Discovery of 3,5-Dimethyl-4-Sulfonyl-1 H-Pyrrole-Based Myeloid Cell Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral Bioavailability

Article abstract of DOI:10.1021/acs.jmedchem.1c00682

Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer therapy. Herein, we describe the rational design, synthesis, and structure-activity relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1 inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition (52%@30 μM) led to the discovery of the most potent compound 40 with high affinity (Kd = 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds). Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-1-dependent manner. 40 exhibited favorable physicochemical properties and pharmacokinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively, these findings implicate that compound 40 is a promising antitumor agent that deserves further preclinical evaluations.

Full text of DOI:10.1021/acs.jmedchem.1c00682

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Article
Discovery of 3,5-Dimethyl-4-Sulfonyl‑1H‑Pyrrole-Based Myeloid Cell
Leukemia 1 Inhibitors with High Affinity, Selectivity, and Oral
Bioavailability
Peng-Ju Zhu, Ze-Zhou Yu, Yi-Fei Lv, Jing-Long Zhao, Yuan-Yuan Tong, Qi-Dong You,*
and Zheng-Yu Jiang*
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ABSTRACT: Myeloid cell leukemia 1 (Mcl-1) protein is a key negative regulator of
apoptosis, and developing Mcl-1 inhibitors has been an attractive strategy for cancer
therapy. Herein, we describe the rational design, synthesis, and structure−activity
relationship study of 3,5-dimethyl-4-sulfonyl-1H-pyrrole-based compounds as Mcl-1
inhibitors. Stepwise optimizations of hit compound 11 with primary Mcl-1 inhibition
(52%@30 μM) led to the discovery of the most potent compound 40 with high affinity (Kd
= 0.23 nM) and superior selectivity over other Bcl-2 family proteins (>40,000 folds).
Mechanistic studies revealed that 40 could activate the apoptosis signal pathway in an Mcl-
1-dependent manner. 40 exhibited favorable physicochemical properties and pharmaco-
kinetic profiles (F% = 41.3%). Furthermore, oral administration of 40 was well tolerated to
effectively inhibit tumor growth (T/C = 37.3%) in MV4-11 xenograft models. Collectively,
these findings implicate that compound 40 is a promising antitumor agent that deserves
further preclinical evaluations.
chronic lymphocytic leukemia (CLL), and targeting Bcl-2 family
proteins proves to be a practical strategy for cancer therapy.
Mcl-1 has been identified as the largest member with 350
amino acid residues among anti-apoptotic proteins.14 Of note,
Mcl-1 also differs from other anti-apoptotic members in that it
possesses four PEST domains composed of proline (P),
glutamate (E), serine (S), and threonine (T) residues.23 Such
domains are targets of E3 ubiquitin-ligases, which promote the
degradation of Mcl-1.24 The binding groove of Mcl-1 is
composed of P1−P4 pockets. In the binding mode of Mcl-1
and Bim, four conserved hydrophobic residues h1−h4 (Ile58,
Leu62, Ile65, and Phe69) of the Bim BH3 domain protrude into
four hydrophobic pockets (P1−P4) of the Mcl-1 (Figure 1)
binding groove, respectively.25 Additionally, there is a salt-
bridge interaction between Asp67 of the Bim BH3 domain and
Arg263 of Mcl-1.26 Small molecules that mimic the hot spots of
BH3-only proteins can occupy the BH3 binding groove of Mcl-
1, which relieves pro-apoptotic proteins to induce tumor cell
apoptosis.27 Therefore, developing BH3 mimetic Mcl-1
inhibitors is a promising strategy for cancer therapy.28−30
INTRODUCTION
■
The evasion of apoptosis is one of the main reasons that results
in cancer.1 B-cell lymphoma-2 (Bcl-2) family proteins,
composed of pro-apoptotic proteins (Bax and Bak), anti-
apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1), and
BH3-only proteins (Bid, Noxa, and Puma),2 have long been
identified as pivotal apoptosis regulators.3 In apoptosis signaling,
dimerization of pro-apoptotic members Bax and Bak can form
oligomers to depolarize the outer mitochondrial membrane
(OMM),4 which can release mitochondrial proteins to induce
apoptosis.5 Mcl-1 was first found by analyzing gene expression of
the ML-1 human myeloid leukemia cell line.6 Like other anti-
apoptotic members, Mcl-1 contains a binding groove compris-
ing Bcl-2 homology (BH) 1-3 domains.7 Such a groove can bind
to the BH3 domains of pro-apoptotic members,8 which disturbs
the formation of Bax/Bak homo/heterodimers and further
inhibits apoptosis.9,10 Mcl-1 is important for the survival and
development of normal cells, such as embryogenesis,11
maintenance of hepatocytes,12 B and T-cells,13 and neuron
development.14 However, overexpression of Mcl-1 has been
widely observed in cancers, such as chronic myeloid leukemia
(CML),15 multiple myeloma (MM),16 non-small cell lung
cancer (NSCLC),17 and colon cancer.18 There is also evidence
showing that Mcl-1 is closely associated with cancer relapse,19
immortalization,20 and drug resistance.21 Therefore, targeting
Mcl-1 is an attractive strategy for cancer therapy. In 2016,
venetoclax (Bcl-2 selective inhibitor) reported by AbbVie22 was
approved by the FDA for the treatment of 17p gene deficiency
Received: April 14, 2021
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the first orally efficacious Mcl-1 inhibitor AMG176 (6) with a
macrocyclic structure. AMG176 has also been promoted into
the clinical trial (NCT02675452). Apart from that, compounds
with other skeletons have also been identified as potent Mcl-1
inhibitors.61−66 Geneste’s group developed thieno[2,3-d]-
pyrimidine-based Mcl-1 inhibitors S63845 (7)67 and MIK665
(8)68 with excellent in vivo potency. To date, the phase I clinical
trial of MIK665 has been completed (NCT02979366).
However, most Mcl-1 inhibitors suffer druggability issues like
poor membrane permeability,47 low oral bioavailability,38 and
big molecular weight. Such problems hinder potent Mcl-1
inhibitors from coming into clinical trials and further from being
approved for cancer therapy. As pan Bcl-2 inhibition leads to
undesired side effects,69 selectivity against Mcl-1 is another issue
that deserves consideration. Herein, we describe the design,
synthesis, and structure−activity relationships (SARs) of a series
of pyrrole-based Mcl-1 inhibitors. Replacing the indole core of 2
with a pyrrole ring yielded hit compound 11 with primary Mcl-1
inhibitory potency (52%@30 μM). Further optimization of 11
led to the discovery of the most potent compound 40 (Mcl-1, Ki
= 20 nM). 40 possessed high Mcl-1 binding affinity (Kd = 0.23
nM) and remarkable selectivity over other Bcl-2 family members
(Bcl-2, Bcl2A1, Bcl-xL, and Bcl-w, Kd > 10,000 nM). In addition,
40 exhibited ideal cellular activity and oral bioavailability (F% =
41.3%). In balb/c nude mice loaded with MV4-11 xenografts, 40
showed desired antitumor effects through oral administration
(T/C = 37.3%).
Figure 1. Binding mode of Mcl-1 and Bim BH3 (PDB 2NL9).
The large and shallow binding groove makes Mcl-1
intrinsically difficult to be targeted.31 Several groups have
developed peptide Mcl-1 inhibitors.32−35 However, during the
last decade, medicinal chemists have contributed more to
developing Mcl-1 small molecule inhibitors.36−38 As shown in
Figure 2, Zhang’s team has reported a series of phenalene-based
Mcl-1 inhibitors,39−41 of which the most potent 1 showed a Ki of
5 nM.42 Indole-based Mcl-1 inhibitors have also been
investigated in depth. In 2008, Abbott’s work pioneered the
discovery of indole Mcl-1 inhibitors.43,44 In 2013, Fesik’s team
discovered an indole-based Mcl-1 inhibitor 2 by using the
fragment merging method.45 Further modifications46−48 led to
VU661013 (3)49 with an ideal in vivo antitumor efficacy.50 In
2015, AbbVie identified A1210477 (4) as an indole-based Mcl-
1 inhibitor,51,52 which is widely used as an in vitro chemical
tool.27,53 Based on A1210477, some other Mcl-1 inhibitors with
an indole core were developed.54,55 Recently, the macro-
cyclization strategy has been widely used in the discovery of
Mcl-1 inhibitors.56 In 2018, AstraZeneca reported the macro-
cyclic clinical candidate AZD5991 (5)57 (NCT03218683) with
excellent antitumor efficacy.58,59 In 2018, Amgen60 discovered
RESULTS AND DISCUSSION
■
Discovery of Initial Hit 11 as an Mcl-1 Inhibitor. In 2013,
through the fragment-based drug design, Fesik’s team45
reported a series of indole-based Mcl-1 inhibitors, of which
the most potent compound 2 showed nanomolar Mcl-1
inhibitory potency (Ki = 55 nM) and proper selectivity over
Bcl-2 (Ki = 870 nM) and Bcl-xL (Ki > 15,000 nM). The deep P2
pocket and polar Arg263 differ the binding groove of Mcl-1 from
that of other Bcl-2 family members.45,70 As shown in Figure 3A,
Figure 2. Representative Mcl-1 inhibitors.
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Figure 3. Design of lead compound 11. (A) Binding mode of compound 2 (PDB 4HW2). (B) SAR study of the linker substitution position. (C)
Binding mode of 10 (PDB 6B4U) and design of 11.
the complex of 2/Mcl-1 (PDB 4HW2) shows that the 4-atom
linker of 2 protrudes into the P2 pocket and the indole-2-
carboxyl acid group forms key polar interaction with Arg263. In
their SAR study, tethering a 4-atom linker to the NH of the
indole core (9b) retained the Mcl-1 inhibitory activity of the
corresponding 3-substituted indole compound (9a) (Figure
3B). Compound 10 was reported to bind to Mcl-1 with high
affinity.51 The binding mode of 10 (PDB 6B4U) demonstrates
that the morpholine ring is solvent-exposed and the phenyl ring
is kept out of the P2 pocket due to the rigid indole core.
Combining these findings, in our design, the pyrrole-2-
carboxylic acid group was kept for maintaining the key polar
interaction with Agr263. The 3,5-dimethyl-4-chlorine phenyl-
linked 4-atom linker has been fully reported to be suitable for P2
pocket binding.47,49 Consequently, this moiety was kept and
attached to the NH group of the pyrrole core to occupy the P2
pocket. For locating the solvent-exposed phenyl ring of 10 in the
P2 pocket, a flexible methylene linker was introduced between
the 4-methyl phenyl group and the pyrrole core. Compound 11
was then designed and prepared with a moderate Mcl-1
inhibition ratio (52%@30 μM) determined by a fluorescence
polarization (FP)-based assay (Figure 3C).
Table 1. Effects of Linkers on Mcl-1 Inhibition
Optimizations of the Linker between the 4-Methyl
Phenyl Group and the Pyrrole Core. According to our
design, the linker between the 4-methyl phenyl group and the
pyrrole core was vital for locating the phenyl group into the P2
pocket. To explore a preferable linker for potency, 12−15 with
various linkers were prepared. The FP assay was used to evaluate
the Mcl-1 inhibitory activities of our compounds. AZD5991 and
A1210477 were selected as positive controls. As shown in Table
1, the introduction of the rigid carbonyl linker (12) and the
sulfone linker (13) improved Mcl-1 inhibitory activity.
However, two-atom linkers (14, 15) showed abolished potency.
The primary SAR results indicated that the conformation and
length of the sulfone linker were the most favorable. As a result,
compound 13 with a sulfone linker was selected for further
modifications.
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Then, we investigated the binding mode of 13 with Mcl-1 by
molecular docking. As shown in Figure 4, as expected, the
Table 2. Effects of Pyrrole Substituents on Mcl-1 Inhibition
Figure 4. Proposed binding mode of compound 13 with Mcl-1 using
molecular docking. The docking site was derived from the position of
the small-molecular ligand cocrystallized in the binding site of Mcl-1
(PDB 4HW2). The ligand is represented as sticks. The hydrogen bonds
are represented by green dashed lines, and the π−σ and π−alkyl
interactions are represented by pink dashed lines. The carbon atoms of
small molecules and Mcl-1 residues are colored cyan and green,
respectively.
pyrrole-2-carboxyl group formed a hydrogen bond with Arg263.
The 3,5-dimethyl-4-chlorine phenyl-linked 4-atom linker
protruded into the P2 pocket. The docking study of 13 also
gave the reason for the advantage of the sulfone linker for
potency: the sulfone linker located the phenyl ring into the P2
pocket. Consequently, the phenyl ring could form additional
hydrophobic interactions with Ala227 and Met231.
Reinspection of the binding mode of 13 gave an idea that
substitution at the 5-position of the pyrrole core could form
more interactions in the P2 pocket (Figure 4). 3,5-Dimethyl
pyrrole-based compound 16 was then prepared. As shown in
Table 2, 16 showed better performance compared to 13.
However, 3,5-diethyl pyrrole-based compound 17 was not
preferred. Only replacement of the 5-methyl group of 16 with an
ethyl group (18) impaired potency. In contrast, replacement of
the 3-methyl group of 16 with an ethyl group (19) retained
comparable activity with 16. Both removal of 5-methyl
substitution at the pyrrole core (20) and further extension at
the 5-position (21) were unfavorable. These results demon-
strated that the appropriate size of 5-substitution was important
for Mcl-1 inhibition and the methyl substituent was optimal.
Further extension at the 3-position of 19 (22) retained potency.
To further confirm the advantage of the sulfone linker for
activity, analogues 23 with a flexible sulfide linker and 24 with a
2-atom length linker were prepared. As shown in Table 2, the
sulfide linker (23) showed a minor decreased potency compared
to the sulfone linker (16). Introduction of a 2-atom linker (24)
remarkably impaired Mcl-1 inhibition.
Then, the binding mode of 16 was elucidated by the docking
study. As shown in Figure 5, The 5-methyl of the pyrrole core
inserted into a narrow cavity and formed additional hydrophobic
interactions with Phe228, Met231, and Phe270 in the P2 pocket,
which led to an improvement in activity.
Effects of Ring Transformation at the Sulfone Linker
on Mcl-1 Inhibition. Then, we evaluated the effects of various
rings at the sulfone linker on Mcl-1 inhibitory activity. Replacing
the phenyl ring with more polar pyridyl rings (25, 26) exhibited
a more than 10-fold decreased potency. The thienyl ring (27)
showed improved activity, while the bicyclic naphthyl ring (28)
Figure 5. Proposed binding mode of 16 with Mcl-1 using molecular
docking. The docking site was derived from the position of the small-
molecular ligand cocrystallized in the binding site of Mcl-1 (PDB
4HW2). The ligand is represented as sticks. The hydrogen bonds are
represented by green dashed lines, and the π−σ and π−alkyl
interactions are represented by pink dashed lines. The carbon atoms
of small molecules and Mcl-1 residues are colored cyan and green,
respectively.
showed weaker activity compared to 16. The cycloalkane
analogs are inactive (29, 30), which suggested that the π-system
of the rings attached to the sulfone linker was necessary for Mcl-
1 inhibition (Table 3).
In addition to Mcl-1 inhibition activity, we also predicted
drug-like properties of all compounds by ADMET 10.0 software
for avoiding potential druggability issues (Supporting Informa-
tion, Table S1). Most compounds possessed proper predicated
solubility, absorption property, and CYP inhibition profiles.
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38 with 3-Br substitution exhibiting improved inhibitory
activity. Potency was improved by substitutions at the 4-
position, as exemplified by compounds 40−43. The most potent
compounds, 40 with 4-acetyl substitution and 43 with 4-OH
substitution, showed about 10-fold improved potency.
Table 3. SAR Study Focused on Rings on the Sulfone Linker
Compounds with various groups at the 4-position were then
prepared (44−55). Generally, as shown in Table 4, the phenyl
ring can tolerate a variety of substituents at the 4-position. 44
with 4-ethyl substitution exhibited a minor increased potency
over 31. Meanwhile, disubstitution on the phenyl ring led to
compounds 56−57 with abolished activity.
The most potent compounds of this set, 40 and 43, possessed
suitable predicated ADME properties, except the poor
predicated membrane permeability of 43 (Supporting Informa-
tion, Table S1).
The binding modes of 40 and 43 were investigated by
docking. As depicted in Figure 6, compared with 16, 40 and 43
possessed similar binding modes. In addition, the 4-acetyl group
of 40 formed a hydrogen bond with Lys234 and the 4-OH of 43
formed a hydrogen bond with Ala227. The hydrogen bonds may
contribute to the 10-fold improved Ki of both compounds.
In Vitro Selectivity Evaluation. Representative com-
pounds 27, 40, 41, 42, 43, and 44 were selected for testing
their selectivity over other Bcl-2 family members (Bcl-2, Bcl-xL,
and Bfl-1) by an FP-based assay or time-resolved fluorescence
resonance energy transfer (TR-FRET) based assay. The results
implicated that all compounds showed promising selectivity as
no significant inhibition against other Bcl-2 family members was
observed at a concentration of 10 μM (Table 5). 40 was selected
for the binding selectivity assay by DiscoverX’s Bcl-2scan
technology.71 As shown in Figure 7, 40 possessed high Mcl-1
binding affinity with a Kd of 0.23 nM, whereas no binding was
observed against Bcl-2, Bcl-xL, Bcl-w, and Bcl2A1 (Kd ≥ 10,000
Thus, the detailed SAR study of the substituted phenyl ring was
carried out to enhance the activity.
Modifications on the Substituents of the Phenyl Ring.
First, we compared the substituent position effects of the phenyl
ring. A library of compounds (31−43) with different
substitution patterns on the phenyl ring was prepared. As
shown in Table 4, 31 with a non-substituted phenyl ring
possessed similar potency to 4-methyl-substituted 16. Sub-
stitutions at the 2-position and 3-position (32−37 and 39)
yielded similar or decreased potency compared to 31 except for
Table 4. SAR Study Focused on Substitution on the Phenyl Ring
cpd
R
Ki SE (μM)
cpd
R
Ki SE (μM)
16
31
32
33
34
35
36
37
38
39
40
41
42
43
44
4-CH3
H
2-COCH3
2-OCH3
2-Br
0.17 0.04
0.19 0.03
0.50 0.13
0.43 0.12
>10
0.15 0.02
0.55 0.16
0.20 0.05
0.060 0.013
0.15 0.04
0.020 0.001
0.070 0.010
0.040 0.005
0.018 0.002
0.070 0.020
45
46
47
48
49
50
51
52
53
54
55
56
57
4-isopropyl
4-tert-butyl
4-OEt
4-Ph
4-COPh
4-OPh
4-F
4-Cl
4-NO2
4-COOH
4-NH2
0.17 0.03
0.34 0.09
0.14 0.03
0.13 0.03
0.38 0.05
0.20 0.03
0.26 0.10
0.18 0.03
0.42 0.08
0.42 0.07
0.34 0.06
>10
2-OH
3-COCH3
3-OCH3
3-Br
3-OH
4-COCH3
4-OCH3
4-Br
4-OH
4-ethyl
2,6-diMe
3,5-diMe
>10
<0.001
0.006 0.001
AZD5991
A1210477
E
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Figure 8. Bcl-2 could not accommodate 40 due to the shallow and
narrow P2 pocket. (A) Overlay of compound 2 (green) and 40
(purple). (B) Comparison of P2 pockets of Mcl-1 and Bcl-2.
Figure 6. Purposed binding modes of 40 (A) and 43 (B) with Mcl-1
using molecular docking. The docking site was derived from the
position of the small-molecular ligand cocrystallized in the binding site
of Mcl-1 (PDB 4HW2). The ligand is represented as sticks. Hydrogen
bonds are represented by green dashed lines, and π−σ and π−alkyl
interactions are represented by pink dashed lines. The carbon atoms of
small molecules and Mcl-1 residues are colored purple and green,
respectively.
In Vitro Anticancer Effects. After confirming the Mcl-1-
targeting ability in vitro, 27, 40, 41, 42, 43, and 44 were then
tested for their antiproliferative activities against the tumor cell
lines sensitive to Mcl-1 inhibitors (H929, MV4-11, SK-BR-3,
and NCI-H23)53,67,72 and the insensitive K562 cell line49,53
through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazo-
lium bromide colourimetric (MTT) assay. A1210477 and
AZD5991 were selected as positive controls. As shown in Table
6, all compounds showed submicromolar activities against the
MM cell line H929. 27, 40, 41, 42, and 44 showed better in vitro
antitumor efficacy compared to the known Mcl-1 inhibitor
A1210477. In spite of the high Mcl-1 inhibitory activity, 43 did
not perform that potency against cancer cell lines in vitro. The
result may attribute to its poor permeability resulted by 4-OH.
In the acute myeloid leukemia (AML) cell line MV4-11, all
compounds showed decreased activities while 40 retained the
submicromolar potency. Decreased potencies were observed in
breast cancer cell line SK-BR-3 and NSCLC cell line NCI-H23
of all compounds, which was in line with other Mcl-1 inhibitors’
performance in solid cancer cell lines.67,68 40, 41, 42, and 44
showed ideal selectivity against CML cell line K562 (IC50 > 30
μM).
