Design of Bcl-2 and Bcl-xL inhibitors with subnanomolar binding affinities based upon a new scaffold

Article abstract of DOI:10.1021/jm300178u

Employing a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of the anti-apoptotic proteins Bcl-2 and Bcl-xL. An initial lead compound with a new scaffold was designed based upon the crystal structure of Bcl-xL an

Full text of DOI:10.1021/jm300178u

Article
pubs.acs.org/jmc
Design of Bcl-2 and Bcl-xL Inhibitors with Subnanomolar Binding
Affinities Based upon a New Scaffold†
Haibin Zhou,‡,§ Jianfang Chen,‡,§ Jennifer L. Meagher,∥,§ Chao-Yie Yang,‡,§ Angelo Aguilar,‡,§ Liu Liu,‡,§
Longchuan Bai,‡,§ Xin Cong,‡ Qian Cai,‡ Xueliang Fang,‡ Jeanne A. Stuckey,∥ and Shaomeng Wang*,‡
‡Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry and ∥Life Sciences
Institute, University of Michigan, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0934, United States
S
* Supporting Information
ABSTRACT: Employing a structure-based strategy, we have designed a new class of potent small-molecule inhibitors of the
anti-apoptotic proteins Bcl-2 and Bcl-xL. An initial lead compound with a new scaffold was designed based upon the crystal
structure of Bcl-xL and U.S. Food and Drug Administration (FDA) approved drugs and was found to have an affinity of 100 μM
for both Bcl-2 and Bcl-xL. Linking this weak lead to another weak-affinity fragment derived from Abbott’s ABT-737 led to an
improvement of the binding affinity by a factor of >10 000. Further optimization ultimately yielded compounds with sub-
nanomolar binding affinities for both Bcl-2 and Bcl-xL and potent cellular activity. The best compound (21) binds to Bcl-xL and
Bcl-2 with Ki < 1 nM, inhibits cell growth in the H146 and H1417 small-cell lung cancer cell lines with IC50 values of 60−90 nM,
and induces robust cell death in the H146 cancer cell line at 30−100 nM.
anti- and pro-apoptotic Bcl-2 members can antagonize the anti-
death function of anti-apoptotic Bcl-2 proteins, and this in turn
can overcome the apoptosis resistance of cancer cells mediated
by the overexpression of these anti-apoptotic Bcl-2 proteins.5,6
Design of potent nonpeptide cell-permeable small-molecule in-
hibitors with the ability to block the protein−protein interac-
tions involving the Bcl-2 family of proteins has been intensely
pursued in the past decade as a novel cancer therapeutic strat-
egy, and a number of laboratories have reported the design and
characterization of nonpeptide small-molecule inhibitors.7−12
Among all the reported Bcl-2/Bcl-xL inhibitors, compound 1
(ABT-737, Figure 1) is arguably the most potent compound.13
Compound 1 binds to Bcl-2, Bcl-xL, and Bcl-w with very high
affinities (Ki <1 nM) and also shows a very high specificity over
Mcl-1 and A1.13 Its analogue, 2 (ABT-263, Figure 1), has been
advanced into phase I/II clinical trials for the treatment of
human cancer.14,15 Recently, another class of potent Bcl-2/
Bcl-xL inhibitors, exemplified by compound 3 (Figure 1), was
designed starting from the chemical structure of compound 1.16
INTRODUCTION
■
Resistance to apoptosis is a hallmark of human cancer,1 and target-
ing key apoptosis regulators with the goal of promoting apoptosis
is an exciting therapeutic strategy for cancer treatment.2,3
The Bcl-2 protein family is a class of key apoptosis regulators
and consists of both anti-apoptotic proteins, including Bcl-2, Bcl-xL,
and Mcl-1, and pro-apoptotic proteins, such as BID, BIM, BAD,
BAK, BAX, and NOXA.4 The anti-apoptotic Bcl-2 and Bcl-xL
proteins are overexpressed in many different types of human tumor
samples and cancer cell lines, and this overexpression confers
resistance of cancer cells to current cancer treatments.5,6 The
anti-apoptotic proteins inhibit apoptosis via heterodimerization
with pro-apoptotic Bcl-2 family proteins.5,6 Despite their struc-
tural similarities, these anti-death Bcl-2 proteins display a certain
binding specificity on pro-death Bcl-2 proteins.5,6 For example,
while Bcl-2 and Bcl-xL bind to BIM and BAD proteins with high
affinities, they have very weak affinities for NOXA. In contrast,
Mcl-1 binds to BIM and NOXA with high affinities but has a very
weak affinity for BAD. These data suggest that the pro-apoptotic
proteins have nonredundant roles in the regulation of apoptosis.
It has been proposed that potent nonpeptide small molecules
designed to block the protein−protein interactions between
Received: February 8, 2012
Published: March 26, 2012
© 2012 American Chemical Society
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Figure 1. Chemical structures of previously reported potent and specific Bcl-2/Bcl-xL inhibitors.
In this paper, we report our structure-based design of highly
potent and specific small-molecule inhibitors of Bcl-2/Bcl-xL,
started from a novel chemical scaffold designed based upon
FDA-approved drugs and the crystal structures of Bcl-xL
complexed with its inhibitors.
a pharmacophore search was made in an in-house three-
dimensional database of 1410 U.S. Food and Drug Adminis-
tration (FDA) approved drugs. Eleven compounds were
identified and were grouped into three classes on the basis of
their scaffolds (Figure 3). Our initial efforts focused on the
second class of compounds, which all contain the bis-aryl-
substituted five-membered heterocyclic template and include
the well-known drugs Lipitor and Celecoxib (Figure 3).
To explore the possible binding models of Lipitor and
Celexocib with Bcl-xL, we performed computational docking of
both drugs to Bcl-xL using the crystal structure adopted by Bcl-
xL in its complex with the BAD BH3 peptide. As can be seen in
Figure 4A ,B, the hydrophobic groups of Lipitor mimic Y105
and L109 of the BAD BH3 peptide in its interaction with Bcl-
xL, whereas the two phenyl groups of Celecoxib mimic the
interaction between L109 and M112 of the BAD BH3 peptide
with Bcl-xL. Figure 4A,B suggested that the bis-aryl-substituted
five-membered heterocycle scaffold could mimic two of three
critical hydrophobic residues in the BAD BH3 peptide.
On the basis of these analyses, compound 4 (Figure 4C),
containing a 3,4-diphenyl-1H-pyrrole-2-carboxamide scaffold,
was designed. Since it was critical to determine the crystal struc-
ture of our initial lead compound complexed with Bcl-xL for
subsequent optimization efforts, we have decided to attach two
soluble groups in compound 4 to facilitate crystallographic studies.
Docking studies suggested that compound 4 can effectively
interact with the deep hydrophobic pocket in site 1.
RESULTS AND DISCUSSION
■
Structure-Based Design of a New Chemical Scaffold
to Target Bcl-xL. The crystal structure of Bcl-xL complexed
with the BAD BH3 peptide17 reveals that the peptide interacts
with two large binding pockets in Bcl-xL, shown in Figure 2.
Figure 2. Crystal structure of Bcl-xL with five key residues of BAD
BH3 peptide at the binding site. Centroids of hydrophobic pharma-
cophores are shown as spheres. The pharmacophore model based on
three residues at site 1 binding pocket (purple spheres within red
circle) was used in pharmocophore search.
Compound 4 was synthesized and evaluated for its binding to
Bcl-xL and Bcl-2 proteins in fluorescence polarization (FP) assays.
