na [M
+ Na]+ calcd, 580.0561; obs., 580.0540.
N-(2-(3-(3-Nitrophenyl)ureido)ethyl)-5-(2-(benzyloxy)phenyl)-
thiophene-2-sulfonamide (TJ-C7). Overall yield: 25.4%, purity:
98.9%. 1H NMR (400 MHz, acetone-d6): δ 8.56−8.53 (m, 2H),
7.78−7.71 (m, 3H), 7.59 (d, J = 4.0 Hz, 1H), 7.55−7.52 (m, 3H), 7.46
(d, J = 8.4 Hz, 1H), 7.43−7.48 (m, 2H), 7.33 (td, J = 8.0, 1.6 Hz, 2H),
7.21 (d, J = 8.0 Hz, 1H), 7.02 (t, J = 7.6 Hz, 1H), 6.84−6.81 (m, 1H),
6.17−6.14 (m, 1H), 5.32 (s, 2H), 3.43−3.39 (m, 2H), 3.20−3.16 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 156.1, 155.4, 149.5, 145.8,
142.7, 141.2, 137.5, 131.7, 130.7, 130.4, 129.4, 128.9, 128.9, 128.7,
125.5, 124.5, 122.5, 122.1, 116.7, 114.3, 113.0, 71.3, 44.6, 40.3; ESI-
MS: m/z 553.0 [M + H]+, 574.9 [M + Na]+; ESI-HRMS
C26H24N4O6S2Na [M + Na]+ calcd, 575.1029; obs., 575.1028.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C8). Overall yield: 41.8%,
purity: 98.5%. 1H NMR (400 MHz, acetone-d6): δ 9.55 (s, 1H),
8.14−8.12 (m, 2H), 7.89−7.88 (m, 2H), 7.79 (dd, J = 8.0, 1.2 Hz, 1H),
7.75 (m, 1H), 7.61 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.54 (d,
J = 7.6 Hz, 2H), 7.41 (t, J = 7.6 Hz, 2H), 7.33 (td, J = 8.0, 1.6 Hz, 2H),
7.22 (d, J = 8.4 Hz, 1H), 7.03 (t, J = 7.2 Hz, 1H), 6.88−6.86 (m, 1H),
5.34 (s, 2H), 3.87−3.82 (m, 2H), 3.35−3.31 (m, 2H); 13C NMR (100
MHz, acetone-d6): δ 182.1, 155.4, 146.6, 146.0, 143.8, 141.0, 137.5,
131.9, 130.8, 129.4, 128.9, 128.9, 128.7, 125.5, 125.1, 122.5, 122.2,
122.0, 114.3, 71.3, 44.6, 43.2; ESI-MS: m/z 568.9 [M + H]+, 590.9 [M
+ Na]+; ESI-HRMS C26H24N4O5S3Na [M + Na]+ calcd, 591.0801;
obs., 591.0784.
N-(2-(3-(3-Nitrophenyl)thioureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C9). Overall yield: 46.1%,
purity: 96.5%. 1H NMR (400 MHz, acetone-d6): δ 9.41 (s, 1H), 8.62
(s, 1H), 7.94 (d, J = 7.6 Hz, 1H), 7.89 (d, J = 8.0 Hz, 1H), 7.79 (d, J =
7.6 Hz, 1H), 7.64−7.60 (m, 2H), 7.57−7.53 (m, 4H), 7.40 (t, J = 7.6
Hz, 2H), 7.35−7.31 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 7.03 (t, J = 7.6
Hz, 1H), 6.88 (t, J = 7.6 Hz, 1H), 5.34 (s, 2H), 3.85−3.81 (m, 2H),
3.33−3.29 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.6, 155.4,
149.0, 145.9, 141.6, 141.1, 137.4, 131.8, 130.8, 130.4, 129.7, 129.4,
128.9, 128.9, 128.7, 125.5, 122.5, 122.2, 119.5, 118.4, 114.3, 71.3, 44.6,
43.3; ESI-MS: m/z 568.9 [M + H]+, 591.0 [M + Na]+; ESI-HRMS
C26H24N4O5S3Na [M + Na]+ calcd, 591.0801; obs., 591.0811.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C10). Overall yield: 40.4%,
purity: 99.6%. 1H NMR (400 MHz, acetone-d6): δ 8.38 (s, 1H), 7.78
(dd, J = 8.0, 1.2 Hz, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.55−7.51 (m, 5H),
7.40 (t, J = 7.6 Hz, 2H), 7.33 (td, J = 8.0, 1.2 Hz, 2H), 7.23 (d, J = 8.0
Hz, 1H), 7.04 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 1.6 Hz, 1H), 6.78 (m, 1H),
6.13−6.10 (m, 1H), 5.33 (s, 2H), 3.40−3.36 (m, 2H), 3.18−3.14 (m,
10273
https://doi.org/10.1021/acs.jmedchem.1c00690
J. Med. Chem. 2021, 64, 10260−10285

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
2H); 13C NMR (100 MHz, acetone-d6): δ 155.8, 155.4, 145.9, 143.8,
141.2, 137.5, 135.4, 131.7, 130.8, 129.4, 128.9, 128.9, 128.7, 125.5,
122.5, 122.2, 121.5, 116.8, 114.3, 71.3, 44.5, 40.3; ESI-MS: m/z597.8
[M + Na]+; ESI-HRMS C26H23Cl2N3O4S2Na [M + Na]+ calcd,
598.0399; obs., 598.0385.
N-(2-(3-(4-Methoxyphenyl)ureido)ethyl)-5-(2-(benzyloxy)-
phenyl)thiophene-2-sulfonamide (TJ-C11). Overall yield: 46.1%,
purity: 96.6%. 1H NMR (400 MHz, acetone-d6): δ 7.85 (s, 1H), 7.78
(dd, J = 7.6, 1.2 Hz, 1H), 7.58 (d, J = 4.0 Hz, 1H), 7.53−7.51 (m, 3H),
7.39 (t, J = 7.2 Hz, 2H), 7.34−7.32 (m, 3H), 7.22 (d, J = 8.4 Hz, 1H),
7.04 (t, J = 7.6 Hz, 1H), 6.92 (m, 1H), 6.79−6.77 (m, 2H), 5.92−5.90
(m, 1H), 5.32 (s, 2H), 3.71 (s, 3H), 3.37−3.32 (m, 2H), 3.14−3.10 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 157.1, 155.9, 155.4, 145.8,
141.3, 137.5, 134.2, 131.7, 130.7, 129.4, 129.0, 128.9, 128.6, 125.5,
122.6, 122.2, 121.2, 114.6, 114.3, 71.3, 55.6, 45.2, 40.3; ESI-MS: m/z
538.0 [M + H]+, 560.0 [M + Na]+; ESI-HRMS C27H27N3O5S2Na [M +
Na]+ calcd, 560.1284; obs., 560.1266.
8.4, 2.0 Hz, 1H), 3.79−3.75 (m, 5H), 3.28−3.24 (m, 2H); 13C NMR
(100 MHz, acetone-d6): δ 182.3, 163.0, 161.3, 151.7, 144.0, 142.6,
140.1, 132.3, 131.5, 130.8, 130.4, 127.2, 127.1, 126.9, 126.4, 122.8,
118.3, 116.8, 112.0, 110.4, 55.6, 44.6, 43.7; ESI-MS: m/z 583.0 [M −
H]−; ESI-HRMS C26H23N4O6S3 [M − H]− calcd, 583.0785; obs.,
583.0795.
N-(2-(3-(3-Chlorophenyl)thioureido)ethyl)-5-(2-(4-
nitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-C17). Overall
yield: 18.1%, purity: 98.0%. 1H NMR (400 MHz, acetone-d6): δ 9.05
(s, 1H), 8.29−8.25 (m, 2H), 7.98 (dd, J = 8.0, 1.6 Hz, 1H), 7.63 (s, 1H),
7.61 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.54 (td, J = 8.0, 1.6
Hz, 1H), 7.46−7.42 (m, 2H), 7.35−7.30 (m, 2H), 7.28 (dd, J = 8.0, 1.2
Hz, 1H), 7.21−7.17 (m, 2H), 7.16 (td, J = 7.2, 2.0 Hz, 1H), 6.95−6.92
(m, 1H), 3.79−3.74 (m, 2H), 3.29−3.24 (m, 2H); 13C NMR (100
MHz, acetone-d6): δ 182.6, 163.0, 151.7, 144.0, 142.6, 141.2, 134.6,
132.3, 131.6, 131.1, 130.3, 127.2, 127.1, 126.9, 126.4, 125.5, 124.3,
122.8, 122.8, 118.3, 44.6, 43.4; ESI-MS: m/z 587.0 [M − H]−; ESI-
HRMS C25H20Cl1N4O5S3 [M − H]− calcd, 587.0290; obs., 587.0285.
N-(2-(3-(4-Methoxyphenyl)ureido)ethyl)-5-(2-(4-nitrophenoxy)-
phenyl)thiophene-2-sulfonamide (TJ-C18). Overall yield: 16.7%,
N-(2-(3-(4-Chlorophenyl)ureido)ethyl)-5-(2-(4-aminophenoxy)-
phenyl)thiophene-2-sulfonamide (TJ-C12). Overall yield: 13.6%,
1
purity: 95.7%. H NMR (400 MHz, methanol-d4): δ 7.69 (dd, J =
1
8.0, 1.6 Hz, 1H), 7.47 (d, J = 4.0 Hz, 1H), 7.46 (d, J = 4.0 Hz, 1H),
7.21−7.14 (m, 3H), 7.08−7.06 (m, 2H), 7.01 (t, J = 7.2 Hz, 1H), 6.73
(d, J = 8.0 Hz, 1H), 6.71−6.69 (m, 2H), 6.66−6.64 (m, 2H), 3.20−3.17
(m, 2H), 2.99−2.96 (m, 2H); 13C NMR (100 MHz, methanol-d4): δ
157.9, 156.1, 148.7, 146.4, 145.4, 141.2, 139.7, 132.5, 130.9, 129.6,
129.2, 128.0, 125.8, 124.2, 124.0, 121.6, 121.3, 118.6, 117.8, 44.4, 40.5;
ESI-MS: m/z 541.0 [M − H]−; ESI-HRMS calcd. for C25H22ClN4O4S2
[M − H]− requires, 541.0776; found, 541.0795.
purity: 96.6%. H NMR (400 MHz, acetone-d6): δ 8.27−8.25 (m,
2H), 7.95 (dd, J = 8.0, 1.6 Hz, 1H), 7.80 (s, 1H), 7.58 (d, J = 4.0 Hz,
1H), 7.54 (d, J = 4.0 Hz, 1H), 7.51 (m, 1H), 7.43 (td, J = 7.2, 1.2 Hz,
1H), 7.34−7.32 (m, 2H), 7.27 (dd, J = 8.0, 1.2 Hz, 1H), 7.18−7.16 (m,
2H), 6.98 (m, 1H), 6.81−6.79 (m, 2H), 5.87 (m, 1H), 3.74 (s, 3H),
3.31−3.28 (m, 2H), 3.10−3.08 (m, 2H); 13C NMR (100 MHz,
acetone-d6): δ 163.0, 155.9, 151.7, 144.0, 142.7, 134.3, 132.3, 131.5,
130.4, 127.2, 127.1, 126.9, 122.8, 120.9, 118.3, 114.7, 55.6, 45.2, 40.1;
ESI-MS: m/z 567.0 [M − H]−; ESI-HRMS C26H23N4O7S2 [M − H]−
calcd, 567.1014; obs., 567.1031.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-(4-
aminophenoxy)phenyl)thiophene-2-sulfonamide (TJ-C13). Overall
yield: 13.6%, purity: 98.7%. 1H NMR (400 MHz, methanol-d4): δ 7.94
(s, 2H), 7.72 (dd, J = 8.0, 1.2 Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.53 (d,
J = 4.0 Hz, 1H), 7.42 (s, 1H), 7.20 (td, J = 7.6, 1.2 Hz, 1H), 7.04 (t, J =
7.6 Hz, 1H), 6.79−6.73 (m, 5H), 3.32−3.30 (m, 2H), 3.12−3.09 (m,
2H); 13C NMR (100 MHz, methanol-d4): δ 157.3, 156.0, 148.8, 146.4,
145.4, 143.2, 141.2, 133.5, 133.1, 132.8, 132.6, 132.4, 130.9, 129.0,
126.1, 125.7, 124.1, 124.0, 123.4, 121.6, 118.8, 118.7, 117.8, 115.5, 44.1,
40.5; ESI-MS: m/z 643.0 [M − H]−; ESI-HRMS calcd. for
C27H21N4O4F6S2 [M − H]− requires, 643.0914; found, 643.0911.
N-(2-(3-(3,5-Dimethylphenyl)ureido)ethyl)-5-(2-(4-
aminophenoxy)phenyl)thiophene-2-sulfonamide (TJ-C14). Overall
yield: 19.0%, purity: 98.1%. 1H NMR (400 MHz, methanol-d4): δ 7.69
(dd, J = 8.0, 1.2 Hz, 1H), 7.47 (d, J = 4.0 Hz, 1H), 7.46 (d, J = 4.0 Hz,
1H), 7.16 (t, J = 7.6 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.83 (s, 2H), 6.74
(d, J = 8.0 Hz, 1H), 6.71−6.69 (m, 2H), 6.67−6.64 (m, 2H), 6.52 (s,
1H), 3.20−3.17 (m, 2H), 2.99−2.96 (m, 2H), 2.12 (s, 6H); 13C NMR
(100 MHz, methanol-d4): δ 158.3, 156.0, 149.2, 146.3, 144.9, 141.3,
140.4, 139.4, 132.5, 130.9, 129.2, 125.9, 125.2, 124.3, 124.1, 121.5,
118.8, 118.2, 118.2, 44.5, 40.5, 21.5; ESI-MS: m/z535.1 [M − H]−;
ESI-HRMS calcd for C27H27N4O4S2 [M − H]− requires, 535.1479;
found, 535.1480.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-5-(2-(4-
nitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-C15). Overall
yield: 13.6%, purity: 98.4%. 1H NMR (400 MHz, acetone-d6): δ 9.48
(s, 1H), 8.31 (s, 2H), 8.29−8.25 (m, 2H), 7.97 (dd, J = 8.0, 1.6 Hz, 1H),
7.74 (m, 1H), 7.71 (s, 1H), 7.61 (d, J = 4.0 Hz, 1H), 7.58 (d, J = 4.0 Hz,
1H), 7.53 (td, J = 7.6, 1.2 Hz, 1H), 7.43 (td, J = 7.6, 1.2 Hz, 1H), 7.27
(d, J = 8.0 Hz, 1H), 7.21−7.18 (m, 2H), 7.17 (m, 1H), 3.80−3.76 (m,
2H), 3.30−3.26 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.7,
163.0, 151.7, 144.2, 144.1, 142.7, 142.5, 132.5, 132.4, 132.2, 131.8,
131.6, 130.3, 127.2, 127.1, 126.9, 126.4, 125.7, 123.6, 123.0, 122.8,
118.3, 117.7, 44.6, 43.0; ESI-MS: m/z 689.1 [M − H]−; DART-HRMS
C27H21N4O5F6S3 [M + H]+ calcd, 691.0573; obs., 691.0553.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C19).
1
Overall yield: 32.7%, Purity: 97.6%. H NMR (400 MHz, acetone-
d6): δ 9.55 (s, 1H), 8.45 (s, 1H), 8.15−8.12 (m, 2H), 7.88−7.86 (m,
2H), 7.79 (dd, J = 8.0, 1.6 Hz, 1H), 7.75−7.74 (m, 1H), 7.63 (d, J = 4.0
Hz, 1H),7.58 (d, J = 4.0 Hz, 1H), 7.31(dd, J = 8.0, 1.6 Hz, 1H), 7.25−
7.18 (m, 2H), 7.05−6.99 (m, 3H), 6.88 (m, 1H), 6.82 (td, J = 7.6, 1.6
Hz, 1H), 5.30 (s, 2H), 3.87−3.83 (m, 2H), 3.37−3.33 (m, 2H); 13C
NMR (100 MHz, acetone-d6): δ 182.0, 158.4, 155.4, 146.6, 146.0,
143.8, 141.0, 139.1, 131.9, 130.8, 130.5, 128.9, 125.5, 125.1, 122.5,
122.1, 119.5, 115.8, 115.3, 114.3, 71.0, 44.6, 43.2; ESI-MS: m/z 583.1
[M − H]−; ESI-HRMS C26H23N4O6S3 [M − H]− calcd, 583.0774; obs.,
584.0775.
N-(2-(3-(3-Nitrophenyl)thioureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C20).
1
Overall yield: 38.8%, purity: 98.7%. H NMR (400 MHz, acetone-
d6): δ 9.47 (s, 1H), 8.63 (s, 1H), 8.50 (s, 1H), 7.95−7.87 (m, 2H), 7.80
(d, J = 7.6 Hz, 1H), 7.71−7.52 (m, 4H), 7.32 (t, J = 7.6 Hz, 1H), 7.25−
7.18 (m, 2H), 7.05−6.99 (m, 3H), 6.91 (m, 1H), 6.82 (d, J = 7.2 Hz,
1H), 5.30 (s, 2H), 3.86−3.81 (m, 2H), 3.35−3.31 (m, 2H); 13C NMR
(100 MHz, acetone-d6): δ 158.5, 155.4, 149.0, 146.0, 141.7, 141.1,
139.1, 131.9, 130.8, 130.5, 130.4, 128.9, 125.5, 122.5, 122.1, 119.5,
118.3, 115.8, 115.3, 114.3, 71.1, 44.6, 43.3; ESI-MS: m/z 583.2 [M −
H]−; ESI-HRMS C26H23N4O6S3 [M − H]− calcd, 583.0774; obs.,
583.0775.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C21).