40 Effectively Induced Apoptosis in an Mcl-1-Depend-
ent Manner. As 40 showed ideal target affinity and
antiproliferative activities, it was selected for further biological
evaluation. A coimmunoprecipitation (Co-ip) experiment was
performed to evaluate the on-target Mcl-1 inhibition of 40 in
living cells. H929 cells were treated with increasing concen-
trations of 40 or positive control AZD5991 (0.05 μM),
respectively. The Mcl-1-Bak complex was monitored by western
blot (WB) analysis. The results (Figure 9A) demonstrated that
Table 5. In Vitro Selectivity Evaluation
a
b
a
cpd
Mcl-1 (Ki SE, μM)
Bcl-2
7.54
Bcl-xL
Bfl-1
27
40
41
42
43
44
0.080 0.020
0.020 0.001
0.070 0.010
0.040 0.005
0.018 0.002
0.070 0.020
37
NA
27
13
NA
36
NA
10.00
14.16
11.64
NA
1.57
1.82
4.14
NA
12.14
8.77
a
b
FP, inhibition@10 μM (%). TR-FRET, inhibition@10 μM (%).
nM). 40 exhibited hugely improved selectivity (>40,000 folds)
over Bcl-2 compared to compound 2 (Mcl-1, Ki = 55 nM; Bcl-2,
Ki = 870 nM). The remarkably improved selectivity of 40 could
be illustrated by the overlay of 40 and 2 (Figure 8A). The
sulfone linker constrained the angle between the phenyl group
and the pyrrole core. As a result, the 4-(phenylsulfonyl)-1H-
pyrrole core of 40 occupied the entire upper part of the P2
pocket. Also, the proper angle forced the 4-atom linker of 40 to
insert into the lower part of Mcl-1’s P2 pocket deeper than that
of 2. The P2 pocket of Bcl-2 was narrower and shallower than
that of Mcl-1 (Figure 8B). Consequently, the P2 pocket of Bcl-2
could hardly accommodate 40, which led to the high selectivity
of 40.
Figure 7. Binding affinities of 40 to Mcl-1 and other Bcl-2 family members.
F
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a
Table 6. Antiproliferative Activities against Different Cell Lines
cpd
H929
MV4-11
SK-BR-3
NCI-H23
K562
27
40
41
42
43
44
0.70 0.12
0.36 0.09
0.39 0.09
0.48 0.17
1.39 0.31
0.49 0.05
0.90 0.29
0.036 0.008
2.30 0.45
0.70 0.07
1.56 0.24
1.70 0.63
2.02 0.28
2.59 0.31
1.55 0.47
0.024 0.006
5.13 0.66
2.84 0.66
6.27 1.89
5.60 1.21
7.02 1.07
3.92 1.65
4.31 1.29
>30
3.10 1.73
3.02 1.35
9.72 2.33
3.84 1.24
2.16 0.75
5.41 1.26
13.5 2.81
0.19 0.04
11.3 1.76
>30
>30
>30
14.8 3.07
>30
A1210477
AZD5991
>30
29.6 5.3
a
Values shown are the mean IC50 SD (μM, n = 3).
40 could displace Bak protein from Mcl-1 in a concentration-
dependent manner. To verify if compound 40 could induce
apoptosis in cells, an annexin-V and propidium iodide (PI)
double staining assay was applied in H929 cells at concen-
trations of 1 and 5 μM. As shown in Figure 9B−G, 40 and
positive control A1210477 significantly induced apoptosis in a
concentration-dependent manner. Apoptosis ratio analysis
demonstrated that 40 was more potent in inducing apoptosis
than A1210477. To further prove that 40 could activate
apoptosis signaling, effects of 40 on the poly ADP-ribose
polymerase (PARP) cleavage and caspase activation were
evaluated in H929 cells and also in K562 cells to assess off-
target effects. Both cell lines were incubated with 40 and
A1210477 at concentrations of 0, 0.1, 0.5, 1, and 5 μM. The
amount of cleaved PARP (cPARP) was evaluated by the WB
assay. As shown in Figure 9H,I, 40 and A1210477 remarkably
upregulated PARP cleavage in H929 cells in a concentration-
dependent manner. However, the same treatment in K562 cells
did not induce comparable PARP cleavage. In caspase activation
assay, K562 cells and H929 cells were exposed to 40 and
A1210477 at indicated concentrations for 4 h and the caspase
activation folds were determined by caspase assay kits. As shown
in Figure 9J,K, 40 concentration-dependently activated caspase
3/7 in H929 cells, which is more potent than A1210477.
However, caspase activation was not observed in K562 cells after
the same treatment of 40 and A1210477. Collectively, these
results showed that 40 activated the apoptosis in an Mcl-1-
dependent manner.
Drug-Like Properties Evaluation of 40. After confirming
that 40 possessed desired cellular activity, drug-like properties
evaluations were carried out. As shown in Table 7, 40 showed
favorable water solubility and permeability in the parallel
artificial membrane permeability assay (PAMPA). CYP
inhibition determination showed that 40 had no pronounced
inhibition against five major isozymes at a concentration of 10
μM (Table 7). In further evaluations, 40 exhibited acceptable
stability in simulated intestinal fluid (SIF), simulated gastric
fluid (SGF), mouse and human plasma, and rat and human liver
microsomes (RLM and HLM) (Table 8). In the Caco-2 cell
model, 40 showed moderate permeability and no significant
efflux was observed. In addition, 40 displayed no hERG channel
inhibition (Table 8 and Supporting Information, Figure S1). In
vivo pharmacokinetic (PK) profiles of 40 were evaluated in SD
rats. Intravenous (iv) administration of 40 at a dose of 3 mg/kg
showed an acceptable half-life of 2.3 h. Of note, 40 showed
favorable bioavailability (F% = 41.3%) by oral (po) admin-
istration (Figure 10). Taken together, 40 exhibited good
druglike properties.
mice loaded with MV4-11 xenograft. The clinical candidate
AZD5991 was selected as a positive control. After po
administration of 40 (n = 6, 60 mpk) and intraperitoneal (ip)
administration of 40 (n = 6, 20 mpk) and AZD5991 (n = 6, 10
mpk) every two days for 14 days, as shown in Figure 11A and
Figure S2, po administration of 40 showed desired in vivo tumor
growth inhibition activity (T/C = 37.30%) and ip admin-
istration of 40 shared equal antitumor activity (T/C = 5.52%)
with positive control AZD5991 (T/C = 5.18%). Moreover, the
administration of 40 did not induce significant changes in body
weight and organ weight (Figure 11B,C). Hematoxylin−eosin
(H&E) staining revealed that 40 could induce necrosis of MV4-
11 tumor tissues but showed no effect on hearts, livers, or
kidneys (Figure 11D). To ascertain the apoptosis pathway
activation of 40 in vivo, the WB assay was used to evaluate PARP
cleavage in tumor biopsies of each group. As shown in Figure
11E, compared with the vehicle group, administration of 40 and
AZD5991 could activate cleavage of PARP. Consistent with
antitumor efficacy and WB results, increased TUNEL staining
was observed in isolated tumors of treated groups (Figure 11F
and Supporting Information, Figure S3). These results
demonstrated that 40 was well tolerated and orally efficacious
in the MV4-11 xenograft model.
Chemistry. Target compounds 11 and 12 were synthesized
according to Scheme 1. In the presence of K2CO3, the reaction
of commercially available 4-chloro-3,5-dimethylphenol (58)
with 1,3-dibromopropane by nucleophilic substitution yielded
intermediate 59. With AlCl3 as the Lewis acid, the Friedel−
Crafts acylation reaction of starting material methyl 1H-pyrrole-
2-carboxylate 60 and 4-methylbenzoyl chloride in DCM gave
intermediate 61. Carbonyl reduction of 61 by triethylsilane in
THF under nitrogen yielded intermediate 62. In the presence of
Cs2CO3, 61 and 62 reacted with 59 to obtain 64 and 63 in DMF.
Final hydrolysis of 63 and 64 by NaOH in THF/MeOH gave
target compounds 11 and 12.
Target compound 13 and analogue 20 were synthesized
according to Scheme 2 from commercially available 65a-b. With
Ag2CO3 as the catalyst, in the presence of methyl isocyanate,
silver-catalyzed isocyanide-alkyne cycloaddition of 65a-b
yielded 66a-b in 1,4-dioxane.73 Subsequently, in the presence
of NaH, the electrophilic substitution reaction of 66a-b and 59
gave 67a-b. Methyl ester groups of 67a-b were hydrolyzed by
NaOH (2 M) in MeOH/THF to yield target compounds 13 and
20.
Target compounds 14 and 15 were synthesized according to
Scheme 3. In the presence of Cs2CO3, starting material ethyl 4-
nitro-1H-pyrrole-2-carboxylate 68 reacted with 59 to give 69.
The nitro group of 69 was reduced to amino group by tin(II)
dichloride dehydrate (SnCl2·2H2O) in EtOH and subsequent
sulfonylation with TosCl gave intermediate 70. Hydrolysis of 70
In Vivo Antitumor Efficacy of 40. Then, in vivo antitumor
efficacy evaluation of 40 was carried out on balb/c nude female
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Figure 9. 40 induced apoptosis in an Mcl-1-dependent manner. (A) Co-ip experiment of 40 and AZD5991 in H929 cells. (B−G) Flow cytometry
assay of 40 and A1210477 in H929 cells at concentrations of 1 μM and 5 μM. (H,I) WB assay of 40 and A1210477 in H929 cells and K562 cells at
concentrations of 0, 0.1, 0.5, 1, and 5 μM. (J,K) Caspase activation abilities of 40 and A1210477 in H929 cells and K562 cells at concentrations of 0,
0.1, 0.5, 1, and 5 μM. The values shown are the means SEM (n = 3 independent observations), ns = no significant, ***p < 0.001, ****p < 0.0001 one-
way ANOVA with Tukey−Kramer posttest.
by NaOH (2 M) in MeOH/THF gave target compound 14.
Nitro reduction of 69 by SnCl2 and subsequent tert-
butyloxycarbonyl (Boc) protection of the amino group gave
compound 71. In the presence of NaH, the electrophilic
substitution reaction of 71 and 4-methylbenzyl bromide yielded
72. Subsequent deprotection of the Boc group by CF3COOH
gave 73 and hydrolysis of 73 yielded target compound 15.
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a
Target compounds 17−19 and 21−22 were synthesized
according to Scheme 5. Starting material diethyl malonate 78
was first converted to diethyl oximinomalonate (79) through a
reported method,74 and then in the presence of Zn, 79 reacted
with diethyl oxalate 80a to give intermediate 81a. The reaction
of 79 and 80b or 80c yielded 81b-c or 81d-e. Further reaction
with p-toluenethiol, nucleophilic substitution with 59, oxidation
by m-CPBA, and hydrolysis were as depicted in Scheme 4, which
gave target compounds 17−19 and 21 and 22.
Target compounds 27 and 28 were synthesized according to
Scheme 6. With AlCl3 as the Lewis acid, Friedel−Crafts
acylation of the starting material ethyl 3,5-dimethyl-1H-
pyrrole-2-carboxylate and 2-thiophenesulfonyl chloride (85a)
or 1-naphthalenesulfonyl chloride (85b) in PhCl gave
intermediates 86a-b. Further nucleophilic substitution with 59
and hydrolysis of 87a-b were the same as depicted in Scheme 4,
which yielded target compounds 27 and 28.
Table 7. PAMPA Permeability Data , Solubility, and CYP450
Isozyme Inhibition Assay of 40
b
parameters
40
PAMPA permeability
solubility (μg/mL)
52.6 1.10
32.3
CYP450 isozymes: 1A2, 2C9, 2C19, Inhibition rates (%): 19.30, 26.04, 2.85,
2D6, 3A4
9.13, 29.36
a
Average of two runs
standard deviation (SD). PAMPA
permeability values were determined at pH 7.4 (10−6 cm/s).
b
Inhibitory rates (%) against the CYP450 enzymes at a concentration
of 10 μM. 7-ethoxycoumarin, sulfaphenazole, omeprazole, prom-
ethazine, and fluconazole were positive controls for CYP1A2,
CYP2C9, CYP2C19, CYP2D6, and CYP3A4, respectively
a
a
Table 8. Stability in SIF , SGF , Mouse, and Human
Plasma , in Vitro Metabolic Stability in RLM and HLM,
Caco-2 Data, and hERG Channel Inhibitory Activity of 40
b
parameters
40
The target compounds 32, 35, 36, 39, 40, 43, 48−50, and 54
were synthesized according to Scheme 7. With CuSO4·5H2O as
a catalyst, aryl iodides 88a−j reacted with ethanedithiol to give
aryl thiols 89a−j in the presence of Cs2CO3 in DMSO under
nitrogen. Further coupling with 59, sulfide oxidation, and
hydrolysis were the same as depicted in Scheme 4, which yielded
target compounds 32, 36, 40, 48−50, and 54 and intermediates
93h−j. Deprotection of the benzyl group of 93h−j by Pd/C and
hydrogen in EtOAc yielded target compounds 35, 39, and 43.
SIF
SGF
mouse plasma
human plasma
85.19
84.50
100.00
96.50
0.75
31.40
58.11
0.83
67.50
64.00
36.2
0.1
> 30
RLM T1/2 (hr)
RLM Clint (μL/min/mg)
RLM 30 min remaining (%)
HLM T1/2 (hr)
HLM Clint (μL/min/mg)
HLM 30 min remaining (%)
Caco-2 permeability (nm/s) (A → B)
efflux ratio
CONCLUSIONS
■
In this paper, we described the discovery of a series of Mcl-1
inhibitors with 3,5-dimethyl-4-sulfonyl-1H-pyrrole core. The
sulfone linker and the 5-methyl group of the pyrrole core
furnished inhibitors with improved Mcl-1 inhibition. Further
modifications on the phenyl ring yielded 40 with high Mcl-1
binding affinity. The sulfone linker constrained the structure to a
proper conformation, which led to the remarkable selectivity
over other Bcl-2 family members. The in vitro antitumor assay
demonstrated that 40 inhibited the growth of H929 and MV4-
11 cells with appropriate potency. 40 was then validated to
induce apoptosis in the H929 cell line in an Mcl-1-dependent
manner. Further studies showed that 40 also possessed good
druglike properties, including appropriate PK profiles and no
inhibition toward hERG and CYPs. In MV4-11 xenografts, oral
administration of 40 was well tolerated to significantly inhibit
tumor growth. We hope our work is useful for developing more
potent Mcl-1 inhibitors and promote cancer therapy targeting
Mcl-1.
hERG (IC50, μM)
a
b
Remaining (%) after 6 h of incubation. Remaining (%) after 2 h of
incubation.
Target compounds 16, 23−26, 29−31, 33 and 34, 37 and 38,
41 and 42, 44−47, 51−53, and 55−57 were synthesized
according to Scheme 4. Commercially available mercaptans
74a−v were first activated by SO2Cl2 in DCM and then reacted
with ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate to give
intermediates 75a−v. 75a−v further coupled with 59 to yield
76a−v. Hydrolysis of 76a by NaOH (2 M) in THF/EtOH
yielded target compound 23. Oxidation of thio bonds of 76a−v
by 3-chloroperbenzoic acid (m-CPBA) yielded compounds
77a−v. Hydrolysis of 77a-v by NaOH (2 M) in EtOH/THF
yielded target compounds 16, 24−26, 29−31, 33−34, 37 and
38, 41 and 42, 44−47, 51−53, and 56−57. Reduction of 53 by
Pd/C under hydrogen in EtOAc yielded target compound 55.
Figure 10. In vivo PK parameters of 40. The values shown are the means. Bars represent SEM.
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Figure 11. Antitumor efficacy of compound 40 in nude mice loaded with MV4-11 xenografts. (A) Tumor volume change. The values shown are the
means SEM **p < 0.01, and ****p < 0.0001, one-way ANOVA with the Tukey−Kramer posttest. (B) Body weight change. (C) Organ weight
change. The values shown are the means. Bars represent SEM. (D) Representative HE staining images of tumor tissues and organ tissues. Scale bar 200
μM. (E) WB assay of tumor tissues. (n = 2 for each group). (F) Representative images of tunel assay of tumor tissues. Scale bar 200 μM.
purification by silica gel column chromatography (petroleum ether/
EXPERIMENTAL SECTION
■
ethyl acetate = 500:1 to 300:1 v/v) gave 59. Colorless liqiud, 6.2 g,
General Procedures. The synthesis of target compounds is shown
yield: 75%. 1H NMR (300 MHz, Chloroform-d) δ 6.68 (s, 2H), 4.11 (t,
in Schemes 1−7. Reactions were monitored by thin-layer chromatog-
J = 7.1 Hz, 2H), 3.56 (t, J = 7.1 Hz, 2H), 2.32 (s, 6H), 2.19 (p, J = 7.1
raphy (TLC) on 0.25 mm silica gel plates (GF254) and visualized
Hz, 2H). ESI-MS m/z: 277.0 [M + 1]+.
under UV light. Melting points were determined with a Melt-Temp II
Methyl-4-(4-methylbenzoyl)-1H-pyrrole-2-carboxylate (61).
At 0 °C, to a solution of 4-methylbenzoyl chloride (5 g, 32 mmol) and
DCM (40 mL), AlCl3 (4.4 g, 32 mmol) was added under nitrogen. The
mixture was stirred for 10 min, and 60 (2 g, 16 mmol) was added. The
reaction was then allowed to stir at room temperature for 2 h under
nitrogen. After the completion of the reaction, 100 mL of water was
1
apparatus. The H NMR and 13C NMR spectra were measured on a
Bruker AV-300 instrument using deuterated solvents with tetrame-
thylsilane as the internal standard. ESI-mass and high-resolution mass
spectra (HRMS) were recorded on a Water Q-Tof micro mass
spectrometer. The purity (≥95%) of the compounds was verified by
HPLC performed on a Shimadzu C18 (4.6 mm × 150 mm, 3.5 μm)
carefully added to the mixture. DCM (80 mL) was added to the mixture
and stirred for 15 min. After filtration, the organic layer was isolated and
the inorganic layer was extracted with DCM (2 times). The combined
organic layer was then washed with brine and dried with Na2SO4.
Removal of solvent under reduced pressure yielded the crude product.
Further purification by silica gel column chromatography (petroleum
ether/ethyl acetate = 30:1 to 15:1 v/v) gave 61. White powder, 2.6 g,
yield: 68%, 1H NMR (300 MHz, Chloroform-d) δ 10.37 (s, 1H), 7.80
(d, J = 7.9 Hz, 2H), 7.60 (s, 1H), 7.40 (s, 1H), 7.31 (d, J = 7.6 Hz, 2H),
3.92 (s, 3H), 2.46 (s, 3H). ESI-MS m/z: 244.1 [M + 1]+.
column using a mixture of 90:10 or 80:20 solvent methanol/water with
1‰ TFA at a flow rate of 0.5 mL/min and peak detection at 254 nm.
5-(3-Bromopropoxy)-2-chloro-1,3-dimethylbenzene (59).
To a mixture of K2CO3 (8.3 g, 60 mmol, 2 equiv), 1,3-dibromopropane
(6 mL, 60 mmol, 2 equiv), and CH3CN (40 mL), 58 (4.7 g, 30 mmol, 1
equiv) was added and the mixture was stirred for 5 h at 90 °C. After
cooling to room temperature, the reaction mixture was diluted with
water (100 mL) and extracted with EtOAc (3 times). The combined
organic layer was then washed with brine and dried over Na2SO4.
Removal of solvent in vacuo yielded a crude product. Further
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a
Scheme 1. Synthesis of Target Compounds 11 and 12
a
Reagents and conditions: (a) 1,3-dibromopropane, K2CO3, CH3CN, 90 °C, 3 h, 75%. (b) 4-methylbenzoyl chloride, AlCl3, DCM, N2, 0 °C−r.t. 2
h, 68%. (c) triethylsilane, THF, N2, r.t. 2 h, 65%. (d) 59, Cs2CO3, DMF, 60 °C, 1 h, 84−89%. (e) NaOH (2 M), THF/MeOH = 1:1, 50 °C,
overnight, 78−95%.
a
Scheme 2. Synthesis of Target Compound 13 and 20
a
Reagents and conditions: (a) methyl isocyanate, Ag2CO3, 1,4-dioxane, 80 °C, 30 min, 18−25%. (b) 59, NaH, DMF, 0 °C−r.t. 0.5 h, 35−42%. (c)
NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 62−72%.
Methyl-4-(4-methylbenzyl)-1H-pyrrole-2-carboxylate (62).
To a solution of 61 (1 g, 4.1 mmol) and THF (30 mL), triethylsilane
(0.93 g, 8.2 mmol) was added under nitrogen. The solution was then
allowed to stir for 2 h at room temperature under nitrogen. Further
purification by silica gel column chromatography (petroleum ether/
ethyl acetate = 80:1 to 40:1 v/v) gave 62, white powder, 0.61 g, yield:
65% 1H NMR (300 MHz, Chloroform-d) δ 9.13 (s, 1H), 7.14 (s, 4H),
6.76 (d, J = 5.6 Hz, 2H), 3.86 (s, 3H), 3.82 (s, 2H), 2.36 (s, 3H). ESI-
MS m/z: 247.1 [M + NH4]+.
Methyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-(4-
methylbenzyl)-1H-pyrrole-2-carboxylate (63). To a solution of
62 (1 g, 4.4 mmol) and DMF (20 mL), Cs2CO3 (2.5 g, 8 mmol) was
added. The mixture was stirred for 10 min at 60 °C and subsequently,
59 (2.2 g, 8 mmol) was added. The mixture was allowed to stir for
another 1 h. After cooling to room temperature, the reaction mixture
was then diluted in 60 mL of water. The mixture was extracted with
EtOAc (3 times). The combined organic layer was then washed with
brine and dried with Na2SO4. Removal of solvent under reduced
pressure yielded a crude product. Further purification by silica gel
column chromatography (petroleum ether/ethyl acetate = 80:1 to 30:1
v/v) gave 63, white powder, yield: 89%. 1H NMR (300 MHz,
Chloroform-d) δ 7.11 (s, 4H), 6.86 (s, 1H), 6.73 (s, 1H), 6.68 (s, 2H),
4.50 (t, J = 6.6 Hz, 2H), 3.88 (t, J = 5.5 Hz, 2H), 3.85 (s, 3H), 3.76 (s,
2H), 2.42 (s, 6H), 2.38 (s, 3H), 2.27 (p, J = 6.0 Hz, 2H). ESI-MS m/z:
464.1 [M + K]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(4-methyl-
benzyl)-1H-pyrrole-2-carboxylic Acid (11). To a solution of 63
(500 mg, 1.2 mmol) in MeOH/THF (5 mL/5 mL) was added 1 mL of
NaOH (2 M). The solution was stirred overnight at 50 °C. After
completion of the reaction, the mixture was quenched with 1 M
hydrochloric acid to pH 2 and then extracted with ethyl acetate (3
times). The organic layer was washed with brine and dried with
Na2SO4. Removal of the solvent and recrystallization from ethyl
acetate/n-hexane gave title compound 11 as a white solid, yield: 78%;
1
mp: 201−203 °C. H NMR (300 MHz, DMSO-d6) δ 12.10 (s, 1H),
7.04 (s, 4H), 6.82 (d, J = 1.6 Hz, 1H), 6.77 (s, 2H), 6.64 (d, J = 1.9 Hz,
1H), 4.38 (t, J = 6.6 Hz, 2H), 3.82 (t, J = 6.0 Hz, 2H), 3.64 (s, 2H), 2.31
(s, 6H), 2.26 (s, 3H), 2.10 (p, J = 6.3 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 162.24, 156.94, 138.94, 136.94, 134.99, 129.25, 128.68,
128.06, 125.53, 122.60, 121.89, 118.27, 115.11, 64.97, 45.46, 32.35,
30.87, 20.91. HRMS (ESI): calcd for C24H26ClNO3 [M-H]− 410.1523,
found 410.1525. HPLC (90:10 methanol:water with 1‰ TFA): tR =
8.51 min, 98.5%.
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a
Scheme 3. Synthesis of Target Compound 14 and 15
a
Reagents and conditions: (a) 59, Cs2CO3, DMF, 60 °C, 1 h, 75%. (b) SnCl2·2H2O, N2, EtOH, 60 °C, 4 h. (c) Pyridine, TosCl, r.t. 1 h, 38%. (d)
NaOH (2 M), THF/MeOH = 1:1, 50 °C, overnight, 43−64%. (e) (Boc)2O, EtOH, r.t. 1 h, 46%. (f) 4-Methylbenzyl bromide, NaH, r.t. 0.5 h, 73%.
(g) CF3COOH, r.t. 1.5 h, 95%.
a
Scheme 4. Synthesis of Target Compounds 16, 23−26, 29−31, 33 and 34, 37 and 38, 41 and 42, 44−47, 51−53, and 55−57
a
Reagents and conditions: (a) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, SO2Cl2, Et3N, DCM, r.t. 45 min, 56−85%; (b) 59, Cs2CO3, DMF, 50
°C, 1 h, 78−85%; (c) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 63−85%. (d) m-CPBA, DCM, r.t. 0.5 h, 55−70%; (e) Pd/C, H2, EA,
50 °C, 3 h, 95%.
sized according to the procedure of 63, white solid, yield 84%; 1H NMR
(300 MHz, Chloroform-d) 7.64 (d, J = 7.2 Hz, 2H), 7.47 (s, 1H), 7.38
Methyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-(4-
methylbenzoyl)-1H-pyrrole-2-carboxylate (64). 64 was synthe-
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a
Scheme 5. Synthesis of Target Compounds 17−19 and 21 and 22
a
Reagents and conditions: (a) AcOH, NaNO2, 0 °C−r.t. overnight; (b) AcONa, AcOH, Zn, r.t.−80 °C, 3 h, 36−43%. (c) p-Toluenethiol, SO2Cl2,
Et3N, DCM, r.t. 45 min, 45−62%; (d) 59, Cs2CO3, DMF, 50 °C, 1 h, 57−72%; (e) m-CPBA, DCM, r.t. 0.5 h, 45−56%; (f) NaOH (2 M),
C2H5OH/THF = 1:1, 50 °C, overnight, 74−86%.
a
Scheme 6. Synthesis of Target Compounds 27 and 28
a
Reagents and conditions: (a) ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate, N2, AlCl3, PhCl, 90 °C, 2 h, 59−68%; (b) 59, Cs2CO3, DMF, 50 °C, 1
h, 63−75%; (c) NaOH (2 M), C2H5OH/THF = 1:1, 50 °C, overnight, 54−63%.
(s, 1H), 7.14 (d, J = 7.5 Hz, 2H), 6.64 (s, 2H), 4.58 (t, J = 6.4 Hz, 2H),
3.87 (d, J = 7.6 Hz, 5H), 2.42 (s, 3H), 2.36 (s, 6H), 2.33−2.21 (m, 2H).
ESI-MS m/z: 462.2 [M + Na]+.
brine and dried with Na2SO4. Removal of solvent under reduced
pressure yielded a crude product. Further purification by silica gel
column chromatography (petroleum ether/ethyl acetate = 20:1 to 5:1
1
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(4-methyl-
benzoyl)-1H-pyrrole-2-carboxylic Acid (12). 12 was synthesized
from 64 according to the procedure of 9, white powder, yield: 95%. mp:
174−175 °C, 1H NMR (300 MHz, Chloroform-d) δ 7.68 (d, J = 8.2 Hz,
2H), 7.66 (s, 1H), 7.48 (s, 1H), 7.17 (d, J = 7.8 Hz, 2H), 6.67 (s, 2H),
4.62 (t, J = 5.7 Hz, 2H), 3.92 (t, J = 4.8 Hz, 2H), 2.46 (s, 3H), 2.38 (s,
6H), 2.34 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 188.74, 162.00,
156.85, 142.42, 136.97, 136.48, 134.95, 129.37, 129.10, 125.57, 124.12,
122.39, 119.05, 115.08, 64.85, 46.57, 30.33, 21.53, 20.90. HRMS (ESI):
calcd for C24H24ClNO4 [M + Na]+ 448.1292, found 448.1293. HPLC
(90:10 methanol:water with 1‰ TFA): tR = 6.97 min, 98.5%.
Methyl-4-tosyl-1H-pyrrole-2-carboxylate (66a). A mixture of
65a (500 mg, 3 mmol), Ag2CO3 (0.08 g, 0.3 mmol), and dioxane (20
mL) was charged with methyl isocyanate (1 equiv). The mixture was
heated to 80 °C and stirred for 1 h. Then, the mixture was cooled to
room temperature, charged with 60 mL water, and extracted with
EtOAc (3 times). The combined organic layer was then washed with
v/v) gave 66a. Yellow oil, 150 mg, yield: 18%. H NMR (300 MHz,
DMSO-d6) δ 12.87 (s, 1H), 7.80 (d, J = 8.3 Hz, 2H), 7.64 (s, 1H), 7.37
(d, J = 8.0 Hz, 2H), 7.03 (s, 1H), 3.77 (s, 3H), 2.35 (s, 3H). ESI-MS m/
z: 280.1 [M + 1]+.
Methyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-
tosyl-1H-pyrrole-2-carboxylate (67a). At 0 °C, to a solution of 66a
(0.150 g, 0.5 mmol) and distilled DMF (15 mL), NaH (0.04 g, 1 mmol)
was added in portions. After the effervescence of the mixture, 59 (0.3 g,
1 mmol) was added and the reaction was stirred at room temperature
for 0.5 h. After the consumption of 66a monitored by TLC, 45 mL
water was carefully added to the mixture and the mixture was extracted
with EtOAc (3 times). The combined organic layer was then washed
with brine and dried over Na2SO4. Removal of solvent under reduced
pressure yielded a crude product. Further purification by silica gel
column chromatography (petroleum ether/ethyl acetate = 70:1 to 20:1
1
v/v) gave 67a. Yellow oil, 0.1 mg, yield: 42%. H NMR (300 MHz,
Chloroform-d) δ 7.75 (d, J = 7.6 Hz, 2H), 7.69 (d, J = 1.5 Hz, 1H), 7.28
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Scheme 7. Synthesis of Target Compounds 32, 35, 36, 39, 40, 43, 48−50, and 54
a
Reagents and conditions: (a) ethanedithiol, CuSO4·5H2O, Cs2CO3, DMSO, N2, 90 °C, 4 h, 76−85%; (b) ethyl 3,5-dimethyl-1H-pyrrole-2-
carboxylate, SO2Cl2, Et3N, DCM, r.t. 45 min, 54−75%; (c) 59, Cs2CO3, DMF, 50 °C, 1 h, 75−85%; (d) NaOH (2 M), C2H5OH/THF = 1:1, 50
°C, overnight, 42−80%; (e) m-CPBA, DCM, r.t. 0.5 h, 63−84%; (f) Pd/C, H2, EA, 50 °C, 3 h, 63−67%.
(d, J = 8.0 Hz, 2H), 6.95 (d, J = 1.5 Hz, 1H), 6.69 (s, 2H), 4.15 (t, J = 7.1
Hz, 2H), 4.06 (t, J = 7.1 Hz, 2H), 3.90 (s, 3H), 2.40 (s, 3H), 2.32 (s,
6H), 2.08 (p, J = 7.1 Hz, 2H). ESI-MS m/z: 476.1 [M + 1]+.
7.1 Hz, 2H), 4.05 (t, J = 7.1 Hz, 2H), 2.34 (s, 3H), 2.33 (s, 6H), 2.08 (p,
J = 7.2 Hz, 2H), 1.35 (t, J = 8.0 Hz, 3H). ESI-MS m/z: 505.1 [M + H]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
methylphenyl)sulfonamido)-1H-pyrrole-2-carboxylic Acid
(14). 14 was synthesized from 70 according to the hydrolysis procedure
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-methyl-4-
tosyl-1H-pyrrole-2-carboxylic Acid (13). 13 was synthesized from
67a according to the hydrolysis procedure of 11. White powder, yield:
68%; mp: 194−196 °C. 1H NMR (300 MHz, Chloroform-d) δ 12.89 (s,
1H), 7.81 (s, 1H), 7.72 (d, J = 6.8 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H),
7.05 (s, 1H), 6.71 (s, 2H), 4.46 (t, J = 5.1 Hz, 2H), 3.83 (t, J = 6.2 Hz,
2H), 2.35 (s, 3H), 2.28 (s, 6H), 2.17−2.08 (m, 2H). HRMS (ESI):
calcd for C23H24ClNO5S [M + NH4]+ 479.1407, found 479.1402.
HPLC (80:20 methanol:water with 1‰ TFA): tR = 8.14 min, 99.8%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-methyl-4-
tosyl-1H-pyrrole-2-carboxylic Acid (20). Starting from 67b, 20 was
synthesized according to the procedure of 11. White powder, yield:
10%; mp: 194−196 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.71 (d,
J = 8.2 Hz, 2H), 7.54 (s, 1H), 7.26 (d, J = 8.2 Hz, 2H), 6.64 (s, 2H), 4.54
(t, J = 6.5 Hz, 2H), 3.83 (t, J = 5.5 Hz, 2H), 2.48 (s, 3H), 2.44 (s, 3H),
2.38 (s, 6H), 2.25 (p, J = 6.1 Hz, 2H). HRMS (ESI): calcd for
C24H26ClNO5S [M + Na]+ 498.1118, found 498.1116. HPLC (80:20
methanol:water with 1‰ TFA): tR = 9.08 min, 98.8%.
1
of 9. Pale yellow solid, yield: 64%; mp: 179−181 °C. H NMR (300
MHz, DMSO-d6) δ 9.76 (s, 1H), 7.58 (d, J = 7.6 Hz, 2H), 7.29 (d, J =
7.6 Hz, 2H), 6.89 (s, 1H), 6.75 (s, 2H), 6.48 (s, 1H), 4.45−4.29 (m,
2H), 3.78 (s, 2H), 2.31 (s, 9H), 2.03 (s, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 160.64, 156.81, 143.24, 137.14, 136.96, 129.86, 129.73,
127.16, 125.56, 121.39, 119.44, 115.20, 111.92, 67.50, 51.48, 25.60,
21.35, 20.91. HRMS (ESI): calcd for C23H25ClN2O5S [M-H]−
475.1094, found 475.1093. HPLC (90:10 methanol:water with 1‰
TFA): tR = 6.85 min, 98.1%.
Ethyl-4-((tert-butoxycarbonyl)amino)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-1H-pyrrole-2-carboxylate (71). Re-
duction of 69 was depicted in the synthesis of 70, and the crude product
was used in the next step directly. 0.6 g (1.7 mmol) of the crude product
was dissolved in EtOH (20 mL), and (Boc)2O (0.65 g, 3.4 mmol) was
added. The solution was stirred at room temperature for 1 h. Upon
completion of the reaction, the solution was concentrated under
reduced pressure and purification by silica gel column chromatography
(petroleum ether/ethyl acetate = 80:1 to 20:1 v/v), which gave 71 as a
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-nitro-
1H-pyrrole-2-carboxylate (69). 69 was synthesized from 68
1
1
according to the procedure of 63, yield 75%; H NMR (300 MHz,
yellow oil, 0.42 g, yield: 46%, H NMR (300 MHz, Chloroform-d) δ
Chloroform-d) δ 7.67 (d, J = 2.0 Hz, 1H), 7.49 (d, J = 2.0 Hz, 1H), 6.65
(s, 2H), 4.61 (t, J = 6.8 Hz, 2H), 4.34 (q, J = 7.1 Hz, 2H), 3.95 (t, J = 5.6
Hz, 2H), 2.38 (s, 6H), 2.31 (p, J = 6.5 Hz, 2H), 1.40 (t, J = 7.1 Hz, 3H).
ESI-MS m/z: 381.1 [M + H]+.
7.72 (d, J = 1.5 Hz, 1H), 6.70 (s, 1H), 6.67 (d, J = 1.5 Hz, 1H), 6.61 (s,
2H), 4.34 (t, J = 7.7 Hz, 2H), 4.28 (q, J = 8.0 Hz, 2H), 4.22 (t, J = 7.5
Hz, 2H), 2.31 (s, 6H), 1.98 (p, J = 7.6 Hz, 2H), 1.50 (s, 9H), 1.36 (t, J =
8.1 Hz, 3H). ESI-MS m/z: 451.2 [M + H]+.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-((4-
methylphenyl)sulfonamido)-1H-pyrrole-2-carboxylate (70).
To a solution of 69 (1.5 g, 4.1 mmol) and EtOH (50 mL), SnCl2·
2H2O (4.6 g, 20 mmol) was added under nitrogen. The mixture was
then allowed to stir at 60 °C for 4 h under nitrogen. Upon the
consumption of 69, the solution was cooled to room temperature and
quenched with aqueous NaOH (2 M) to pH 9. DCM (70 mL) was
added, and the mixture was stirred for 15 min. The organic layer was
isolated after the undissolved white solid was filtered. The aqueous
phase was then extracted with DCM (2 times). The combined organic
layer was then washed with brine and dried with Na2SO4. Removal of
solvent under reduced pressure yielded a crude product as a deep brown
oil without further purification. 0.6 g (1.7 mmol) of the crude product
was dissolved in pyridine (15 mL), and p-toluenesulfonyl chloride (0.65
g, 3.4 mmol) was added. The solution was stirred at room temperature
for 1 h. Upon completion of the reaction, the solution was concentrated
under reduced pressure, and the crude product 70 was purified by silica
gel column chromatography (petroleum ether/ethyl acetate = 60:1 to
Ethyl-4-((tert-butoxycarbonyl)(4-methylbenzyl)amino)-1-
(3-(4-chloro-3,5-dimethylphenoxy)propyl)-1H-pyrrole-2-car-
boxylate (72). To a solution of 71 (0.4 g, 0.9 mmol) and DMF (15
mL), NaH (0.08 g, 2 mmol) was added in portions. After the
effervescence of the mixture, 4-methylbenzyl bromide (0.36 g, 2 mmol)
was added and the reaction was stirred at room temperature for 0.5 h.
After the consumption of 71 monitored by TLC, 45 mL of water was
carefully added to the mixture and then the mixture was extracted with
EtOAc (3 times). The combined organic layer was then washed with
brine and dried over Na2SO4. The combined organic layer was
concentrated under reduced pressure and further purification by silica
gel column chromatography (petroleum ether/ethyl acetate = 100:1 to
1
40:1 v/v) gave 72. Light yellow oil, 0.4 g, yield: 73%. H NMR (300
MHz, Chloroform-d) 1H NMR (300 MHz, Chloroform-d) δ 7.26 (d, J
= 1.5 Hz, 1H), 7.19 (d, J = 7.6 Hz, 2H), 7.11 (d, J = 7.5 Hz, 2H), 6.85 (d,
J = 1.5 Hz, 1H), 6.69 (s, 2H), 5.24 (s, 2H), 4.28 (q, J = 7.5 Hz, 2H), 4.04
(dt, J = 10.0, 7.1 Hz, 4H), 2.33 (s, 3H), 2.32 (s, 6H), 2.11 (p, J = 7.1 Hz,
2H), 1.47 (s, 9H), 1.34 (t, J = 8.0 Hz, 3H). ESI-MS m/z: 555.2 [M +
H]+.
1
20:1 v/v) as yellow oil, 0.63 g, yield: 38%, H NMR (300 MHz,
Chloroform-d) δ 7.62 (s, 1H), 7.32 (d, J = 7.5 Hz, 2H), 7.15 (d, J = 8.0
Hz, 2H), 6.92 (s, 1H), 6.68 (s, 2H), 4.29 (q, J = 8.0 Hz, 2H), 4.14 (t, J =
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-4-((4-
methylbenzyl)amino)-1H-pyrrole-2-carboxylate (73). 72 (0.3 g,
N
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0.56 mmol) was dissolved in CF3COOH (5 mL) and the solution was
stirred for 1.5 h at room temperature. Upon the consumption of 72,
saturated aqueous NaHCO3 was added carefully in portions to pH 8.
The mixture was extracted with EtOAc (3 times). The combined
organic layer was then washed with saturated aqueous NaHCO3 and
brine and then dried with Na2SO4. Removal of solvent under reduced
pressure gave 73 as light purple oil, 0.23 g, Yield: 95%, 1H NMR (300
MHz, Chloroform-d) δ 7.34 (d, J = 1.5 Hz, 1H), 7.23 (d, J = 1.5 Hz,
1H), 7.18 (d, J = 6.6 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 6.68 (s, 2H),
4.52 (s, 2H), 4.26 (q, J = 8.0 Hz, 2H), 4.12−4.02 (m, 4H), 2.34 (s, 3H),
2.32 (s, 6H), 2.10 (p, J = 7.1 Hz, 2H), 1.35 (t, J = 8.0 Hz, 3H). ESI-MS
m/z: 455.2 [M + H]+.
The combined organic layer was concentrated under reduced pressure
and further purification by silica gel column chromatography
(petroleum ether/ethyl acetate = 20:1 to 4:1 v/v) gave 77a. Light
yellow oil, yield: 56%. 1H NMR (300 MHz, Chloroform-d) δ 7.77 (d, J
= 8.2 Hz, 2H), 7.30 (d, J = 8.7 Hz, 2H), 6.70 (s, 2H), 4.32 (q, J = 7.1 Hz,
2H), 4.11 (t, J = 5.8 Hz, 2H), 3.64 (t, J = 6.4 Hz, 2H), 2.44 (s, 3H), 2.39
(s, 12H), 2.37−2.30 (m, 2H), 1.38 (t, J = 7.1 Hz, 3H). ESI-MS m/z:
540.2 [M + Na]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-tosyl-1H-pyrrole-2-carboxylic Acid (16). 16 was synthesized
from 77a according to the hydrolysis procedure of 23. White powder,
172 mg, Yield: 57%, mp: 183−184 °C 1H NMR (300 MHz,
chloroform-d) δ 12.87 (s, 1H), 7.67 (d, J = 8.0 Hz, 2H), 7.35 (d, J =
7.7 Hz, 2H), 6.73 (s, 2H), 4.41 (t, J = 6.8 Hz, 2H), 3.87 (t, J = 4.8 Hz,
2H), 2.54 (s, 3H), 2.36 (s, 6H), 2.28 (s, 6H), 2.03 (s, 2H). 13C NMR
(75 MHz, DMSO-d6) δ 162.57, 156.70, 143.59, 141.31, 139.07, 136.94,
130.22, 127.93, 126.26, 125.55, 121.07, 119.06, 115.01, 65.16, 42.82,
30.22, 21.36, 20.86, 11.96, 11.02. HRMS (ESI): calcd for
C25H28ClNO5S [M + Na]+ 512.1274, found 512.1269. HPLC (80:20
methanol:water with 1‰ TFA): tR = 11.93 min, 99.6%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
methylbenzyl)amino)-1H-pyrrole-2-carboxylic Acid (15). 15
was synthesized from 73 according to the hydrolysis procedure of 11.