Compound 4 has Ki values of 78 μM for Bcl-2 and 138 μM for
Bcl-xL (Table 1). Although in a typical drug discovery program such
weak affinities would seem to disqualify it as a useful lead compound,
it does possess an attractive druglike core structure and excellent
aqueous solubility, and it was proven to be an excellent starting point
in our design of new and potent Bcl-2/Bcl-xL inhibitors.
Site 1 is a deep, well-defined binding pocket, while site 2 is more
exposed to solvents. We decided to focus on site 1 for the design
of initial lead compounds with novel chemical scaffolds.
Site 1 of Bcl-xL interacts with Y105, L109, and M112, three
hydrophobic residues of the BAD BH3 peptide. The distances
between centers of mass of the side chains of any two of these
three residues are between 5.5 and 7.4 Å (Figure 2). These
three closely clustered hydrophobic residues in the BAD BH3
peptide offer a 3D pharmacophore template, which we used to
search for new scaffolds. A pharmacophore model was con-
structed from these three hydrophobic residues and the struc-
tural information, which consists of two aromatic rings and one
hydrophobic group. The distance between the centers of the
two aromatic rings was defined as 5 1 Å, and the distance
between the center of each of the aromatic rings and the center
of mass of the hydrophobic group was set to 6 1 Å. We were
particularly interested in identifying scaffolds with good
pharmacological and toxicological properties, and accordingly,
To provide a solid structural basis for our subsequent structure-
based optimization of 4, we determined its crystal structure, at a
resolution of 1.7 Å, in a complex with Bcl-xL (Figure 4D). This
shows that 4 indeed binds to the large, deep hydrophobic pocket
in site 1 of Bcl-xL, supporting both our modeling prediction and
our design premise.
Structure-Based Design of Potent Bcl-2/Bcl-xL Inhib-
itors. For a compound to achieve a high binding affinity for
Bcl-xL, it may be necessary to occupy both sites 1 and 2 in the
protein.13 Since 4 occupies only site 1, a second fragment,
capable of occupying site 2, is needed. We used the crystal
structure of 1 complexed with Bcl-xL18 to identify a fragment
that could be accommodated in site 2 (red circle in Figure 4D).
Superposition of the crystal structure of 4 and the crystal structure
of 1 in its complex with Bcl-xL showed that the core structure in 4
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Figure 3. Three classes of scaffolds identified based on the pharmacophores in the Bad BH3 peptide. The three-dimensional pharmocophore model
is based on two aromatic rings (shown in blue) and one hydrophobic group (shown in red). The distance between the aromatic rings was set
to 5 1 Å, and the distances between the aromatic ring and hydrophobic groups were set to 6 1 Å.
and the p-chlorobiphenyl fragment in 1 (Figure 5A) both occupy
site 1. It is also evident that fragment 5 occupies site 2 in Bcl-xL
(Figure 5A,B). Therefore, we reasoned that linking compounds 4
and 5 could yield new compounds with high affinity for Bcl-xL.
Compound 5 was synthesized by the published method19 and
found to have very weak affinities (IC50 > 100 μM) for Bcl-xL and
Bcl-2 proteins in our FP-based assays. These data further showed
that interacting with either site 1 or site 2 is insufficient to yield
compounds with high affinities for Bcl-xL and Bcl-2, and occupation
of both sites is needed to achieve high affinities for Bcl-xL and Bcl-2.
Therefore, we decided to link 4 and 5 together with a proper linker
for the design of potent inhibitors of Bcl-xL and Bcl-2.
Analysis of the modeled structure of 5 complexed with Bcl-xL
and the crystal structures of 1 and 4 complexed with Bcl-xL
suggested that the meta position of the unsubstituted phenyl
ring in 4 and the sulfonamido nitrogen atom of 5 are sites at
which 4 and 5 could be linked together for the design of potent
Bcl-xL inhibitors (Figure 5B). The distance between this ring in
4 and the sulfonamido nitrogen atom of 5 is 8.2 Å (Figure 5B).
Compound 6, designed to make use of the linker in 1 (Figures 5C
and 6), which is 10.6 Å in length, was found to have Ki = 2.0 nM for
Bcl-2 and Ki < 1 nM for Bcl-xL, and is thus >10 000 times more
potent than either 4 or 5. The dramatically improved binding
affinities of 6 over those of 4 and 5 supported our design strategy.
Compounds 1 and 6 have similar affinities for Bcl-xL, but 6 is
less potent than 1 toward Bcl-2 (Table 1). To further optimize
the binding affinities of 6, we next tested linkers with various
lengths, flexibility, orientation, and chemical properties (Figures
5C and 6). Modeling suggested that removal of the carbonyl
group in the N-acylsulfonamide in 6 may lead to further im-
provement in the binding affinity (Figure S1 in Supporting
Information). Removal of this carbonyl group from 6 gives 7, in
which the linker has been shortened from 10.6 to 9.9 Å. The
affinity of 7 for both Bcl-2 and Bcl-xL (Ki < 1 nM) is very sim-
ilar to that of 1. Replacement of the piperazine in 7 by a triazole
aromatic ring shortens the linker to 9.0 Å, giving 8, which also
binds to both Bcl-2 and Bcl-xL with Ki < 1 nM. Replacement of
the piperazine ring in the linker in 6 with a rigid ethynyl group
leads to 9, which has a linker length of 9.1 Å and binds to both
Bcl-2 and Bcl-xL with Ki < 1 nM. Removal of the carbonyl
group from the N-acylsulfonamide group in 9 leads to 10,
which has a linker length of 8.3 Å and binds to Bcl-2 and Bcl-xL
with Ki = 1.5 nM and 1.7 nM, respectively.
Hence, compounds 7, 8, and 9 bind to Bcl-2 and Bcl-xL with
Ki <1 nM and are as potent as 1 toward both proteins. The
binding data show that tethering 4 and 5, two weak Bcl-2/
Bcl-xL inhibitors, with linkers approximately 9.0 Å in length
produced highly potent Bcl-2/Bcl-xL inhibitors, whose affinities
for Bcl-2/Bcl-xL are 4 orders of magnitude better than those of
4 or 5. When the linker is longer (10.6 Å in 6) or shorter (8.3 Å
in 10), the resulting compounds are less potent. To investigate
the influence of linker flexibility, we synthesized 11, with a more
flexible linker than that in 7, and found that 11 is at least 10 times
less potent than 7.
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Figure 4. Structure-based design of a new scaffold as the initial lead compound 4 and its crystal structure in complex with Bcl-xL. (A, B) Rank 1 pose of
(A) Lipitor and (B) Celecoxib with Bcl-xL, using the BAD BH3 peptide-bound Bcl-xL structure (PDB ID 2BZW). (C) Identification of the core
scaffold from the FDA-approved drugs database for a new class of Bcl-2/Bcl-xL inhibitors. (D) Co-crystal structure of 4 in complex with Bcl-xL (1.7 Å).