1
Overall yield: 29.0%, purity: 96.9%. H NMR (400 MHz, acetone-
d6): δ 8.74 (s, 1H), 8.47 (s, 1H), 8.12 (s, 2H), 7.77 (d, J = 7.2 Hz, 1H),
7.60 (d, J = 4.0 Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.50 (s, 1H), 7.32 (t, J
= 7.6 Hz, 1H), 7.23−7.18 (m, 2H), 7.04−6.98 (m, 3H), 6.83−6.76 (m,
2H), 6.27 (m, 1H), 5.28 (s, 2H), 3.44−3.40 (m, 2H), 3.23−3.18 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 158.5, 156.0, 155.4, 145.9,
143.4, 141.3, 139.1, 132.8, 132.5, 132.1, 131.8, 131.8, 130.7, 130.5,
128.9, 125.5, 122.5, 122.1, 119.5, 118.4, 115.9, 115.4, 114.8, 114.3, 71.1,
44.4, 40.4; ESI-MS: m/z 658.0 [M − H]−; DART-HRMS
C28H24N3O5F6S2 [M + H]+ calcd, 660.1056; obs., 660.1045.
N-(2-(3-(3-Methoxyphenyl)thioureido)ethyl)-5-(2-(4-
nitrophenoxy)phenyl)thiophene-2-sulfonamide (TJ-C16). Overall
yield: 17.2%, purity: 97.6%. 1H NMR (400 MHz, acetone-d6): δ 8.90
(s, 1H), 8.28−8.26 (m, 2H), 7.98 (d, J = 7.2 Hz, 1H), 7.60 (d, J = 4.0
Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.54−7.52 (m, 1H), 7.44 (t, J = 7.6
Hz, 1H), 7.34 (m, 1H), 7.28 (d, J = 8.0 Hz, 1H), 7.25−7.18 (m, 3H),
7.02 (s, 1H), 6.98−6.95 (m, 1H), 6.88 (d, J = 8.0 Hz, 1H), 6.74 (dd, J =
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C22).
1
Overall yield: 21.5%, purity: 98.7%. H NMR (400 MHz, DMSO-
10274
https://doi.org/10.1021/acs.jmedchem.1c00690
J. Med. Chem. 2021, 64, 10260−10285

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
d6): δ 9.46 (s, 1H), 9.02 (s, 1H), 7.91−7.88 (m, 1H), 7.83 (dd, J = 7.6,
1.6 Hz, 1H), 7.67 (d, J = 4.0 Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.44 (d, J
= 2.0 Hz, 2H), 7.33 (td, J = 7.6, 1.6 Hz, 1H), 7.21−7.15 (m, 2H), 7.05−
7.01 (m, 2H), 6.90 (t, J = 7.6 Hz, 1H), 6.85 (m, 1H), 6.70 (dd, J = 8.0,
1.6 Hz, 1H), 6.42−6.39 (m, 1H), 5.28 (s, 2H), 3.21−3.16 (m, 2H),
2.96−2.92 (m, 2H); 13C NMR (100 MHz, DMSO-d6): δ 157.5, 154.7,
153.9, 144.0, 142.9, 139.9, 137.9, 133.9, 130.8, 129.9, 129.5, 127.9,
124.8, 121.2, 121.0, 120.1, 118.0, 115.6, 114.9, 114.3, 113.5, 69.8, 42.8;
ESI-MS: m/z 591.8 [M + H]+; DART-HRMS C26H24Cl2N3O5S2 [M +
H]+ calcd, 592.0529; obs., 592.0517.
2.17 (m, 2H), 2.02−1.98 (m, 2H), 1.69−1.63 (m, 2H), 1.52−1.46 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 155.9, 154.6, 146.1, 143.5,
141.1, 132.8, 132.5, 132.1, 131.8, 131.7, 130.7, 128.8, 125.8, 125.1,
123.1, 122.9, 121.7, 118.4, 114.8, 77.0, 68.9, 44.4, 40.4, 33.0, 29.6; ESI-
MS: m/z 650.1 [M − H]−; ESI-HRMS C27H28N3O5F6S2 [M + H]+
calcd, 652.1369; obs., 652.1373.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-5-(2-(((1r,4r)-4-
hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C28).
Overall yield: 25.8%, purity: 95.2%. 1H NMR (400 MHz, acetone-d6): δ
8.39 (s, 1H), 7.79 (dd, J = 8.0, 1.6 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H),
7.56 (d, J = 4.0 Hz, 1H), 7.51 (d, J = 2.0 Hz, 2H), 7.34 (t, J = 7.2 Hz,
1H), 7.20 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.96 (t, J = 2.0
Hz, 1H), 6.82 (m, 1H), 6.12 (m, 1H), 4.60 (m, 1H), 3.75−3.72 (m,
2H), 3.41−3.37 (m, 2H), 3.22−3.17 (m, 2H), 2.21−2.15 (m, 2H),
2.02−1.98 (m, 2H), 1.68−1.65 (m, 2H), 1.50−1.46 (m, 2H); 13C
NMR (100 MHz, acetone-d6): δ 155.9, 154.6, 146.1, 143.8, 141.0,
135.4, 131.6, 130.7, 128.8, 125.0, 122.9, 121.7, 121.5, 116.9, 114.8, 77.0,
68.9, 44.6, 40.3, 33.0, 29.6; ESI-MS: m/z 582.0 [M − H]−; ESI-HRMS
C25H28Cl2N3O5S2 [M + H]+ calcd, 584.0842; obs., 584.0843.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-5-(2-(((1r,4r)-4-
hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C29).
Overall yield: 21.9%, purity: 98.1%. 1H NMR (400 MHz, acetone-d6): δ
9.56 (s, 1H), 8.15−8.13 (m, 2H), 7.90−7.87 (m, 2H), 7.81−7.79 (m,
2H), 7.61 (d, J = 4.0 Hz, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.34 (td, J = 7.6,
1.6 Hz, 1H), 7.21 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.92−
6.89 (m, 1H), 4.64−4.58 (m, 1H), 3.87−3.83 (m, 2H), 3.75−3.71 (m,
2H), 3.39−3.35 (m, 2H), 2.22−2.18 (m, 2H), 2.02−1.98 (m, 2H),
1.70−1.66 (m, 2H), 1.49−1.46 (m, 2H); 13C NMR (100 MHz,
acetone-d6): δ 182.1, 154.6, 146.7, 146.2, 143.9, 140.9, 131.8, 130.7,
128.8, 125.2, 125.1, 122.9, 122.1, 121.7, 114.8, 77.0, 68.9, 44.6, 43.2,
33.1, 29.7; ESI-MS: m/z 575.1 [M − H]−; ESI-HRMS C25H29N4O6S3
[M + H]+ calcd, 577.1244; obs., 577.1244.
N-(2-(3-(4-Methoxyphenyl)ureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C23).
1
Overall yield: 23.8%, purity: 96.3%. H NMR (400 MHz, acetone-
d6): δ 8.66 (s, 1H), 7.87 (s, 1H), 7.79 (dd, J = 8.0, 1.6 Hz, 1H), 7.60 (d, J
= 4.0 Hz, 1H), 7.53 (d, J = 4.0 Hz, 1H), 7.35−7.27 (m, 3H), 7.23−7.19
(m, 2H), 7.05−7.01 (m, 2H), 6.97 (d, J = 7.6 Hz, 1H), 6.89−6.86 (m,
1H), 6.80 (dd, J = 8.0, 1.6 Hz, 1H), 6.78−6.74 (m, 2H), 5.99−5.96 (m,
1H), 5.26 (s, 2H), 3.71 (s, 3H), 3.39−3.34 (m, 2H), 3.18−3.13 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 158.6, 157.3, 156.1, 155.4,
145.8, 141.3, 139.0, 133.9, 131.6, 130.7, 130.4, 128.9, 125.4, 122.6,
122.1, 121.6, 119.5, 116.0, 115.5, 114.7, 114.2, 71.1, 55.6, 45.2, 40.3;
ESI-MS: m/z 575.9 [M + Na]+; ESI-HRMS C27H28N3O6S2 [M + H]+
calcd, 554.1414; obs., 554.1401.
N-(2-(3-(3,5-Dimethylphenyl)ureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C24).
1
Overall yield: 25.7%, purity: 95.3%. H NMR (400 MHz, acetone-
d6): δ 8.60 (s, 1H), 7.89 (s, 1H), 7.76 (dd, J = 7.6, 1.6 Hz, 1H), 7.57 (d, J
= 4.0 Hz, 1H), 7.53 (d, J = 4.0 Hz, 1H), 7.32 (td, J = 7.6, 1.6 Hz, 1H),
7.22−7.17 (m, 2H), 7.04−7.00 (m, 4H), 6.96 (d, J = 7.6 Hz, 1H),
6.85−6.80 (m, 2H), 6.56 (s, 1H), 6.03−6.02 (m, 1H), 5.24 (s, 2H),
3.39−3.34 (m, 2H), 3.18−3.14 (m, 2H), 2.16 (s, 6H); 13C NMR (100
MHz, acetone-d6): δ 158.6, 156.9, 155.4, 145.8, 141.3, 140.9, 139.0,
138.8, 131.6, 130.7, 130.4, 128.9, 125.4, 124.3, 122.6, 122.1, 119.5,
117.2, 115.9, 115.5, 114.2, 71.1, 45.1, 40.2, 21.5; ESI-MS: m/z 551.6 [M
+ H]+; ESI-HRMS C28H28N3O5S2 [M − H]− calcd, 550.1465; obs.,
550.1465.
N-(2-(3-(4-Nitrophenyl)ureido)ethyl)-5-(2-(((1r,4r)-4-
hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C30).
Overall yield: 15.0%, purity: 99.5%. 1H NMR (400 MHz, acetone-d6): δ
8.73 (s, 1H), 8.11−8.08 (m, 2H), 7.79 (dd, J = 7.6, 1.6 Hz, 1H), 7.68−
7.66 (m, 2H), 7.59 (d, J = 4.0 Hz, 1H), 7.56 (d, J = 4.0 Hz, 1H), 7.33
(td, J = 7.6, 1.6 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.00 (td, J = 7.6, 0.8
Hz, 1H), 6.83−6.80 (m, 1H), 6.25−6.22 (m, 1H), 4.62−4.56 (m, 1H),
3.75−3.69 (m, 2H), 3.44−3.39 (m, 2H), 3.24−3.20 (m, 2H), 2.21−
2.17 (m, 2H), 2.02−1.97 (m, 2H), 1.71−1.51 (m, 2H), 1.51−1.42 (m,
2H); 13C NMR (100 MHz, acetone-d6): δ 155.6, 154.6, 147.8, 146.1,
142.3, 141.1, 131.6, 130.7, 128.8, 125.7, 125.0, 122.9, 121.7, 117.9,
114.8, 77.0, 68.9, 44.5, 40.3, 33.0, 29.7; ESI-MS: m/z 559.1 [M − H]−;
ESI-HRMS C25H29N4O7S2 [M + H]+ calcd, 561.1472; obs., 561.1473.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-5-(2-
(((1s,4s)-4-hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfona-
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-5-(2-
((3-hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C25).
1
Overall yield: 37.4%, purity: 96.3%. H NMR (400 MHz, acetone-
d6): δ 9.64 (s, 1H), 8.52 (s, 1H), 8.33 (s, 2H), 7.84−7.79 (m, 2H), 7.71
(s, 1H), 7.63 (d, J = 4.0 Hz, 1H), 7.59 (d, J = 4.0 Hz, 1H), 7.32 (td, J =
7.6, 1.6 Hz, 1H), 7.25−7.19 (m, 2H), 7.05−6.99 (m, 3H), 6.93−6.90
(m, 1H), 6.82 (dd, J = 8.0, 1.6 Hz, 1H), 5.30 (s, 2H), 3.87−3.82 (m,
2H), 3.36−3.31 (m, 2H); 13C NMR (100 MHz, acetone-d6): δ 182.6,
158.5, 155.4, 146.1, 142.7, 141.0, 139.1, 132.1, 131.9, 131.8, 130.8,
130.5, 128.9, 125.7, 125.5, 123.5, 123.0, 122.5, 122.1, 119.5, 117.6,
115.8, 115.3, 114.3, 71.1, 44.7, 43.1; ESI-MS: m/z 673.9 [M − H]−;
DART-HRMS C28H24N3O4F6S3 [M + H]+ calcd, 676.0828; obs.,
676.0812.
1
mide (TJ-C31). Overall yield: 21.4%, purity: 96.1%. H NMR (400
MHz, acetone-d6): δ 9.54 (s, 1H), 8.32 (s, 2H), 7.82−7.79 (m, 2H),
7.71 (s, 1H), 7.62 (d, J = 4.0 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H), 7.35 (td,
J = 7.6, 1.6 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.01 (td, J = 7.6, 0.8 Hz,
1H), 6.95−6.92 (m, 1H), 4.71−4.70 (m, 1H), 3.94−3.93 (m, 1H),
3.86−3.81 (m, 3H), 3.39−3.34 (m, 2H), 2.14−2.07 (m, 2H), 1.85−
1.73 (m, 6H); 13C NMR (100 MHz, acetone-d6): δ 182.7, 154.6, 146.3,
142.8, 141.0, 132.4, 132.1, 131.8, 131.8, 131.5, 130.8, 129.2, 125.7,
125.3, 123.5, 123.0, 121.7, 117.6, 114.8, 75.4, 67.6, 44.6, 43.2, 31.4,
28.0; ESI-MS: m/z 668.0 [M + H]+; ESI-HRMS C27H28N3O4F6S3 [M +
H]+ calcd, 668.1141; obs., 668.1141.
N-(2-(3-(3-Chlorophenyl)thioureido)ethyl)-5-(2-((3-
hydroxybenzyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C26).
1
Overall yield: 38.8%, purity: 95.1%. H NMR (400 MHz, acetone-
d6): δ 9.14 (s, 1H), 8.50 (s, 1H), 7.81 (dd, J = 7.6, 1.2 Hz, 1H), 7.66 (d, J
= 2.0 Hz, 1H), 7.63 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.54−
7.51 (m, 1H), 7.36−7.19 (m, 5H), 7.14 (d, J = 8.0 Hz, 1H), 7.06−6.99
(m, 3H), 6.94−6.91 (m, 1H), 6.82 (dd, J = 7.6, 2.0 Hz, 1H), 5.30 (s,
2H), 3.85−3.80 (m, 2H), 3.33−3.29 (m, 2H); 13C NMR (100 MHz,
acetone-d6): δ 182.5, 158.4, 155.4, 145.9, 141.1, 139.0, 134.5, 131.8,
131.0, 130.7, 130.5, 128.9, 125.5, 125.4, 124.2, 122.7, 122.5, 122.1,
119.5, 115.8, 115.3, 114.3, 71.0, 44.7, 43.5; ESI-MS: m/z 572.0 [M −
H]−; ESI-HRMS C26H25Cl1N3O4S3 [M − H]− calcd, 574.0690; obs.,
574.0686.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-
(((1s,4s)-4-hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfona-
1
mide (TJ-C32). Overall yield: 18.0%, purity: 95.7%. H NMR (400
MHz, acetone-d6): δ 8.73 (s, 1H), 8.12 (s, 2H), 7.78 (dd, J = 8.0, 1.6 Hz,
1H), 7.59 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.51 (s, 1H),
7.34 (td, J = 7.6, 1.6 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.00 (td, J = 7.6,
0.8 Hz, 1H), 6.85−6.82 (m, 1H), 6.29 (m, 1H), 4.71−4.68 (m, 1H),
3.96−3.95 (m, 1H), 3.86−3.85 (m, 1H), 3.44−3.39 (m, 2H), 3.26−
3.21 (m, 2H), 2.13−2.06 (m, 2H), 1.84−1.70 (m, 6H); 13C NMR (100
MHz, acetone-d6): δ 155.9, 154.6, 146.2, 143.5, 141.2, 132.8, 132.5,
132.2, 131.9, 131.8, 130.8, 129.2, 125.9, 125.3, 123.1, 123.0, 121.6,
118.4, 114.8, 75.4, 67.6, 44.5, 40.3, 31.4, 28.0; ESI-MS: m/z 650.1 [M −
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-5-(2-
(((1r,4r)-4-hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfonamide
1
(TJ-C27). Overall yield: 27.0%, purity: 98.8%. H NMR (400 MHz,
acetone-d6): δ 8.79 (s, 1H), 8.13 (s, 2H), 7.79 (dd, J = 8.0, 1.6 Hz, 1H),
7.60 (d, J = 4.0 Hz, 1H), 7.57 (d, J = 4.0 Hz, 1H), 7.51 (s, 1H), 7.33 (td,
J = 7.6, 1.6 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H), 7.00 (td, J = 7.6, 0.8 Hz,
1H), 6.86−6.83 (m, 1H), 6.30−6.28 (m, 1H), 4.61−4.59 (m, 1H),
3.76−3.66 (m, 2H), 3.45−3.40 (m, 2H), 3.24−3.20 (m, 2H), 2.21−
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Article
H]−; ESI-HRMS C27H28N3O5F6S2 [M + H]+ calcd, 652.1369; obs.,
652.1371.
5.40 (s, 1H), 3.31 (d, J = 5.1 Hz, 2H), 3.06 (d, J = 5.3 Hz, 2H), 2.19 (s,
3H); 13C NMR (100 MHz, CDCl3): δ 171.29, 155.45, 154.49, 149.15,
144.68, 142.13, 141.18, 141.10, 139.93, 133.84, 132.67, 132.22, 131.89,
131.56, 131.11, 129.98, 124.41, 123.99, 121.60, 118.67, 115.58, 110.57,
106.47, 43.41, 39.71, 21.22; ESI-MS: m/z 698.00 [M + Na]+; ESI-
HRMS calcd for C28H24O6N3F6S2 [M + H]+, 676.1004.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-5-(2-(((1s,4s)-4-
hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C33).