1
Brown powder, yield: 43%. mp: 157−158 °C. H NMR (300 MHz,
DMSO-d6) δ 7.20 (d, J = 7.9 Hz, 2H), 7.09 (d, J = 7.9 Hz, 2H), 6.77 (s,
2H), 6.37 (d, J = 2.1 Hz, 1H), 6.28 (d, J = 2.1 Hz, 1H), 4.29 (t, J = 6.5
Hz, 2H), 3.94 (s, 2H), 3.83 (t, J = 6.0 Hz, 2H), 2.31 (s, 6H), 2.27 (s,
3
3H), 2.06 (p, J = 6.2, 2H). C NMR (75 MHz, DMSO) δ 162.20,
156.96, 138.08, 136.94, 135.86, 135.66, 129.09, 127.78, 125.48, 119.55,
115.18, 114.39, 106.25, 65.02, 49.99, 45.05, 30.96, 21.12, 20.92. HRMS
(ESI): calcd for C24H27ClN2O3 [M + H]+ 427.1788, found 427.1781.
HPLC (90:10 methanol:water with 1‰ TFA): tR = 7.25 min, 98.2%.
Ethyl-3,5-dimethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate
(75a). At 0 °C, to a solution of p-toluenethiol (74a, 1.5 g, 12 mmol),
Et3N (5 drops), and distilled DCM (20 mL), SO2Cl2 (1.6 g, 12 mmol)
was carefully added in portions. The solution was allowed to stir for 10
min at room temperature. Ethyl 3,5-dimethyl-1H-pyrrole-2-carboxylate
(1.1 g, 6 mmol) was then added, and the solution was stirred for another
45 min. Upon the consumption of ethyl 3,5-dimethyl-1H-pyrrole-2-
carboxylate, the reaction solution was concentrated under reduced
pressure and purification by silica gel column chromatography
(petroleum ether/ethyl acetate = 70:1 to 20:1 v/v) gave 75a, white
solid, 73%, 1H NMR (300 MHz, Chloroform-d) δ 9.23 (s, 1H), 7.06 (d,
J = 8.0 Hz, 2H), 6.94 (d, J = 8.3 Hz, 2H), 4.39 (q, J = 7.1 Hz, 2H), 2.36
(s, 3H), 2.34 (s, 3H), 2.32 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). ESI-MS m/
z: 288.1 [M-H]−.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-di-
methyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate (76a). 76a was
synthesized from 75a according to the procedure of 63, colorless oil,
yield: 75%; 1H NMR (300 MHz, Chloroform-d) δ 7.03 (d, J = 8.1 Hz,
2H), 6.89 (d, J = 8.2 Hz, 2H), 6.65 (s, 2H), 4.54 (t, J = 5.6 Hz, 2H), 4.35
(q, J = 7.1 Hz, 2H), 3.93 (t, J = 5.7 Hz, 2H), 2.40 (s, 6H), 2.38 (s, 3H),
2.37 (s, 3H), 2.35 (s, 3H), 2.24 (p, J = 6.2 Hz, 2H), 1.42 (t, J = 7.1 Hz,
3H). ESI-MS m/z: 486.2 [M + 1]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-(p-tolylthio)-1H-pyrrole-2-carboxylic Acid (23). To a solution
of 76a (380 mg, 0.78 mmol) in EtOH/THF (10 mL/10 mL), NaOH (2
M, 3 mL) was added. The mixture was stirred overnight at 50 °C. After
the completion of the reaction, the mixture was then acidified with 1 M
HCl and extracted with ethyl acetate. The organic layer was washed
with brine and dried with Na2SO4. Removal of solution and further
recrystallization from EtOH/n-hexane gave title compound 23. Yellow
solid, Yield: 85%; mp: 125−127 °C. 1H NMR (300 MHz, Chloroform-
d) δ 7.40 (d, J = 8.1 Hz, 2H), 7.29 (d, J = 6.1 Hz, 2H), 6.63 (s, 2H), 4.49
(t, J = 6.7 Hz, 2H), 3.88 (t, J = 5.3 Hz, 2H), 2.42 (s, 3H), 2.38 (s, 3H),
2.36 (s, 9H), 2.18 (m, J = 12.3, 6.4 Hz, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 162.72 , 156.75 , 141.18 , 136.95 , 135.52 , 134.31 , 132.79
, 130.05 , 125.54 , 125.27 , 119.64 , 114.97 , 107.66 , 64.90 , 43.21 , 30.57
, 20.88 , 20.80 , 12.74 , 10.91. HRMS (ESI): calcd for C25H28ClNO3S
[M−H]− 456.1400, found 456.1399. HPLC (80:20 methanol: water
with 1‰ TFA): tR = 14.8 min, 98.6%.
Starting from corresponding thiol compounds 74b−v, target
compounds 24−26, 29−31, 33−34, 37 and 38, 41 and 42, 44−47,
51−53, and 56−57 were synthesized analogously according to 16.
4-(Benzylsulfonyl)-1-(3-(4-chloro-3,5-dimethylphenoxy)-
propyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (24). Light
1
yellow solid. Yield: 22%; mp: 144−146 °C. H NMR (300 MHz,
Chloroform-d) δ 7.32 (s, 3H), 7.12 (d, J = 6.7 Hz, 2H), 6.65 (s, 2H),
4.45 (s, 2H), 4.29 (s, 2H), 3.93 (s, 2H), 2.59 (s, 3H), 2.39 (s, 6H), 2.10
(s, 2H), 2.02 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 162.69, 156.65,
140.35, 136.93, 131.40, 129.91, 129.17, 128.53, 128.37, 125.55, 120.54,
115.81, 115.08, 65.05, 62.79, 42.54, 30.32, 20.86, 11.71, 10.17. HRMS
(ESI): calcd for C25H28ClNO5S [M + Na]+ 512.1274, found 512.1266.
HPLC (80:20 methanol:water with 1‰ TFA): tR = 9.79 min, 97.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-(pyridin-2-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (25). Pink
solid, Yield: 15%; mp: 183−185 °C. 1H NMR (300 MHz, DMSO-d6) δ
12.74 (s, 1H), 8.64 (d, J = 3.7 Hz, 1H), 8.08 (s, 2H), 7.63 (s, 1H), 6.73
(s, 2H), 4.42 (s, 2H), 3.87 (s, 2H), 2.53 (s, 3H), 2.39 (s, 3H), 2.28 (s,
6H), 2.03 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 168.07, 164.81,
164.27, 158.54, 141.08, 138.73, 131.16, 131.03, 129.70, 127.39, 123.16,
120.38, 118.86, 118.56, 116.93, 67.15, 44.69, 32.05, 22.56, 13.62, 12.84.
HRMS (ESI): calcd for C23H25ClN2O5S [M + H]+ 477.1251, found
477.1242. HPLC (80:20 methanol:water with 1‰ TFA): tR = 6.49
min, 98.1%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-(pyridin-4-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (26). Pur-
ple solid, Yield: 13%; mp: 179−181 °C. 1H NMR (300 MHz,
Chloroform-d) δ 8.74 (d, J = 2.7 Hz, 2H), 7.50 (d, J = 5.2 Hz, 2H), 6.62
(s, 2H), 4.52 (t, J = 6.8 Hz, 2H), 3.90 (t, J = 5.3 Hz, 2H), 2.41 (s, 3H),
2.36 (s, 6H), 2.32 (s, 3H), 2.21 (s, 2H). 13C NMR (75 MHz, DMSO-
d6) δ 162.53, 156.72, 155.92, 151.80, 150.45, 139.61, 137.00, 128.69,
121.17, 119.74, 119.53, 115.04, 65.12, 42.84, 30.33, 20.90, 11.60, 10.58.
HRMS (ESI): calcd for C23H25ClN2O5S [M + Na]+ 499.1070, found
499.1094. HPLC (90:10 methanol:water with 1‰ TFA): tR = 6.07
min, 97.0%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(cyclohexyl-
sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (29).
White solid, yield: 27%; mp: 159−161 °C. 1H NMR (300 MHz,
Chloroform-d) δ 6.61 (s, 2H), 4.53 (s, 2H), 3.87 (s, 2H), 3.40 (s, 1H),
2.61 (d, J = 13.0 Hz, 6H), 2.34 (s, 6H), 2.18 (s, 2H), 2.07−1.52 (m,
10H). HRMS (ESI): calcd for C24H32ClNO5S [M + Na]+ 504.1587,
found 504.1580. HPLC (80:20 methanol:water with 1‰ TFA): tR =
9.16 min, 96.5%.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-di-
methyl-4-tosyl-1H-pyrrole-2-carboxylate (77a). At 0 °C, to a
solution of 77a (0.8 g, 1 mmol) and distilled DCM (20 mL), 3-
chloroperbenzoic acid (m-CPBA, 85%, 0.4 g, 2 mmol) was added. The
solution was allowed to stir at room temperature for 0.5 h and then
quenched with 20 mL of saturated aqueous NaHCO3. The organic layer
was isolated and the aqueous phase was extracted with DCM (2 times).
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-(cyclopen-
tylsulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic Acid (30).
White solid, yield: 13%; mp: 164−166 °C. 1H NMR (300 MHz,
Chloroform-d) δ 6.61 (s, 2H), 4.53 (s, 2H), 3.87 (s, 2H), 3.40 (s, 1H),
2.61 (d, J = 13.0 Hz, 6H), 2.34 (s, 6H), 2.18 (s, 2H), 2.01−1.60 (m,
8H). HRMS (ESI): calcd for C23H30ClNO5S [M + NH4]+ 485.1877,
O
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found 485.1877. HPLC (90:10 methanol:water with 1‰ TFA): tR =
9.10 min, 99.4%.
3H), 2.27 (s, 3H), 2.18 (s, 6H), 1.94 (s, 2H). 13C NMR (75 MHz,
DMSO-d6) δ 162.52, 156.71, 143.29, 139.57, 136.97, 132.96, 128.30,
127.98, 127.18, 125.57, 121.34, 118.10, 115.05, 65.20, 60.21, 42.94,
30.18, 20.90, 11.94, 11.09. HRMS (ESI): calcd for C24H25BrClNO5S
[M + Na]+ 578.0203, found 578.0198. HPLC (80:20 methanol:water
with 1‰ TFA): tR = 8.64 min, 98.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
ethylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic
Acid (44). White solid, yield: 12%; mp: 181−183 °C. 1H NMR (300
MHz, Chloroform-d) δ 7.79 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz,
2H), 6.63 (s, 2H), 4.52 (t, J = 7.0 Hz, 2H), 3.90 (t, J = 5.5 Hz, 2H),
2.81−2.70 (m, 2H), 2.68 (s, 3H), 2.59 (s, 3H), 2.36 (s, 6H), 2.17 (t, J =
6.3 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ
162.56, 156.77, 149.58, 141.66, 139.10, 136.96, 129.10, 127.97, 126.36,
125.61, 121.19, 119.14, 115.13, 65.31, 42.87, 30.29, 28.43, 20.84, 15.41,
11.95, 11.07. HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+
526.1431, found 526.1424. HPLC (80:20 methanol:water with 1‰
TFA): tR = 15.28 min, 98.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-(phenylsulfonyl)-1H-pyrrole-2-carboxylic Acid (31). White
1
solid, yield: 28%; mp: 186−188 °C. H NMR (300 MHz, Chloro-
form-d) δ 7.94−7.86 (m, 2H), 7.61−7.49 (m, 3H), 6.63 (s, 2H), 4.53
(s, 2H), 3.89 (s, 2H), 2.68 (s, 3H), 2.60 (s, 3H), 2.36 (s, 6H), 2.18 (s,
2H). 13C NMR (75 MHz, DMSO-d6) δ 162.56, 156.68, 144.04, 139.30,
136.93, 133.21, 129.82, 128.03, 126.20, 125.56, 121.16, 118.67, 114.99,
65.12, 42.87, 30.21, 42.87, 30.21, 20.88, 11.96, 11.04. HRMS (ESI):
calcd for C24H26ClNO5S [M + Na]+ 498.1118, found 498.1114. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 12.11 min, 99.2%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((2-
methoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (33). White solid, yield: 28%; mp: 198−200 °C. H NMR
(300 MHz, DMSO-d6) δ 12.76 (s, 1H), 7.95 (d, J = 7.4 Hz, 1H), 7.67−
7.55 (m, 1H), 7.13 (d, J = 5.1 Hz, 2H), 6.73 (s, 2H), 4.44 (s, 2H), 3.90
(s, 2H), 3.72 (s, 3H), 2.52 (s, 3H), 2.26 (d, J = 8.3 Hz, 9H), 2.04 (s,
2H). 13C NMR (75 MHz, DMSO-d6) δ 162.67, 157.09, 156.64, 140.36,
136.93, 135.46, 130.52, 128.76, 128.43, 125.53, 120.34, 118.45, 115.03,
113.52, 65.08, 56.29, 42.61, 30.27, 20.83, 11.81, 10.93. HRMS (ESI):
calcd for C25H28ClNO6S [M + NH4]+ 523.1670, found 523.1667.
HPLC (90:10 methanol:water with 1‰ TFA): tR = 7.37 min, 99.5%.
4-((2-Bromophenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
isopropylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (45). White solid, Yield: 23%; mp: 178−180 °C. H NMR
(300 MHz, DMSO-d6) δ 12.88 (s, 1H), 7.69 (d, J = 8.2 Hz, 2H), 7.40
(d, J = 8.3 Hz, 2H), 6.72 (s, 2H), 4.39 (t, J = 6.8 Hz, 2H), 3.86 (t, J = 5.4
Hz, 2H), 2.92 (dt, J = 13.5, 7.0 Hz, 1H), 2.54 (s, 3H), 2.35 (s, 3H), 2.26
(s, 6H), 2.08−1.97 (m, 2H), 1.16 (d, J = 6.9 Hz, 6H). HRMS (ESI):
calcd for C27H32ClNO5S [M + Na]+ 540.1587, found 540.1577. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 20.33 min, 99.6%.
4-((4-(tert-Butyl)phenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (34). White solid, Yield: 25%; mp: 171−173 °C. H NMR
(300 MHz, Chloroform-d) δ 8.24 (d, J = 7.7 Hz, 1H), 7.60 (t, J = 7.4 Hz,
1H), 7.51 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 7.5 Hz, 1H), 6.60 (s, 2H),
4.57−4.40 (m, 2H), 3.80 (t, J = 5.1 Hz, 2H), 2.42 (s, 3H), 2.37 (s, 6H),
2.35 (s, 3H), 2.23−2.11 (m, 2H). 13C NMR (75 MHz, DMSO-d6) δ
162.58, 156.57, 142.66, 139.86, 136.92, 133.83, 132.36, 128.91, 127.96,
125.55, 120.60, 119.09, 118.48, 114.93, 64.61, 42.44, 30.25, 20.88,
11.49, 10.85. HRMS (ESI): calcd for C24H25BrClNO5S [M + Na]+
578.0203, found 578.0195. HPLC (90:10 methanol:water with 1‰
TFA): tR = 9.49 min, 97.3%.
1
ylic Acid (46). White solid, yield: 31%; mp: 187−189 °C. H NMR
(300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.48
(d, J = 8.3 Hz, 2H), 6.65 (s, 2H), 4.31 (s, 2H), 3.79 (s, 2H), 2.47 (s,
3H), 2.28 (s, 3H), 2.18 (s, 6H), 1.94 (s, 2H), 1.17 (s, 9H). 13C NMR
(75 MHz, DMSO-d6) δ 162.58, 156.74, 156.29, 141.34, 139.17, 136.98,
127.90, 126.73, 126.12, 125.56, 121.15, 119.01, 115.10, 65.24, 42.87,
35.29, 31.17, 30.24, 20.92, 12.03, 11.10. HRMS (ESI): calcd for
C28H34ClNO5S [M + Na]+ 554.1744, found 554.1736. HPLC (80:20
methanol:water with 1‰ TFA): tR = 25.54 min, 99.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((3-
methoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (37). White solid, yield: 22%; mp: 159−161 °C. H NMR
(300 MHz, DMSO-d6) δ 12.91 (s, 1H), 7.55−7.45 (m, 1H), 7.34 (d, J =
8.2 Hz, 1H), 7.31−7.16 (m, 2H), 6.77 (s, 2H), 4.42 (s, 2H), 3.90 (d, J =
11.5 Hz, 2H), 3.80 (s, 3H), 2.55 (s, 3H), 2.39 (s, 3H), 2.28 (s, 6H), 2.04
(s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.53, 159.84, 156.64,
145.30, 139.33, 136.90, 131.15, 128.02, 125.53, 121.16, 118.80, 118.56,
118.25, 114.94, 111.08, 65.12, 55.92, 42.85, 30.17, 20.82, 11.95, 11.01.
HRMS (ESI): calcd for C25H28ClNO6S [M + NH4]+ 523.1670, found
523.1663. HPLC (80:20 methanol:water with 1‰ TFA): tR = 8.25
min, 96.1%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
ethoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic
1
Acid (47). White solid, yield: 8%; mp: 183−185 °C. H NMR (300
MHz, DMSO-d6) δ 12.87 (s, 1H), 7.71 (d, J = 8.8 Hz, 2H), 7.05 (d, J =
8.8 Hz, 2H), 6.73 (s, 2H), 4.48−4.34 (m, 2H), 4.08 (q, J = 7.3, 6.7 Hz,
2H), 3.87 (t, J = 5.6 Hz, 2H), 2.54 (s, 3H), 2.36 (s, 3H), 2.28 (s, 6H),
2.02 (s, 2H), 1.33 (t, J = 6.9 Hz, 3H). 13C NMR (75 MHz, DMSO-d6) δ
162.60, 162.17, 156.77, 138.78, 136.97, 135.83, 128.47, 127.83, 125.61,
121.09, 119.67, 115.29, 115.12, 65.31, 64.20, 42.82, 30.33, 20.83, 14.81,
11.93, 11.04. HRMS (ESI): calcd for C26H30ClNO6S [M + Na]+
542.1380, found 542.1373. HPLC (80:20 methanol:water with 1‰
TFA): tR = 12.22 min, 99.5%.
4-((3-Bromophenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (38). White solid, yield: 28%; mp: 181−183 °C. H NMR
(300 MHz, DMSO-d6) δ 12.75 (s, 1H), 7.73−7.64 (m, 2H), 7.51−7.37
(m, 2H), 6.77 (s, 2H), 4.43 (s, 2H), 3.89 (s, 2H), 2.37 (s, 3H), 2.31 (s,
6H), 2.12 (s, 3H), 2.06 (d, J = 7.0 Hz, 2H). HRMS (ESI): calcd for
C24H25BrClNO5S [M + Na]+ 578.0203, found 578.0195. HPLC (90:10
methanol:water with 1‰ TFA): tR = 9.93 min, 96.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
fluorophenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic
Acid (51). White solid, yield: 21%; mp: 158−160 °C. 1H NMR (300
MHz, DMSO-d6) δ 12.83 (s, 1H), 7.76 (d, J = 5.3 Hz, 1H), 7.65 (dd, J =
16.2, 8.5 Hz, 2H), 7.29 (t, J = 8.8 Hz, 1H), 6.63 (s, 2H), 4.32 (t, J = 6.9
Hz, 2H), 3.77 (t, J = 4.9 Hz, 2H), 2.44 (s, 3H), 2.27 (s, 3H), 2.18 (s,
6H), 1.94 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 161.78, 156.80,
143.26, 137.09, 136.94, 129.85, 127.10, 125.52, 122.31, 120.78, 120.50,
115.18, 112.03, 64.87, 45.50, 30.72, 21.34, 20.91. HRMS (ESI): calcd
for C24H25ClFNO5S [M + Na]+ 516.1024, found 516.1021. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 10.37 min, 95.5%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
chlorophenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carboxylic
Acid (52). White solid, Yield: 14%; mp: 165−167 °C. 1H NMR (300
MHz, DMSO-d6) δ 12.93 (s, 1H), 7.80 (d, J = 8.5 Hz, 2H), 7.62 (d, J =
8.7 Hz, 2H), 6.73 (s, 2H), 4.42 (t, J = 8.1 Hz, 2H), 3.87 (t, J = 4.7 Hz,
2H), 2.54 (s, 3H), 2.36 (s, 3H), 2.28 (s, 6H), 2.04 (s, 2H). 13C NMR
(75 MHz, DMSO-d6) δ 162.55, 156.69, 142.87, 139.52, 138.19, 136.94,
129.97, 128.22, 128.02, 125.58, 121.34, 118.18, 115.00, 65.17, 42.94,
30.20, 20.87, 11.95, 11.08. HRMS (ESI): calcd for C24H25Cl2NO5S [M
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
methoxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (41). White solid, yield: 16%; mp: 180−182 °C. H NMR
(300 MHz, DMSO-d6) δ 12.79 (s, 1H), 7.72 (d, J = 8.6 Hz, 2H), 7.06
(d, J = 8.6 Hz, 2H), 6.73 (s, 2H), 4.45−4.35 (m, 2H), 3.87 (t, J = 4.3 Hz,
2H), 3.81 (s, 3H), 2.53 (s, 3H), 2.36 (s, 3H), 2.28 (s, 6H), 2.02 (s, 2H).