Table 1. Binding Affinities of Our Designed Compounds for Bcl-2 and Bcl-xL Proteins in Fluorescence Polarization Assays and
Inhibition of Cell Growth in Two Cancer Cell Lines
binding affinities
cell growth inhibition, IC50 SD
Bcl-2 (FP)
Bcl-xL (FP)
Mcl-1
(μM)
compd
IC50 SD (nM)
Ki SD (nM)
IC50 SD (nM)
Ki SD (nM)
IC50 SD (μM)
H146
H1417
>10
4
213 16 (μM)
>100 (μM)
8.7 1.9
78.0 5.9 (μM)
453 25 (μM)
238 14 (μM)
6.1 1.5
138 7.6 (μM)
>100
>100
>10
>10
>10
>10
>10
>10
>10
>10
>1
>10
>10
>10
5
75.0 4.2 (μM)
>10
6
2.0 0.5
<0.6
<1
>10
7
1.3 0.7
6.2 2.2
<1
2.0 1.1
>10
1.8 0.07
>10
8
1.4 0.8
<0.6
4.8 1.1
<1
9
1.9 0.6
<0.6
5.7 0.7
<1
>10
>10
18
19
12
13
1
6.6 2.2
1.5 0.3
15.4 6.0
8.5 1.0
21.8 9.0
<0.6
12.6 3.1
44.1 5.5
7.6 1.6
1.7 0.9
11.3 0.6
<1
>10
>10
60.6 23.1
33.9 3.8
85.3 34.8
>10
>10
2.5 0.8
>10
3.3 1.4
>10
88.3 14.4
24.7 4.0
<1
2
0.2
6
2
0.097 0.030
>10
0.13 0.05
>10
14
15
>100 (μM)
547 (μM)
166 (μM)
5.7 0.1 (μM)
>100
>100
534 105 (μM)
138 27 (μM)
19 0.3 (μM)
>10 000
7.5 0.7
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Figure 5. Computational structure-based design of a new class of Bcl-2/Bcl-xL inhibitors by linking two fragments with weak binding affinities. (A)
Superposition of the crystal structure of 4 (green) onto the crystal structure of 1 (yellow) in complex with Bcl-xL (B) Measurement of the distance
(8.2 Å) between 4 and 5. The 1-bound Bcl-xL structure was used in the surface representation. (C) Chemical structures of 5 and the new designed
Bcl-2/Bcl-xL inhibitors with different linkers.
Figure 6. Chemical structures of designed Bcl-2 and Bcl-xL inhibitors.
To examine binding specificity, we evaluated the binding of
these compounds to Mcl-1. Similar to 1, compounds 6, 7, 8, 9,
10 and 11 all were found to bind to Mcl-1 with IC50 > 10 μM,
thus showing very high specificity over Mcl-1 (Table 1).
Changing the attachment position of the linker from the
meta to the para position of the phenyl ring in 7 results in 12
(Figure 6), which is >10 times less potent than 7 in binding to
Bcl-2 but is equipotent with 7 in its binding to Bcl-xL (Table 1).
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Figure 7. Chemical structures of 7 and its analogues.
Table 2. Binding Affinities of Our Designed Compounds for Bcl-2 and Bcl-xL Proteins in Fluorescence Polarization Assays and
Inhibition of Cell Growth in Two Small-Cell Lung Cancer Cell Lines
binding affinities
Bcl-2 (FP)
Bcl-xL(FP)
Mcl-1
cell growth inhibition, IC50 SD (μM)
compd
IC50 + SD (nM)
Ki SD (nM)
IC50 SD (nM)
Ki SD (nM)
IC50 SD (μM)
H146
H1417
7
1.3 0.7
0.6 0.2
5.3 0.6
82.6 19.5
45.8 26.1
1.7 1.0
4.1 0.7
<0.6
6.2 2.2
4.9 1.2
6.3 0.4
34.4 3.5
12.6 5.4
3.6 1.1
7.5 1.3
<1
>10
>10
>2
2.0 1.1
1.8 0.07
0.65 0.51
0.65 0.15
>10
16
17
18
19
20
21
1
<0.6
<1
0.43 0.25
0.40 0.31
>10
1.2 0.2
21.1 5.0
11.6 6.8
<0.6
<1
8.3 1.0
1.7 0.6
<1
>2
>2
4.9 3.2
7.3 2.8
>2
0.34 0.01
0.061 0.009
0.097 0.03
0.552 0.089
0.090 0.003
0.13 0.05
0.8 0.2
<0.6
<1
>2
2
0.2
6
2
<1
>1
We also synthesized 13 (Figure 6), an analogue of 1 lacking
the benzamido carbonyl group. This compound binds to Bcl-2
and Bcl-xL with Ki values of 18 nM and 17 nM, respectively,
an affinity >10 times weaker than that of 1. Hence, the carbonyl
group in 1 evidently contributes significantly to its binding
affinities for Bcl-2 and Bcl-xL. By contrast, removal of the corre-
sponding carbonyl group in 6 enhances the binding affinity for Bcl-2
by 6 times while having no effect on the binding to Bcl-xL.
Compounds 14 and 15, fragments of 7 (Figure 6), were syn-
thesized and their binding to Bcl-2 and Bcl-xL was examined. Both
14 and 15 bind to Bcl-2 and Bcl-xL with weak affinities (Table 1).
As a single agent, 1 was shown to be very effective in inhibition
of cancer cell growth in cancer cell lines such as small-cell lung
cancer cell lines H146 and H1417, with high levels of Bcl-2/Bcl-xL
but low levels of Mcl-1. Since 7 and several other compounds bind
to Bcl-2 and Bcl-xL with very high affinities, show high specificity
over Mcl-1 and have the same binding profiles as 1, we evaluated
their ability, in comparison to 1, to inhibit cell growth in the H146
and H1417 cancer cell lines with the results shown in Table 1.
Consistent with their weak binding affinities, four compounds
(4, 5, 14, and 15) have poor activities (IC50 > 10 μM) in inhibi-
tion of cell growth in these two cancer cell lines. Although com-
pounds 6, 8, 9, 10, and 11 all have high affinities for both Bcl-2
and Bcl-xL, they also have weak cellular activities (IC50 > 10 μM),
suggesting that these compounds may suffer from poor cell per-
meability. Removal of the carbonyl group from the linker in 6
to give 7 significantly improves both the binding affinities for
Bcl-2 and Bcl-xL and the cellular activity. Compound 7 inhibits
cell growth in these two cancer cell lines and has IC50 values of
approximately 2.0 μM against both cancer cell lines. In contrast,
removal of the corresponding carbonyl group in 1, which re-
sults in compound 13, is detrimental to binding affinities for
both Bcl-2 and Bcl-xL, as well as to the cell growth inhibitory
activities. The binding and cellular data thus identify 7 as a
promising lead compound for further optimization, and ac-
cordingly, we focused next on 7 to further investigate the structure−
activity relationships (SAR) for this class of compounds.
Structure−Activity Relationships of Compound 7.
Modeling suggested that the dihydroxybutyl side chain in 7
lacks any specific interactions with Bcl-xL (Figure S1B in Supporting
Information). Removal of this dihydroxybutyl side chain yields
16 (Figure 7), which binds to Bcl-2 and Bcl-xL with Ki < 1 nM.
It inhibits cell growth in the H146 and H1417 cancer cell lines
with IC50 values of 0.43 μM and 0.65 μM, respectively, and is
thus 3−5 times more potent than 7 (Table 2). The data thus
show that truncation of the dihydroxybutyl side chain in 7 to a
methyl group is accompanied by retention of the high binding
affinities for Bcl-2/Bcl-xL and significant improvement in its
cellular activities in both the H146 and H1417 cancer cell lines.
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Figure 8. Binding models of (A) 20 and (B) 21 with Bcl-xL. The Bcl protein from the crystal structure between 1 and Bcl-xL was used in the
docking simulations. The highest ranked poses of both compounds were selected as the binding models. The ethyl group added to 20 is denoted by
the red circle. Residues of Bcl-xL at the binding site are labeled.
Figure 9. (A) Cell death induction by 20 and 21 in the H146 cancer cell line. Cells were treated for 24 h and cell death was analyzed by trypan blue
assay. (B) Induction of cleavage of PARP and caspase-3 by 20 and 21 in the H146 cell line. Cells were treated for 24 h, and caspase-3 and PARP
were probed by Western blotting.