Overall yield: 21.7%, purity: 95.7%. 1H NMR (400 MHz, acetone-d6): δ
9.55 (s, 1H), 8.16−8.13 (m, 2H), 7.90−7.88 (m, 2H), 7.81−7.79 (m,
2H), 7.61 (d, J = 4.0 Hz, 1H), 7.60 (d, J = 4.0 Hz, 1H), 7.34 (td, J = 7.6,
1.6 Hz, 1H), 7.19 (d, J = 8.0 Hz, 1H), 7.01 (t, J = 7.6 Hz, 1H), 6.94−
6.91 (m, 1H), 4.71−4.67 (m, 1H), 3.92−3.91 (m, 1H), 3.87−3.82 (m,
3H), 3.40−3.35 (m, 2H), 2.13−2.06 (m, 2H), 1.86−1.70 (m, 6H); 13C
NMR (100 MHz, acetone-d6): δ 182.1, 154.5, 146.7, 146.2, 143.9,
140.9, 131.8, 130.8, 129.1, 125.3, 125.1, 122.9, 122.1, 121.6, 114.7, 75.4,
67.5, 44.5, 43.3, 31.4, 27.9; ESI-MS: m/z 575.0 [M − H]−; ESI-HRMS
C25H29N4O6S3 [M + H]+ calcd, 577.1244; obs., 577.1247.
N-(2-(3-(4-Nitrophenyl)ureido)ethyl)-5-(2-(((1s,4s)-4-
hydroxycyclohexyl)oxy)phenyl)thiophene-2-sulfonamide (TJ-C34).
Overall yield: 19.4%, purity: 99.3%. 1H NMR (400 MHz, acetone-d6): δ
8.75 (s, 1H), 8.11−8.09 (m, 2H), 7.77 (dd, J = 8.0, 1.6 Hz, 1H), 7.68−
7.65 (m, 2H), 7.57 (m, 2H), 7.33 (td, J = 7.6, 1.6 Hz, 1H), 7.18 (d, J =
8.0 Hz, 1H), 7.00 (t, J = 7.6 Hz, 1H), 6.86−6.83 (m, 1H), 6.30−6.27
(m, 1H), 4.69−4.66 (m, 1H), 3.96−3.95 (m, 1H), 3.86−3.85 (m, 1H),
3.43−3.39 (m, 2H), 3.26−3.22 (m, 2H), 2.10−2.07 (m, 2H), 1.84−
1.73 (m, 6H); 13C NMR (100 MHz, acetone-d6): δ 155.7, 154.5, 147.7,
146.1, 142.3, 141.1, 131.7, 130.7, 129.2, 125.6, 125.2, 123.0, 121.6,
118.0, 114.7, 75.4, 67.5, 44.6, 40.2, 31.4, 28.0; ESI-MS: m/z 559.0 [M −
H]−; ESI-HRMS C25H29N4O7S2 [M + H]+ calcd, 561.1472; obs.,
561.1477.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-3-(2-(3,4-
dimethoxyphenoxy)-4-methylphenyl)thiophene-2-sulfonamide
1
(FWJ-D5). Overall yield: 18.9%, purity: 98.7%. H NMR (400 MHz,
acetone-d6): δ 8.68 (s, 1H), 8.14 (s, 2H), 7.75 (d, J = 5.1 Hz, 1H), 7.54
(s, 1H), 7.38 (d, J = 7.7 Hz, 1H), 7.19 (d, J = 5.1 Hz, 1H), 6.92 (d, J =
7.7 Hz, 1H), 6.86 (d, J = 8.7 Hz, 1H), 6.76−6.65 (m, 2H), 6.55 (dd, J =
8.7, 2.7 Hz, 1H), 6.46 (t, J = 5.8 Hz, 1H), 6.19 (s, 1H), 3.75 (s, 6H),
3.34 (q, J = 5.9 Hz, 2H), 3.09 (q, J = 6.0 Hz, 2H); 13C NMR (100 MHz,
acetone-d6): δ 156.17, 155.65, 151.18, 146.62, 143.29, 141.52, 140.73,
137.91, 133.01, 132.33, 132.00, 129.51, 125.66, 123.76, 122.86, 118.47,
118.23, 114.70, 113.14, 111.30, 105.46, 56.27, 55.93, 43.96, 40.30,
21.15; ESI-MS: m/z 726.10 [M + Na]+; ESI-HRMS calcd for
C30H28O6N3F6S2 [M + H]+, 704.1310.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-3-(2-(3,4-
dihydroxyphenoxy)-5-isopropylphenyl)thiophene-2-sulfonamide
1
(FWJ-D6). Overall yield: 18.0%, purity: 99.0%. H NMR (400 MHz,
acetone-d6): δ 8.72 (s, 1H), 8.09 (d, J = 23.2 Hz, 3H), 7.74 (d, J = 5.1
Hz, 2H), 7.54 (s, 1H), 7.41 (d, J = 1.6 Hz, 1H), 7.23−7.14 (m, 2H),
6.77 (dd, J = 8.5, 4.1 Hz, 2H), 6.56 (d, J = 2.5 Hz, 1H), 6.39 (dd, J = 8.5,
2.6 Hz, 1H), 6.33 (t, J = 5.7 Hz, 1H), 6.19 (s, 1H), 3.31 (dd, J = 11.3, 5.6
Hz, 2H), 3.02 (dd, J = 11.9, 6.0 Hz, 2H), 2.89 (dd, J = 13.8, 6.8 Hz, 1H),
1.24 (d, J = 6.9 Hz, 6H); 13C NMR (100 MHz, acetone-d6): δ 155.4,
154.1, 150.3, 146.3, 142.9, 141.8, 141.6, 137.7, 132.7, 132.1, 131.8,
130.5, 129.3, 128.2, 125.5, 125.0, 122.8, 118.0, 117.6, 115.9, 114.5,
110.8, 107.7, 43.8, 39.9, 33.7, 23.9; ESI-MS: m/z 704.15 [M + H]+; ESI-
HRMS calcd for C30H28O6N3F6S2 [M + H]+, 704.1320.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-3-(2-
(3,4-dihydroxyphenoxy)-4-methylphenyl)thiophene-2-sulfonamide
1
(FWJ-D1). Overall yield: 19.1%, purity: 96.1%. H NMR (400 MHz,
CDCl3): δ 7.82 (s, 2H), 7.68 (s, 1H), 7.55 (d, J = 5.1 Hz, 1H), 7.23 (d, J
= 7.7 Hz, 1H), 7.10 (d, J = 5.0 Hz, 1H), 6.93 (d, J = 7.5 Hz, 1H), 6.78−
6.72 (m, 2H), 6.51 (d, J = 2.3 Hz, 1H), 6.33−6.27 (m, 1H), 3.70 (s,
2H), 3.15 (s, 2H), 2.29 (s, 3H); 13C NMR (100 MHz, CDCl3): δ
179.52, 152.03, 149.94, 148.42, 140.44, 138.15, 137.74, 136.85, 133.94,
131.66, 129.42, 128.45, 126.96, 125.78, 124.44, 122.60, 122.02, 119.16,
118.43, 116.86, 112.81, 106.63, 45.47, 42.32, 21.64; ESI-MS: m/z
690.00 [M − H]−; ESI-HRMS calcd for C28H22O5N3F6S3 [M − H]−,
690.0641.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-3-(2-(3,4-
dimethoxyphenoxy)-5-isopropylphenyl)thiophene-2-sulfonamide
1
(FWJ-D7). Overall yield: 18.0%, purity: 97.1%. H NMR (400 MHz,
acetone-d6): δ 8.13 (s, 2H), 7.75 (d, J = 5.1 Hz, 1H), 7.53 (s, 1H), 7.42
(d, J = 2.1 Hz, 1H), 7.20 (dd, J = 12.2, 3.6 Hz, 2H), 6.86 (d, J = 8.7 Hz,
1H), 6.77 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 2.6 Hz, 1H), 6.54 (dd, J = 8.7,
2.7 Hz, 1H), 3.74 (d, J = 2.2 Hz, 6H), 3.30 (t, J = 5.7 Hz, 2H), 3.04 (t, J
= 6.0 Hz, 2H), 2.92 (dd, J = 13.8, 6.9 Hz, 1H), 1.24 (d, J = 6.9 Hz, 6H);
13C NMR (101 MHz, acetone-d6): δ 155.6, 154.2, 151.2, 146.53, 143.2,
141.6, 138.1, 132.8, 132.2, 131.9, 130.7, 128.4, 125.6, 125.2, 122.9,
118.1, 117.7, 114.5, 113.1, 111.1, 105.3, 56.2, 55.9, 43.9, 40.2, 33.83,
24.0; ESI-MS: m/z 770.15 (M + K)+; ESI-HRMS calcd for
C32H32O6N3F6S2 [M + H]+, 732.1628.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-3-(2-
(3,4-dihydroxyphenoxy)-5-isopropylphenyl)thiophene-2-sulfona-
1
mide (FWJ-D2). Overall yield: 14.1%, purity: 98.0%. H NMR (400
MHz, acetone-d6): δ 9.51 (s, 1H), 8.31 (s, 2H), 8.01 (s, 1H), 7.80 (s,
1H), 7.77 (d, J = 5.1 Hz, 1H), 7.71 (d, J = 11.3 Hz, 2H), 7.40 (d, J = 2.2
Hz, 1H), 7.24−7.18 (m, 2H), 6.78 (t, J = 8.3 Hz, 2H), 6.56 (d, J = 2.8
Hz, 1H), 6.48−6.34 (m, 2H), 3.73 (dd, J = 11.9, 5.9 Hz, 2H), 3.16 (q, J
= 6.1 Hz, 2H), 2.93 (dd, J = 13.8, 6.9 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H);
13C NMR (100 MHz, acetone-d6): δ 181.7, 153.6, 149.7, 145.8, 142.3,
141.8, 141.3, 141.2, 137.1, 132.1, 131.1, 130.8, 130.0, 128.8, 127.7,
124.5, 117.1, 115.4, 110.2, 107.2, 43.7, 42.0, 33.1, 23.4; ESI-MS: m/z
718.05 [M − H]−; ESI-HRMS calcd for C30H26O5N3F6S3 [M − H]−,
718.0955.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-3-(2-(3,4-dihydroxy-
phenoxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D8).
1
Overall yield: 18.2%, purity: 95.0%. H NMR (400 MHz, acetone-
d6): δ 8.37 (s, 1H), 8.06 (s, 1H), 7.78−7.72 (m, 2H), 7.53 (d, J = 1.7 Hz,
2H), 7.35 (d, J = 7.7 Hz, 1H), 7.17 (d, J = 5.1 Hz, 1H), 7.00 (s, 1H),
6.90 (d, J = 7.7 Hz, 1H), 6.78 (d, J = 8.6 Hz, 1H), 6.67 (s, 1H), 6.57 (d, J
= 2.7 Hz, 1H), 6.40 (dt, J = 11.7, 4.3 Hz, 2H), 6.07 (s, 1H), 3.30 (q, J =
5.9 Hz, 2H), 3.04 (q, J = 5.9 Hz, 2H), 2.26 (s, 3H); 13C NMR (100
MHz, acetone-d6): δ 155.67, 155.02, 149.54, 145.94, 142.98, 141.58,
140.90, 139.98, 137.13, 134.57, 132.30, 131.64, 128.78, 122.88, 122.03,
120.73, 117.69, 116.16, 115.52, 110.60, 107.50, 43.47, 39.53, 20.49;
ESI-MS: m/z 606.05 [M − H]−; ESI-HRMS calcd for
C26H24O6N3Cl2S2 [M + H]+, 608.0478.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)thioureido)ethyl)-3-(2-
(3,4-dimethoxyphenoxy)-5-isopropylphenyl)thiophene-2-sulfona-
1
mide (FWJ-D3). Overall yield: 14.4%, purity: 96.7%. H NMR (400
MHz, acetone-d6): δ 9.48 (s, 1H), 8.30 (s, 2H), 7.77 (d, J = 5.1 Hz, 1H),
7.71 (d, J = 10.4 Hz, 2H), 7.42 (d, J = 2.3 Hz, 1H), 7.23 (q, J = 2.7 Hz,
2H), 6.87 (d, J = 8.7 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 2.7
Hz, 1H), 6.54 (dd, J = 8.7, 2.7 Hz, 1H), 6.48 (s, 1H), 3.74 (dd, J = 9.9,
5.6 Hz, 8H), 3.18 (q, J = 6.1 Hz, 2H), 2.93 (dd, J = 13.8, 6.9 Hz, 1H),
1.25 (d, J = 6.9 Hz, 6H); 13C NMR (101 MHz, acetone-d6): δ 182.4,
154.2, 151.3, 151.1, 146.5, 143.2, 142.4, 141.8, 137.8, 132.9, 131.9,
131.5, 130.7, 129.5, 128.4, 125.3, 123.3, 122.7, 117.8, 117.4, 113.1,
111.0, 105.3, 56.3, 55.9, 44.4, 42.7, 33.8, 24.1; ESI-MS: m/z 746.10 [M
− H]−; ESI-HRMS calcd for C32H32O5N3F6S3 [M + H]+, 748.1401.
N-(2-(3-(3,5-Bis(trifluoromethyl)phenyl)ureido)ethyl)-3-(2-(3,4-
dihydroxyphenoxy)-4-methylphenyl)thiophene-2-sulfonamide
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-3-(2-(3,4-dihydroxy-
phenoxy)-5-isopropylphenyl)thiophene-2-sulfonamide (FWJ-D9).
1
Overall yield: 16.1%, purity: 96.5%. H NMR (400 MHz, acetone-
d6): δ 7.74 (d, J = 5.1 Hz, 1H), 7.52 (d, J = 1.8 Hz, 2H), 7.41 (d, J = 2.2
Hz, 1H), 7.20 (d, J = 5.1 Hz, 1H), 7.16 (dd, J = 8.5, 2.2 Hz, 1H), 7.01 (t,
J = 1.8 Hz, 1H), 6.80−6.73 (m, 2H), 6.60 (d, J = 2.7 Hz, 1H), 6.39 (dd, J
= 8.6, 2.8 Hz, 1H), 3.28 (t, J = 6.0 Hz, 2H), 2.99 (t, J = 6.0 Hz, 2H), 2.91
(dt, J = 13.8, 6.9 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H); 13C NMR (100
MHz, acetone-d6): δ 155.5, 154.1, 150.1, 146.4, 143.3, 142.8, 141.8,
141.5, 137.7, 135.0, 132.6, 130.5, 129.2, 128.2, 124.9, 121.1, 117.5,
116.5, 115.8, 107.7, 43.9, 39.8, 33.7, 23.9; ESI-MS: m/z 658.10 [M +
Na]+; ESI-HRMS calcd for C28H28O6N3Cl2S2 [M + H]+, 636.0788.
1
(FWJ-D4). Overall yield: 18.7%, purity: 97.9%. H NMR (400 MHz,
CDCl3): δ 7.66 (s, 2H), 7.53−7.42 (m, 4H), 7.24 (s, 1H), 7.12 (d, J =
5.1 Hz, 1H), 6.88 (d, J = 7.7 Hz, 1H), 6.77−6.67 (m, 2H), 6.50 (d, J =
2.7 Hz, 1H), 6.29 (dd, J = 8.6, 2.7 Hz, 1H), 5.88 (s, 1H), 5.80 (s, 1H),
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a
Scheme 3. Synthesis of Compounds FWJ-E3, FWJ-E6, FWJ-E10, and FWJ-E16
a
Reagents and conditions: (a) Pd(OAc)2, K3PO4, Ph2Pcy, THF; (b) Cs2CO3, DMSO; (c) (i)TFA, DCM, (ii)aryl isothiocyanate, DCM; d) BBr3,
DCM.
N-(2-(3-(3,5-Dichlorophenyl)ureido)ethyl)-3-(2-(3,4-dimethoxy-
phenoxy)-5-isopropylphenyl)thiophene-2-sulfonamide (FWJ-D10).
Overall yield: 16.1%, purity: 97.3%. 1H NMR (400 MHz, acetone-d6): δ
8.39 (s, 1H), 7.75 (d, J = 5.1 Hz, 1H), 7.52 (d, J = 1.8 Hz, 2H), 7.42 (d, J
= 2.3 Hz, 1H), 7.24−7.17 (m, 2H), 6.99 (t, J = 1.8 Hz, 1H), 6.86 (d, J =
8.7 Hz, 1H), 6.74 (dd, J = 14.6, 5.6 Hz, 2H), 6.54 (dd, J = 8.7, 2.7 Hz,
1H), 6.38 (t, J = 5.6 Hz, 1H), 6.09 (t, J = 5.5 Hz, 1H), 3.75 (d, J = 3.2
Hz, 6H), 3.27 (q, J = 5.9 Hz, 2H), 3.02 (q, J = 5.9 Hz, 2H), 2.93 (dd, J =
13.8, 6.9 Hz, 1H), 1.24 (t, J = 7.1 Hz, 6H). 13C NMR (100 MHz,
acetone-d6): δ 155.6, 154.2, 151.3, 151.1, 146.5, 143.6, 143.1, 141.6,
138.1, 135.1, 132.8, 130.7, 129.4, 128.4, 125.2, 121.2, 117.6, 116.6,
113.1, 111.1, 105.4, 56.2, 55.9, 44.1, 40.2, 33.8, 24.2; ESI-MS: m/z
662.05 [M − H]−; ESI-HRMS calcd for C30H32O6N3Cl2S2 [M + H]+,
664.1103.
N-(2-(3-(3-Nitrophenyl)thioureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D11). Over-
all yield: 18.2%, purity: 96.3%. 1H NMR (400 MHz, acetone-d6): δ 9.33
(s, 1H), 8.62 (s, 1H), 8.01 (s, 1H), 7.98 (d, J = 8.1 Hz, 1H), 7.90 (d, J =
7.4 Hz, 1H), 7.81 (s, 1H), 7.76 (d, J = 5.1 Hz, 1H), 7.59 (t, J = 8.1 Hz,
1H), 7.54 (s, 1H), 7.36 (d, J = 7.7 Hz, 1H), 7.18 (d, J = 5.1 Hz, 1H),
6.91 (d, J = 7.6 Hz, 1H), 6.78 (d, J = 8.5 Hz, 1H), 6.68 (s, 1H), 6.57 (d, J
= 2.8 Hz, 1H), 6.48 (s, 1H), 6.41 (dd, J = 8.6, 2.8 Hz, 1H), 3.75 (dd, J =
11.7, 5.8 Hz, 2H), 3.19 (dd, J = 12.1, 6.1 Hz, 2H), 2.28 (s, 3H); 13C
NMR (100 MHz, acetone-d6): δ 182.2, 156.1, 149.9, 148.6, 146.3,
142.0, 141.4, 141.2, 140.4, 137.3, 132.7, 132.0, 130.1, 129.5, 129.4,
123.3, 122.4, 119.2, 118.1, 116.0, 111.1, 108.0, 44.2, 42.7, 21.0; ESI-
MS: m/z 622.90 [M + Na]+; ESI-HRMS calcd for C26H24O7N4NaS3
[M + Na]+, 623.0698.