13C NMR (75 MHz, DMSO-d6) δ 162.84, 162.59, 156.72, 138.83,
136.97, 135.89, 128.49, 127.77, 125.56, 120.99, 119.55, 115.06, 114.96,
65.20, 56.11, 42.82, 30.26, 20.88, 11.95, 11.03. HRMS (ESI): calcd for
C25H28ClNO6S [M + Na]+ 528.1224, found 528.1218. HPLC (80:20
methanol:water with 1‰ TFA): tR = 9.52 min, 99.6%.
4-((4-Bromophenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (42). White solid, yield: 17%; mp: 174−176 °C. H NMR
(300 MHz, DMSO-d6) δ 12.83 (s, 1H), 7.65 (dd, J = 16.7, 8.6 Hz, 4H),
6.63 (s, 2H), 4.32 (t, J = 5.6 Hz, 2H), 3.78 (t, J = 5.0 Hz, 2H), 2.43 (s,
P
https://doi.org/10.1021/acs.jmedchem.1c00682
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
+ Na]+ 532.0728, found 532.0726. HPLC (80:20 methanol:water with
1‰ TFA): tR = 15.25 min, 99.9%.
J = 8.2 Hz, 2H), 6.66 (s, 2H), 4.60−4.51 (m, 2H), 4.35 (q, J = 7.1 Hz,
2H), 3.96 (t, J = 5.7 Hz, 2H), 2.81 (dq, J = 15.1, 7.4 Hz, 4H), 2.38 (s,
6H), 2.30 (s, 3H), 2.24 (dd, J = 13.0, 6.9 Hz, 2H), 1.41 (t, J = 7.1 Hz,
3H), 1.14−1.04 (m, 6H). ESI-MS m/z: 514.2 [M + 1]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-((4-nitrophenyl)sulfonyl)-1H-pyrrole-2-carboxylic Acid (53).
1
Yellow solid, yield: 13%; mp: 196−198 °C. H NMR (300 MHz,
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-di-
ethyl-4-tosyl-1H-pyrrole-2-carboxylate (84a). 84a was synthe-
DMSO-d6) δ 12.89 (s, 1H), 8.25 (d, J = 8.7 Hz, 2H), 7.96 (d, J = 8.7 Hz,
2H), 6.63 (s, 2H), 4.33 (t, J = 6.8 Hz, 2H), 3.78 (t, J = 5.2 Hz, 2H), 2.47
(s, 3H), 2.29 (s, 3H), 2.18 (s, 6H), 1.95 (s, 2H). HRMS (ESI): calcd for
C24H25ClN2O7S [M - H]− 519.0993, found 519.0990. HPLC (80:20
methanol:water with 1‰ TFA): tR = 10.45 min, 99.5%.
1
sized according to the procedure of 77a, colorless oil, yield: 56%. H
NMR (300 MHz, Chloroform-d) δ 7.75 (d, J = 8.3 Hz, 2H), 7.28 (d, J =
8.3 Hz, 2H), 6.65 (s, 2H), 4.56−4.45 (m, 2H), 4.32 (q, J = 7.1 Hz, 2H),
3.95 (t, J = 5.6 Hz, 2H), 3.12 (q, J = 7.4 Hz, 2H), 2.97 (q, J = 7.3 Hz,
2H), 2.44 (s, 3H), 2.39 (s, 6H), 2.19 (p, J = 5.8 Hz, 2H), 1.38 (t, J = 7.1
Hz, 3H), 1.26 (t, J = 7.4 Hz, 3H), 1.06 (t, J = 7.3 Hz, 3H). ESI-MS m/z:
568.2 [M + Na]+.
4-((4-Aminophenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylic Acid (55). To a solution of 53 (0.2 g, 0.34 mmol) and EtOAc (10
mL), 5% Pd/C (0.05 equiv) was added. The mixture was stirred at 50
°C for 3 h. Upon the consumption of 53, the raction mixture was cooled
to room temperature and Pd/C was then filtered. The solvent was
evaporated under reduced pressure to give 55 as a gray solid, yield: 15%;
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-diethyl-4-
tosyl-1H-pyrrole-2-carboxylic Acid (17). White powder, yield:
74%; mp: 180−182 °C. 1H NMR (300 MHz, Chloroform-d) δ 7.76 (d,
J = 8.2 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 6.64 (s, 2H), 4.53 (t, J = 7.1
Hz, 2H), 3.93 (t, J = 5.4 Hz, 2H), 3.15 (q, J = 7.4 Hz, 2H), 3.06 (q, J =
7.3 Hz, 2H), 2.45 (s, 3H), 2.37 (s, 6H), 2.21 (p, J = 5.9 Hz, 2H), 1.27 (t,
J = 7.4 Hz, 3H), 1.09 (t, J = 7.3 Hz, 3H). HRMS (ESI): calcd for
C27H32ClNO5S [M + Na]+ 540.1587, found 540.1582. HPLC (80:20
methanol:water with 1‰ TFA): tR = 15.84 min, 99.2%.
1
mp: 156−158 °C. H NMR (300 MHz, DMSO-d6) δ 12.77 (s, 1H),
7.42 (d, J = 8.7 Hz, 2H), 6.75 (s, 2H), 6.58 (d, J = 8.7 Hz, 2H), 6.03 (s,
2H), 4.39 (t, J = 6.9 Hz, 2H), 3.87 (t, J = 5.7 Hz, 2H), 2.51 (s, 3H), 2.35
(s, 3H), 2.28 (s, 6H), 2.01 (dd, J = 8.6, 5.5 Hz, 2H). HRMS (ESI): calcd
for C24H27ClN2O5S [M + Na]+ 513.1227, found 513.1225. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 5.13 min, 99.2%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((2,6-
dimethylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
81b−e were synthesized from the cyclization reaction of 79 and 80b-
c according to the procedure of 81a. Starting from the corresponding
81b−e, 18 and 19 and 21 and 22 were synthesized according to the
procedure of 17.
1
ylic Acid (56). White solid, yield: 25%; mp: 179−181 °C. H NMR
(300 MHz, Chloroform-d) δ 7.32 (t, J = 8.5 Hz, 1H), 7.12 (d, J = 7.6 Hz,
2H), 6.61 (s, 2H), 4.54 (t, J = 6.9 Hz, 2H), 3.89 (t, J = 5.4 Hz, 2H), 2.60
(s, 6H), 2.57 (s, 3H), 2.36 (s, 6H), 2.30 (s, 3H), 2.23−2.14 (m, 2H).
13C NMR (75 MHz, DMSO) δ 164.31, 158.51, 141.62, 140.36, 140.07,
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-5-ethyl-3-
methyl-4-tosyl-1H-pyrrole-2 carboxylic Acid (18). White powder,
yield: 77%; mp: 186−188 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.64
(d, J = 8.9 Hz, 2H), 6.98 (d, J = 8.9 Hz, 2H), 6.63 (s, 2H), 4.29 (t, J = 6.7
Hz, 2H), 4.09 (q, J = 7.1 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.46 (s, 3H),
2.26 (s, 3H), 2.18 (s, 6H), 1.93 (s, 2H), 1.14 (t, J = 7.1 Hz, 3H). HRMS
(ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1425.
HPLC (80:20 methanol:water with 1‰ TFA): tR = 14.70 min, 97.7%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-ethyl-5-
methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (19). White solid,
yield: 83%; mp: 178−180 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.84
(s, 1H), 7.66 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 6.73 (s, 2H),
4.42 (t, J = 6.2, 2H), 3.88 (t, J = 5.0 Hz, 2H), 2.87 (q, J = 7.3 Hz, 2H),
2.54 (s, 3H), 2.35 (s, 3H), 2.28 (s, 6H), 2.04 (dt, J = 10.9, 5.4 Hz, 2H),
0.94 (t, J = 6.8 Hz, 3H). 13C NMR (75 MHz, DMSO) δ 162.39, 156.73,
143.61, 141.54, 139.11, 136.96, 134.67, 130.25, 126.29, 125.54, 120.57,
118.28, 115.05, 65.24, 42.92, 30.20, 21.41, 20.90, 18.83, 16.35, 11.17.
HRMS (ESI): calcd for C26H30ClNO5S [M + Na]+ 526.1431, found
526.1426. HPLC (80:20 methanol:water with 1‰ TFA): tR = 12.78
min, 98.1%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-5-isobutyl-3-
methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (21). White solid,
yield: 79%; mp: 162−164 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.94
(s, 1H), 7.55 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 6.71 (s, 2H),
4.42 (t J = 6.7 Hz, 2H), 3.79 (t, J = 5.6 Hz, 2H), 2.88 (d, J = 7.3 Hz, 2H),
2.33 (s, 3H), 2.26 (s, 6H), 2.24 (s, 3H), 2.00−1.88 (m, 3H), 0.87 (d, J =
6.4 Hz, 6H). HRMS (ESI): calcd for C28H34ClNO5S [M + NH4]+
549.2187, found 549.2185. HPLC (90:10 methanol:water with 1‰
TFA): tR = 12.2 min, 99.4%.
139.99, 138.71, 134.35, 133.57, 127.41, 123.09, 122.47, 116.90, 67.00,
44.31, 32.12, 23.61, 22.57, 12.82, 12.64. HRMS (ESI): calcd for
C26H30ClNO5S [M + Na]+ 526.1431, found 526.1423. HPLC (80:20
methanol:water with 1‰ TFA): tR = 15.33 min, 99.8%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((3,5-
dimethylphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (57). White solid. Yield: 18%; mp: 184−186 °C. H NMR
(300 MHz, Chloroform-d) δ 7.49 (s, 2H), 7.20 (s, 1H), 6.63 (s, 2H),
4.53 (t, J = 7.0 Hz, 2H), 3.91 (t, J = 5.5 Hz, 2H), 2.68 (s, 3H), 2.61 (s,
3H), 2.39 (s, 6H), 2.36 (s, 6H), 2.23−2.13 (m, 2H). HRMS (ESI):
calcd for C26H30ClNO5S [M + Na]+ 526.1431, found 526.1425. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 15.59 min, 96.1%.
Ethyl-3,5-diethyl-1H-pyrrole-2-carboxylate (81a). 78 (8 g; 50
mmol) was first added to an aqueous solution of NaNO2 (10.3 g in 25
mL, 150 mmol). After stirring for 24 h at 0 °C, the solution was allowed
to reach room temperature. After extracting the aqueous layer several
times with DCM, the combined organic layers were dried over MgSO4
and then evaporated to dryness. The resulting crude product was
dissolved in acetic acid (50 mL) and added dropwise to a stirred
solution of 80a (4.4 g, 44 mmol) in acetic acid (50 mL). A mixture of Zn
(10 g; 156 mmol) and AcONa (12.7 g; 156 mmol) was added
alternatively. The mixture was then stirred at 80 °C for 2 h, poured into
water (300 mL), and extracted with DCM (3 times) The combined
organic layer was concentrated under reduced pressure and purification
by silica gel column chromatography (petroleum ether/ethyl acetate =
50:1 to 20:1 v/v) gave 81a, light yellow powder, yield: 43%. 1H NMR
(300 MHz, Chloroform-d) δ 8.82 (s, 1H), 5.99−5.89 (m, 1H), 4.35 (q,
J = 7.1 Hz, 2H), 2.82 (q, J = 7.5 Hz, 2H), 2.66 (q, J = 7.6 Hz, 2H), 1.40
(t, J = 7.1 Hz, 3H), 1.31 (t, J = 7.6 Hz, 3H), 1.24 (t, J = 7.7 Hz, 3H). ESI-
MS m/z: 194.1 [M-1]−.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3-isobutyl-5-
methyl-4-tosyl-1H-pyrrole-2-carboxylic Acid (22). White solid,
yield: 86%; mp: 149−151 °C. 1H NMR (300 MHz, DMSO-d6) δ 12.89
(s, 1H), 7.59 (d, J = 7.8 Hz, 2H), 7.31 (d, J = 7.7 Hz, 2H), 6.70 (s, 2H),
4.51−4.33 (m, 2H), 3.81 (t, J = 5.1 Hz, 2H), 2.82 (d, J = 6.9 Hz, 2H),
2.46 (s, 3H), 2.32 (s, 3H), 2.25 (s, 6H), 2.07−1.96 (m, 2H), 1.83−1.73
(m, 1H), 0.76 (d, J = 6.3 Hz, 6H). 13C NMR (75 MHz, DMSO-d6) δ
166.33, 156.20, 143.26, 141.29, 140.55, 137.19, 136.92, 129.55, 126.56,
126.23, 120.24, 119.11, 114.26, 64.48, 43.10, 33.24, 30.81, 30.38, 22.28,
21.53, 20.96, 11.47. HRMS (ESI): calcd for C28H34ClNO5S [M +
NH4]+ 549.2187, found 549.2180. HPLC (80:20 methanol:water with
1‰ TFA): tR = 16.92 min, 99.3%.
Ethyl-3,5-diethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate
(82a). 81a was synthesized according to the procedure of 75a, yellow
1
powder, yield: 62%. H NMR (300 MHz, Chloroform-d) δ 9.30 (s,
1H), 7.04 (d, J = 7.8 Hz, 2H), 6.93 (d, J = 8.0 Hz, 2H), 4.40 (q, J = 7.1
Hz, 2H), 2.82 (q, J = 7.3 Hz, 2H), 2.75 (q, J = 7.5, 6.0 Hz, 2H), 2.31 (s,
3H), 1.42 (t, J = 7.1 Hz, 3H), 1.23 (t, J = 7.6 Hz, 3H), 1.09 (t, J = 7.3 Hz,
3H). ESI-MS m/z: 318.3 [M + 1]+.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-di-
ethyl-4-(p-tolylthio)-1H-pyrrole-2-carboxylate (83a). 83a was
synthesized according to the procedure of 63, colorless oil, yield: 72%.
1H NMR (300 MHz, Chloroform-d) δ 7.01 (d, J = 8.4 Hz, 2H), 6.89 (d,
Ethyl-3,5-dimethyl-4-(thiophen-2-ylsulfonyl)-1H-pyrrole-2-
carboxylate (86a). To a solution of 85a (2 g, 11 mmol) and PhCl (40
mL), AlCl3 (1.5 g, 11 mmol) was added under nitrogen at 0 °C. The
Q
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Article
mixture was srirred for 10 min and ethyl 3,5-dimethyl-1H-pyrrole-2-
carboxylate (1 g, 6 mmol) was added. The mixture was then allowed to
stir at 90 °C for 2 h under nitrogen. After completing the reaction
monitored by TLC, 100 mL of water was carefully added to the mixture.
The mixture was added 40 mL DCM and stirred for 15 min. After
filtration, the organic layer was isolated and the aqueous phase was
extracted with DCM (2 times). The combined organic layer was then
washed with brine and dried with Na2SO4. Removal of solvent under
reduced pressure yielded crude product. Further purification by silica
gel column chromatography (petroleum ether/ethyl acetate = 40:1 to
2H), 4.35 (q, J = 7.1 Hz, 2H), 3.93 (t, J = 5.6 Hz, 2H), 2.56 (s, 3H), 2.36
(s, 6H), 2.32 (s, 6H), 2.25 (p, J = 6.5 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H).
ESI-MS m/z: 536.1 [M + Na]+.
Ethyl-4-((4-acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylate (92a). 92a was synthesized according to the procedure of 75a,
yellow solid, yield: 65%. 1H NMR (300 MHz, Chloroform-d) δ 8.06 (d,
J = 7.0 Hz, 2H), 7.96 (d, J = 7.0 Hz, 2H), 6.64 (s, 2H), 4.51 (t, J = 6.8
Hz, 2H), 4.37−4.25 (m, 2H), 3.92 (t, J = 5.0 Hz, 2H), 2.67 (s, 6H), 2.51
(s, 3H), 2.38 (s, 6H), 2.17 (p, J = 5.5 Hz, 2H), 1.38 (t, J = 7.7 Hz, 3H).
ESI-MS m/z: 546.2 [M + H]+.
1
10:1 v/v) gave 86a, yellow powder, 2.6 g, yield: 68%. H NMR (300
4-((4-Acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylic Acid (40). 40 was synthesized from 92a according to the hydrolysis
procedure of 16, yellow powder, Yield: 42%, mp: 215−216 °C, H
MHz, Chloroform-d) δ 9.42 (s, 1H), 7.68 (dd, J = 3.7, 1.3 Hz, 1H), 7.60
(dd, J = 5.0, 1.3 Hz, 1H), 7.09 (dd, J = 4.9, 3.8 Hz, 1H), 4.36 (q, J = 7.1
Hz, 2H), 2.64 (s, 3H), 2.58 (s, 3H), 1.40 (t, J = 7.1 Hz, 3H). ESI-MS m/
z: 312.0 [M - 1]−.
1
NMR (300 MHz, DMSO-d6) δ 12.93 (s, 1H), 8.07 (d, J = 8.3 Hz, 2H),
7.90 (d, J = 8.3 Hz, 2H), 6.71 (s, 2H), 4.40 (t, J = 6.7 Hz, 2H), 3.85 (t, J
= 5.9 Hz, 2H), 2.59 (s, 3H), 2.54 (s, 3H), 2.35 (s, 3H), 2.25 (s, 6H),
2.01 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 197.73, 162.57, 156.74,
150.93, 139.28, 137.99, 137.00, 129.13, 128.67, 125.59, 125.09, 121.06,
120.48, 115.06, 65.13, 42.77, 30.37, 27.29, 20.89, 11.57, 10.56. HRMS
(ESI): calcd for C26H28ClNO6S [M + Na]+ 540.1224, found 540.1217.
HPLC (90:10 methanol:water with 1‰ TFA): tR = 7.50 min, 99.8%.
Starting from corresponding iodobenzenes 88b−g, 32, 36, 48−50,
and 54 were synthesized according to the procedure of 40.
Ethyl-1-(3-(4-chloro-3,5-dimethylphenoxy)propyl)-3,5-di-
methyl-4-(thiophen-2-ylsulfonyl)-1H-pyrrole-2-carboxylate
(87a). 87a was synthesized according to the procedure of 63, yellow
powder, yield: 63%. 1H NMR (300 MHz, Chloroform-d) δ 7.63 (dd, J =
3.7, 1.3 Hz, 1H), 7.57 (dd, J = 5.0, 1.3 Hz, 1H), 7.07 (dd, J = 5.0, 3.8 Hz,
1H), 6.63 (s, 2H), 4.51 (t, J = 7.1 Hz, 2H), 4.32 (q, J = 7.1 Hz, 2H), 3.90
(t, J = 5.6 Hz, 2H), 2.65 (s, 3H), 2.59 (s, 3H), 2.38 (s, 6H), 2.17 (p, J =
6.0 Hz, 2H), 1.39 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 510.1 [M + 1]+.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-(thiophen-2-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (27).
Starting from 87a, 27 was synthesized through the hydrolysis procedure
of 23. Brown solid, yield: 54%; mp: 178−180 °C. 1H NMR (300 MHz,
DMSO-d6) δ 12.91 (s, 1H), 7.95 (s, 1H), 7.65 (s, 1H), 7.15 (s, 1H),
6.73 (s, 2H), 4.42 (s, 2H), 3.87 (s, 2H), 2.54 (s, 3H), 2.44 (s, 3H), 2.28
(s, 6H), 2.03 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.56, 156.72,
146.06, 139.14, 136.98, 133.85, 131.75, 128.43, 127.90, 125.58, 121.24,
119.64, 115.08, 65.19, 42.94, 30.22, 20.90, 11.99, 11.15. HRMS (ESI):
calcd for C22H24ClNO5S2 [M + Na]+ 504.0682, found 504.0686. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 16.32 min, 97.9%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-(naphthalen-1-ylsulfonyl)-1H-pyrrole-2-carboxylic Acid (28).
Starting from 85b, 28 was synthesized according to the procedure of 27,
white solid, yield: 63%; mp: 184−186 °C. 1H NMR (300 MHz, DMSO-
d6) δ 12.88 (s, 1H), 8.43−8.33 (m, 1H), 8.24 (d, J = 8.2 Hz, 1H), 8.16
(d, J = 7.2 Hz, 1H), 8.11−8.01 (m, 1H), 7.68 (t, J = 7.8 Hz, 1H), 7.60
(dd, J = 6.3, 3.3 Hz, 2H), 6.70 (s, 2H), 4.44 (t, J = 6.7 Hz, 2H), 3.84 (t, J
= 5.5 Hz, 2H), 2.61 (s, 3H), 2.27 (s, 6H), 2.24 (s, 3H), 2.10−1.99 (m,
2H). HRMS (ESI): calcd for C28H28ClNO5S [M + Na]+ 548.1274,
found 548.1266. HPLC (80:20 methanol:water with 1‰ TFA): tR =
13.96 min, 99.6%.
1-(4-Mercaptophenyl)ethan-1-one (89a). To a mixture of 4-
iodoacetophenone (88a, 5 g, 20 mmol), CuSO4·5H2O (0.25 g, 1
mmol), Cs2CO3 (32.4 g, 100 mmol), and DMSO (70 mL), 1,2-
ethanedithiol (3.7 g, 40 mmol) was added under nitrogen at room
temperature. The mixture was stirred in the preheated oil bath at 90 °C
for 4 h. After being cooled to room temperature, the reaction mixture
was carefully quenched by aqueous HCl (1 N) to pH 2. EtOAc (80 mL)
was added, and the mixture was stirred for another 10 min. The organic
layer was separated and washed with brine, dried over MgSO4, and
concentrated under vacuum. The crude product was further purified by
column chromatography (petroleum ether/ethyl acetate = 50:1 to 10:1
v/v) to provide 89a as a light yellow powder, yield: 76%. 1H NMR (300
MHz, Chloroform-d) δ 7.93 (d, J = 8.6 Hz, 2H), 7.59 (d, J = 8.5 Hz,
2H), 2.60 (s, 3H). ESI-MS m/z: 150.9 [M-1]−.