The N-[3-(4-methylpiperazin-1-yl)propyl]amide side chain
in 16 was used to improve the aqueous solubility. We inves-
tigated the effect of modifications of this side chain in 16 on
binding to Bcl-2/Bcl-xL. Compound 17, in which the N-[3-
(4-methylpiperazin-1-yl)propyl]amide side chain in 16 was
truncated to an N-methylcarbamoyl group (Figure 7), binds
to Bcl-2 with Ki = 1.2 nM and to Bcl-xL with Ki < 1 nM.
Compounds 16 and 17 thus have similar potencies in inhibition of
cell growth against both the H146 and H1417 cell lines. Compound
18, obtained by removal of the N-methylcarbamoyl group from 17,
has a much weaker binding affinity for both Bcl-2 and Bcl-xL than 16
and 17. It also has very weak cellular activity in both H146 and
H1417 cancer cell lines with IC50 values >10 μM (Table 2). These
data show that the amide group in 16 and 17 plays a role in
binding to Bcl-2 and Bcl-xL and in inhibition of cell growth.
Replacement of the N-methylcarbamoyl substituent on the
pyrrole ring in 17 by a carbethoxyl group results in 19 (Figure 7),
which binds to Bcl-2 10 times less potently than 17, but 19 is only
slightly less potent than 17 in binding to Bcl-xL. It has IC50 values
of 4.9 μM and 7.3 μM in inhibition of cell growth in H146 and
H1417 cancer cell lines, respectively, and is thus 10 times less
potent than 17 (Table 2).
binds to Bcl-2 and Bcl-xL with high affinities, with Ki < 1 nM,
and has IC50 values of 0.34 μM and 0.55 μM in inhibition of
cell growth against the H146 and H1417 cancer cell lines,
respectively.
In an effort to further improve the binding affinities and es-
pecially the cellular activity of 20, we performed docking sim-
ulations of 20 based on the crystal structures of 4 and 1 com-
plexed with Bcl-xL. The predicted binding model for 20
(Figure 8A) suggested that there is an unoccupied small
hydrophobic pocket close to the 5-position of the pyrrole core
in 20. Accordingly, we designed and synthesized 21, in which
an additional ethyl group was introduced to the 5-position of
the pyrrole ring in 20 (Figures 7 and 8B). Compound 21 binds
to both Bcl-2 and Bcl-xL with very high affinities (Ki < 1 nM),
actually exceeding the lower limits of the assays, and it has an
improved cell-growth inhibitory activity against the H146 and
H1417 cancer cell lines, with IC50 values of 61 nM and 90 nM,
respectively. Hence, 21 is as potent as 1, both in binding to Bcl-2
and Bcl-xL and in inhibition of cell growth against both the H146
and H1417 cancer cell lines.
Further Evaluation of Compounds 20 and 21. We next
evaluated the ability of 20 and 21 to induce cell death in the
H146 cell line (Figure 9A). Both compounds effectively induce
cell death in a dose-dependent manner in H146 cells as
Replacement of the N-methylcarbamoyl substituent on the
pyrrole ring of 17 by a carboxyl substituent generates 20, which
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a
Scheme 1. Synthesis of Compound 4
a
Reagents and conditions: (a) (i) K2CO3, MeOH, reflux; (ii) CNCH2COOEt, t-BuOK. (b) (S)-4-(2-Iodoethyl)-2,2-dimethyl-1,3-dioxolane, K2CO3.
(c) (i) KOH, H2O/THF/MeOH; (ii) 3-(4-methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM; (iii) 4 M HCl in dioxane, MeOH.
a
Scheme 2. Synthesis of Compound 6
a
Reagents and conditions: (a) (i) K2CO3, MeOH, reflux; (ii) CNCH2COOEt, t-BuOK. (b) (S)-4-(2-Iodoethyl)-2,2-dimethyl-1,3-dioxolane,
K2CO3. (c) (i) KOH, H2O/THF/MeOH; (ii) 3-(4-methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM. (d) (i) 27, Pd(dba)2, tri-tert-
butylphosphine, sodium tert-butoxide, DMF, toluene; (ii) 4 M HCl in dioxane, MeOH, 10 min.
determined in a trypan blue assay, with 21 being more potent than
20. Compound 21 induces substantial cell death at 30−100 nM
and >70% cell death in 24 h at 300 nM.
We further tested 20 and 21 in the H146 cell line for their
ability to induce cleavage of poly(ADP-ribose) polymerase (PARP)
and caspase-3, two biochemical markers of apoptosis (Figure 9B),
and found that 21 is more potent than 20 and can effectively
induce cleavage of PARP and caspase-3 in 24 h at concen-
trations as low as 100 nM. These data are consistent with their
activities in the cell growth assay.
Synthesis. The synthesis of the initial lead compound (4)
is outlined in Scheme 1. Briefly, benzaldehyde, 4-chlorophenyl
cyanide, and K2CO3 were heated in methanol to generate 2-(4-
chlorophenyl)-3-phenylacrylonitrile, which was used in a 2 + 3
cycloaddition reaction with ethyl isocyanoacetate to produce
pyrrole 22.20 Alkylation of 22 with (S)-4-(2-iodoethyl)-2,2-
dimethyl-1,3-dioxolane in the presence of K2CO3 at 60 °C in
N,N-dimethylformamide (DMF) gave 23, hydrolysis of which
afforded the corresponding acid, which was coupled to 1-(3-amino-
propyl)-4-methylpiperazine. Removal of the acetal protecting group
with HCl produced 4.
The synthesis of compound 6 is shown in Scheme 2. Com-
pounds 24, 25, and 26 were synthesized, employing the same
strategy as in Scheme 1, and intermediate 27 was prepared as
described previously.19 Compound 6 was formed by palladium-
catalyzed amination21 of 26 with 27 in the presence of Pd(dba)2
and tri-tert-butylphosphine, followed by acetal deprotection.
Compounds 7, 13, 14, and 15 were prepared as shown in
Scheme 3. Commercially available 4-fluoro-3-nitrobenzene-1-
sulfonyl chloride was treated with the corresponding aniline in
pyridine at 0 °C to produce the sulfonamides 28 and 29. (R)-
N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine was used to
displace the fluorine in 28 and the Boc group was removed to
give compound 15. Using the same strategy, 14 was synthesized
from 29. Palladium-catalyzed amination was again employed to
couple 15 and 26, and the resulting compound was deprotected
to generate compound 7. Reductive amination of 15 with 4′-
chloro-[1, 1′-biphenyl]-2-carbaldehyde was employed in the
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a
Scheme 3. Synthesis of Compounds 7, 13, 14, and 15
a
Reagents and conditions: (a) tert-Butyl 4-(4-aminophenyl)piperazine-1-carboxylate, pyridine. (b) (i) (R)-N1,N1-Dimethyl-4-(phenylthio)butane-
1,3-diamine, DIEA, DMF; (ii) TFA, CH2Cl2. (c) (i) 26, Pd(dba)2, tri-tert-butylphosphine, sodium tert-butoxide, DMF, toluene; (ii) 4 M HCl in
dioxane, MeOH, 10 min. (d) 4′-Chloro-[1,1′-biphenyl]-2-carbaldehyde, Na(OAc)3BH, ClCH2CH2Cl. (e) Aniline, pyridine. (f) (R)-N1,N1-Dimethyl-
4-(phenylthio)butane-1,3-diamine, DIEA, DMF.
a
Scheme 4. Synthesis of Compounds 9 and 10
a
Reagents and conditions: (a) 4-Ethynylbenzoic acid, EDCI, DMAP, CH2Cl2. (b) (i) 26, Pd(PPh3)4, CuI, Et3N, DMF; (ii) 4 M HCl in dioxane,
MeOH, 10 min. (c) (i) 4-Ethynylaniline, pyridine; (ii) (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DlPEA, DMF.
presence of sodium triacetoxyborohydride and 1,2-dichloro-
ethane to give 13.
addition24 in a mixture of water and t-butanol in the presence of
CuSO4·5H2O and (+)-sodium L-ascorbate. The acetal group
in the resulting triazole was removed with HCl to produce
compound 8.