N-(2-(3-(3-Nitrophenyl)ureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D12). Over-
all yield: 18.7%, purity: 95.7%. 1H NMR (400 MHz, acetone-d6): δ 8.57
(t, J = 2.1 Hz, 1H), 8.52 (s, 1H), 8.06 (s, 1H), 7.82−7.77 (m, 1H), 7.74
(d, J = 5.9 Hz, 3H), 7.51 (t, J = 8.2 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H),
7.17 (d, J = 5.1 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 6.77 (d, J = 8.6 Hz,
1H), 6.67 (s, 1H), 6.57 (d, J = 2.8 Hz, 1H), 6.44−6.37 (m, 2H), 6.10 (s,
1H), 3.33 (dd, J = 11.9, 5.9 Hz, 2H), 3.06 (dd, J = 12.0, 6.0 Hz, 2H),
2.25 (s, 3H); 13C NMR (100 MHz, acetone-d6): δ 155.66, 155.24,
149.53, 148.75, 145.94, 141.87, 141.58, 140.90, 139.97, 137.17, 132.29,
131.66, 129.65, 128.76, 123.78, 122.88, 122.04, 117.67, 115.90, 115.51,
112.33, 110.59, 107.51, 43.51, 39.54, 20.48; ESI-MS: m/z 583.10 [M −
H]−; ESI-HRMS calcd for C26H23O8N4S2 [M − H]−, 583.0961.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D13). Over-
all yield: 18.0%, purity: 97.1%. 1H NMR (400 MHz, acetone-d6): δ 9.51
(s, 1H), 8.19 (d, J = 9.2 Hz, 2H), 8.01 (s, 1H), 7.90 (d, J = 9.2 Hz, 2H),
7.82 (s, 1H), 7.76 (d, J = 5.1 Hz, 1H), 7.67 (s, 1H), 7.36 (d, J = 7.7 Hz,
1H), 7.18 (d, J = 5.1 Hz, 1H), 6.92 (d, J = 7.5 Hz, 1H), 6.78 (d, J = 8.6
Hz, 1H), 6.68 (s, 1H), 6.57 (d, J = 2.8 Hz, 1H), 6.49 (s, 1H), 6.41 (dd, J
= 8.6, 2.8 Hz, 1H), 3.76 (dd, J = 11.7, 5.8 Hz, 2H), 3.20 (dd, J = 12.0, 6.0
Hz, 2H), 2.28 (s, 3H); 13C NMR (100 MHz, acetone-d6): δ 181.8,
156.3, 150.1, 146.6, 146.5, 143.7, 142.1, 141.6, 140.6, 137.5, 132.9,
132.2, 129.6, 124.9, 123.5, 122.5, 122.0, 118.2, 116.1, 111.2, 108.2, 44.5,
42.7, 21.1; ESI-MS: m/z 623.05 [M + Na]+; ESI-HRMS calcd for
C26H24O7N4NaS3 [M + Na]+, 623.0698.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-5-isopropylphenyl)thiophene-2-sulfonamide (FWJ-D14).
1
Overall yield: 14.3%, purity: 97.6%. H NMR (400 MHz, acetone-
d6): δ 9.62 (s, 1H), 8.18 (d, J = 9.0 Hz, 2H), 8.05 (s, 1H), 7.92 (d, J = 8.9
Hz, 2H), 7.77 (dd, J = 14.0, 5.5 Hz, 3H), 7.40 (s, 1H), 7.21 (t, J = 7.1
Hz, 2H), 6.78 (dd, J = 8.5, 3.7 Hz, 2H), 6.58 (d, J = 2.4 Hz, 1H), 6.47−
6.33 (m, 2H), 3.74 (d, J = 5.4 Hz, 2H), 3.22−3.13 (m, 2H), 2.91 (dd, J =
13.7, 6.8 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H); 13C NMR (100 MHz,
acetone-d6): δ 181.3, 153.6, 149.7, 145.8, 142.3, 141.1, 132.1, 130.0,
128.8, 127.7, 124.5, 124.2, 121.1, 117.1, 115.4, 110.2, 107.3, 43.7, 42.0,
33.1, 23.4; ESI-MS: m/z 629.10 [M + H]+; ESI-HRMS calcd for
C28H29O7N4S3 [M + H]+, 629.1190.
N-(2-(3-(4-Nitrophenyl)thioureido)ethyl)-3-(2-(3,4-dimethoxy-
phenoxy)-5-isopropylphenyl)thiophene-2-sulfonamide (FWJ-D15).
Overall yield: 15.9%, purity: 97.9%. 1H NMR (400 MHz, acetone-d6): δ
9.52 (s, 1H), 8.18 (d, J = 9.1 Hz, 2H), 7.89 (d, J = 9.1 Hz, 2H), 7.80−
7.73 (m, 1H), 7.69 (s, 1H), 7.41 (s, 1H), 7.23 (d, J = 5.1 Hz, 2H), 6.88
(d, J = 8.7 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H),
6.57−6.45 (m, 2H), 3.75 (d, J = 4.9 Hz, 8H), 3.18 (dd, J = 11.8, 5.9 Hz,
2H), 2.92 (dt, J = 14.0, 7.0 Hz, 1H), 1.25 (d, J = 6.9 Hz, 6H); 13C NMR
(101 MHz, acetone-d6): δ 181.8, 154.2, 151.3, 151.1, 146.5, 143.2,
141.8, 137.9, 132.9, 130.7, 129.6, 128.4, 125.2, 125.0, 121.9, 117.8,
113.1, 111.0, 105.3, 56.3, 56.0, 44.5, 42.7, 33.8, 24.1; ESI-MS: m/z
657.15 [M + H]+; ESI-HRMS calcd for C30H33O7N4S3 [M + H]+,
658.1504.
N-(2-(3-(4-Nitrophenyl)ureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D16). Over-
all yield: 17.8%, purity: 93.6%. 1H NMR (400 MHz, acetone-d6): δ 8.72
(s, 1H), 8.15 (d, J = 9.2 Hz, 2H), 8.05 (s, 1H), 7.79 (s, 1H), 7.74 (d, J =
5.1 Hz, 1H), 7.71 (d, J = 9.2 Hz, 2H), 7.36 (d, J = 7.7 Hz, 1H), 7.18 (d, J
= 5.1 Hz, 1H), 6.90 (d, J = 7.7 Hz, 1H), 6.77 (d, J = 8.6 Hz, 1H), 6.66 (s,
1H), 6.58 (d, J = 2.8 Hz, 1H), 6.41 (dd, J = 8.5, 2.7 Hz, 2H), 6.17 (s,
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1H), 3.33 (dd, J = 11.8, 5.9 Hz, 2H), 3.05 (q, J = 5.9 Hz, 2H), 2.25 (s,
3H); 13C NMR (100 MHz, acetone-d6): δ 156.68, 155.73, 150.49,
147.89, 146.94, 142.58, 142.52, 141.89, 140.97, 138.14, 133.30, 132.66,
129.78, 125.82, 123.83, 122.97, 118.60, 118.24, 116.50, 111.62, 108.59,
44.33, 40.58, 21.49; ESI-MS: m/z 607.15 [M + Na]+; ESI-HRMS calcd
for C26H25O8N4S2 [M + H]+, 585.1109.
FWJ-E11, FWJ-E13, FWJ-E15, and FWJ-E18. These compounds were
dissolved in 3 mL of DCM. Then, 10 equiv of BBr3 was added dropwise
in an ice-water bath. The mixture was stirred for 1 h. After that, water
was slowly added to quench the reaction. 3 mL of CH2Cl2 was used to
extract the residue three times. The demethylated products were
collected via evaporation, filtration, and concentration, that is, FWJ-E1,
FWJ-E4, FWJ-E7, FWY-E9, FWJ-E12, FWJ-E14, and FWJ-E17.
Using a similar synthetic route, compound 16 (55 mg, 0.2927 mmol)
reacted with compound 13 (100 mg, 0.2439 mmol) to obtain the
mediate product 17. 3,4-dimethoxyphenol (16 mg, 0.1018 mmol), 17
(40 mg, 0.0849 mmol), and DMSO (1 mL) were added to the flask.
Then, cesium carbonate (33 mg, 0.1018 mmol) was added, and the
solution was heated and stirred at 110 °C for 1 h. After that, water (3
mL) and dichloromethane (3 mL) were added to extract three times. It
was further purified by chromatography (EtOAc/petroleum ether 1:5)
to obtain 18 as a yellow solid.
N-(2-(3-(3-Methoxyphenyl)thioureido)ethyl)-3-(2-(3,4-dihydroxy-
phenoxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D17).
1
Overall yield: 17.6%, purity: 97.3%. H NMR (400 MHz, acetone-
d6): δ 8.92 (s, 1H), 8.05 (s, 1H), 7.76 (dd, J = 14.7, 5.1 Hz, 2H), 7.34
(dd, J = 14.1, 6.4 Hz, 2H), 7.24 (t, J = 8.1 Hz, 1H), 7.17 (d, J = 5.1 Hz,
1H), 7.03 (s, 1H), 6.90 (t, J = 6.7 Hz, 2H), 6.77 (dd, J = 15.7, 8.9 Hz,
2H), 6.67 (s, 1H), 6.58 (d, J = 2.6 Hz, 1H), 6.49−6.36 (m, 2H), 3.82−
3.70 (m, 5H), 3.17 (dd, J = 12.0, 6.1 Hz, 2H), 2.28 (s, 3H); ESI-MS: m/
z 584.05 [M − H]−; ESI-HRMS calcd for C27H26O6N3S3 [M − H]−,
584.0991.
N-(2-(3-(3-Methoxyphenyl)ureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D18). Over-
all yield: 17.6%, purity: 95.2%. 1H NMR (400 MHz, acetone-d6): δ 8.16
(s, 1H), 8.02 (s, 1H), 7.73 (d, J = 5.1 Hz, 1H), 7.68 (s, 1H), 7.36 (d, J =
7.7 Hz, 1H), 7.22 (s, 1H), 7.17 (d, J = 5.1 Hz, 1H), 7.11 (t, J = 8.1 Hz,
1H), 6.90 (d, J = 7.7 Hz, 2H), 6.77 (d, J = 8.6 Hz, 1H), 6.67 (s, 1H),
6.58 (d, J = 2.8 Hz, 1H), 6.52 (dd, J = 8.2, 2.1 Hz, 1H), 6.45−6.34 (m,
2H), 5.96 (s, 1H), 3.74 (s, 3H), 3.31 (q, J = 5.9 Hz, 2H), 3.02 (q, J = 6.0
Hz, 2H), 2.25 (s, 3H); 13C NMR (100 MHz, acetone-d6): δ 160.9,
156.3, 156.2, 150.1, 146.5, 142.2, 142.1, 141.5, 140.6, 137.8, 132.9,
132.3, 129.9, 129.3, 123.5, 122.6, 118.3, 116.2, 111.3, 111.2, 108.1,
108.0, 104.9, 55.1, 44.4, 40.1, 21.1; ESI-MS: m/z 592.15 [M + Na]+.
N-(2-(3-(4-Methoxyphenyl)thioureido)ethyl)-3-(2-(3,4-dihydroxy-
phenoxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D19).
Compound 18 (1.4 mmol) reacted with DCM (10 mL) and TFA
(1.5 mL) at room temperature for 5 h. The residue was treated with
potassium carbonate saturated solution and extracted by 10 mL of
water and 10 mL of CH2Cl2 three times. The intermediate compound
reacted with isocyanate or 1.1 equiv of isocyanate or isothiocyanate to
produce FWJ-E3, FWJ-E6, and FWJ-E16. The demethylated product
FWJ-E10 was obtained as the previous protocol.
N-(4-Nitrophenyl)-4-((3-(2-(3,4-dihydroxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carbothioamide (FWJ-E1).
1
Overall yield: 31.4%, purity: 96.4%. H NMR (400 MHz, acetone-
d6): δ 8.15 (d, J = 9.1 Hz, 2H), 7.89 (d, J = 5.1 Hz, 1H), 7.68−7.61 (m,
2H), 7.48 (dd, J = 7.6, 1.6 Hz, 1H), 7.37−7.30 (m, 1H), 7.29 (d, J = 5.1
Hz, 1H), 7.09 (t, J = 7.1 Hz, 1H), 6.79 (t, J = 8.1 Hz, 2H), 6.63 (d, J =
2.8 Hz, 1H), 6.45 (dd, J = 8.6, 2.8 Hz, 1H), 4.07−4.01 (m, 4H), 3.09−
3.00 (m, 4H); 13C NMR (101 MHz, Acetone-d6): δ 182.7, 157.1, 149.2,
148.0, 146.8, 142.9, 142.7, 134.3, 133.4, 130.9, 130.7, 124.8, 124.5,
123.6, 123.5, 122.0, 116.3, 116.2, 112.0, 108.8, 48.9, 46.1; ESI-MS: m/z
611.00 [M − H]−; ESI-HRMS calcd for C27H23O7N4S3 [M − H]−,
611.0724.
1
Overall yield: 18.7%, purity: 91.8%. H NMR (400 MHz, acetone-
d6): δ 8.75 (s, 1H), 8.13 (s, 2H), 7.73 (d, J = 5.1 Hz, 1H), 7.36 (d, J = 7.7
Hz, 1H), 7.19 (dd, J = 17.6, 7.0 Hz, 3H), 7.06 (d, J = 5.3 Hz, 1H), 6.94−
6.87 (m, 3H), 6.79 (d, J = 8.6 Hz, 1H), 6.67 (s, 1H), 6.58 (d, J = 2.8 Hz,
1H), 6.46 (s, 1H), 6.41 (dd, J = 8.6, 2.8 Hz, 1H), 3.79 (s, 3H), 3.73 (q, J
= 6.1 Hz, 2H), 3.15 (q, J = 6.1 Hz, 2H), 2.28 (s, 3H); ESI-MS: m/z
584.05 [M − H]−; ESI-HRMS calcd for C27H26O6N3S3 [M − H]−,
584.0989.
N-(2-(3-(4-Methoxyphenyl)ureido)ethyl)-3-(2-(3,4-dihydroxyphe-
noxy)-4-methylphenyl)thiophene-2-sulfonamide (FWJ-D20). Over-
all yield: 18.2%, purity: 98.3%. 1H NMR (400 MHz, acetone-d6): δ 8.26
(s, 1H), 7.81 (s, 1H), 7.72 (d, J = 5.1 Hz, 1H), 7.61 (s, 1H), 7.36 (d, J =
7.8 Hz, 1H), 7.31 (d, J = 9.0 Hz, 2H), 7.18 (d, J = 5.1 Hz, 1H), 6.89 (d, J
= 7.8 Hz, 1H), 6.82 (d, J = 9.0 Hz, 2H), 6.76 (d, J = 8.6 Hz, 1H), 6.67 (s,
1H), 6.59 (d, J = 2.8 Hz, 1H), 6.45−6.37 (m, 2H), 5.90 (s, 1H), 3.74 (s,
3H), 3.30 (q, J = 6.0 Hz, 2H), 3.01 (q, J = 6.0 Hz, 2H), 2.25 (s, 3H); 13C
NMR (100 MHz, acetone-d6): δ 156.18, 155.58, 149.34, 145.89,
141.64, 140.75, 139.90, 133.05, 132.29, 131.64, 128.64, 122.82, 121.93,
120.72, 117.59, 115.38, 113.79, 110.51, 107.46, 54.69, 43.89, 39.49,
20.48; ESI-MS: m/z 592.15 [M + Na]+; ESI-HRMS calcd for
C27H28O7N3S2 [M + H]+, 570.1362.
N-(4-Nitrophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carbothioamide (FWJ-E2).
1
Overall yield: 31.4%, purity: 98.0%. H NMR (400 MHz, acetone-
d6): δ 9.23 (s, 1H), 8.15 (d, J = 9.1 Hz, 2H), 7.90 (d, J = 5.1 Hz, 1H),
7.63 (d, J = 9.1 Hz, 2H), 7.49 (dd, J = 7.6, 1.5 Hz, 1H), 7.38−7.29 (m,
2H), 7.12 (t, J = 7.2 Hz, 1H), 6.90−6.76 (m, 3H), 6.60 (dd, J = 8.7, 2.7
Hz, 1H), 4.10−3.99 (m, 4H), 3.77 (s, 3H), 3.74 (s, 3H), 3.12−3.03 (m,
4H); 13C NMR (100 MHz, acetone-d6): δ 182.83, 156.85, 151.33,
150.48, 148.02, 147.06, 142.95, 133.47, 133.38, 130.98, 130.73, 125.17,
124.55, 123.33, 122.30, 116.75, 113.36, 111.98, 106.07, 56.15, 48.83,
46.07; ESI-MS: m/z 639.05 [M − H]−; ESI-HRMS calcd for
C29H27O7N4S3 [M − H]−, 639.1066.