4-((2-Acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (32). Yellow solid, yield: 9%; mp: 180−182 °C. H NMR
(300 MHz, DMSO-d6) δ 12.95 (s, 1H), 7.67 (dd, J = 47.1, 19.2 Hz,
4H), 6.77 (s, 2H), 4.44 (s, 2H), 3.92 (s, 2H), 2.45 (s, 6H), 2.25 (d, J =
15.6 Hz, 9H), 2.06 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 202.53,
162.59, 156.77, 141.23, 140.23, 139.75, 137.00, 133.48, 130.35, 128.40,
127.21, 125.59, 121.12, 118.14, 115.14, 65.13, 42.76, 32.18, 30.36,
20.93, 11.83, 11.09. HRMS (ESI): calcd for C26H28ClNO6S [M +
NH4]+ 535.1664, found 535.1661. HPLC (90:10 methanol:water with
1‰ TFA): tR = 23.10 min, 97.2%.
4-((3-Acetylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (36). Pale yellow solid, yield: 14%; mp: 172−174 °C. H
NMR (300 MHz, DMSO-d6) δ 12.98 (s, 1H), 8.25 (d, J = 12.4 Hz, 2H),
8.05 (d, J = 7.8 Hz, 1H), 7.76 (t, J = 7.7 Hz, 1H), 6.75 (s, 2H), 4.45 (t, J
= 6.2 Hz, 2H), 3.90 (t, J = 4.5 Hz, 2H), 2.65 (s, 3H), 2.59 (s, 3H), 2.42
(s, 3H), 2.30 (s, 6H), 2.07 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ
197.13, 162.53, 156.68, 144.68, 139.61, 137.88, 136.94, 133.04, 130.75,
130.41, 128.03, 125.57, 124.96, 121.38, 118.11, 114.99, 65.14, 42.96,
30.21, 27.21, 20.87, 11.98, 11.10. HRMS (ESI): calcd for
C26H28ClNO6S [M + NH4]+ 535.1664, found 535.1660. HPLC
(80:20 methanol:water with 1‰ TFA): tR = 7.76 min, 99.6%.
4-([1,1′-Biphenyl]-4-ylsulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1
ylic Acid (48). White solid, Yield: 35%; mp: 158−160 °C. H NMR
(300 MHz, DMSO-d6) δ 12.93 (s, 1H), 7.93−7.37 (m, 9H), 6.74 (s,
2H), 4.42 (s, 2H), 3.88 (s, 2H), 2.59 (s, 3H), 2.41 (s, 3H), 2.26 (s, 6H),
2.05 (s, 2H). 13C NMR (75 MHz, DMSO-d6) δ 162.58, 156.76, 144.82,
142.91, 139.33, 138.88, 136.97, 129.53, 128.98, 128.06, 127.50, 126.92,
125.63, 121.34, 118.86, 115.11, 65.30, 42.93, 30.30, 20.84, 12.02, 11.15.
HRMS (ESI): calcd for C30H30ClNO5S [M + H]+ 552.1611, found
552.1603. HPLC (90:10 methanol:water with 1‰ TFA): tR = 17.4
min, 98.2%.
4-((4-Benzoylphenyl)sulfonyl)-1-(3-(4-chloro-3,5-
Ethyl-4-((4-acetylphenyl)thio)-3,5-dimethyl-1H-pyrrole-2-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
carboxylate (90a). 90a was synthesized according to the procedure of
1
1
ylic Acid (49). Light yellow solid, yield: 16%; mp: 146−148 °C. H
75a, yellow solid. Yield: 54%. H NMR (300 MHz, Chloroform-d) δ
NMR (300 MHz, Chloroform-d) δ 7.99 (d, J = 8.4 Hz, 2H), 7.90 (d, J =
8.4 Hz, 2H), 7.85−7.79 (m, 2H), 7.68 (t, J = 7.4 Hz, 1H), 7.54 (t, J = 7.6
Hz, 2H), 6.63 (s, 2H), 4.55 (t, J = 6.9 Hz, 2H), 3.91 (t, J = 5.4 Hz, 2H),
2.71 (s, 3H), 2.61 (s, 3H), 2.35 (s, 6H), 2.19 (s, 2H). 13C NMR (75
MHz, DMSO-d6) δ 195.13, 162.54, 156.75, 147.06, 140.99, 139.80,
136.97, 136.63, 133.67, 130.88, 130.22, 129.12, 128.21, 126.47, 125.63,
121.58, 117.99, 115.11, 65.31, 43.01, 30.26, 20.83, 11.99, 11.16. HRMS
(ESI): calcd for C31H30ClNO6S [M + NH4]+ 597.1821, found
9.60 (s, 1H), 7.84 (d, J = 8.4 Hz, 2H), 7.07 (d, J = 8.3 Hz, 2H), 4.41 (q, J
= 7.1 Hz, 2H), 2.59 (s, 3H), 2.36 (s, 3H), 2.32 (s, 3H), 1.44 (t, J = 7.1
Hz, 3H). ESI-MS m/z: 316.2 [M−1]−.
Ethyl-4-((4-acetylphenyl)thio)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylate (91a). 91a was synthesized according to the procedure of 63,
yellow oil, yield: 75%. 1H NMR (300 MHz, Chloroform-d) δ 7.79 (d, J
= 8.5 Hz, 2H), 7.01 (d, J = 8.4 Hz, 2H), 6.64 (s, 2H), 4.58 (t, J = 6.9 Hz,
R
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597.1815. HPLC (90:10 methanol:water with 1‰ TFA): tR = 15.33
min, 99.9%.
°C for 3 h. Upon the consumption of 91h, the reaction mixture was
cooled to room temperature and Pd/C was then filtered. The solvent
was evaporated under reduced pressure to give 43 as a gray solid, yield:
96%; mp: 177−179 °C. 1H NMR (300 MHz, DMSO-d6) δ 7.65 (d, J =
8.7 Hz, 2H), 6.91 (d, J = 8.7 Hz, 2H), 6.77 (s, 2H), 4.43 (t, J = 6.7 Hz,
2H), 3.89 (t, J = 5.5 Hz, 2H), 2.55 (s, 3H), 2.39 (s, 3H), 2.31 (s, 6H),
2.05 (dt, J = 13.0, 6.6 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ
162.64, 161.74, 156.74, 138.59, 136.98, 134.22, 128.69, 127.70, 125.56,
120.94, 119.94, 116.16, 115.07, 65.23, 42.76, 30.29, 20.89, 11.96, 11.02.
HRMS (ESI): calcd for C24H26ClNO6S [M + Na]+ 514.1067, found
514.1061. HPLC (80:20 methanol:water with 1‰ TFA): tR = 6.33
min, 99.2%.
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-3,5-dimethyl-
4-((4-phenoxyphenyl)sulfonyl)-1H-pyrrole-2-carboxylic Acid
(50). White solid, yield: 15%; mp: 164−166 °C. 1H NMR (300
MHz, Chloroform-d) δ 7.82 (d, J = 8.6 Hz, 2H), 7.44 (t, J = 7.8 Hz, 2H),
7.26 (t, J = 7.3 Hz, 1H), 7.06 (dd, J = 15.6, 8.3 Hz, 4H), 6.63 (s, 2H),
4.53 (t, J = 6.6 Hz, 2H), 3.91 (t, J = 5.0 Hz, 2H), 2.68 (s, 3H), 2.59 (s,
3H), 2.36 (s, 6H), 2.24−2.12 (m, 2H). 13C NMR (75 MHz, DMSO-d6)
δ 162.58, 161.19, 156.77, 155.20, 139.07, 138.20, 136.98, 130.80,
128.84, 127.94, 125.63, 125.40, 121.23, 120.56, 119.20, 118.10, 115.12,
65.32, 42.89, 30.31, 20.85, 11.96, 11.09. HRMS (ESI): calcd for
C30H30ClNO6S [M + NH4]+ 585.1826, found 585.1819. HPLC (80:20
methanol:water with 1‰ TFA): tR = 22.46 min, 99.2%.
Starting from 1-(benzyloxy)-2-iodobenzene (88j) and 1-(benzyl-
oxy)-3-iodobenzene (88i), 35 and 39 were obtained through the
procedure of 43.
4-((4-Carboxyphenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((2-
1
ylic Acid (54). Light purple solid, yield: 15%; mp: 183−185 °C. H
hydroxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
NMR (300 MHz, DMSO-d6) δ 13.34 (s, 1H), 8.11 (d, J = 8.4 Hz, 2H),
7.93 (d, J = 8.3 Hz, 2H), 6.76 (s, 2H), 4.45 (s, 2H), 3.89 (s, 2H), 2.58 (s,
3H), 2.40 (s, 3H), 2.30 (s, 6H), 2.07 (s, 2H). 13C NMR (75 MHz,
DMSO) δ 166.48, 162.48, 156.73, 147.55, 139.73, 136.96, 135.00,
130.74, 128.16, 126.50, 125.63, 121.50, 117.99, 115.09, 65.31, 42.98,
30.23, 20.82, 11.90, 11.12. HRMS (ESI): calcd for C25H26ClNO7S [M
+ NH4]+ 537.1462, found 537.1461. HPLC (80:20 methanol:water
with 1‰ TFA): tR = 6.63 min, 97.7%.
1
ylic Acid (35). White solid, yield: 10%; mp: 175−177 °C. H NMR
(300 MHz, Chloroform-d) δ 9.44 (s, 1H), 7.44 (d, J = 7.7 Hz, 2H), 7.03
(d, J = 8.2 Hz, 1H), 6.94 (t, J = 7.6 Hz, 1H), 6.63 (s, 2H), 4.55 (t, J = 7.0
Hz, 2H), 3.90 (t, J = 5.4 Hz, 2H), 2.68 (s, 3H), 2.57 (s, 3H), 2.37 (s,
6H), 2.20 (dt, J = 12.1, 5.9 Hz, 2H). 13C NMR (75 MHz, DMSO-d6) δ
162.73, 158.73, 156.73, 156.22, 140.24, 139.05, 136.98, 135.05, 129.23,
128.71, 125.55, 120.18, 118.89, 117.80, 115.12, 112.59, 65.17, 42.58,
21.50, 20.91, 11.91, 11.26. HRMS (ESI): calcd for C24H26ClNO6S [M
+ NH4]+ 509.1513, found 509.1505. HPLC (80:20 methanol:water
with 1‰ TFA): tR = 14.95 min, 99.3%.
4-(Benzyloxy)benzenethiol (89h). 89h was synthesized from 1-
(benzyloxy)-4-iodobenzene (88h) according to the procedure of 89a,
white solid. Yield: 68%. 1H NMR (300 MHz, Chloroform-d) δ 7.45−
7.41 (m, 2H), 7.39−7.34 (m, 3H), 7.33 (d, J = 7.4 Hz, 2H), 6.81 (d, J =
7.5 Hz, 2H), 5.03 (s, 2H), 3.73 (s, 1H). ESI-MS m/z: 215.1 [M-H]−.
Ethyl-4-((4-(benzyloxy)phenyl)thio)-3,5-dimethyl-1H-pyr-
role-2-carboxylate (90h). 90h was synthesized from 4-(benzyloxy)-
benzenethiol according to the procedure of 75a, yellow solid. Yield:
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((3-
hydroxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylic Acid (39). Gray solid, yield: 12%; mp: 173−175 °C. 1H NMR (300
MHz, DMSO-d6) δ 12.91 (s, 1H), 10.18 (s, 1H), 7.39 (t, J = 7.8 Hz,
1H), 7.22 (d, J = 9.9 Hz, 2H), 7.02 (d, J = 8.0 Hz, 1H), 6.76 (s, 2H),
4.44 (s, 2H), 3.91 (s, 2H), 2.56 (s, 3H), 2.41 (s, 3H), 2.31 (s, 6H), 2.05
(dd, J = 12.3, 6.3 Hz, 2H). HRMS (ESI): calcd for C24H26ClNO6S [M +
NH4]+ 509.1513, found 509.1507. HPLC (80:20 methanol:water with
1‰ TFA): tR = 6.47 min, 97.1%.
1
73%. H NMR (300 MHz, Chloroform-d) δ 9.27 (s, 1H), 7.51−7.35
(m, 5H), 7.01 (d, J = 8.8 Hz, 2H), 6.88 (d, J = 8.8 Hz, 2H), 5.05 (s, 2H),
4.38 (q, J = 7.1 Hz, 2H), 2.38 (s, 3H), 2.36 (s, 3H), 1.42 (t, J = 7.1 Hz,
3H). ESI-MS m/z: 380.1 [M−H]−.
Protein Purification. GST-tagged proteins Mcl-1 (residues 171−
327), Bcl-2 (residues 1−205) and A1/Bfl-1 (residues1−151) were
expressed from the pGEX-4 T-1 vector in E. coli BL21 (DE3) cells.
Protein expression was induced by 1.0 mM IPTG at 11 °C for 8 h. Cells
were lysed by an ultrasonic cracker in 25 mM Tris pH 8.0 buffer
containing 300 mM NaCl, 0.1% Tween 20, and 0.1% mercaptoethanol.
Proteins were further purified on a GSTtrap column (5 cm) in 50 mM
Tris pH 8.0 buffer, with 200 mM NaCl, 10 mM reduced glutathione,
and 1 mM DTT. Lastly, 10% SDS-PAGE was used for confirming the
molecular weight of the purified proteins.
FP-Based Competition Assay. An FP competition assay was used
to evaluate the inhibitory activities of the compounds against Mcl-1,75
Bcl-2,68 and Bfl-1.76 Different concentrations of compounds were
prepared by serial dilution in assay buffer (Mcl-1: 20 mM Tris, 150 mM
NaCl, 3 mM DTT, pH = 7.5; Bfl-1 and Bcl-2: 55 mM Hepes, 274 mM
NaCl, 1.48 mM Na2HPO4, pH = 7.0). FITC-Bak-BH3 was chosen as a
probe for Mcl-1 (10 nM final). Fam-Bid was chosen as probes for Bcl-2
and Bfl-1, and the final probe concentrations were 0.66 and 0.33 nM,
respectively. The protein final concentrations for Mcl-1, Bfl-1, and Bcl-2
were 20, 13.3, and 20 nM, respectively. The experiments were
conducted in 384-well black flat-bottomed polystyrene plates (Corning
#3575). For each assay, equal volumes of the diluted compounds (20
μL), proteins (20 μL), and probes (20 μL) were added to the wells in an
orderly fashion. Plates were rocked for 30 min at room temperature in
the dark, and then the FP values were detected using a SpectraMax
multimode microplate reader (Molecular Devices) with excitation and
emission wavelengths at 485 and 535 nm, respectively. For each assay,
the FP values of blank controls (probes only) were recorded as Pmin; the
FP values of negative controls (probes and proteins) were recorded as
Ethyl-4-((4-(benzyloxy)phenyl)thio)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylate (91h). 91h was synthesized according to the procedure of 63,
colorless oil, yield: 81%. 1H NMR (300 MHz, Chloroform-d) δ 7.45−
7.35 (m, 5H), 6.95 (d, J = 8.9 Hz, 2H), 6.85 (d, J = 8.9 Hz, 2H), 6.64 (s,
2H), 5.03 (s, 2H), 4.55 (t, J = 7.0 Hz, 2H), 4.33 (q, J = 7.1 Hz, 2H), 3.92
(t, J = 5.6 Hz, 2H), 2.37 (s, 6H), 2.37 (s, 3H), 2.36 (s, 3H), 2.23 (p, J =
6.2 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 578.2 [M + H]+.
Ethyl-4-((4-(benzyloxy)phenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylate (92h). 92h was synthesized according to the procedure of 77a,
white powder, yield: 63%. 1H NMR (300 MHz, Chloroform-d) δ 7.81
(d, J = 8.9 Hz, 2H), 7.44 (d, J = 4.2 Hz, 5H), 7.04 (d, J = 8.9 Hz, 2H),
6.64 (s, 2H), 5.14 (s, 2H), 4.49 (t, J = 7.1 Hz, 2H), 4.31 (q, J = 7.1 Hz,
2H), 3.92 (t, J = 5.5 Hz, 2H), 2.66 (s, 3H), 2.51 (s, 3H), 2.38 (s, 6H),
2.17 (p, J = 6.0 Hz, 2H), 1.38 (t, J = 7.1 Hz, 3H). ESI-MS m/z: 610.2 [M
+ H]+.
4-((4-(Benzyloxy)phenyl)sulfonyl)-1-(3-(4-chloro-3,5-
dimethylphenoxy)propyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylic acid (93h). To a solution of 92h (380 mg, 0.62 mmol) in EtOH/
THF (10 mL/10 mL) was added NaOH (2 M, 3 mL). The mixture was
stirred overnight at 50 °C. After completion of the reaction, the mixture
was then acidified with 1 M HCl and extracted with ethyl acetate. The
organic layer was washed with brine and dried with Na2SO4. Removal of
solution and further recrystallization from EtOH/nhexane gave title
compound 93h. White powder, yield: 80%. 1H NMR (300 MHz,
Chloroform-d) δ 7.83 (d, J = 8.6 Hz, 2H), 7.46−7.39 (m, 5H), 7.06 (d, J
= 8.6 Hz, 2H), 6.64 (s, 2H), 5.15 (s, 2H), 4.53 (t, J = 6.8 Hz, 2H), 3.92
(t, J = 5.2 Hz, 2H) 2.68 (s, 3H), 2.60 (s, 3H), 2.37 (s, 6H), 2.19 (p, J =
6.7 Hz, 2H). ESI-MS m/z: 604.1 [M + Na]+.
P
max, and the FP values of test wells (compounds, probes, and protein)
1-(3-(4-Chloro-3,5-dimethylphenoxy)propyl)-4-((4-
hydroxyphenyl)sulfonyl)-3,5-dimethyl-1H-pyrrole-2-carbox-
ylic Acid (43). To a solution of 93h (0.2 g, 0.34 mmol) and EtOAc (10
mL), 5% Pd/C (0.05 equiv) was added. The mixture was stirred at 50
were recorded as Ptest. The inhibition rates of the compounds at each
concentration point were calculated using the equation as follows:
inhibition rate (%) = [1 − (Ptest − Pmin)/(Pmax − Pmin)] × 100%, and the
S
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IC50 values were calculated using Graphpad Prism 6.0 software. Ki was
calculated as previously reported.47,77
WB Analysis. PARP cleavage induction of the compounds was
determined by the WB assay. After treatment with serially diluted 40
and A1210477 (TOPSCIENCE, T2632, China) , the H929 cells or
K562 cells were washed with PBS and lysed in RIPA lysis buffer. The
lysates were centrifuged at 12,000 rpm for 15 min at 4 °C, and the
supernatants were collected. The protein concentration was deter-
mined using the BCA protein assay kit (KeyGen BioTech, KGA902,
China). Equal amounts of the total protein extracts were subjected to
SDS-PAGE and then transferred onto NC membranes (PALL, 66485,
USA). After blocking the nonspecific binding sites with 5% nonfat milk
for 1 h, membranes were incubated at 4 °C overnight with the primary
antibody anti-cPARP (Abcam, ab32064, UK). After washing with
TBST for 10 min three times, the membranes were treated with an IgG-
HRP secondary antibody (Abcam, ab6721, UK) at 37 °C for 1−2 h and
scanned using the Odyssey infrared imaging system (SYNGENE,
G:BOXChemiXR5, UK).
Caspase 3/7 Activation Assay. Caspase 3/7 activation was
analyzed according to the previous report.78 H929 or K562 Cells were
dispensed in a 96-well plate and incubated with 40 and A1210477
(TOPSCIENCE, T2632, China) at indicated concentrations for 4 h.
After removal of compounds, cells were rinsed by culture medium and 1
μL/mL Caspase 3/7 assay buffer (KeyGen BioTech, KGA S037−50,
China) was added. The mixture was incubated at room temperature in
the dark for 30 min. Luminescence was measured using a multifunc-
tional microplate reader (Molecular Devices, Spectramax M3, USA).
PAMPA Evaluation. Membrane permeability coefficients were
evaluated via double-sink PAMPA on a PAMPA Explorer instrument
(pION). The detailed experimental procedures have been reported
previously.79 Briefly, the test compound was diluted with donor buffer
at pH 7.4 and placed in the donor side. The test compound was allowed
to permeate to the acceptor side through the artificial membrane over 4
h incubation at 25 °C. After incubation, the “sandwich” plate was
separated. The donor and acceptor solutions were then measured with a
UV plate reader, and the permeability value was calculated by pION
software.
CYP450 Inhibition Assay. The CYP450 inhibition activities of 40
on the five major isozymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6,
and CYP3A4) were evaluated. The assay was conducted in phosphate
buffer (100 mM, pH 7.4). For each assay, equal volumes (25 μL) of
diluted microsomes (0.2 mg/mL final), tested compounds or positive
control inhibitors (10 μM final, DMSO as the negative control), and
specific substrates of the isozymes (10 μM final) were preincubated at
37 °C for 5 min. Then, 25 μL of NADPH (1 mM, final) was added to
initiate the reaction. After 20 min of incubation, 100 μL of cold
acetonitrile was added to terminate the reaction. The samples were
centrifuged, and the supernatants were analyzed by LC−MS/MS to
determine the metabolites. The inhibition rate was calculated using the
following equation: inhibition rate (%) = [1 − (formation of the
metabolite in the presence of tested compound)/(formation of the
metabolite of negative control)] × 100%.