Compounds 9 and 10, in which the linker is an ethynyl
group, were synthesized as shown in Scheme 4. Compound 5
was synthesized by the published procedure.19 Compound 5
was then coupled with 4-ethynylbenzoic acid in the presence of
1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide (EDCI) to
give compound 30. Pd(0)-catalyzed coupling22 of the iodide 26
with the terminal alkyne 30, followed by deprotection of the
acetal with HCl, yielded compound 9. Compound 10 was syn-
thesized by a procedure similar to that for compound 9.
Scheme 5 shows synthesis of compound 8 with a triazole ring
as the linker. CuI/L-proline-catalyzed coupling reaction23 of aryl
iodide 26 with sodium azide was carried out at 70 °C in
dimethyl sulfoxide (DMSO) to produce 32. This aryl azide 32
and alkyne 31 were joined by a CuI-catalyzed Huisgen cyclo-
The linear synthetic strategy shown in Scheme 6 was em-
ployed to prepare compounds 12 and 11. Compounds 33 and
34 were synthesized by the strategy described in Scheme 1.
With L-proline as a promotor, Ullmann-type C−N bond forma-
tion reaction25 of 34 with 1-(p-nitrophenyl)piperazine provided
intermediate 35, and subsequent hydrolysis of the ethyl ester
and coupling with 1-(3-aminopropyl)-4-methylpiperazine in the
presence of EDCI gave 36. Hydrogenation of the nitro group
in 36 gave the aniline, which was treated with 4-fluoro-3-
nitrobenzene-1-sulfonyl chloride in pyridine. Subsequent
displacement of the fluoro group with (R)-N1,N1-dimethyl-4-
(phenylthio)butane-1,3-diamine and acetal deprotection
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a
Scheme 5. Synthesis of Compound 8
a
Reagents and conditions: (a) NaN3, CuI, L-proline, NaOH, DMSO, 70 °C. (b) (i) 31, CuSO4·5H2O, sodium L-ascorbate, H2O/t-BuOH; (ii) 4 M
HCl in dioxane, MeOH, 10 min.
a
Scheme 6. Synthesis of Compounds 12 and 11
a
Reagents and conditions: (a) (i) K2CO3, MeOH, reflux; (ii) CNCH2COOEt, t-BuOK. (b) (S)-(2-iodoethyl)-2,2-dimethyl-1,3-dioxolane, K2CO3.
(c) 1-(4-Nitrophenyl)piperazine, CuI, L-proline, K2CO3, 80 °C, 2 h. (d) (i) KOH, H2O/THF/MeOH; (ii) 3-(4-methylpiperazin-1-yl)propan-1-
amine, EDCI, HOBt, DIEA, DCM. (e) (i) H2, Pd/C, (ii) 4-fluoro-3-nitrobenzene-1-sulfonyl chloride, pyridine; (iii) (R)-N1,N1-dimethyl-4-
(phenylthio)butane-1,3-diamine, DlPEA, DMF. (iv) 4 M HCl in dioxane, MeOH. (f) N1-(4-Nitrophenyl)ethane-1,2-diamine, CuI, L-proline, K2CO3,
80 °C, overnight.
a
Scheme 7. Synthesis of Compounds 16−20
a
Reagents and conditions: (a) MeI, K2CO3. (b) 1-(4-Nitrophenyl)piperazine, CuI, L-proline, K2CO3, 80 °C, 2 h. (c) (i) KOH, H2O/THF/MeOH,
reflux; (ii) H2, Pd/C; (iii) 4-fluoro-3-nitrobenzene-1-sulfonyl chloride, pyridine; (iv) (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DIPEA,
DMF. (d) 3-(4-Methylpiperazin-1-yl)propan-1-amine, EDCI, HOBt, DIEA, DCM. (e) Methylamine, EDCI, HOBt, DIEA, DCM. (f) TFA, CH2Cl2.
(g) EtOH, N,N′-diisopropylcarbodiimide, 4-(dimethylamino)pyridine, THF.
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a
Scheme 8. Synthesis of Compound 21
a
Reagents and conditions: (a) (i) NBS, DMF; (ii) MeI, K2CO3. (b) 1-(4-Nitrophenyl)piperazine, CuI, L-proline, K2CO3, 80 °C, 2 h. (c)
Ethynyltrimethylsilane, Pd(PPh3)4, CuI, Et3N, DMF, 85 °C. (d) KOH, dioxane, EtOH, H2O, reflux, 2 h. (e) (i) H2, Pd/C; (ii) 4-fluoro-3-
nitrobenzene-1-sulfonyl chloride, pyridine; (iii) (R)-N1,N1-dimethyl-4-(phenylthio)butane-1,3-diamine, DlPEA, DMF.
with HCl produced 12. Compound 11 was synthesized
similarly.
The nature of the linker plays a key role in achieving high
binding affinities for Bcl-2/Bcl-xL and potent cell growth
inhibitory activity against cancer cells. Further structure−
activity relationship studies of 7 yielded a very promising
lead compound, 21, which binds to Bcl-2 and Bcl-xL with Ki
values <1 nM, exceeding the limits of the binding assays.
Compound 21 achieves IC50 values of 60 and 90 nM against
the H146 and H1417 cancer cell lines and induces robust cell
death in the H146 cancer cell line at 30 nM. It is now the
subject of further optimization, the results of which will be
reported in future publications.
The syntheses of compounds 16−20 are outlined in Scheme 7.
Methylation with MeI and subsequent Ullmann-type C−N
bond formation reaction of 24 yielded compound 39. Com-
pound 20 was prepared from 39 by a strategy similar to that
used for 12. The amides 16 and 17 were produced by coupling
the acid 20 to the corresponding amines by use of EDCI and
hydroxybenzotriazole (HOBt). Treatment of 20 with trifluoro-
acetic acid (TFA) afforded the decarboxylated compound 18.
The ester 19 was synthesized by condensation of ethanol and
the acid 20 with N,N′-diisopropylcarbodiimide in the presence
of 4-(dimethylamino)pyridine.
Compound 21 was prepared as shown in Scheme 8. Bromi-
nation of 24 with N-bromosuccinimide (NBS) in DMF and
subsequent methylation afforded 40, which was coupled to
1-(p-nitrophenyl)piperazine to give the intermediate 41.
Sonogashira coupling of 41 with ethynyltrimethylsilane in the
presence of CuI and Pd(PPh3)4 yielded 42. Hydrolysis of the
ethyl ester and simultaneous removal of the trimethylsilane
protecting group in 42 afforded 43. Reduction of the nitro and
ethynyl groups in 43 with H2 and Pd/C as the catalyst gave a
product which, subjected to the same strategy as described in
Scheme 7, yielded compound 21.
EXPERIMENTAL SECTION
■
General Chemistry Information. Unless otherwise stated,
all reactions were performed under nitrogen atmosphere in dry
solvents under anhydrous conditions. Reagents were used as
supplied without further purification unless otherwise noted.
NMR spectra were acquired at a proton frequency of 300 MHz,
and chemical shifts are reported in parts per million (ppm)
relative to an internal standard. The final products were purified
by a C18 reverse phase semipreparative HPLC column with
solvent A (0.1% TFA in water) and solvent B (0.1% TFA in
CH3CN) as eluents.