N-(4-Nitrophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)-5-
nitrophenyl)thiophen-2-yl)sulfonyl)piperazine-1-carbothioamide
1
(FWJ-E3). Overall yield: 72.1%, purity: 96.5%. H NMR (400 MHz,
acetone-d6): δ 9.25 (s, 1H), 8.38 (d, J = 2.8 Hz, 1H), 8.25 (dd, J = 9.2,
2.8 Hz, 1H), 8.15 (d, J = 9.1 Hz, 2H), 8.04 (d, J = 5.1 Hz, 1H), 7.62 (d, J
= 9.1 Hz, 2H), 7.47 (d, J = 5.1 Hz, 1H), 6.98−6.88 (m, 3H), 6.75 (dd, J
= 8.7, 2.7 Hz, 1H), 4.13−4.07 (m, 4H), 3.79 (s, 3H), 3.78 (s, 3H),
3.16−3.10 (m, 4H); 13C NMR (100 MHz, acetone-d6): δ 182.96,
162.55, 151.62, 148.71, 148.20, 148.05, 142.27, 140.67, 134.71, 133.12,
131.96, 128.95, 126.56, 125.21, 124.63, 123.38, 115.81, 113.36, 113.01,
106.54, 56.35, 48.86, 46.26; ESI-MS: m/z 683.95 [M − H]−; ESI-
HRMS calcd for C29H26O9N5S3 [M − H]−, 684.0896.
N-(4-Nitrophenyl)-4-((3-(2-(3,4-dihydroxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carboxamide (FWJ-E4). Overall
yield: 30.4%, purity: 94.5%. 1H NMR (400 MHz, acetone-d6): δ 8.14
(d, J = 9.3 Hz, 2H), 7.87 (d, J = 5.1 Hz, 1H), 7.73 (d, J = 9.3 Hz, 2H),
7.48 (dd, J = 7.6, 1.6 Hz, 1H), 7.37−7.31 (m, 1H), 7.28 (d, J = 5.1 Hz,
1H), 7.09 (t, J = 7.5 Hz, 1H), 6.77 (t, J = 8.7 Hz, 2H), 6.58 (d, J = 2.7
Hz, 1H), 6.42 (dd, J = 8.6, 2.8 Hz, 1H), 3.64−3.56 (m, 4H), 2.99−2.93
(m, 4H); 13C NMR (100 MHz, acetone-d6): δ 157.04, 154.52, 149.00,
147.40, 146.71, 142.66, 142.55, 134.15, 133.37, 133.22, 130.65, 130.51,
125.04, 124.69, 121.79, 119.19, 119.11, 116.13, 116.07, 111.79, 108.66,
The general procedure for the synthesis of compounds FWJ-E1−
FWJ-E18 (see Schemes 2 and 3) is described as follows:
Compound 12 (2.93 mmol), 13 (2.44 mmol), palladium acetate
(0.49 mmol), diphenylcyclohexylphosphine (0.98 mmol), and
potassium phosphate (7.32 mmol) were added into a 100 mL flask.
After full deoxidation, THF (25 mL) and water (5 mL) were added to
dissolve the reactants. The solution was then heated under reflux for 6 h.
The residue was concentrated and extracted by water (20 mL) and ethyl
acetate (20 mL) three times. It was further purified by chromatography
(EtOAc/petroleum ether 1:10) to obtain compound 14.
Compound 14 (1.7 mmol) reacted with DCM (10 mL) and TFA
(1.7 mL) at room temperature for 10 h. The residue was treated with
potassium carbonate saturated solution and extracted by 15 mL of
water and 20 mL of CH2Cl2 three times to obtain compound 15.
Compound 15 (∼50 mg), CH2Cl2 (3 mL), and isocyanate or 1.1
equiv of isocyanate or isothiocyanate were added into a 25 mL flask.
The mixture was stirred at room temperature until the reaction is
complete. The residue was purified by chromatography (EtOAc/
petroleum ether 1:3) to obtain compounds FWJ-E2, FWJ-E5, FWJ-E8,
10278
https://doi.org/10.1021/acs.jmedchem.1c00690
J. Med. Chem. 2021, 64, 10260−10285

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
46.17, 44.28; ESI-MS: m/z 619.10 [M + Na]+; ESI-HRMS calcd for
C27H23O8N4S2 [M − H]−, 595.0872.
N-(3, 5-Bis(trifluoromethyl)phenyl)-4-((3-(2-(3, 4-
dimethoxyphenoxy)phenyl)thiophen-2-yl)sulfonyl)piperazine-1-
1
carbothioamide (FWJ-E11). Overall yield: 30.8%, purity: 96.6%. H
N-(4-Nitrophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carboxamide (FWJ-E5). Overall
yield: 30.4%, purity: 97.2%. 1H NMR (400 MHz, acetone-d6): δ 8.65 (s,
1H), 8.18−8.10 (m, 2H), 7.88 (d, J = 5.1 Hz, 1H), 7.75 (dd, J = 7.2, 5.1
Hz, 2H), 7.49 (dd, J = 7.6, 1.6 Hz, 1H), 7.40−7.27 (m, 2H), 7.12 (t, J =
7.1 Hz, 1H), 6.81 (dd, J = 13.6, 8.5 Hz, 2H), 6.74 (d, J = 2.7 Hz, 1H),
6.56 (dd, J = 8.7, 2.7 Hz, 1H), 3.74 (s, 6H), 3.64−3.56 (m, 4H), 3.02−
2.94 (m, 4H); 13C NMR (100 MHz, acetone-d6): δ 157.11, 154.69,
151.52, 150.52, 147.86, 147.27, 142.91, 142.83, 134.62, 133.65, 133.56,
131.03, 130.89, 125.41, 125.35, 122.45, 119.32, 116.74, 113.48, 112.21,
106.24, 56.51, 56.24, 46.51, 44.63; ESI-MS: m/z 647.15 [M + Na]+;
ESI-HRMS calcd for C29H28O8N4NaS2 [M + Na]+, 647.1254.
N-(4-Nitrophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)-5-
nitrophenyl)thiophen-2-yl)sulfonyl)piperazine-1-carboxamide
NMR (400 MHz, acetone-d6): δ 9.20 (s, 1H), 8.08 (s, 2H), 7.90 (d, J =
5.1 Hz, 1H), 7.73 (s, 1H), 7.49 (dd, J = 7.6, 1.6 Hz, 1H), 7.39−7.33 (m,
1H), 7.31 (d, J = 5.1 Hz, 1H), 7.12 (t, J = 7.5 Hz, 1H), 6.87−6.75 (m,
3H), 6.60 (dd, J = 8.6, 2.7 Hz, 1H), 4.12−4.06 (m, 4H), 3.75 (s, 3H),
3.72 (s, 3H), 3.12−3.05 (m, 4H); 13C NMR (100 MHz, acetone-d6): δ
183.07, 157.22, 151.71, 150.87, 147.42, 144.06, 143.29, 134.53, 133.82,
133.72, 131.93, 131.60, 131.33, 131.08, 125.57, 125.45, 122.66, 117.15,
113.70, 112.33, 106.41, 56.67, 56.46, 48.82, 46.41; ESI-MS: m/z 754.05
[M + Na]+; ESI-HRMS calcd for C31H27O5N3F6NaS3 [M + Na]+,
754.0915.
N-(3, 5-Bis(trifluoromethyl)phenyl)-4-((3-(2-(3, 4-
dihydroxyphenoxy)phenyl)thiophen-2-yl)sulfonyl)piperazine-1-
carboxamide (FWJ-E12). Overall yield: 29.3%, purity: 96.0%. 1H
NMR (400 MHz, acetone-d6): δ 8.65 (s, 1H), 8.18 (s, 2H), 8.06 (s,
1H), 7.87 (d, J = 5.1 Hz, 1H), 7.77 (s, 1H), 7.58 (s, 1H), 7.48 (dd, J =
7.6, 1.6 Hz, 1H), 7.39−7.31 (m, 1H), 7.27 (d, J = 5.1 Hz, 1H), 7.10 (t, J
= 7.5 Hz, 1H), 6.79 (d, J = 8.3 Hz, 1H), 6.74 (d, J = 8.5 Hz, 1H), 6.57 (d,
J = 2.8 Hz, 1H), 6.42 (dd, J = 8.6, 2.8 Hz, 1H), 3.66−3.54 (m, 4H),
3.03−2.94 (m, 4H); 13C NMR (100 MHz, acetone-d6): δ 157.44,
155.06, 149.70, 146.94, 143.44, 143.15, 142.86, 134.73, 133.73, 133.61,
132.49, 132.17, 131.11, 130.96, 126.01, 125.37, 123.31, 122.36, 119.98,
116.81, 116.51, 112.26, 108.92, 46.59, 44.61; ESI-MS: m/z 686.00 [M
− H]−; ESI-HRMS calcd for C29H22O6N3F6S2 [M − H]−, 686.0869.
N-(3, 5-Bis(trifluoromethyl)phenyl)-4-((3-(2-(3, 4-
dimethoxyphenoxy)phenyl)thiophen-2-yl)sulfonyl)piperazine-1-
carboxamide (FWJ-E13). Overall yield: 31.8%, purity: 95.7%. 1H
NMR (400 MHz, acetone-d6): δ 8.63 (s, 1H), 8.20 (s, 2H), 7.87 (d, J =
5.1 Hz, 1H), 7.58 (s, 1H), 7.49 (dd, J = 7.6, 1.6 Hz, 1H), 7.39−7.31 (m,
1H), 7.30 (d, J = 5.1 Hz, 1H), 7.12 (td, J = 7.5, 1.0 Hz, 1H), 6.80 (dd, J =
8.5, 4.6 Hz, 2H), 6.74 (d, J = 2.7 Hz, 1H), 6.57 (dd, J = 8.7, 2.7 Hz, 1H),
3.73 (s, 6H), 3.64−3.57 (m, 4H), 3.03−2.96 (m, 4H); 13C NMR (100
MHz, acetone-d6): δ 157.26, 154.95, 151.69, 150.72, 147.42, 143.63,
143.07, 137.74, 133.79, 133.70, 132.53, 132.21, 131.17, 131.04, 125.54,
122.60, 119.87, 116.95, 113.58, 112.32, 106.35, 56.61, 56.32, 46.63,
44.60; ESI-MS: m/z 738.00 [M + Na]+; ESI-HRMS calcd for
C31H28O6N3F6S2 [M + H]+, 716.1323.
1
(FWJ-E6). Overall yield: 73.9%, purity: 95.4%. H NMR (400 MHz,
acetone-d6): δ 8.69 (s, 1H), 8.38 (d, J = 2.8 Hz, 1H), 8.25 (dd, J = 9.2,
2.9 Hz, 1H), 8.14 (d, J = 9.3 Hz, 2H), 8.01 (d, J = 5.1 Hz, 1H), 7.73 (d, J
= 9.3 Hz, 2H), 7.45 (d, J = 5.1 Hz, 1H), 6.93 (dd, J = 8.9, 5.1 Hz, 2H),
6.87 (d, J = 2.7 Hz, 1H), 6.71 (dd, J = 8.7, 2.7 Hz, 1H), 3.78 (s, 3H),
3.78 (s, 3H), 3.69−3.62 (m, 4H), 3.08−3.01 (m, 4H); 13C NMR (100
MHz, acetone-d6): δ 162.62, 154.54, 151.64, 148.64, 148.21, 147.70,
142.73, 142.26, 140.46, 134.98, 133.07, 131.82, 128.88, 126.56, 125.29,
119.19, 115.65, 113.32, 112.97, 106.50, 56.33, 56.22, 46.51, 44.49; ESI-
MS: m/z 667.95 [M − H]−; ESI-HRMS calcd for C29H26O10N5S2 [M −
H]−, 668.1128.
N-(3-Nitrophenyl)-4-((3-(2-(3,4-dihydroxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carboxamide (FWJ-E7). Overall
yield: 31.1%, purity: 98.5%. 1H NMR (400 MHz, acetone-d6): δ 8.47 (s,
1H), 7.91−7.80 (m, 3H), 7.54−7.45 (m, 2H), 7.38−7.31 (m, 1H), 7.28
(d, J = 5.1 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.76 (dd, J = 15.0, 8.4 Hz,
2H), 6.56 (d, J = 2.7 Hz, 1H), 6.43 (dd, J = 8.5, 2.7 Hz, 1H), 3.64−3.55
(m, 4H), 3.01−2.93 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 156.6,
154.4, 148.5, 147.2, 144.7, 142.0, 142.0, 138.8, 133.7, 133.5, 132.4,
130.3, 129.8, 129.7, 126.9, 122.6, 121.3, 119.0, 115.9, 115.4, 114.2,
113.2, 107.6, 45.7, 43.7; ESI-MS: m/z 595.05 [M − H]−; ESI-HRMS
calcd for C27H23O8N4S2 [M − H]−, 595.0960.
N-(3-Nitrophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carboxamide (FWJ-E8). Overall
yield: 31.1%, purity: 97.1%. 1H NMR (400 MHz, acetone-d6): δ 8.52
(dd, J = 9.2, 7.1 Hz, 2H), 7.86 (ddd, J = 19.9, 8.6, 1.7 Hz, 3H), 7.56−
7.47 (m, 2H), 7.35 (dd, J = 12.3, 5.0 Hz, 1H), 7.30 (d, J = 5.1 Hz, 1H),
7.12 (t, J = 7.5 Hz, 1H), 6.80 (dd, J = 12.2, 8.5 Hz, 2H), 6.74 (d, J = 2.7
Hz, 1H), 6.57 (dd, J = 8.7, 2.7 Hz, 1H), 3.73 (s, 6H), 3.64−3.55 (m,
4H), 3.02−2.92 (m, 4H); 13C NMR (100 MHz, CDCl3): δ 155.5,
154.1, 149.9, 149.8, 148.3, 145.6, 142.1, 140.3, 133.0, 132.6, 132.5,
130.1, 129.8, 129.5, 125.6, 124.3, 122.1, 117.6, 116.8, 114.4, 111.7,
110.8, 104.4, 56.2, 56.0, 45.3, 43.6; ESI-MS: m/z 647.15 [M + Na]+;
ESI-HRMS calcd for C29H28O8N4NaS2 [M + Na]+, 647.1245.
N-(3, 5-Bis(trifluoromethyl)phenyl)-4-((3-(2-(3, 4-
dihydroxyphenoxy)phenyl)thiophen-2-yl)sulfonyl)piperazine-1-
N-(4-Chlorophenyl)-4-((3-(2-(3,4-dihydroxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carbothioamide (FWJ-E14).
1
Overall yield: 25.9%, purity: 97.2%. H NMR (400 MHz, acetone-
d6): δ 7.88 (d, J = 5.1 Hz, 1H), 7.48 (dd, J = 10.9, 5.0 Hz, 2H), 7.41 (d, J
= 8.8 Hz, 1H), 7.38−7.26 (m, 6H), 7.09 (t, J = 7.5 Hz, 1H), 6.79 (dd, J =
8.4, 4.0 Hz, 2H), 6.64 (d, J = 2.7 Hz, 1H), 6.47 (dd, J = 8.5, 2.7 Hz, 1H),
4.06−3.95 (m, 4H), 3.07−2.96 (m, 4H); 13C NMR (100 MHz,
acetone-d6): δ 183.23, 157.22, 146.63, 142.83, 140.37, 133.53, 133.33,
130.89, 130.71, 130.64, 128.73, 128.09, 127.24, 121.94, 116.33, 116.15,
112.18, 108.84, 48.46, 46.09; ESI-MS: m/z 624.05 [M + Na]+; ESI-
HRMS calcd for C27H24O5N3ClNaS3 [M − H]−, 624.0461.
N-(4-Chlorophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carbothioamide (FWJ-E15).
1
1
Overall yield: 25.9%, purity: 95.6%. H NMR (400 MHz, acetone-
carbothioamide (FWJ-E9). Overall yield: 30.2%, purity: 95.0%. H
d6): δ 8.83 (s, 1H), 7.89 (d, J = 5.1 Hz, 1H), 7.49 (dd, J = 7.6, 1.6 Hz,
1H), 7.38−7.25 (m, 6H), 7.11 (t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.7 Hz,
1H), 6.80 (dd, J = 11.1, 5.5 Hz, 2H), 6.60 (dd, J = 8.7, 2.7 Hz, 1H),
4.07−3.96 (m, 4H), 3.76 (s, 3H), 3.74 (s, 3H), 3.08−2.97 (m, 4H); 13C
NMR (100 MHz, acetone-d6): δ 183.42, 156.97, 150.46, 147.08,
142.88, 133.53, 133.45, 130.98, 130.78, 128.76, 127.11, 122.29, 116.71,
113.37, 112.09, 106.13, 56.36, 56.19, 48.46, 46.13; ESI-MS: m/z 652.05
[M + Na]+; ESI-HRMS calcd for C29H28O5N3ClNaS3 [M + Na]+,
652.0773.
N-(4-Chlorophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)-5-
nitrophenyl)thiophen-2-yl)sulfonyl)piperazine-1-carbothioamide
(FWJ-E16). Overall yield: 73.2%, purity: 95.2%. 1H NMR (400 MHz,
acetone-d6): δ 8.87 (s, 1H), 8.40 (d, J = 2.8 Hz, 1H), 8.25 (dd, J = 9.2,
2.8 Hz, 1H), 8.03 (d, J = 5.1 Hz, 1H), 7.47 (d, J = 5.1 Hz, 1H), 7.31 (dd,
J = 21.3, 8.9 Hz, 4H), 6.98−6.89 (m, 3H), 6.75 (dd, J = 8.7, 2.7 Hz, 1H),
4.11−4.03 (m, 4H), 3.80 (s, 3H), 3.78 (s, 3H), 3.13−3.05 (m, 4H); 13C
NMR (100 MHz, DMSO-d6): δ 181.5, 161.4, 150.0, 147.2, 146.6,
140.8, 139.7, 139.5, 132.7, 132.4, 132.2, 128.4, 127.8, 126.6, 126.20,
NMR (400 MHz, acetone-d6): δ 8.08 (s, 2H), 7.90 (d, J = 5.1 Hz, 1H),
7.72 (s, 1H), 7.48 (dd, J = 7.6, 1.6 Hz, 1H), 7.39−7.31 (m, 1H), 7.28 (d,
J = 5.1 Hz, 1H), 7.10 (t, J = 7.5 Hz, 1H), 6.78 (dd, J = 14.9, 8.4 Hz, 2H),
6.61 (d, J = 2.8 Hz, 1H), 6.44 (dd, J = 8.6, 2.8 Hz, 1H), 4.14−4.00 (m,
4H), 3.14−3.02 (m, 4H); ESI-MS: m/z 702.00 [M − H]−; ESI-HRMS
calcd for C29H22O5N3F6S3 [M − H]−, 702.0619.