HLM and RLM Stability. Compound 40 was preincubated with
human liver microsomes (0.2 mg/mL) or rat liver microsomes (0.5 mg/
mL) for 5 min at 37 °C in phosphate buffer (100 mM, pH 7.4). Then, 1
mM NADPH was added to initiate the reaction. After incubation for
different times (0, 15, 30, 60, and 120 min) at 37 °C, cold acetonitrile
was added to precipitate the protein. Then, the samples were
centrifuged, and the supernatants were analyzed by LC−MS/MS.
Human and Mouse Plasma Stability. 40 was dissolved in DMSO
at 10 mM and 4 μL of compound solution was then incubated with 996
μL of prewarmed mouse or human plasma in 37 °C. 5-fold volumes of
cold acetonitrile were added to stop the reaction at 0, 0.5, 1, and 2 h.
The samples were centrifuged, the supernatant was transferred to a new
96-well plate, and the samples were mixed with purity water (v:v = 1:2).
The compound concentration was measured by LC−MS−MS.
Caco-2 Permeability. Caco-2 cells purchased from ATCC were
seeded onto polyethylene membranes (PET) in 24-well Corning
transwell plates at 1 × 105 cells/cm2, and the medium was refreshed
every other day until the 21st day for confluent cell monolayer
formation. The standard transport buffer in the study was HBSS at pH
7.4. Before and after the transport studies, the monolayer integrity was
TR-FRET Based Assay. The Bcl-xL inhibitory ratio was assessed by
time-resolved fluorescence resonance energy transfer (TR-FRET)
technology48 using a recombinant Bcl-xL and a peptide-ligand
substrate. All of the reactions were conducted at room temperature.
The 20 μL reaction mixture in buffer contains Bcl-xL, the indicated
amount of the inhibitor (10 μM final), ligand, and the reaction dyes.
The reaction mixture was incubated for 120 min before reading the TR-
FRET signal. For the background, the ligand was replaced with assay
buffer. Fluorescence signals for both the donor and acceptor dyes were
measured using a Tecan Infinite M1000 plate reader. TR-FRET was
recorded as the ratio of the fluorescence of the acceptor and the donor
dyes (acceptor/donor).
Molecular Docking. Compounds 13, 16, 40, and 43 were
imported to Discovery Studio (DS) 4.0, and the 3D conformation
was generated by the “Prepare Ligands” protocol at pH 7.0. Then, the
compound was energy minimized in a CHARMm force field for
docking. The Mcl-1-ligand complex (PDB ID: 4HW2) downloaded
from the Protein Data Bank was chosen for docking studies. Protein
residues around the original ligand (radius = 8.0 Å) completely covered
the BH3 binding cavity. Libdock was used to dock 13, 16, 40, and 43
into the BH3 binding groove. The top 10 of 100 poses generated were
selected for analyzing binding interactions.
BCL2scan Binding Assays. Kd values against Mcl-1, Bcl-2, Bcl-xL,
Bcl-w, and Bcl2A1 of 40 were determined by Discovery X’s BCL2scan
technology. Compounds interacting with the Bcl-2 family proteins
prevented binding of these proteins to an immobilized known peptide
ligand and reduced the amount of target protein captured on the solid
support. Dissociation constants (Kds) for test compound-Bcl-2 family
protein interactions were calculated by measuring the amount of target
protein captured on the solid support. Assay conditions were optimized
for the measurement of true thermodynamic test compound Kd values.
MTT Assay. Antiproliferative activities of the compounds against
different cancer cell lines were determined using MTT assays. Cells
were seeded into 96-well plates at 5000 cells per well and incubated for
24 h. Then, the cells were treated with either serial dilutions of the
compounds or DMSO for 72 h. MTT solution (5 mg/mL) was added,
and the plates were incubated at 37 °C for another 4 h. After removing
the solution in the wells of the plates, 150 μL of DMSO was added to
dissolve the MTT formazan crystals. The absorbance values (OD
values) were determined using the Elx800 absorbance microplate
reader (BioTek, Vermont, USA) at 570 nm. Cell viability inhibition
rates of the compounds at each concentration point were calculated
using the equation as follows: inhibition rate (%) = [1 − (ODtest
−
ODblank)/(ODcontrol − ODblank)] × 100%. The IC50 values were
calculated using Graphpad Prism 6.0 software.
Co-ip Experiment. H929 cells were administered with different
concentrations of 40, DMSO or AZD5991 (Biochempartner,
BCP23513, China) for 4 h. Next, the cells were shaken in RIPA buffer
(Thermo Fisher Scientific, 78501, US) for 45 min on ice. The
supernatant was obtained from centrifugation at 12,000 rpm for 20 min.
Then, 5 μL of the Mcl-1 antibody (Cell Signaling Technology, D35A5,
US) was added into 1 mL of the supernatant. The mixture was shaken at
4 °C overnight. Then, Protein A/G PLUS-Agarose (Santa Cruz
Biotechnology, sc-2003, US) was used to precipitate the Mcl-1 complex
after incubation for 4 h. The beads were centrifuged and washed with
RIPA buffer four times and subjected to SDS-PAGE following with WB
analysis. The primary antibodies used in WB analysis were the Mcl-1
antibody (Cell Signaling Technology, D35A5, US) and Bak antibody
(Abcam, Y164, UK).
Apoptosis Assay by Annexin V/Propidium Iodide Staining.
H929 cells were seeded in a 6-well plate and incubated with 40 and
A1210477 (TOPSCIENCE, T2632, China) of indicated concen-
trations for 48 h. After incubation, cells were collected and washed with
PBS buffer. Cells were then dispersed with 500 μL of binding buffer per
tube and then 5 μL of Annexin V-FITC and 5 μL of propidium iodide
were added. After incubation for 15 min at 20 °C in the dark, apoptosis
was analyzed by flow cytometry in a flow cytometer (Becton-Dickinson
, FACS Calibur, US).
T
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evaluated by measuring transepithelial electrical resistance (TEER).
The pre-transport and post-transport TEER were required to be no less
than 200 Ω·cm2. The standard transport buffer in the study was HBSS
at pH 7.4. 40 was diluted with transport buffer from a 10 mM stock
solution to a concentration of 2 or 10 μM. The bidirectional
permeability of the 40 was investigated by adding the compound
solution either in the apical chamber or the basolateral chamber. The
efflux ratio was used to identify whether 40 was the Pgp substrate or not.
40 and the reference compound were quantified by LC−MS/MS
analysis.
In Vivo Pharmacokinetics Study. All animal experiments were
performed in accordance with the protocols approved by the
Institutional Animal Care and Use Committee of China Pharmaceutical
University. The pharmacokinetics study of 40 was conducted in SD rats
(200-250 g), which were purchased from Jinan Pengyue Laboratory
Animal Breeding Co. Ltd. The rats were randomly divided into two
groups (n = 6 for each group). The two groups were administered
compound 40 intravenously at 3 mg/kg and orally at 10 mg/kg. Blood
samples (150 μL) were collected into heparinized Eppendorf tubes at
predetermined time points (0, 0.016, 0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 8,
12, and 24 h) and centrifuged immediately at 4 °C and 8000 rpm for 5
min. The plasma samples were analyzed by LC−MS/MS.
In Vivo Antitumor Efficacy Evaluation. All animal experiments
were performed in accordance with the protocols approved by the
Institutional Animal Care and Use Committee of China Pharmaceutical
University. The female balb/c nude mice (7 weeks) were purchased
from GemPharmatech Co. Ltd. The mice were maintained in standard
conditions (12/12 h light/dark cycle, 22 3 °C, relative humidity of
40%) for 3 days and fed with a standard laboratory rodent diet and
water. The mice were then injected subcutaneously with MV4−11 cells
(5 × 106) in the right flank. When the average tumor volume reached
approximately 200 mm3, the mice were randomized into four groups (n
= 6 per group). Administration groups were intraperitoneally or orally
treated every 2 days for 14 days. Tumor growth and body weight were
monitored at the same interval. The mice were given free access to diet
and water during the course of experiments. The tumor volume was
calculated using the formula (smaller diameter)2 × (larger diameter)/2,
and the RTV was calculated accordingly as RTV = Vt/V0, where Vt is the
tumor volume measured at each time point after treatment and V0 is the
tumor volume at the beginning of treatment. The drug efficacy was
assessed by calculating the T/C values on the final day of the study
using the formula T/C = (mean RTVTreated/mean RTVControl) × 100%.
of Pharmacy, China Pharmaceutical University, Nanjing
210009, China; orcid.org/0000-0002-1671-1582;
Email: jiangzhengyucpu@163.com
Authors
Peng-Ju Zhu − State Key Laboratory of Natural Medicines, and
Jiang Su Key Laboratory of Drug Design and Optimization
and Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, Nanjing 210009, China
Ze-Zhou Yu − State Key Laboratory of Natural Medicines, and
Jiang Su Key Laboratory of Drug Design and Optimization
and Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, Nanjing 210009, China
Yi-Fei Lv − State Key Laboratory of Natural Medicines, and
Jiang Su Key Laboratory of Drug Design and Optimization
and Department of Medicinal Chemistry, School of Pharmacy,
China Pharmaceutical University, Nanjing 210009, China
Jing-Long Zhao − State Key Laboratory of Natural Medicines,
and Jiang Su Key Laboratory of Drug Design and
Optimization and Department of Medicinal Chemistry, School
of Pharmacy, China Pharmaceutical University, Nanjing
210009, China
Yuan-Yuan Tong − State Key Laboratory of Natural Medicines,
and Jiang Su Key Laboratory of Drug Design and
Optimization and Department of Medicinal Chemistry, School
of Pharmacy, China Pharmaceutical University, Nanjing
210009, China
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00682
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00682.
This study was supported by Projects 81930100, 81773581, and
81773639 of the National Natural Science Foundation of China,
the Priority Academic Program Development of Jiangsu Higher
Education Institutions, CPU2018GY02 of Double First Class
Innovation Team of China Pharmaceutical University, the
Project Program of State Key Laboratory of Natural Medicines,
China Pharmaceutical University (No. SKLNMZZ202003); the
“Qing Lan” Project of Jiangsu Province, and the Young Elite
Scientists Sponsorship Program by CAST (No.Y-
ESS20180146).
■
sı
Molecular formula strings and SAR data (CSV)
Predicated binding modes of 13, 16, 40, and 43 (ZIP)
Predicated drug-like properties, hERG channel inhibition
assay, hematoxylin and eosin (H&E) staining, TUNEL
1
analysis, statistical analysis and H NMR, 13C NMR,
HRMS and HPLC spectra for representative target
compounds (PDF)
ABBREVIATIONS
■
A1/Bfl1, Bcl-2-related protein A1; Bak, Bcl-2 homologous
antagonist-killer protein; Bax, Bcl-2-associated X protein; Bcl-2,
B-cell lymphoma 2; Bcl-xl, B-cell lymphoma x long isoform; Bcl-
w, Bcl-2-like-2; CML, chronic myeloid leukemia; CYP,
cytochrome P450; Co-ip, coimmunoprecipitation; FP, fluo-
rescence polarization; hERG, human Ether-a-go-go-Related
Gene; H&E, hematoxylin and eosin; HLM, human liver
microsome; Mcl-1, myeloid cell leukemia 1; MM, multiple
myeloma; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenylte-
trazolium bromide; PAMPA, parallel artificial membrane
permeability assay; PARP, poly ADP-ribose polymerase; PPIs,
protein−protein interactions; RLM, rat liver microsome; SAR,
AUTHOR INFORMATION
Corresponding Authors
■
Qi-Dong You − State Key Laboratory of Natural Medicines,
and Jiang Su Key Laboratory of Drug Design and
Optimization and Department of Medicinal Chemistry, School
of Pharmacy, China Pharmaceutical University, Nanjing
210009, China; orcid.org/0000-0002-8587-0122;
Email: youqd@163.com
Zheng-Yu Jiang − State Key Laboratory of Natural Medicines,
and Jiang Su Key Laboratory of Drug Design and
Optimization and Department of Medicinal Chemistry, School
U
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human non-small cell lung cancer cells. Cancer Biol. Ther. 2005, 4, 267−
276.
(18) Tong, J.; Wang, P.; Tan, S.; Chen, D.; Nikolovska-Coleska, Z.;
Zou, F.; Yu, J.; Zhang, L. Mcl-1 degradation is required for targeted
therapeutics to eradicate colon cancer cells. Cancer Res. 2017, 77,
2512−2521.
structure−activity relationships; SGF, simulated gastric fluid;
SIF, simulated intestinal fluid; TR-FRET, time-resolved
fluorescence resonance energy transfer; TUNEL, terminal
deoxynucleotidyl transferase dutp nick end labeling; WB,
western blot
(19) Pepper, C.; Lin, T. T.; Pratt, G.; Hewamana, S.; Brennan, P.;
Hiller, L.; Hills, R.; Ward, R.; Starczynski, J.; Austen, B.; Hooper, L.;
Stankovic, T.; Fegan, C. Mcl-1 expression has in vitro and in vivo
significance in chronic lymphocytic leukemia and is associated with
other poor prognostic markers. Blood 2008, 112, 3807−3817.
(20) Zhou, P.; Qian, L.; Bieszczad, C. K.; Noelle, R.; Binder, M.; Levy,
N. B.; Craig, R. W. Mcl-1 in transgenic mice promotes survival in a
spectrum of hematopoietic cell types and immortalization in the
myeloid lineage. Blood 1998, 92, 3226−3239.
(21) Touzeau, C.; Maciag, P.; Amiot, M.; Moreau, P. Targeting Bcl-2
for the treatment of multiple myeloma. Leukemia 2018, 32, 1899−1907.
(22) 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.
(23) Quinn, B. A.; Dash, R.; Azab, B.; Sarkar, S.; Das, S. K.; Kumar, S.;
Oyesanya, R. A.; Dasgupta, S.; Dent, P.; Grant, S.; Rahmani, M.; Curiel,
D. T.; Dmitriev, I.; Hedvat, M.; Wei, J.; Wu, B.; Stebbins, J. L.; Reed, J.
C.; Pellecchia, M.; Sarkar, D.; Fisher, P. B. Targeting Mcl-1 for the
therapy of cancer. Expert Opin. Invest. Drugs 2011, 20, 1397−1411.
(24) Akgul, C. Mcl-1 is a potential therapeutic target in multiple types
of cancer. Cell. Mol. Life Sci. 2009, 66, 1326−1336.
(25) Lessene, G.; Czabotar, P. E.; Colman, P. M. BCL-2 family
antagonists for cancer therapy. Nat. Rev. Drug. Discov. 2008, 7, 989−
1000.
(26) Fire, E.; Gullá, S. V.; Grant, R. A.; Keating, A. E. Mcl-1-Bim
complexes accommodate surprising point mutations via minor
structural changes. Protein Sci. 2010, 19, 507−519.
(27) Gomez-Bougie, P.; Maiga, S.; Tessoulin, B.; Bourcier, J.; Bonnet,
A.; Rodriguez, M. S.; Le Gouill, S.; Touzeau, C.; Moreau, P.; Pellat-
Deceunynck, C.; Amiot, M. BH3-mimetic toolkit guides the respective
use of BCL2 and MCL1 BH3-mimetics in myeloma treatment. Blood
2018, 132, 2656−2669.
(28) Zhang, Z.; Song, T.; Li, X.; Wu, Z.; Feng, Y.; Xie, F.; Liu, C.; Qin,
J.; Chen, H. Novel soluble myeloid cell leukemia sequence 1 (Mcl-1)
inhibitor (E,E)-2-(benzylaminocarbonyl)-3-styrylacrylonitrile (4g)
developed using a fragment-based approach. Eur. J. Med. Chem. 2013,
59, 141−149.
(29) Zhang, Z.; Liu, C.; Li, X.; Song, T.; Wu, Z.; Liang, X.; Zhao, Y.;
Shen, X.; Chen, H. Fragment-based design, synthesis, and biological
evaluation of N-substituted-5-(4-isopropylthiophenol)-2-hydroxynico-
tinamide derivatives as novel Mcl-1 inhibitors. Eur. J. Med. Chem. 2013,
60, 410−420.
(30) Liu, L.; Liu, R.; Yang, X.; Hou, X.; Fang, H. Design, synthesis and
biological evaluation of tyrosine derivatives as Mcl-1 inhibitors. Eur. J.
Med. Chem. 2020, 191, 112142.
(31) Arkin, M. R.; Wells, J. A. Small-molecule inhibitors of protein-
protein interactions: progressing towards the dream. Nat. Rev. Drug.
Discov. 2004, 3, 301−317.
(32) Horne, W. S.; Boersma, M. D.; Windsor, M. A.; Gellman, S. H.
Sequence-based design of alpha/beta-peptide foldamers that mimic
BH3 domains. Angew. Chem., Int. Ed. Engl. 2008, 47, 2853−2856.
(33) de Araujo, A. D.; Lim, J.; Wu, K.-C.; Xiang, Y.; Good, A. C.;
Skerlj, R.; Fairlie, D. P. Bicyclic helical peptides as dual inhibitors
selective for Bcl2A1 and Mcl-1 proteins. J. Med. Chem. 2018, 61, 2962−
2972.
REFERENCES
■
(1) Green, D. R.; Reed, J. C. Mitochondria and apoptosis. Science
1998, 281, 1309−1312.
(2) Adams, J. M.; Cory, S. The Bcl-2 protein family: arbiters of cell
survival. Science 1998, 281, 1322−1326.
(3) Lutz, R. J. Role of the BH3 (Bcl-2 homology 3) domain in the
regulation of apoptosis and Bcl-2-related proteins. Biochem. Soc. Trans.
2000, 28, 51−56.
(4) Ren, D.; Tu, H.-C.; Kim, H.; Wang, G. X.; Bean, G. R.; Takeuchi,
O.; Jeffers, J. R.; Zambetti, G. P.; Hsieh, J. J.-D.; Cheng, E. H.-Y. BID,
BIM, and PUMA are essential for activation of the BAX- and BAK-
dependent cell death program. Science 2010, 330, 1390−1393.
(5) Kluck, R. M.; Bossy-Wetzel, E.; Green, D. R.; Newmeyer, D. D.
The release of cytochrome c from mitochondria: a primary site for Bcl-2
regulation of apoptosis. Science 1997, 275, 1132−1136.
(6) Kozopas, K. M.; Yang, T.; Buchan, H. L.; Zhou, P.; Craig, R. W.
Mcl-1, a gene expressed in programmed myeloid cell differentiation, has
sequence similarity to Bcl-2. Proc. Natl. Acad. Sci. U. S. A. 1993, 90,
3516−3520.
̀
(7) Herrant, M.; Jacquel, A.; Marchetti, S.; Belhacene, N.; Colosetti,
P.; Luciano, F.; Auberger, P. Cleavage of Mcl-1 by caspases impaired its
ability to counteract Bim-induced apoptosis. Oncogene 2004, 23, 7863−
7873.
(8) Adams, J. M.; Cory, S. Life-or-death decisions by the Bcl-2 protein
family. Trends Biochem. Sci. 2001, 26, 61−66.
(9) Gomez-Bougie, P.; Wuilleme-Toumi, S.; Ménoret, E.; Trichet, V.;
̀
Robillard, N.; Philippe, M.; Bataille, R.; Amiot, M. Noxa up-regulation
and Mcl-1 cleavage are associated to apoptosis induction by bortezomib
in multiple myeloma. Cancer Res. 2007, 67, 5418−5424.
(10) Pierson, W.; Cauwe, B.; Policheni, A.; Schlenner, S. M.;
Franckaert, D.; Berges, J.; Humblet-Baron, S.; Schönefeldt, S.; Herold,
M. J.; Hildeman, D.; Strasser, A.; Bouillet, P.; Lu, L.-F.; Matthys, P.;
Freitas, A. A.; Luther, R. J.; Weaver, C. T.; Dooley, J.; Gray, D. H.;
Liston, A. Antiapoptotic Mcl-1 is critical for the survival and niche-
filling capacity of Foxp3+ regulatory T cells. Nat. Immunol. 2013, 14,
959−965.
(11) Renjini, A. P.; Titus, S.; Narayan, P.; Murali, M.; Jha, R. K.;
Laloraya, M. STAT3 and MCL-1 associate to cause a mesenchymal
epithelial transition. J. Cell Sci. 2014, 127, 1738−1750.
(12) Hikita, H.; Takehara, T.; Shimizu, S.; Kodama, T.; Li, W.; Miyagi,
T.; Hosui, A.; Ishida, H.; Ohkawa, K.; Kanto, T.; Hiramatsu, N.; Yin, X.
M.; Hennighausen, L.; Tatsumi, T.; Hayashi, N. Mcl-1 and Bcl-xL
cooperatively maintain integrity of hepatocytes in developing and adult
murine liver. Hepatology 2009, 50, 1217−1226.
(13) Opferman, J. T.; Letai, A.; Beard, C.; Sorcinelli, M. D.; Ong, C.
C.; Korsmeyer, S. J. Development and maintenance of B and T
lymphocytes requires antiapoptotic MCL-1. Nature 2003, 426, 671−
676.