Ethyl 4-(4-Chlorophenyl)-3-phenyl-1H-pyrrole-2-car-
boxylate (22). A mixture of benzaldehyde (1.06 g, 10 mmol),
4-chlorophenyl cyanide (1.52 g, 10 mmol), and K2CO3 (1.66 g,
12 mmol) was refluxed overnight in MeOH (15 mL) under N2.
The mixture was cooled, poured into H2O (15 mL), and stirred
for 20 min. The precipitate was collected by filtration, washed
with H2O (2 × 15 mL) and petroleum ether (2 × 15 mL),
and air-dried to give 2-(4-chlorophenyl)-3-phenylacrylonitrile.
A solution of this compound and ethyl isocyanoacetate (1.13 g,
10 mmol) in tetrahydrofuran (THF; 20 mL) was added drop-
wise to a stirred solution of potassium t-butoxide (1.34 g,
12 mmol) in DMF (15 mL) at 0 °C under N2. After being stirred
at 0 °C for 1 h, the reaction mixture was diluted with H2O
(20 mL) and extracted with EtOAc (2 × 20 mL). The com-
bined organic fractions were washed with water (20 mL) and
then with brine (20 mL). After removal of the solvent under
vacuum, the residue was purified by flash chromatography on
SUMMARY
■
Employing a computational structure-based design strategy,
we have designed compound 4, which contains a novel druglike
scaffold and binds to one well-defined binding pocket in Bcl-xL.
The binding model of 4 with Bcl-xL was determined experi-
mentally by X-ray crystallography. On the basis of the crystal
structure of 4 and of 1 (ABT-737) in complex with Bcl-xL, we
employed 4 and 5, a large fragment of 1, for the design of new
small-molecule inhibitors that occupy two separate binding
pockets in Bcl-xL. Although both 4 and 5 bind to Bcl-2 and
Bcl-xL with very weak affinities, linking them together with
appropriate linkers yielded compounds with very high affinities
(Ki < 1 nM) for both Bcl-2 and Bcl-xL. Optimization of the
linker between them resulted in 7, which not only binds to Bcl-
2 and Bcl-xL with high affinities (Ki < 1 nM) but also potently
inhibits cell growth in two small-cell lung cancer cell lines,
which are sensitive to potent and specific Bcl-2/Bcl-xL inhib-
itors. Our results demonstrate that linking two molecules
with very weak affinities for Bcl-2/Bcl-xL with appropriate
linkers can result in highly potent Bcl-2/Bcl-xL inhibitors.
1
silica gel to afford 22 (1.7 g, 52% over two steps). H NMR
(300 MHz, CCl3D) δ 9.62 (br s, 1H), 7.32−7.30 (m, 5H),
7.18 (d, J = 8.3 Hz, 2H), 7.11 (d, J = 2.9 Hz, 1H), 7.05 (d,
J = 8.3 Hz, 2H), 4.22 (q, J = 7.1 Hz, 2H), 1.16 (t, J = 7.1 Hz,
3H); 13C NMR (75 MHz, CCl3D) δ 161.4, 134.2, 133.1,
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131.9, 130.8, 129.5, 129.2, 128.4, 127.6, 127.0, 125.5, 120.4,
120.2, 60.4, 14.0.
1H), 7.16 (d, J = 8.5 Hz, 2H), 7.10−6.97 (m, 5H), 4.63−4.56
(m, 1H), 4.46−4.39 (m, 1H), 4.15−4.03 (m, 4H), 3.60 (t, J =
6.7 Hz, 1H), 2.21−2.14 (m, 1H), 2.07−1.99 (m, 1H), 1.47
(s, 3H), 1.38 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CCl3D) δ 161.2, 139.7, 138.1, 135.6, 132.6, 131.9,
129.7, 129.4, 129.3, 128.4, 126.5, 123.0, 119.9, 93.3, 73.1,
69.1, 60.0, 46.8, 35.5, 27.1, 25.7, 13.8; ESI MS m/z 580.1
(M + H)+.
(S)-Ethyl 4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-di-
oxolan-4-yl)ethyl]-3-phenyl-1H-pyrrole-2-carboxylate
(23). Compound 22 (1.7 g 5.2 mmol), (S)-4-(2-iodoethyl)-2,2-
dimethyl-1,3-dioxolane (2.0 g, 7.8 mmol), and K2CO3 (2.2 g,
15.6 mmol) in DMF (15 mL) were heated to 60 °C for 8 h.
The reaction was cooled, diluted with water (20 mL), and ex-
tracted into EtOAc (30 mL, 2 × 20 mL). The combined organic
fractions were washed with water (4 × 10 mL) and then with
brine (10 mL) and dried over Na2SO4. After evaporation of the
solvent, the residue was purified by flash chromatography on
silica gel to provide 23 (2.2 g, 92% yield). 1H NMR (300 MHz,
CCl3D) δ 7.30−7.28 (m, 3H), 7.21−7.18 (m, 2H), 7.13 (d, J =
8.5 Hz, 2H), 7.07 (s, 1H), 6.99 (d, J = 8.5 Hz, 2H), 4.65−4.57
(m, 1H), 4.47−4.38 (m, 1H), 4.17−4.01 (m, 4H), 3.61 (t, J =
7.3 Hz, 1H), 2.23−2.15 (m, 1H), 2.09−1.98 (m, 1H), 1.48 (s,
3H), 1.39 (s, 3H), 0.93 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CCl3D) δ 161.5, 135.8, 133.0, 131.6, 131.4, 130.6, 129.2,
128.3, 127.6, 126.7, 126.3, 123.0, 119.9, 73.2, 69.1, 59.8, 46.8,
35.5, 27.1, 25.7, 13.6.
(S)-4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-dioxo-
lan-4-yl)ethyl]-3-(3-iodophenyl)-N-[3-(4-methylpipera-
zin-1-yl)propyl]-1H-pyrrole-2-carboxamide (26). Potassi-
um hydroxide (0.71 g, 12.6 mmol) was added to a solution
of 25 (2.44 g, 4.2 mmol) in a mixture of THF/MeOH/H2O
(1:1:1, 30 mL) and the solution was refluxed until no start-
ing material was detectable by TLC. After being cooled, the
reaction was neutralized with 1 M HCl and the compound was
extracted with EtOAc. The EtOAc solution was washed with
brine, dried over Na2SO4, and concentrated in vacuum to pro-
duce the crude acid, which was used directly in the next step
without purification. A solution of this acid, 1-(3-aminopropyl)-
4-methylpiperazine (0.86 g, 5.5 mmol), EDCI (1.5 g 6.3 mmol),
HOBt (1.0 g, 6.3 mol), and N,N-diisopropylethylamine (1.46
mL, 8.4 mmol) in DCM (15 mL) was stirred for 8 h and then
concentrated. The residue was purified by flash chromatog-
(S)-4-(4-Chlorophenyl)-1-(3,4-dihydroxybutyl)-N-[3-(4-
methylpiperazin-1-yl)propyl]-3-phenyl-1H-pyrrole-2-
carboxamide (4). Potassium hydroxide (0.2 g, 3.6 mmol) was
added to a solution of 23 (0.54 g, 1.2 mmol) in a mixture of
THF/methanol/water (1:1:1, 10 mL) and the solution was
refluxed until no starting material could be detected by thin-
layer chromatography (TLC). After being cooled, the reaction
was neutralized with 1 M HCl and extracted with EtOAc. The
EtOAc solution was washed with brine, dried over Na2SO4, and
concentrated in vacuum to produce the crude acid, which was
used in the next step without purification. A solution of this
acid, 1-(3-aminopropyl)-4-methylpiperazine (0.23 g, 1.4 mmol),
EDCI (0.35 g 1.8 mmol), HOBt (0.23 g, 1.8 mmol), and
N,N-diisopropylethylamine (0.42 mL, 2.4 mmol) in dichloro-
methane (DCM; 10 mL) was stirred for 8 h and then concen-
trated. The residue was resolved in MeOH (10 mL) and
treated with 1 mL of HCl solution (4 N in dioxane) for
10 min, and then the solvent was removed under vacuum.