N-(3,5-Bis(trifluoromethyl)phenyl)-4-((3-(2-(3,4-dihydroxyphe-
noxy)-5-nitrophenyl)thiophen-2-yl)sulfonyl)piperazine-1-carbo-
1
thioamide (FWJ-E10). Overall yield: 75.4%, purity: 95.8%. H NMR
(400 MHz, acetone-d6): δ 9.26 (s, 1H), 8.37 (d, J = 2.8 Hz, 1H), 8.26
(dd, J = 9.2, 2.8 Hz, 1H), 8.18 (s, 1H), 8.04 (dd, J = 9.7, 5.1 Hz, 4H),
7.72 (s, 1H), 7.44 (d, J = 5.1 Hz, 1H), 6.93 (d, J = 9.2 Hz, 1H), 6.83 (d, J
= 8.5 Hz, 1H), 6.74 (d, J = 2.7 Hz, 1H), 6.56 (dd, J = 8.5, 2.7 Hz, 1H),
4.19−4.10 (m, 4H), 3.18−3.12 (m, 4H); 13C NMR (100 MHz, DMSO-
d6): δ 180.9, 161.7, 146.6, 145.7, 143.4, 142.9, 140.6, 139.7, 132.3,
129.9, 129.6, 127.6, 126.2, 124.5, 123.7, 121.9, 115.9, 114.6, 111.2,
108.7, 47.4, 45.20; ESI-MS: m/z 746.90 [M − H]−; ESI-HRMS calcd
for C29H21O7N4F6S3 [M − H]−, 747.0483.
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123.8, 114.9, 112.3, 112.1, 105.6, 55.7, 47.3, 45.2; ESI-MS: m/z 672.95
[M − H]−; ESI-HRMS calcd for C29H27O7N4ClNaS3 [M + Na]+,
697.0621.
was induced by 0.4 mM isopropyl-b-d-thiogalactopyranoside (IPTG)
at 37 °C for 4 h. Cells were lysed in 25 mM Tris-HCl, pH 8.0 buffer
containing 300 mM NaCl, 5 mM βME, 1% (v/v) Triton X-100, 1×
protease inhibitor cocktail, and 0.1 mg/mL phenylmethanesulfonyl
fluoride (PMSF). His-TEV-Bcl-2 protein was purified from the soluble
fraction using the Ni-NTA resin (QIAGEN) following the
manufacturer’s instructions. The His-TEV-Bcl-2 protein was further
cleaved by TEV protease (0.5 mg/mL, 4 °C, 15 h) to produce Bcl-2
protein.
N-(4-Fluorophenyl)-4-((3-(2-(3,4-dihydroxyphenoxy)-phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carboxamide (FWJ-E17). Over-
all yield: 29.2%, purity: 98.4%. 1H NMR (400 MHz, acetone-d6): δ 8.10
(s, 1H), 7.87 (d, J = 5.1 Hz, 1H), 7.49 (dd, J = 7.6, 1.6 Hz, 1H), 7.46−
7.40 (m, 2H), 7.36−7.28 (m, 2H), 7.08 (dd, J = 7.4, 6.6 Hz, 1H), 6.99
(t, J = 8.8 Hz, 2H), 6.76 (dd, J = 8.0, 6.3 Hz, 2H), 6.54 (d, J = 2.7 Hz,
1H), 6.46 (dd, J = 8.5, 2.8 Hz, 1H), 3.58−3.50 (m, 4H), 2.94−2.86 (m,
4H); 13C NMR (100 MHz, acetone-d6): δ 157.3, 148.8, 142.7, 133.5,
133.4, 130.7, 130.6, 124.5, 122.6, 122.5, 122.4, 121.7, 116.3, 116.2,
115.7, 115.5, 115.3, 112.3, 108.7, 108.6, 46.4, 44.2; ESI-MS: m/z
568.10 [M − H]−; ESI-HRMS calcd for C27H23O6N3FS2 [M − H]−,
568.1023.
N-(4-Fluorophenyl)-4-((3-(2-(3,4-dimethoxyphenoxy)-phenyl)-
thiophen-2-yl)sulfonyl)piperazine-1-carboxamide (FWJ-E18). Over-
all yield: 31.9%, purity: 97.3%. 1H NMR (400 MHz, acetone-d6): δ 8.06
(s, 1H), 7.85 (d, J = 5.1 Hz, 1H), 7.49 (td, J = 7.2, 3.4 Hz, 3H), 7.38−
7.27 (m, 2H), 7.11 (t, J = 7.2 Hz, 1H), 6.99 (t, J = 8.8 Hz, 2H), 6.84−
6.72 (m, 3H), 6.56 (dd, J = 8.7, 2.7 Hz, 1H), 3.73 (s, 3H), 3.72 (s, 3H),
3.59−3.46 (m, 4H), 2.99−2.90 (m, 4H); 13C NMR (100 MHz,
acetone-d6): δ 160.01, 157.64, 156.92, 155.24, 151.19, 150.15, 146.96,
142.56, 137.27, 137.25, 134.35, 133.41, 133.33, 130.72, 130.63, 125.00,
122.12, 121.94, 121.86, 116.34, 115.47, 115.25, 113.14, 112.01, 105.95,
56.20, 55.94, 46.27, 44.23; ESI-MS: m/z 596.00 [M − H]−; ESI-HRMS
calcd for C29H29O6N3FS2 [M + H]+, 598.1480.
Bcl-xL. The Bcl-xL protein used in our study is a special truncated
construction of the full-length protein, with deletion of residues 45−84
on a large loop region and residues 210−233 at the C-terminal
hydrophobic region. It was cloned into the pSJ2 vector (a modified
pET28a vector) at the EcoRI and Xhol sites using the following
oligonucleotides: 5′-TCTCGAATTCATGTCTCAGAGCAA-
C C G G G A - 3 ′
a n d
5 ′ - G G T C C T C G A G T -
CAGCGTTCCTGGCCCTTTCG-3′. The Bcl-xL protein with a N-
terminal 8×His tag was expressed in E. coli BL21 (DE3) cells. Cells were
grown at 37 °C in the LB medium containing 100 μg/mL ampicillin to
an OD600 of 0.6. Protein expression was induced by 0.4 mM IPTG at 20
°C for 16 h. Cells were lysed in 25 mM tris-HCl, pH 8.0 buffer
containing 300 mM NaCl, 5 mM βME, 1% (v/v) Triton X-100, 1×
protease inhibitor cocktail, and 0.1 mg/mL PMSF. His-TEV-Bcl-xL
protein was purified from the soluble fraction using the Ni-NTA resin
(QIAGEN) following the manufacturer’s instructions. The His-TEV-
Bcl-xL protein was further cleaved by TEV protease (0.5 mg/mL, 4 °C,
15 h) to produce Bcl-xL protein.
All NMR spectra were recorded at room temperature on a Bruker
AV-400 400 MHz spectrometer. They were calibrated by using residual
CHCl3 (dH = 7.26 ppm) and CDCl3 (dC = 77.16 ppm), CH3OH (dH
= 3.31 ppm) and CD3OD (dC = 49.00 ppm), acetone (dH = 2.05 ppm)
and acetone-d6 (dC = 29.84, 206.26 ppm), or DMSO (dH = 2.50 ppm)
and DMSO-d6 (dC = 39.52 ppm) as internal references. The following
abbreviations are used to designate multiplicities: s: singlet; d: doublet;
t: triplet; q: quartet; m: multiplet; quint: quintet; br: broad. High-
resolution mass spectrometry (HRMS) was performed on a Bruker
APEXIII 7.0 T ESI-FT mass spectrometer at an emitter voltage of 4000
V. Purity of each final compound was determined by analytical HPLC.
An Agilent 1260 series HPLC (G7114A 1260 VWD detector) with an
Agilent Zorbax Exlipse Plus-C18 (4.6 × 150 mm, 5 μM) reversed-phase
column was used to perform all analytical HPLC analyses. The elution
buffer was a gradient of H2O/CH3OH. HPLC traces for all final
compounds (i.e., TJ-A, TJ-B, TJ-C, FWJ-D, and FWJ-E compounds)
and four lead compounds (i.e., YCW-E5, YCW-E10, YCW-E11, and
YCW-E16) are given in the Supporting Information (Part III). The
purity of all described compounds was ≥95% except for three
compounds (i.e., FWJ-D16: 93.6%; FWJ-D19: 91.8%; FWJ-E4:
94.5%).
The overall yields of all TJ-A, TJ-B, TJ-C, and FWJ-D compounds
and most FWJ-E compounds were calculated from the starting
materials (e.g., 1 and 2 in Scheme 1) as the product of the following
steps. The yields of intermediate 9 (Scheme 1) and 13 (Scheme 2) were
close to 100% (i.e., 98% and 99%, respectively), so they were not
included in the yield calculation. For compounds FWJ-E3, FWJ-E6,
FWJ-E10, and FWJ-E16, shown in Scheme 3, their yields were
calculated from 16, which was purchased directly. Consequently, their
yields were significantly higher than those of the other compounds.
Expression and Purification of Bcl-2, Bcl-xL, and Mcl-1. Bcl-2.
The Bcl-2 protein used in our study has the same construction as the
one (Bcl-2/Bcl-xL, isoform 2) in Fesik et al.’s work,42 which is
composed of amino acid residues 1−34 of human Bcl-2, amino acid
residues 29−44 of human Bcl-xL, and amino acid residues 92−207 of
human Bcl-2. Such a construction has a good solubility in water and
reserves the biological function of human Bcl-2. It was cloned into the
pSJ2 vector at the EcoRI and XhoI sites using the following
oligonucleotides: 5′-TTTTGAATTCATGGCGCACGCAGGTCG-
3′ and 5′-TTTTCTCGAGTCAACGCATGGACG-GGC-3’. Bcl-2
protein with a N-terminal 8× His tag was produced in Escherichia coli
BL21 (DE3) cells. Cells were grown at 37 °C in the LB medium
containing 100 μg/mL ampicillin to an OD600 of 0.6. Protein expression
Mcl-1. The Mcl-1 protein used in our study has the same
construction as the one (hMcl-1BLR) in Colman et al.’s work,43
which is composed of amino acid residues 152−189 of mouse Mcl-1
and 41 amino acid residues 209−327 of human Mcl-1. Such a
construction has a good solubility in water and reserves the biological
function of human Mcl-1. It was cloned into the pSJ2 vector (a modified
pET28a vector) at the HindIII and BamHI sites using the following
oligonucleotides: 5′CCCCAAGCTTTTAACCACCTTC-
CAGGTCTTCAAC-3′ and 5′-TTCCGGATCCATGGAAG-AT-
GATCTGTACCGTCAGTC-3′. The Mcl-1 protein with a N-terminal
8× His tag was expressed in E. coli BL21 (DE3) cells. Cells were grown
at 37 °C in the LB medium containing 100 μg/mL ampicillin to an
OD600 of 0.6. Protein expression was induced by 0.4 mM IPTG at 20 °C
for 16 h. Cells were lysed in 25 mM Tris-HCl, pH 8.0 buffer containing
300 mM NaCl, 5 mM βME, 1% (v/v) Triton X-100, 1× protease
inhibitor cocktail, and 0.1 mg/mL PMSF. His-TEV-Mcl-1 protein was
purified from the soluble fraction using the Ni-NTA resin (QIAGEN)
following the manufacturer’s instructions. The His-TEV-Mcl-1 protein
was further cleaved by TEV protease (0.5 mg/mL, 4 °C, 15 h) to
produce Mcl-1 protein.
In Vitro Binding Assay. In our binding assay, a 26-residue peptide
derived from the BH3 domain on the Bid protein with 5 carboxy-
fluorescein (5-FAM) on the N-terminus, that is, 5-FAM-QEDIIR-
NIARHLAQVGDSMD-RSIPPG, was used as the fluorescence tracer.
This peptide was ordered from the HD Biosciences Cooperation
Company. The desired sequences were verified by amino acid
component analysis and the MS spectrum. The purity of this peptide
was over 95% as verified by HPLC results. Our dose-dependent
saturation experiments determined that this Bid-BH3 peptide bound to
Bcl-xL with Kd = 215 nM, to Bcl-2 with Kd = 215 nM, and to Mcl-1 with
Kd = 160 nM.
In competitive binding experiments, each protein (Bcl-xL, Bcl-2, or
Mcl-1) and the tested compound (in 1% DMSO-d6 solution) were pre-
incubated in the assay buffer (PBS, pH 7.3, 140 mM NaCl, 2.7 mM KCl,
10 mM Na2HPO4, 1.8 mM KH2PO4) at 37 °C for 30 min. 20 μL of a
solution in PBS of the FAM-Bid peptide was then added to the solution
to produce a final volume of 200 μL and incubated at 37 °C for another
20 min. The total concentration of the Bid-BH3 peptide used in the
assay was 10 nM, while the total concentration of protein was set to 5
times the Kd value of the fluorescence tracer, that is, 205 nM in the case
of Bcl-xL, 445 nM in the case of Bcl-2, and 145 nM in the case of Mcl-1.
At last, the solutions were transferred into Corning 384-well, black, flat-
bottom plates (Corning Incorporated) with 60 μL per well and 3 wells
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per sample. The polarization values in millipolarization units (mP) were
measured at an excitation wavelength at 485 nm and an emission
wavelength at 535 nm using the Tecan Genios Pro Injector Reader
(Tecan Group Ltd.). For each experiment, a positive control containing
the protein (Bcl-xL, Bcl-2, or Mcl-1), the Bid-BH3 peptide, and 1% (v/
v) [d6]DMSO and a negative control containing only Bid-BH3 peptide
were included on each assay plate.
Each compound was tested against all three target proteins (Bcl-xL,
Bcl-2, and Mcl-1) at seven different concentrations, that is, 1 nM, 10
nM, 100 nM, 1 μM, 10 μM, 50 μM, and 100 μM. At each concentration,
the average mP value of three parallel measurements was used to
perform nonlinear fitting to obtain the final binding curve. The
nonlinear fitting job was conducted by using the GraphPad Prism
software (version 5, http://www.graphpad.com). The concentration of
the given compound where 50% of the bound peptide was displaced
(IC50) was derived from the binding curve. The competitive inhibition
constant (Ki) of each tested compound was then calculated with the
following equation developed by Wang et al.44 assuming that it forms a
binary complex with the target protein
Data Centre, United Kingdom).45 The receptor used for docking was
extracted from the complex structure formed between human Mcl-1
and a small-molecule inhibitor (PDB entry: 4WMR).46 Both ligand
molecules were sketched with the “Builder” module in the MOE
software (version 2018, Chemical Computing Group, Canada) and
then minimized with the MMFF94x force field. The binding site on
Mcl-1 was defined as all amino acid residues within 10 Å around the
bound ligand. The GOLD program employs a genetic algorithm (GA)
to control the docking process. The main GA parameters used in our
docking job were set as follows: the whole population was placed on 5
separate islands with 100 individuals on each. The total number of GA
operations was 100,000. The probabilities for crossover, mutation, and
migration operations were 0.95, 0.95, and 0.10, respectively. The
ChemPLP scoring function was employed to compute binding scores
during docking.47 A total of 30 binding poses were finally generated for
each ligand molecule, which were clustered with a root-mean-square
deviation (rmsd) cutoff of 2.0 Å. The most reasonable binding pose was
selected among the best-scored ones in each cluster through visual
inspection.
The selected docking poses of YCW-E10 and FWJ-D4 were then
refined through MD simulation by using the AMBER program (version
14, University of California San Francisco).48 Each ligand molecule was
assigned GAFF parameters by using the “Antechamer” module in the
AMBER program. Atomic partial charges on the ligand molecule were
calculated with the BCC method. Mcl-1 protein atoms were assigned
the PARM14 template charges implemented in the AMBER program.
All ionizable residues were set at their default protonation states under a
neutral pH value. The complex structure was soaked in a box of TIP3P
water molecules with a margin of 10 Å along each dimension. The
whole system was neutralized by adding an appropriate number of
counterions. The whole system was then subjected to a multi-step
minimization process: (i) the complex structure was relaxed by 100
cycles of steepest descent minimization and then 4900 cycles of
conjugated gradient minimization, where all heavy atoms on the protein
and the ligand were constrained with a constant of 10.0 kcal mol−1 Å−2.
(ii) The whole complex went through the same minimization process as
the previous step, but no constraint was applied. Then, the system was
gradually heated up from 0 to 300 K in 150 ps, followed by 100 ns MD
simulation at a constant temperature of 300 K and a constant pressure
of 1 atm. During the simulation, electrostatic interactions were
calculated with the PME algorithm, and the distance cutoff of
nonbonded interactions was set as 10 Å. The SHAKE algorithm was
applied to fix the lengths of all chemical bonds connecting hydrogen
atoms. A total of 5000 snapshots were retrieved at a 10 ps interval from
the last 50 ns MD trajectory. These snapshots were grouped into
clusters based on mass-weighted rmsd values calculated on the residues
in the binding site, with a cutoff value of 1.5 Å by using the MMTSB tool
(http://blue11.bch.msu.edu/mmtsb/Main_Page). The snapshot clos-
est to the cluster center in the largest cluster was selected as the
representative model of the given complex. The final binding modes of
YCW-E10 and FWJ-D4 to Mcl-1 are shown in Figure 2b.
The representative conformation of the Mcl-1 protein was used as
the initial structure for hydration site prediction by WATsite.41 Side-
chain conformations of Asn, Gln, and His residues and tautomers and
protonation states of His residues were adjusted by using Reduce.49
Hydrogen atoms were added to the structures by using GROMACS
(v4.6.1).50 The protein was solvated in an octahedron water box using
the SPC water model with a water layer of a minimum of 10 Å between
any protein atom and the edge of the box. MD simulation was
performed by using GROMACS with the AMBER03 force field.48 The
system was equilibrated first for 250 ps with harmonical restrains
applied to all protein atoms (force constant = 1000 kJ mol−1 nm−2).