(14) Thomas, L. W.; Lam, C.; Edwards, S. W. Mcl-1: the molecular
regulation of protein function. FEBS Lett. 2010, 584, 2981−2989.
(15) Aichberger, K. J.; Mayerhofer, M.; Krauth, M. T.; Skvara, H.;
Florian, S.; Sonneck, K.; Akgul, C.; Derdak, S.; Pickl, W. F.; Wacheck,
V.; Selzer, E.; Monia, B. P.; Moriggl, R.; Valent, P.; Sillaber, C.
Identification of Mcl-1 as a BCR/ABL-dependent target in chronic
myeloid leukemia (CML): evidence for cooperative antileukemic
effects of imatinib and mcl-1 antisense oligonucleotides. Blood 2005,
105, 3303−3311.
(16) Steven, L. G.; Klaus, P.; Jean-Luc, H.; Anderson, K. C. Mcl-1
regulation and its role in multiple myeloma. Cell Cycle 2004, 3, 1259−
1262.
(17) Song, L.; Coppola, D.; Livingston, S.; Cress, W. D.; Haura, E. B.
Mcl-1 regulates survival and sensitivity to diverse apoptotic stimuli in
V
https://doi.org/10.1021/acs.jmedchem.1c00682
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(34) Muppidi, A.; Doi, K.; Edwardraja, S.; Drake, E. J.; Gulick, A. M.;
Wang, H. G.; Lin, Q. Rational design of proteolytically stable, cell-
permeable peptide-based selective Mcl-1 inhibitors. J. Am. Chem. Soc.
2012, 134, 14734−14737.
(48) Shaw, S.; Bian, Z.; Zhao, B.; Tarr, J. C.; Veerasamy, N.; Jeon, K.
O.; Belmar, J.; Arnold, A. L.; Fogarty, S. A.; Perry, E.; Sensintaffar, J. L.;
Camper, D. V.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W.
Optimization of potent and selective tricyclic indole diazepinone
myeloid cell leukemia-1 inhibitors using structure-based design. J. Med.
Chem. 2018, 61, 2410−2421.
(49) Lee, T.; Christov, P. P.; Shaw, S.; Tarr, J. C.; Zhao, B.; Veerasamy,
N.; Jeon, K. O.; Mills, J. J.; Bian, Z.; Sensintaffar, J. L.; Arnold, A. L.;
Fogarty, S. A.; Perry, E.; Ramsey, H. E.; Cook, R. S.; Hollingshead, M.;
Davis Millin, M.; Lee, K. M.; Koss, B.; Budhraja, A.; Opferman, J. T.;
Kim, K.; Arteaga, C. L.; Moore, W. J.; Olejniczak, E. T.; Savona, M. R.;
Fesik, S. W. Discovery of potent myeloid cell leukemia-1 (Mcl-1)
inhibitors that demonstrate in vivo activity in mouse xenograft models
of human cancer. J. Med. Chem. 2019, 62, 3971−3988.
(35) Stewart, M. L.; Fire, E.; Keating, A. E.; Walensky, L. D. The
MCL-1 BH3 helix is an exclusive MCL-1 inhibitor and apoptosis
sensitizer. Nat. Chem. Biol. 2010, 6, 595−601.
(36) 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., Jr.; Sebti, S.;
Wang, H. G. Discovery of marinopyrrole A (maritoclax) as a selective
Mcl-1 antagonist that overcomes ABT-737 resistance by binding to and
targeting Mcl-1 for proteasomal degradation. J. Biol. Chem. 2012, 287,
10224−10235.
(50) Ramsey, H. E.; Fischer, M. A.; Lee, T.; Gorska, A. E.; Arrate, M.
P.; Fuller, L.; Boyd, K. L.; Strickland, S. A.; Sensintaffar, J.; Hogdal, L. J.;
Ayers, G. D.; Olejniczak, E. T.; Fesik, S. W.; Savona, M. R. A novel Mcl-
1 inhibitor combined with venetoclax rescues venetoclax-resistant acute
myelogenous leukemia. Cancer. Discov. 2018, 8, 1566−1581.
(51) 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.
(52) Leverson, J. D.; Zhang, H.; Chen, J.; Tahir, S. K.; Phillips, D. C.;
Xue, J.; Nimmer, P.; Jin, S.; Smith, M.; Xiao, Y.; Kovar, P.; Tanaka, A.;
Bruncko, M.; Sheppard, G. S.; Wang, L.; Gierke, S.; Kategaya, L.;
Anderson, D. J.; Wong, C.; Eastham-Anderson, J.; Ludlam, M. J. C.;
Sampath, D.; Fairbrother, W. J.; Wertz, I.; Rosenberg, S. H.; Tse, C.;
Elmore, S. W.; Souers, A. J. Potent and selective small-molecule MCL-1
inhibitors demonstrate on-target cancer cell killing activity as single
agents and in combination with ABT-263 (navitoclax). Cell. Death. Dis.
2015, 6, No. e1590.
(37) Lanning, M. E.; Yu, W.; Yap, J. L.; Chauhan, J.; Chen, L.; Whiting,
E.; Pidugu, L. S.; Atkinson, T.; Bailey, H.; Li, W.; Roth, B. M.; Hynicka,
L.; Chesko, K.; Toth, E. A.; Shapiro, P.; MacKerell, A. D., Jr.; Wilder, P.
T.; Fletcher, S. Structure-based design of N-substituted 1-hydroxy-4-
sulfamoyl-2-naphthoates as selective inhibitors of the Mcl-1 oncopro-
tein. Eur. J. Med. Chem. 2016, 113, 273−292.
(38) Rescourio, G.; Gonzalez, A. Z.; Jabri, S.; Belmontes, B.; Moody,
G.; Whittington, D.; Huang, X.; Caenepeel, S.; Cardozo, M.; Cheng, A.
C.; Chow, D.; Dou, H.; Jones, A.; Kelly, R. C.; Li, Y.; Lizarzaburu, M.;
Lo, M. C.; Mallari, R.; Meleza, C.; Rew, Y.; Simonovich, S.; Sun, D.;
Turcotte, S.; Yan, X.; Wong, S. G.; Yanez, E.; Zancanella, M.; Houze, J.;
Medina, J. C.; Hughes, P. E.; Brown, S. P. Discovery and in vivo
evaluation of macrocyclic Mcl-1 inhibitors featuring an α-hydroxy
phenylacetic acid pharmacophore or bioisostere. J. Med. Chem. 2019,
62, 10258−10271.
(39) Song, T.; Wang, Z.; Ji, F.; Feng, Y.; Fan, Y.; Chai, G.; Li, X.; Li, Z.;
Zhang, Z. Deactivation of Mcl-1 by dual-function small-molecule
inhibitors targeting the bcl-2 homology 3 domain and facilitating mcl-1
ubiquitination. Angew. Chem., Int. Ed. Engl. 2016, 55, 14250−14256.
(40) Wang, Z.; He, N.; Guo, Z.; Niu, C.; Song, T.; Guo, Y.; Cao, K.;
Wang, A.; Zhu, J.; Zhang, X.; Zhang, Z. Proteolysis targeting chimeras
for the selective degradation of Mcl-1/Bcl-2 derived from nonselective
target binding ligands. J. Med. Chem. 2019, 62, 8152−8163.
(41) Wang, Z.; Guo, Z.; Song, T.; Zhang, X.; He, N.; Liu, P.; Wang, P.;
Zhang, Z. Proteome-wide identification of on- and off-targets of Bcl-2
inhibitors in native biological systems by using affinity-based probes
(afbps). ChemBioChem 2018, 19, 2312−2320.
(42) Zhang, Z.; Wu, G.; Xie, F.; Song, T.; Chang, X. 3-Thiomorpholin-
8-oxo-8H-acenaphtho[1,2-b]pyrrole-9-carbonitrile (S1) based mole-
cules as potent, dual inhibitors of B-cell lymphoma 2 (Bcl-2) and
myeloid cell leukemia sequence 1 (Mcl-1): structure-based design and
structure-activity relationship studies. J. Med. Chem. 2011, 54, 1101−
1105.
(53) Xiao, Y.; Nimmer, P.; Sheppard, G. S.; Bruncko, M.; Hessler, P.;
Lu, X.; Roberts-Rapp, L.; Pappano, W. N.; Elmore, S. W.; Souers, A. J.;
Leverson, J. D.; Phillips, D. C. Mcl-1 is a key determinant of breast
cancer cell survival: validation of Mcl-1 dependency utilizing a highly
selective small molecule inhibitor. Mol. Cancer Ther. 2015, 14, 1837−
1847.
(54) Akca̧ y, G.; Belmonte, M. A.; Aquila, B.; Chuaqui, C.; Hird, A. W.;
Lamb, M. L.; Rawlins, P. B.; Su, N.; Tentarelli, S.; Grimster, N. P.; Su,
Q. Inhibition of Mcl-1 through covalent modification of a noncatalytic
lysine side chain. Nat. Chem. Biol. 2016, 12, 931−936.
(55) Papatzimas, J. W.; Gorobets, E.; Maity, R.; Muniyat, M. I.;
MacCallum, J. L.; Neri, P.; Bahlis, N. J.; Derksen, D. J. From inhibition
to degradation: targeting the antiapoptotic protein myeloid cell
leukemia 1 (Mcl-1). J. Med. Chem. 2019, 62, 5522−5540.
(43) Bruncko, M.; Song, X.; Tao, Z.; Kunzer, A. R. 7-nonsubstituted
Indole Mcl-1 Inhibitors. WO2008130970Al, 2008.
(56) Johannes, J. W.; Bates, S.; Beigie, C.; Belmonte, M. A.; Breen, J.;
Cao, S.; Centrella, P. A.; Clark, M. A.; Cuozzo, J. W.; Dumelin, C. E.;
Ferguson, A. D.; Habeshian, S.; Hargreaves, D.; Joubran, C.; Kazmirski,
S.; Keefe, A. D.; Lamb, M. L.; Lan, H.; Li, Y.; Ma, H.; Mlynarski, S.;
Packer, M. J.; Rawlins, P. B.; Robbins, D. W.; Shen, H.; Sigel, E. A.;
Soutter, H. H.; Su, N.; Troast, D. M.; Wang, H.; Wickson, K. F.; Wu, C.;
Zhang, Y.; Zhao, Q.; Zheng, X.; Hird, A. W. Structure based design of
non-natural peptidic macrocyclic Mcl-1 inhibitors. ACS Med. Chem.
Lett. 2017, 8, 239−244.
(44) Elmore, S. W.; Souers, A. J.; Bruncko, M.; Song, X.; Wang, X.;
Hasvold, L. A.; Wang, L.; Kunzer, A. R.; Park, C.; Wendt, M. D.; Tao, Z.
7-sonsubstituted Indole Mcl-1 Inhibitors. WO2008131000A2, 2008.
(45) Friberg, A.; Vigil, D.; Zhao, B.; Daniels, R. N.; Burke, J. P.; Garcia-
Barrantes, P. M.; Camper, D.; Chauder, B. A.; Lee, T.; Olejniczak, E. T.;
Fesik, S. W. Discovery of potent myeloid cell leukemia 1 (Mcl-1)
inhibitors using fragment-based methods and structure-based design. J.
Med. Chem. 2013, 56, 15−30.
(46) Burke, J. P.; Bian, Z.; Shaw, S.; Zhao, B.; Goodwin, C. M.; Belmar,
J.; Browning, C. F.; Vigil, D.; Friberg, A.; Camper, D. V.; Rossanese, O.
W.; Lee, T.; Olejniczak, E. T.; Fesik, S. W. Discovery of tricyclic indoles
that potently inhibit Mcl-1 using fragment-based methods and
structure-based design. J. Med. Chem. 2015, 58, 3794−3805.
(47) Pelz, N. F.; Bian, Z.; Zhao, B.; Shaw, S.; Tarr, J. C.; Belmar, J.;
Gregg, C.; Camper, D. V.; Goodwin, C. M.; Arnold, A. L.; Sensintaffar,
J. L.; Friberg, A.; Rossanese, O. W.; Lee, T.; Olejniczak, E. T.; Fesik, S.
W. Discovery of 2-indole-acylsulfonamide myeloid cell leukemia 1
(Mcl-1) inhibitors using fragment-based methods. J. Med. Chem. 2016,
59, 2054−2066.
(57) 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.
(58) Sale, M. J.; Minihane, E.; Monks, N. R.; Gilley, R.; Richards, F.
M.; Schifferli, K. P.; Andersen, C. L.; Davies, E. J.; Vicente, M. A.;
W
https://doi.org/10.1021/acs.jmedchem.1c00682
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Ozono, E.; Markovets, A.; Dry, J. R.; Drew, L.; Flemington, V.; Proia,
T.; Jodrell, D. I.; Smith, P. D.; Cook, S. J. Targeting melanoma’s MCL1
bias unleashes the apoptotic potential of BRAF and ERK1/2 pathway
inhibitors. Nat. Commun. 2019, 10, 5167.
(59) Zhang, H.; Nakauchi, Y.; Köhnke, T.; Stafford, M.; Bottomly, D.;
Thomas, R.; Wilmot, B.; McWeeney, S. K.; Majeti, R.; Tyner, J. W.
Integrated analysis of patient samples identifies biomarkers for
venetoclax efficacy and combination strategies in acute myeloid
leukemia. Nat. Cancer. 2020, 1, 826−839.
(70) Li, X.; Dou, J.; You, Q.; Jiang, Z. Inhibitors of BCL2A1/Bfl-1
protein: Potential stock in cancer therapy. Eur. J. Med. Chem. 2021, 220,
113539.
(71) Chang, J.; Wang, Y.; Shao, L.; Laberge, R. M.; Demaria, M.;
Campisi, J.; Janakiraman, K.; Sharpless, N. E.; Ding, S.; Feng, W.; Luo,
Y.; Wang, X.; Aykin-Burns, N.; Krager, K.; Ponnappan, U.; Hauer-
Jensen, M.; Meng, A.; Zhou, D. Clearance of senescent cells by ABT263
rejuvenates aged hematopoietic stem cells in mice. Nat. Med. 2016, 22,
78−83.
(72) Munkhbaatar, E.; Dietzen, M.; Agrawal, D.; Anton, M.;
Jesinghaus, M.; Boxberg, M.; Pfarr, N.; Bidola, P.; Uhrig, S.;
Höckendorf, U.; Meinhardt, A. L.; Wahida, A.; Heid, I.; Braren, R.;
Mishra, R.; Warth, A.; Muley, T.; Poh, P. S. P.; Wang, X.; Fröhling, S.;
Steiger, K.; Slotta-Huspenina, J.; van Griensven, M.; Pfeiffer, F.; Lange,
S.; Rad, R.; Spella, M.; Stathopoulos, G. T.; Ruland, J.; Bassermann, F.;
Weichert, W.; Strasser, A.; Branca, C.; Heikenwalder, M.; Swanton, C.;
McGranahan, N.; Jost, P. J. MCL-1 gains occur with high frequency in
lung adenocarcinoma and can be targeted therapeutically. Nat.
Commun. 2020, 11, 4527.
(73) Liu, J.; Fang, Z.; Zhang, Q.; Liu, Q.; Bi, X. Silver-catalyzed
isocyanide-alkyne cycloaddition: a general and practical method to
oligosubstituted pyrroles. Angew. Chem. Int. Ed. Engl. 2013, 52, 6953−
6957.
(60) Caenepeel, S.; Brown, S. P.; Belmontes, B.; Moody, G.; Keegan,
K. S.; Chui, D.; Whittington, D. A.; Huang, X.; Poppe, L.; Cheng, A. C.;
Cardozo, M.; Houze, J.; Li, Y.; Lucas, B.; Paras, N. A.; Wang, X.;
Taygerly, J. P.; Vimolratana, M.; Zancanella, M.; Zhu, L.; Cajulis, E.;
Osgood, T.; Sun, J.; Damon, L.; Egan, R. K.; Greninger, P.;
McClanaghan, J. D.; Gong, J.; Moujalled, D.; Pomilio, G.; Beltran, P.;
Benes, C. H.; Roberts, A. W.; Huang, D. C.; Wei, A.; Canon, J.; Coxon,
A.; Hughes, P. E. AMG 176, a selective Mcl-1 inhibitor, is effective in
hematologic cancer models alone and in combination with established
therapies. Cancer. Discov. 2018, 8, 1582−1597.
(61) Lu, X.; Liu, Y. C.; Orvig, C.; Liang, H.; Chen, Z. F. Discovery of a
Copper-Based Mcl-1 Inhibitor as an Effective Antitumor Agent. J. Med.
Chem. 2020, 63, 9154−9167.
(62) Abulwerdi, F.; Liao, C.; Liu, M.; Azmi, A. S.; Aboukameel, A.;
Mady, A. S.; 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.
(63) 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.
(64) Lu, X.; Liu, Y. C.; Orvig, C.; Liang, H.; Chen, Z. F. Discovery of β-
carboline copper(II) complexes as Mcl-1 inhibitor and in vitro and in
vivo activity in cancer models. Eur. J. Med. Chem. 2019, 181, 111567.
(65) 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.
(66) Petros, A. M.; Swann, S. L.; Song, D.; Swinger, K.; Park, C.;
Zhang, H.; Wendt, M. D.; Kunzer, A. R.; Souers, A. J.; Sun, C.
Fragment-based discovery of potent inhibitors of the anti-apoptotic
MCL-1 protein. Bioorg. Med. Chem. Lett. 2014, 24, 1484−1488.
(67) 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.;
Smith, J.; Pedder, C.; Graham, C.; Studeny, A.; Lysiak-Auvity, G.;
Girard, A. M.; Grave, F.; Segal, D.; Riffkin, C. D.; Pomilio, G.; Galbraith,
L. C.; 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.; 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.
(74) Kattamuri, P. V.; Yin, J.; Siriwongsup, S.; Kwon, D. H.; Ess, D. H.;
̈
Li, Q.; Li, G.; Yousufuddin, M.; Richardson, P. F.; Sutton, S. C.; Kurti,
L. Practical singly and doubly electrophilic aminating agents: a new,
more sustainable platform for carbon-nitrogen bond formation. J. Am.
Chem. Soc. 2017, 139, 11184−11196.
(75) Cohen, N. A.; Stewart, M. L.; Gavathiotis, E.; Tepper, J. L.;
Bruekner, S. R.; Koss, B.; Opferman, J. T.; Walensky, L. D. A
competitive stapled peptide screen identifies a selective small molecule
that overcomes MCL-1-dependent leukemia cell survival. Chem. Biol.
2012, 19, 1175−1186.
(76) Kump, K. J.; Miao, L.; Mady, A. S. A.; Ansari, N. H.; Shrestha, U.
K.; Yang, Y.; Pal, M.; Liao, C.; Perdih, A.; Abulwerdi, F. A.;
Chinnaswamy, K.; Meagher, J. L.; Carlson, J. M.; Khanna, M.;
Stuckey, J. A.; Nikolovska-Coleska, Z. Discovery and characterization of
2,5-substituted benzoic acid dual inhibitors of the anti-apoptotic Mcl-1
and Bfl-1 proteins. J. Med. Chem. 2020, 63, 2489−2510.
(77) 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. Structural insights
into the design of small molecule inhibitors that selectively antagonize
Mcl-1. J. Med. Chem. 2010, 53, 2314−2318.
(78) Szlavik, Z.; Ondi, L.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Radics,
G.; Murray, J.; Davidson, J.; Chen, I.; Davis, B.; Hubbard, R. E.; Pedder,
C.; Dokurno, P.; Surgenor, A.; Smith, J.; Robertson, A.; LeToumelin-
Braizat, G.; Cauquil, N.; Zarka, M.; Demarles, D.; Perron-Sierra, F.;
Claperon, A.; Colland, F.; Geneste, O.; Kotschy, A. Structure-guided
discovery of a selective Mcl-1 inhibitor with cellular activity. J. Med.
Chem. 2019, 62, 6913−6924.
(79) Wu, Y.; Jiang, Z.; Li, Z.; Gu, J.; You, Q.; Zhang, X. Click
chemistry-based discovery of [3-hydroxy-5-(1h-1,2,3-triazol-4-yl)-
picolinoyl]glycines as orally active hypoxia-inducing factor prolyl
hydroxylase inhibitors with favorable safety profiles for the treatment of
anemia. J. Med. Chem. 2018, 61, 5332−5349.
(68) Szlavik, Z.; Csekei, M.; Paczal, A.; Szabo, Z. B.; Sipos, S.; Radics,
G.; Proszenyak, A.; Balint, B.; Murray, J.; Davidson, J.; Chen, I.;
Dokurno, P.; Surgenor, A. E.; Daniels, Z. M.; Hubbard, R. E.; Le
Toumelin-Braizat, G.; Claperon, A.; Lysiak-Auvity, G.; Girard, A. M.;
Bruno, A.; Chanrion, M.; Colland, F.; Maragno, A. L.; Demarles, D.;
Geneste, O.; Kotschy, A. Discovery of S64315, a potent and selective
Mcl-1 inhibitor. J. Med. Chem. 2020, 63, 13762−13795.
(69) Zhu, P. J.; Yu, Z. Z.; You, Q. D.; Jiang, Z. Y. Myeloid cell
leukemin-1 inhibitors: a growing arsenal for cancer therapy. Drug
Discovery Today 2020, 25, 1873−1882.
X
https://doi.org/10.1021/acs.jmedchem.1c00682
J. Med. Chem. XXXX, XXX, XXX−XXX