The residue was then purified by HPLC to provide 4 (0.5 g,
1
raphy on silica gel to afford 26 (2.26 g, 78% in two steps). H
NMR (300 MHz, CCl3D) δ 7.66−7.64 (m, 2H), 7.16−7.13 (m,
3H), 7.05 (t, J = 7.8 Hz, 1H), 6.95−6.93 (m, 3H), 5.59 (t, J =
5.1 Hz, 1H), 4.47−4.43 (m, 1H), 4.38−4.31 (m, 1H), 4.11−
4.00 (m, 2H), 3.55 (t, J = 7.1 Hz, 1H), 3.26−3.19 (m, 2H),
2.35−1.97 (m, 15H), 1.49−1.43 (m, 5H), 1.34 (s, 3H); 13C
NMR (75 MHz, CCl3D) δ 161.5, 139.3, 137.0, 136.5, 132.8,
131.8, 130.4, 129.9, 129.2, 128.4, 124.3, 124.1, 123.1, 122.1,
94.6, 73.2, 69.1, 55.8, 55.0, 53.0, 46.2, 46.0, 38.0, 35.7, 27.0,
26.1, 25.7; ESI MS m/z 691.6 (M + H)+.
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-{3-[4-
(4-{[(4-{[(S)-4-(dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitrophenyl)sulfonyl]carbamoyl}phenyl)-
piperazin-1-yl]phenyl}-N-[3-(4-methylpiperazin-1-yl)-
propyl]-1H-pyrrole-2-carboxamide (6). Pd(dba)2 (3.5 mg,
0.006 mmol), tri-tert-butylphosphine (1 M in toluene, 4.8 μL),
and sodium tert-butoxide (18 mg, 0.18 mmol) were added to a
stirred slurry of 26 (83 mg, 0.12 mmol) and 27 (88 mg, 0.14
mmol) in a mixture of toluene/DMF (1:1, 4 mL) at room tem-
perature under N2. The mixture was heated to 70 °C and moni-
tored by thin-layer chromatography. After complete consump-
tion of starting materials, the reaction mixture was filtered through
Celite and concentrated. The residue was resolved in MeOH
(5 mL) and treated with 0.2 mL of HCl solution (4 M in
dioxane) for 10 min, and then the solvent was removed in
vacuum. Purification of the residue by HPLC afforded 6 (39 mg,
1
79%). H NMR (300 MHz, CD3OD) δ 7.24−7.20 (m, 3H),
7.09−6.89 (m, 5H), 6.88 (d, J = 8.5 Hz, 2H), 4.28−4.16 (m,
2H), 3.50−3.35 (m, 11H), 3.12−3.04 (m, 2H), 2.85 (s, 3H),
2.82−2.75 (m, 2H), 1.95−1.89 (m, 1H), 1.68−1.63 (m, 3H);
13C NMR (75 MHz, CD3OD) δ 165.6, 136.2, 134.9, 132.6,
131.8, 130.5, 129.7, 129.2, 128.4, 126.6, 126.1, 124.9, 123.6, 70.2,
67.3, 55.5, 51.6, 49.9, 46.2, 43.4, 37.3, 36.4, 25.1; ESI MS m/z
525.8 (M + H)+.
Ethyl 4-(4-Chlorophenyl)-3-(3-iodophenyl)-1H-pyr-
role-2-carboxylate (24). Compound 24 was prepared by a pro-
cedure similar to that used for compound 22. The yield was
49% in two steps. 1H NMR (300 MHz, CCl3D) δ 9.48 (s, 1H),
7.74 (s, 1H), 7.64 (d, J = 7.8 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H),
7.14 (d, J = 7.7 Hz, 1H), 7.10 (d, J = 2.9 Hz, 1H), 7.05−7.00
1
29%). H NMR (300 MHz, CD3OD) δ 8.66 (d, J = 2.2 Hz,
1H), 7.93 (dd, J = 2.2, 9.2 Hz, 1H), 7.75 (d, J = 9.0 Hz, 2H),
7.29−6.95 (m, 15H), 6.81 (s, 1H), 6.74 (d, J = 7.4 Hz, 1H),
4.38−4.27 (m, 2H), 4.16−4.13 (m, 1H), 3.54−3.32 (m, 11H),
3.24−3.08 (m, 14H), 2.84 (s, 6H), 2.82 (s, 3H), 2.61 (t, J = 7.1
Hz, 2H), 2.27−2.14 (m, 2H), 2.03−2.00 (m, 1H), 1.78−1.75
(m, 1H), 1.66−1.61 (m, 2H); 13C NMR (75 MHz, CD3OD) δ
167.2, 165.4, 155.8, 152.1, 148.5, 137.2, 136.2, 135.6, 135.0,
132.5, 132.2, 131.5, 130.6, 130.4, 130.1, 129.7, 127.8, 127.5,
126.8, 126.0, 124.9, 123.7, 123.5, 121.7, 119.7, 116.5, 115.7,
114.8, 70.2, 67.3, 55.9, 55.4, 53.0, 52.4, 50.7, 50.2, 48.0, 46.2,
43.6, 43.5, 39.3, 37.6, 36.4, 30.1, 25.9; ESI MS m/z 1135.6
(M + H)+.
(m, 3H), 4.22 (q, J = 7.1 Hz, 2H), 1.20 (t, J = 7.1 Hz, 3H); 13
C
NMR (75 MHz, CCl3D) δ 161.2, 139.7, 136.4, 135.9, 132.6,
132.2, 130.0, 129.5, 129.3, 128.5, 127.1, 125.5, 120.42, 120.39,
93.3, 60.6, 14.1.
(S)-Ethyl 4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-di-
oxolan-4-yl)ethyl]-3-(3-iodophenyl)-1H-pyrrole-2-car-
boxylate (25). Compound 25 was prepared in 87% yield from
1
24 by a procedure similar to that used with compound 23. H
NMR (300 MHz, CCl3D) δ 7.67 (s, 1H), 7.62 (d, J = 7.8 Hz,
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t-Butyl 4-[4-(4-Fluoro-3-nitrophenylsulfonamido)-
phenyl]piperazine-1-carboxylate (28). 4-Fluoro-3-nitro-
benzene-1-sulfonyl chloride (312 mg, 1.3 mmol) was added
to t-butyl 4-(4-aminophenyl)piperazine-1-carboxylate (360 mg,
1.3 mmol) in pyridine (10 mL) at 0 °C. The mixture was stirred
at 0 °C for 30 min and then concentrated under vacuum. The
residue was purified by flash chromatography on silica gel to
(d, J = 8.7 Hz, 2H), 4.40 (s, 2H), 4.08 (m. 1H), 3.38−3.31 (m,
3H), 3.21−3.08 (m, 9H), 2.84 (s, 6H), 2.25−2.15 (m, 2H); 13
C
NMR (75 MHz, CD3OD) δ 148.6, 147.9, 144.4, 139.7, 136.2,
135.3, 134.4, 132.6, 132.3, 132.2, 132.1, 131.6, 131.4, 130.2,
130.1, 130.0, 128.0, 127.8, 127.6, 127.5, 124.3, 118.6, 116.2,
58.0, 55.9, 52.8, 52.4, 47.5, 43.5, 39.6, 30.1; ESI MS m/z 785.8
(M + H)+.