Then, a total of 2 ns MD simulation was performed at a constant
temperature of 300 K and a constant pressure of 1 atm. The last 1 ns of
the trajectory generated 1000 snapshots for identifying the water
molecule locations by using a quality threshold (QT) clustering
algorithm. All the above jobs were executed on a DELL T7810 desktop
workstation (dual Intel Xeon E5-2620 v3 processors, 16 GB memory,
and Intel C602 motherboard) running RedHat Linux.
[I]50
Ki =
[L]50
Kd
[P]0
+
+ 1
(
)
Kd
In this equation, [I]50 and [L]50 denote the concentrations of the free
small-molecule inhibitor (I) and the free fluorescence-labeled peptide
ligand (L) at 50% inhibition, respectively, and [P]0 is the concentration
of the free protein (P) before the inhibitor is added, that is, 0%
inhibition. In order to solve this equation, one needs to supply the total
concentration of the protein used in the assay ([P]T), the total
concentration of the fluorescence-labeled ligand used in the assay
([L]T), the dissociation constant (Kd) between these two species, and
the IC50 value of the tested compound as inputs.
15N-Heteronuclear Single-Quantum Coherence Spectrosco-
py. The human Mcl-1 protein (172−327aa) was used in this
experiment. The pET28a-His-hMcl-1 vector donated by Zhang’s
Group (Dalian University of Technology) was expressed in E. coli
BL21(DE3) cells. Cells were grown at 37 °C in the M9 medium
containing 15N-ammonium chloride and Kana. Protein expression was
induced by addition of 0.4 mM IPTG at 37 °C for 4 h. Cells were lysed
in 25 mM tris(hydroxymethyl)aminomethane (Tris)-HCl buffer at pH
7.0 containing 100 mM NaCl and 0.1 mg mL−1 PMSF. 15N-labeled His-
hMcl-1 protein was purified from the soluble fraction by using the Ni-
NTA resin (Qiagen). The 15N-labeled His-hMcl-1 protein was further
purified on Superdex75 (GE Healthcare) in phosphate-buffered saline
(PBS) solution.
15N-heteronuclear single-quantum coherence (HSQC) NMR
spectra were recorded on the Agilent 800 MHz NMR spectrometer
at 25 °C. The 15N-labeled His-hMcl-1 protein was dissolved in 20 mM
sodium phosphate buffer at pH 6.5, with 1 mM DTT, 1% DMSO-d6,
and 10% D2O. The final concentration of the protein was 0.15 mM.
Then, HSQC NMR spectra of the protein at its free state were recorded.
After that, the tested compounds dissolved in DMSO-d6 (final
concentration: 0.3 mM) were added into the above solution, which
was incubated at 4 °C for 1 h and then centrifuged. HSQC NMR
spectra of the protein-compound complex were recorded. The resulting
NMR spectra were processed and analyzed by using the Sparky software
(http://nmrfam.wisc.edu/nmrfam-sparky-distribution/). The HSQC
spectrum of free Mcl-1, which is publicly available from the Biological
Magnetic Resonance Data Bank (http://www.bmrb.wisc.edu/; access
number: 19654), was used as the reference for the assignment of
chemical shifts. The Ω-value computed as the following equation was
used to quantify the chemical shift of each residue on the two-
dimensional HSQC spectra.
Ω = (0.2Δδ15N)2 + (Δδ1H)2
Molecular Modeling. Binding modes to Mcl-1 for compounds
YCW-E10 and FWJ-D4 were predicted by molecular modeling. As the
first step, a rough binding mode was generated by using the molecular
docking program GOLD (version 2018, Cambridge Crystallographic
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Cytotoxicity Assay. The cytotoxicity activities of our compounds
were tested by a refined MTT assay, that is, the CCK8 cell viability
assay, on four cell lines. These four cell lines included human cervical
tumor cell Hela, two leukemia tumor cells, HL-60 and RS4;11, and
human renal epithelial HEK293T cells as a control. All cell lines were
originally obtained from the American Type Culture Collection
(ATCC, Manassas, VA) and were cultured in Dulbecco’s modified
Eagle’s medium (HEK293T) and the RPMI-1640 medium (Hela, HL-
60, RS4;11) with 10% fetal bovine serum (FBS) and supplemented with
1% penicillin−streptomycin (P/S). Cell Counting Kit-8 was bought
from MesGen (Cat. MG6432, MesGen, China) for the cytotoxicity
assay. In each test, 100 mL of a suspension of 8000 HeLa/HEK293T
cells and 50 mL of a suspension of 16,000 HL-60/RS4;11 cells were
seeded in each well on 96-well plates. They were treated with the tested
compounds of five concentrations (2.5, 5, 10, 20, and 40 μM) and
incubated in a CO2 incubator for 48 h at 37 °C. After that, the CCK-8
solution (10 μL) was added to each well. Plates were incubated for an
additional 2 h at 37 °C. The supernatant was carefully removed, and
DMSO (100 mL) was added to dissolve the formazan crystals. The
absorbance at 450 nm was then recorded by using a SpectraMax-190
microplate reader (Molecular Devices, USA). The absorbance at the
same wavelength for the well with only the medium or the well with the
medium and cells was defined as the blank value or control value,
respectively. The cell viability was calculated as the following equation:
subjected to electrophoresis on 10% sodium dodecylsulfate poly-
acrylamide gels by using a Bio-Rad mini-gel apparatus and blotted to
polyvinylidene difluoride membranes (Millipore, MA, USA) through
electrotransfer. Membranes were blocked for 1 h with 5% nonfat dry
milk in Tris-buffered saline containing 0.1% Tween-20 (TBST) and
then incubated with antibodies (mouse monoclonal anti-caspase-9
antibodies at a 1:1000 dilution, rabbit monoclonal anti-caspase-3
antibodies at a 1:1000 dilution, rabbit monoclonal anti-PARP
antibodies at a 1:1000 dilution, and mouse monoclonal anti-Actin
antibodies at a 1:2000 dilution) overnight at 4 °C. Primary antibodies
were labeled with goat anti-mouse immunoglobulin G (IgG) at a
1:5000 dilution or goat anti-rabbit IgG at a 1:5000 dilution conjugated
with horseradish peroxidase (Pierce) and shocked on a shaking table for
1 h at room temperature. Immunoreactive bands were detected by
incubation of the membranes with enhanced chemiluminescence
(ECL) reagents.
Co-Immunoprecipiation. MCF-7 cells were incubated at 37 °C for
24 h with ABT-199 (1 μM), A-1210477 (3 μM), FWJ-D4 (10 μM),
FWJ-D5 (10 μM), or YCW-E16 (10 μM). Cells were harvested and
then lysed on ice in IP buffer (10 mM HEPES, pH 7.4, 50 mM NaCl, 5
mM MgCl2, 1 mM EGTA, 5% glycerol, 0.5% Triton X-100)
supplemented with 1% protease/phosphatase inhibitor for 30 min.
After centrifugation (15 min, 12,000g, 4 °C), supernatants were
collected and normalized to the protein content. 2 μg of the anti-Bim
antibody was added to an input volume of 200 μL with 5−10 mg/mL
protein. After shaking overnight at 4 °C, 20 μL of protein G sepharose
beads (GE Healthcare, Madison, WI, USA) was added and the samples
incubated for 2 h at 4 °C with constant rotation. Immunocomplexes
were washed three times with IP buffer. Immunoprecipitated proteins
were analyzed by western blot using the anti-Mcl-1 antibody or anti-
Bcl-2 antibody. Proteins were detected with an ECL kit.
Anti-tumor Effect on the Xenograft Mouse Model. All
procedures for this animal experiment were approved by the
Institutional Animal Care and Use Committee of SMOC (Shanghai
Model Organisms Center, Inc.) with IACUC no. 2018-0004. To
develop xenograft tumors, 5 × 106 RS4;11 cells with 50% Matrigel (BD
Biosciences, United States) were injected subcutaneously in the right
abdomen of 6-week-old, male NOD/SCID mice (N = 37), which were
obtained from the Shanghai Model Organisms Center, Inc. (Shanghai,
China). The width and length of the tumors were measured 2−3 times
each week using an electronic caliper. Tumor volume was calculated as
volume = (length × width2)/2. When tumors reached approximately
250 mm3, mice were size-matched into different dosing groups and
control groups, where each dosing group consisted of eight individuals
and each control group consisted of five individuals. Compounds ABT-
199 and FWJ-D5 were formulated in 60% polyethylene glycol 400
(Sangon Biotech, China) and administrated at the doses and schedules
shown in Figure 9 (i.e., ABT-199 at a dose of 12.5 mg/kg; FWJ-D5 at
doses of 12.5, 25, and 50 mg/kg). Weights of the mice were measured
weekly. Tumor tissues were placed in liquid nitrogen for cryo-
sectioning. Then, sections were analyzed by immunohistochemical
staining. Proliferation was detected using the Ki-67 antibody (ABcam,
USA). Images were captured with a Leica SP8 microscope (Leica,
Germany).
cell viability (%)
(absorbance (compound) − absorbance(blank))
(absorbance (control) − absorbance(blank))
=
× 100%
If necessary, the concentration at 50% inhibition (IC50) of each
tested compound was derived through nonlinear fitting of the average
inhibition rates read from three independent wells by using the
Graphpad Prism software (version 5, www.graphpad.com).
Apoptotic Mechanism Studies. Cell Culture and Treatment of
Compounds. RS4;11 cells were cultured in the RPMI-1640 medium
supplemented with 10% (v/v) FBS, 100 g mL−1 streptomycin, and 100
IU mL−1 penicillin. Cells were incubated under a 5% CO2 humidified
atmosphere at 37 °C. Cells were seeded into a 75 cm2 Corning cell
culture flask (for mitochondria isolation) or 6- or 12-well cell culture
plates (for drug stimulation) and maintained by addition of a new
medium every day. Cells were plated at a density of 5 × 105 mL 24 h
before compound treatment. Before mitochondria isolation or drug
inducement of normal cells, cell viability was confirmed to be >95% by
using the trypan blue exclusion assay. ABT-199 and other tested
compounds were dissolved in DMSO to a final concentration lower
than 0.1%. The control cells were treated with the same volume of the
vehicle. The medium was cultured in the presence of multiple doses of
ABT-199 (0.125, 0.25, 0.5, 1.0, and 2.0 μM) or tested compounds (2.5,
5.0, 10, 20, and 40 μM) for 24 h. Three parallel experiments were
conducted for each compound under the same conditions.
Flow Cytometry Analysis (Mitodamage Staining). RS4;11 cells
were incubated with tested compounds for 24−48 h. They were
measured by flow cytometry with a MitoDamage Kit (FCCH100106,
Merck Millipore). Cell pellets were suspended in 1× assay buffer with
MitoSense Red Dye 1:50 and Annexin V stock solution 1:20 at a
concentration of 1 × 105 cells/200 μL for 15 min in a 37 °C CO2
incubator. Washing was done two more times with 200 μL of 1× assay
buffer, and cells were resuspended in each well with 200 μL of 1× assay
buffer. 5 μL of the 7-AAD reagent was added to each well. A total of
10,000 events were obtained by using the green channel for Annexin V-
FITC, the RED2 channel for MitoSence, and the red channel for 7-
AAD. Data presented in this work were representative of those obtained
from at least three independent experiments done in triplicate.
ASSOCIATED CONTENT
■
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00690.
Information of the predicted hydration sites in the Mcl-1
binding pocket, flow cytometric analysis of three selected
compounds, and HPLC traces for the reported com-
pounds (PDF)
West Blot Detection of Caspase-9 and Caspase-3 Activation.
RS4;11 cells were lysed in the lysis buffer [1% Triton X-100, 50 mM
Tris (pH 7.4), and 150 mM NaCl, containing protease inhibitors
(Complete, Roche)] maintained on ice and vortexed gently every 10
min. The sample was centrifuged for 15 min at 12,000g and 4 °C to get
rid of cell debris. The protein concentration was assayed by using
bicinchoninic acid (Pierce). The cytosolic protein (40 mg) was then
Molecular formula strings of all reported compounds
(CSV)
Structural files of the predicted binding modes of YCW-
E10 and FWJ-D4 with Mcl-1 (ZIP)
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Article
Author Contributions
Statement on the ethnicity of all animal experiments
performed in this work (PDF)
⊥Y.L., W.F., and Q.G. equal contributions to this work.
Notes
The authors declare no competing financial interest.
AUTHOR INFORMATION
■
Corresponding Authors
ACKNOWLEDGMENTS
■
Zhichao Zhang − State Key Laboratory of Fine Chemicals,
Zhang Dayu School of Chemistry, Dalian University of
Technology, Dalian, Liaoning 116024, People’s Republic of
China; orcid.org/0000-0002-2774-2384;
This study was financially supported by the National Natural
Science Foundation of China (grant nos. 81725022, 81430083,
21472227, 21661162003, 21673276, and 21472226), the
Ministry of Science and Technology of China (National Key
Research Program, grant no. 2016YFA0502302), and the
Chinese Academy of Sciences (Strategic Priority Research
Program, grant no. XDB20000000).
Email: zczhang@dlut.edu.cn
Ran Hong − CAS Key Laboratory of Synthetic Chemistry of
Natural Substances, Chinese Academy of Sciences, Shanghai
200032, People’s Republic of China; orcid.org/0000-
0002-7602-539X; Email: rhong@sioc.ac.cn
REFERENCES
■
Renxiao Wang − Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, People’s
Republic of China; State Key Laboratory of Bioorganic and
Natural Products Chemistry, Center for Excellence in
Molecular Synthesis, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, People’s
Republic of China; Shanxi Key Laboratory of Innovative Drugs
for the Treatment of Serious Diseases Basing on Chronic
Inflammation, College of Traditional Chinese Medicines,
Shanxi University of Chinese Medicine, Taiyuan, Shanxi
030619, People’s Republic of China; orcid.org/0000-
0003-0485-0259; Email: wangrx@fudan.edu.cn
(1) Kerr, J. F. R.; Wyllie, A. H.; Currie, A. R. Apoptosis: a basic
biological phenomenon with wide-ranging implications in tissue
kinetics. Br. J. Cancer 1972, 26, 239−257.
(2) Anderson, M. H.; Becker, J. C.; Straten, P. Regulators of apoptosis:
suitable targets for immune therapy of cancer. Nat. Rev. Drug Discovery
2005, 4, 399−409.
(3) Cotter, T. G. Apoptosis and cancer: the genesis of a research field.
Nat. Rev. Cancer 2009, 9, 501−507.
(4) Hanahan, D.; Weinberg, R. A. Hallmarks of cancer: the next
generation. Cell 2011, 144, 646−674.
(5) Czabotar, P. E.; Lessene, G.; Strasser, A.; Adams, J. M. Control of
apoptosis by the BCL-2 protein family: implications for physiology and
therapy. Nat. Rev. Mol. Cell Biol. 2014, 15, 49−63.
(6) Petros, A. M.; Nettesheim, D. G.; Wang, Y.; Olejniczak, E. T.;
Meadows, R. P.; Mack, J.; Swift, K.; Matayoshi, E. D.; Zhang, H.;
Thompson, C. B.; Fesik, S. W. Rationale for Bcl-xL/Bad peptide
complex formation from structure, mutagenesis, and biophysical
studies. Protein Sci. 2000, 9, 2528−2534.
(7) Cory, S.; Huang, D. C. S.; Adams, J. M. The Bcl-2 family: roles in
cell survival and oncogenesis. Oncogene 2003, 22, 8590−8607.
(8) Konopleva, M.; Zhao, S.; Hu, W.; Jiang, S.; Snell, V.; Weidner, D.;
Jackson, C. E.; Zhang, X.; Champlin, R.; Estey, E.; Reed, J. C.; Andreeff,
M. The anti-apoptotic genes Bcl-X(L) and Bcl-2 are over-expressed and
contribute to chemoresistance of non-proliferating leukaemic CD34+
cells. Br. J. Haematol. 2002, 118, 521−534.
(9) Palve, V.; Mallick, S.; Ghaisas, G.; Kannan, S.; Teni, T.
Overexpression of Mcl-1L splice variant is associated with poor
prognosis and chemoresistance in oral cancers. PLoS One 2014, 9,
No. e111927.
(10) Lessene, G.; Czabotar, P. E.; Colman, P. M. BCL-2 family
antagonists for cancer therapy. Nat. Rev. Drug Discovery 2008, 7, 989−
1000.
(11) Delbridge, A. R. D.; Grabow, S.; Strasser, A.; Vaux, D. L. Thirty
years of BCL-2: translating cell death discoveries into novel cancer
therapies. Nat. Rev. Cancer 2016, 16, 99−109.
(12) Garner, T. P.; Lopez, A.; Reyna, D. E.; Spitz, A. Z.; Gavathiotis, E.
Progress in targeting the BCL-2 family of proteins. Curr. Opin. Chem.
Biol. 2017, 39, 133−142.
(13) Souers, A. J.; Leverson, J. D.; Boghaert, E. R.; Ackler, S. L.;
Catron, N. D.; Chen, J.; Dayton, B. D.; Ding, H.; Enschede, S. H.;
Fairbrother, W. J.; Huang, D. C. S.; Hymowitz, S. G.; Jin, S.; Khaw, S. L.;
Kovar, P. J.; Lam, L. T.; Lee, J.; Maecker, H. L.; Marsh, K. C.; Mason, K.
D.; Mitten, M. J.; Nimmer, P. M.; Oleksijew, A.; Park, C. H.; Park, C.-
M.; Phillips, D. C.; Roberts, A. W.; Sampath, D.; Seymour, J. F.; Smith,
M. L.; Sullivan, G. M.; Tahir, S. K.; Tse, C.; Wendt, M. D.; Xiao, Y.; Xue,
J. C.; Zhang, H.; Humerickhouse, R. A.; Rosenberg, S. H.; Elmore, S. W.
ABT-199, a potent and selective BCL-2 inhibitor, achieves antitumor
activity while sparing platelets. Nat. Med. 2013, 19, 202−208.
(14) Nguyen, M.; Marcellus, R. C.; Roulston, A.; Watson, M.; Serfass,
L.; Murthy Madiraju, S. R.; Goulet, D.; Viallet, J.; Belec, L.; Billot, X.;
Acoca, S.; Purisima, E.; Wiegmans, A.; Cluse, L.; Johnstone, R. W.;
Beauparlant, P.; Shore, G. C. Small molecule obatoclax (GX15-070)
Authors
Yan Li − Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, People’s
Republic of China; State Key Laboratory of Bioorganic and
Natural Products Chemistry, Center for Excellence in
Molecular Synthesis, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, Shanghai 200032, People’s
Republic of China; orcid.org/0000-0002-8259-2470
Wenjie Fan − State Key Laboratory of Bioorganic and Natural
Products Chemistry, Center for Excellence in Molecular
Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, People’s Republic of
China
Qineng Gong − Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, People’s
Republic of China
Jie Tian − State Key Laboratory of Bioorganic and Natural
Products Chemistry, Center for Excellence in Molecular
Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, People’s Republic of
China
Mi Zhou − Department of Medicinal Chemistry, School of
Pharmacy, Fudan University, Shanghai 201203, People’s
Republic of China
Qing Li − State Key Laboratory of Bioorganic and Natural
Products Chemistry, Center for Excellence in Molecular
Synthesis, Shanghai Institute of Organic Chemistry, Chinese
Academy of Sciences, Shanghai 200032, People’s Republic of
China
Laura B. Uwituze − State Key Laboratory of Fine Chemicals,
Zhang Dayu School of Chemistry, Dalian University of
Technology, Dalian, Liaoning 116024, People’s Republic of
China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00690
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antagonists MCL-1 and overcomes MCL-1-mediated resistance to
apoptosis. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 19512−19517.
(15) Tse, C.; Shoemaker, A. R.; Adickes, J.; Anderson, M. G.; Chen, J.;
Jin, S.; Johnson, E. F.; Marsh, K. C.; Mitten, M. J.; Nimmer, P.; Roberts,
L.; Tahir, S. K.; Xiao, Y.; Yang, X.; Zhang, H.; Fesik, S.; Rosenberg, S.
H.; Elmore, S. W. ABT-263: a potent and orally bioavailable Bcl-2
family inhibitor. Cancer Res. 2008, 68, 3421−3428.
(16) Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Armstrong, R.
C.; Augeri, D. J.; Belli, B. A.; Bruncko, M.; Deckwerth, T. L.; Dinges, J.;
Hajduk, P. J.; Joseph, M. K.; Kitada, S.; Korsmeyer, S. J.; Kunzer, A. R.;
Letai, A.; Li, C.; Mitten, M. J.; Nettesheim, D. G.; Ng, S.; Nimmer, P.
M.; O’Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed, J. C.; Shen, W.;
Tahir, S. K.; Thompson, C. B.; Tomaselli, K. J.; Wang, B.; Wendt, M.
D.; Zhang, H.; Fesik, S. W.; Rosenberg, S. H. An inhibitor of Bcl-2
family proteins induces regression of solid tumors. Nature 2005, 435,
677−681.
(17) Oliver, C. L.; Miranda, M. B.; Shangary, S.; Land, S.; Wang, S.;
Johnson, D. E. (-)-Gossypol acts directly on the mitochondria to
overcome Bcl-2- and Bcl-X(L)-mediated apoptosis resistance. Mol.
Cancer Ther. 2005, 4, 23−31.
(18) Porter, J.; Payne, A.; de Candole, B.; Ford, D.; Hutchinson, B.;
Trevitt, G.; Turner, J.; Edwards, C.; Watkins, C.; Whitcombe, I.; Davis,
J.; Stubberfield, C. Tetrahydroisoquinoline amide substituted phenyl
pyrazoles as selective Bcl-2 inhibitors. Bioorg. Med. Chem. Lett. 2009, 19,
230−233.
(19) Wu, Y.; Lakhani, N. J.; Boyer, M.; Zhou, Q.; Rasco, D. W.;
Huang, Y.; Men, L.; Li, Y.; Xia, Z.; Wang, H.; Ji, J.; Lu, B.; He, Z.; Dong,
Q.; Yang, D.; Zhai, Y. A phase I study of novel Bcl-2/Bcl-xL inhibitor
APG-1252 in patients with advanced SCLC or other solid tumor. J.
Thorac. Oncol. 2018, 13, S1048−S1049.
(20) Caenepeel, S. R.; Belmontes, B.; Sun, J.; Coxon, A.; Moody, G.;
Hughes, P. E. Abstract 2027: preclinical evaluation of AMG176, a novel,
potent and selective Mcl-1 inhibitor with robust anti-tumor activity in
Mcl-1 dependent cancer models. Cancer Res. 2017, 77, 2027.
(21) Konopleva, M.; Contractor, R.; Tsao, T.; Samudio, I.; Ruvolo, P.
P.; Kitada, S.; Deng, X.; Zhai, D.; Shi, Y.-X.; Sneed, T.; Verhaegen, M.;
Soengas, M.; Ruvolo, V. R.; McQueen, T.; Schober, W. D.; Watt, J. C.;
Jiffar, T.; Ling, X.; Marini, F. C.; Harris, D.; Dietrich, M.; Estrov, Z.;
McCubrey, J.; May, W. S.; Reed, J. C.; Andreeff, M. Mechanisms of
apoptosis sensitivity and resistance to the BH3 mimetic ABT-737 in
acute myeloid leukemia. Cancer Cell 2006, 10, 375−388.
(22) van Delft, M. F.; Wei, A. H.; Mason, K. D.; Vandenberg, C. J.;
Chen, L.; Czabotar, P. E.; Willis, S. N.; Scott, C. L.; Day, C. L.; Cory, S.;
Adams, J. M.; Roberts, A. W.; Huang, D. C. S. The BH3 mimetic ABT-
737 targets selective Bcl-2 proteins and efficiently induces apoptosis via
Bak/Bax if Mcl-1 is neutralized. Cancer Cell 2006, 10, 389−399.
(23) Ashkenazi, A.; Fairbrother, W. J.; Leverson, J. D.; Souers, A. J.
From basic apoptosis discoveries to advanced selective BCL-2 family
inhibitors. Nat. Rev. Drug Discovery 2017, 16, 273−284.
Smith, J.; Pedder, C.; Graham, C.; Studeny, A.; Lysiak-Auvity, G.;
Girard, A.-M.; Gravé, F.; Segal, D.; Riffkin, C. D.; Pomilio, G.;
Galbraith, L. C. A.; Aubrey, B. J.; Brennan, M. S.; Herold, M. J.; Chang,
C.; Guasconi, G.; Cauquil, N.; Melchiore, F.; Guigal-Stephan, N.;
Lockhart, B.; Colland, F.; Hickman, J. A.; Roberts, A. W.; Huang, D. C.
S.; Wei, A. H.; Strasser, A.; Lessene, G.; Geneste, O. The MCL1
inhibitor S63845 is tolerable and effective in diverse cancer models.
Nature 2016, 538, 477−482.
(30) Lee, T.; Bian, Z.; Zhao, B.; Hogdal, L. J.; Sensintaffar, J. L.;
Goodwin, C. M.; Belmar, J.; Shaw, S.; Tarr, J. C.; Veerasamy, N.;
Matulis, S. M.; Koss, B.; Fischer, M. A.; Arnold, A. L.; Camper, D. V.;
Browning, C. F.; Rossanese, O. W.; Budhraja, A.; Opferman, J.; Boise, L.
H.; Savona, M. R.; Letai, A.; Olejniczak, E. T.; Fesik, S. W. Discovery
and biological characterization of potent myeloid cell leukemia-1
inhibitors. FEBS Lett. 2017, 591, 240−251.
(31) 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. Structure-guided design of a series of MCL-1 inhibitors
with high affinity and selectivity. J. Med. Chem. 2015, 58, 2180−2194.
(32) Abulwerdi, F.; Liao, C.; Liu, M.; Azmi, A. S.; Aboukameel, A.;
Mady, A. S. A.; Gulappa, T.; Cierpicki, T.; Owens, S.; Zhang, T.; Sun,
D.; Stuckey, J. A.; Mohammad, R. M.; Nikolovska-Coleska, Z. A novel
small-molecule inhibitor of Mcl-1 blocks pancreatic cancer growth in
vitro and in vivo. Mol. Cancer Ther. 2014, 13, 565−575.
(33) Doi, K.; Li, R.; Sung, S.-S.; Wu, H.; Liu, Y.; Manieri, W.;
Krishnegowda, G.; Awwad, A.; Dewey, A.; Liu, X.; Amin, S.; Cheng, C.;
Qin, Y.; Schonbrunn, E.; Daughdrill, G.; Loughran, T. P.; Sebti, S.;
Wang, H.-G. Discovery of marinopyrrole A (Maritoclax) as a selective
Mcl-1 antagonist that overcomes ABT-737 resistence by binding to and
targeting Mcl-1 for proteasomal degradation. J. Biol. Chem. 2012, 287,
10224−10235.
(34) Richard, D. J.; Lena, R.; Bannister, T.; Blake, N.; Pierceall, W. E.;
Carlson, N. E.; Keller, C. E.; Koenig, M.; He, Y.; Minond, D.; Mishra, J.;
Cameron, M.; Spicer, T.; Hodder, P.; Cardone, M. H. Hydroxyquino-
line-derived compounds and analoguing of selective Mcl-1 inhibitors
using a functional biomarker. Bioorg. Med. Chem. 2013, 21, 6642−6649.
(35) Bernardo, P. H.; Sivaraman, T.; Wan, K.-F.; Xu, J.;
Krishnamoorthy, J.; Song, C. M.; Tian, L.; Chin, J. S. F.; Lim, D. S.
W.; Mok, H. Y. K.; Yu, V. C.; Tong, J. C.; Chai, C. L. L. Structural
insights into the design of small molecule inhibitors that selectively
antagonize Mcl-1. J. Med. Chem. 2010, 53, 2314−2318.
(36) Ding, X.; Li, Y.; Lv, L.; Zhou, M.; Han, L.; Zhang, Z.; Ba, Q.; Li, J.;
Wang, H.; Liu, H.; Wang, R. De novo design, synthesis and evaluation
of benzylpiperazine derivatives as highly selective binders of Mcl-1.
ChemMedChem 2013, 8, 1986−2014.
(37) Tron, A. E.; Belmonte, M. A.; Adam, A.; Aquila, B. M.; Boise, L.
H.; Chiarparin, E.; Cidado, J.; Embrey, K. J.; Gangl, E.; Gibbons, F. D.;
Gregory, G. P.; Hargreaves, D.; Hendricks, J. A.; Johannes, J. W.;
Johnstone, R. W.; Kazmirski, S. L.; Kettle, J. G.; Lamb, M. L.; Matulis, S.
M.; Nooka, A. K.; Packer, M. J.; Peng, B.; Rawlins, P. B.; Robbins, D.
W.; Schuller, A. G.; Su, N.; Yang, W.; Ye, Q.; Zheng, X.; Secrist, J. P.;
Clark, E. A.; Wilson, D. M.; Fawell, S. E.; Hird, A. W. Discovery of Mcl-
1-specific inhibitor AZD5991 and preclinical activity in multiple
myeloma and acute myeloid leukemia. Nat. Commun. 2018, 9, 5341.
(38) Yang, C.; Chen, S.; Zhou, M.; Li, Y.; Li, Y.; Zhang, Z.; Liu, Z.; Ba,
Q.; Li, J.; Wang, H.; Yan, X.; Ma, D.; Wang, R. Development of 3-
phenyl-N-(2-(3-phenylureido)ethyl)-thiophene-2-sulfonamide com-
pounds as inhibitors of antiapoptotic Bcl-2 family proteins.
ChemMedChem 2014, 9, 1436−1452.
(39) Zhou, B.; Li, X.; Li, Y.; Xu, Y.; Zhang, Z.; Zhou, M.; Zhang, X.;
Liu, Z.; Zhou, J.; Cao, C.; Yu, B.; Wang, R. Discovery and development
of thiazolo[3,2[a]pyrimidinone derivatives as general inhibitors of Bcl-
2 family proteins. ChemMedChem 2011, 6, 904−921.
(40) Xu, Y.; Zhou, M.; Li, Y.; Li, C.; Zhang, Z.; Yu, B.; Wang, R.
Characterization of the stereochemical structures of 2H-thiazolo[3,2-
(24) Krajewski, S.; Bodrug, S.; Krajewska, M.; Shabaik, A.; Gascoyne,
R.; Berean, K.; Reed, J. C. Immunohistochemical analysis of Mcl-1
protein in human tissues: Differential regulation of Mcl-1 and Bcl-2
protein production suggests a unique role for Mcl-1 in control of
programmed cell death in vivo. Am. J. Pathol. 1995, 146, 1309−1319.
(25) Brunelle, J. K.; Ryan, J.; Yecies, D.; Opferman, J. T.; Letai, A. Mcl-
1-dependent leukemia cells are more sensitive to chemotherapy than
BCL-2-dependent counterparts. J. Cell Biol. 2009, 187, 429−442.
(26) Han, L.; Wang, R. Rise of the selective inhibitors of anti-apoptotic
Bcl-2 family proteins. ChemMedChem 2013, 8, 1437−1440.
(27) Beekman, A. M.; Howell, L. A. Small-molecule and peptide
inhibitors of the pro-survival protein Mcl-1. ChemMedChem 2016, 11,
802−813.
(28) Negi, A.; Murphy, P. V. Development of Mcl-1 inhibitors for
cancer therapy. Eur. J. Med. Chem. 2021, 210, 113038.
(29) Kotschy, A.; Szlavik, Z.; Murray, J.; Davidson, J.; Maragno, A. L.;
Le Toumelin-Braizat, G.; Chanrion, M.; Kelly, G. L.; Gong, J.-N.;
Moujalled, D. M.; Bruno, A.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos,
S.; Radics, G.; Proszenyak, A.; Balint, B.; Ondi, L.; Blasko, G.;
Robertson, A.; Surgenor, A.; Dokurno, P.; Chen, I.; Matassova, N.;
10284
https://doi.org/10.1021/acs.jmedchem.1c00690
J. Med. Chem. 2021, 64, 10260−10285

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
a]pyrimidine compounds and their binding affinities for anti-apoptotic
Bcl-2 family proteins. ChemMedChem 2013, 8, 1345−1352.
(41) Masters, M. R.; Mahmoud, A. H.; Yang, Y.; Lill, M. A. Efficient
and accurate hydration site profiling for enclosed binding sites. J. Chem.
Inf. Model. 2018, 58, 2183−2188.
(42) Petros, A. M.; Medek, A.; Nettesheim, D. G.; Kim, D. H.; Yoon,
H. S.; Swift, K.; Matayoshi, E. D.; Oltersdorf, T.; Fesik, S. W. Solution
structure of the antiapoptotic protein bcl-2. Proc. Natl. Acad. Sci. U.S.A.
2001, 98, 3012−3017.
(43) Czabotar, P. E.; Lee, E. F.; van Delft, M. F.; Day, C. L.; Smith, B.
J.; Huang, D. C. S.; Fairlie, W. D.; Hinds, M. G.; Colman, P. M.
Structural insights into the degradation of Mcl-1 induced by BH3
domains. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 6217−6222.
(44) Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.; Tomita, Y.;
Li, P.; Roller, P. P.; Krajewski, K.; Saito, N. G.; Stuckey, J. A.; Wang, S.
Development and optimization of a binding assay for the XIAP BIR3
domain using fluorescence polarization. Anal. Biochem. 2004, 332,
261−273.
(45) Jones, G.; Willett, P.; Glen, R. C. Molecular recognition of
receptor sites using a genetic algorithm with a description of
desolvation. J. Mol. Biol. 1995, 245, 43−53.
(46) Clifton, M. C.; Dranow, D. M.; Leed, A.; Fulroth, B.; Fairman, J.
W.; Abendroth, J.; Atkins, K. A.; Wallace, E.; Fan, D.; Xu, G.; Ni, Z. J.;
Daniels, D.; Van Drie, J.; Wei, G.; Burgin, A. B.; Golub, T. R.; Hubbard,
B. K.; Serrano-Wu, M. H. A maltose-binding protein fusion construct
yields a robust crystallography platform for MCL1. PLoS One 2015, 10,
No. e0125010.
(47) Korb, O.; Stutzle, T.; Exner, T. E. Empirical scoring functions for
̈
advanced protein-ligand docking with PLANTS. J. Chem. Inf. Model.
2009, 49, 84−96.
(48) Case, D. A.; Babin, V.; Berryman, J. T.; Betz, R. M.; Cai, Q.;
Cerutti, D. S.; Cheatham, T. E.; Darden, T. A.; Duke, R. E.; Gohlke, H.;
Goetz, A. W.; Gusarov, S.; Homeyer, N.; Janowski, P.; Kaus, J.;
Kolossvary, I.; Kovalenko, A.; Lee, T. S.; LeGrand, S.; Luchko, T.; Luo,
R.; Madej, B.; Merz, K. M.; Paesani, F.; Roe, D. R.; Roitberg, A.; Sagui,
C.; Salomon-Ferrer, R.; Seabra, G.; Simmerling, C. L.; Smith, W.;
Swails, J.; Walker, R. C.; Wang, J.; Wolf, R. M.; Wu, X.; Kollman, P. A.
AMBER 14; University of Carlifornia: San Francisco, 2014.
(49) Word, J. M.; Lovell, S. C.; Richardson, J. S.; Richardson, D. C.
Asparagine and glutamine: using hydrogen atom contacts in the choice
of side-chain amide orientation. J. Mol. Biol. 1999, 285, 1735−1747.
(50) Hess, B.; Kutzner, C.; van der Spoel, D.; Lindahl, E. GROMACS
4: algorithms for highly efficient, load-balanced, and scalable molecular
simulation. J. Chem. Theory Comput. 2008, 4, 435−447.
10285
https://doi.org/10.1021/acs.jmedchem.1c00690
J. Med. Chem. 2021, 64, 10260−10285