1
afford 28 (474 mg, 76%). H NMR (300 MHz, CCl3D) δ 8.43
4-Fluoro-3-nitro-N-phenylbenzenesulfonamide (29).
Compound 29 was prepared in 80% yield by a procedure sim-
ilar to that used to prepare compound 28. 1H NMR (300 MHz,
CCl3D) δ 8.51 (dd, J = 2.3, 6.8 Hz, 1H), 8.02−7.97 (m, 1H),
7.42−7.30 (m, 3H), 7.14−7.11 (m, 2H), 6.92 (s, 1H).
(R)-4-{[4-(Dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitro-N-phenylbenzenesulfonamide (14). Com-
pound 14 was prepared from 29 by a similar procedure as that
(dd, J = 2.2, 6.8 Hz, 1H), 7.95−7.90 (m, 1H), 7.37 (t, J = 9.3 Hz,
1H), 7.01−6.98 (m, 3H), 6.81 (d, J = 8.9 Hz, 2H), 3.59−3.56
(m, 4H), 3.13−3.10 (m, 4H), 1.49 (s, 9H); ESI MS m/z 480.9
(M + H)+.
(R)-4-{[4-(Dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitro-N-[4-(piperazin-1-yl)phenyl]benzene-
sulfonamide (15). DIEA (70 μL, 0.4 mmol) was added to a
solution of 28 (100 mg, 0.21 mmol) and (R)-N1,N1-dimethyl-
4-phenylthio)butane-1,3-diamine (47 mg, 0.21 mmol) in DMF.
The solution was stirred overnight and then concentrated. The
residue was resolved in MeOH (5 mL) and treated with HCl
solution (4 M in dioxane, 4 mL), which was stirred for 5 h and
then concentrated. This residue was purified by HPLC to afford
1
used for compound 15, in 83% yield. H NMR (300 MHz,
CD3OD) δ 8.38 (d, J = 2.2 Hz, 1H), 8.15 (d, J = 9.3 Hz, 1H),
7.62 (dd, J = 2.1, 9.1 Hz, 1H), 7.28−7.23 (m, 2H), 7.18−6.97
(m, 8H), 6.90, (d, J = 9.3 Hz, 1H), 4.11−4.07 (m, 1H), 3.40−
3.33 (m, 1H), 3.23−3.09 (m, 3H), 2.85 (s, 6H), 2.31−2.11 (m,
2H); 13C NMR (75 MHz, CD3OD) δ 147.9, 139.0, 136.1,
134.4, 132.3, 131.5, 130.3, 128.0, 127.9, 127.7, 125.8, 121.8,
116.1, 55.9, 52.3, 43.5, 39.4, 30.1; ESI MS m/z 501.7 (M + H)+.
(R)-N-[(4-{[4-(Dimethylamino)-1-(phenylthio)butan-2-
yl]amino}-3-nitrophenyl)sulfonyl]-4-ethynylbenzamide
(30). A suspension of 5 (400 mg, 0.94 mmol), 4-ethynylbenzoic
acid (138 mg, 0.94 mmol), (dimethylamino)pyridine (DMAP;
230 mg, 1.88 mmol), and EDCI (362 g, 1.88 mmol) in CH2Cl2
(10 mL) was stirred at room temperature for 8 h. The reaction
mixture was washed with saturated NH4Cl (3 × 8 mL) and
concentrated. The crude residue was purified by HPLC to
1
15 (170 mg, 81% over two steps). H NMR (300 MHz,
CD3OD) δ 8.24 (d, J = 2.2 Hz, 1H), 7.56 (dd, J = 2.2, 9.1 Hz,
1H), 7.13−7.10 (m, 2H), 7.04−6.87 (m, 8H), 4.09−4.07
(m, 1H), 3.37−3.27 (m, 10H), 3.21−3.13 (m, 2H), 2.82
(s, 6H), 2.24−2.14 (m, 2H); 13C NMR (75 MHz, CD3OD) δ
149.3, 148.0, 136.2, 134.4, 132.1, 132.0, 131.6, 130.1, 128.0,
127.8, 127.5, 124.4, 118.8, 116.3, 55.9, 52.4, 47.9, 44.7, 43.5,
39.5, 30.1; ESI MS m/z 585.7 (M + H)+.
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-(3-{4-
[4-(4-{[(R)-4-(dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitrophenylsulfonamido)phenyl]piperazin-1-yl}-
phenyl)-N-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyrrole-
2-carboxamide (7). Compound 7 was prepared in 24% yield
from compounds 15 and 26 by a similar procedure as that used
1
provide 30 (365 mg, 70%). H NMR (300 MHz, CD3OD) δ
8.66 (s, 1H), 8.30 (d, J = 9.2 Hz, 1H), 7.89 (d, J = 8.0 Hz, 2H),
7.52 (d, J = 8.0 Hz, 2H), 7.21−7.19 (m, 2H), 7.05−6.89 (m,
4H), 4.16−4.15 (m, 1H), 3.80 (s, 1H), 3.40−3.34 (m, 1H),
3.24−3.13 (m, 3H), 2.86 (s, 6H), 2.30−2.14 (m, 2H); 13C
NMR (75 MHz, CD3OD) δ 165.5, 147.2, 134.8, 134.1, 132.0,
131.6, 130.7, 130.1, 128.7, 128.5, 128.0, 127.4, 126.5, 125.4,
114.6, 81.8, 81.0, 54.5, 51.1, 42.2, 38.0, 28.7; ESI MS m/z 553.8
(M + H)+.
1
to prepare BM-977. H NMR (300 MHz, CD3OD) δ 8.30 (d,
J = 2.2 Hz, 1H), 7.59 (dd, J = 2.3, 9.2 Hz, 1H), 7.28 (t, J = 7.9
Hz, 1H), 7.18−6.98 (m, 15H), 6.91 (d, J = 9.4 Hz, 1H), 6.83 (s,
1H), 6.78 (d, J = 7.5 Hz, 1H), 4.38−4.28 (m, 2H), 4.09−4.08
(m, 1H), 3.55−3.32 (m, 11H), 3.21−3.15 (m, 14H), 2.84 (s,
3H), 2.83 (s, 6H), 2.70−2.65 (m, 2H), 2.25−1.99 (m, 3H),
1.78−1.64 (m, 3H); 13C NMR (75 MHz, CD3OD) δ 165.4,
151.7, 147.9, 137.2, 136.2, 135.0, 134.5, 132.6, 132.2, 131.6,
130.7, 130.5, 130.2, 129.3, 129.2, 128.0, 127.9, 127.7, 126.7,
126.1, 124.8, 124.3, 124.1, 123.5, 120.0, 118.8, 116.8, 116.2,
115.8, 70.2, 67.2, 55.9, 55.4, 53.1, 52.4, 51.0, 50.7, 50.5, 46.2,
43.7, 43.5, 39.6, 37.7, 36.4, 30.1, 25.9; ESI MS m/z 1107.7
(M + H)+.
(R)-N-(4-{4-[(4′-Chloro-[1,1′-biphenyl]-2-yl)methyl]-
piperazin-1-yl}phenyl)-4-{[4-(dimethylamino)-1-(phenyl-
thio)but-2-yl]amino}-3-nitrobenzenesulfonamide (13).
Compound 15 (58.5 mg, 0.1 mmol) and 4′-chloro-[1,1′-biphenyl]-
2-carbaldehyde (21.7 mg, 0.1 mmol) were mixed in 1,2-dichloro-
ethane (5 mL) and then treated with sodium triacetoxy-
borohydride (30 mg, 0.14 mmol). The mixture was stirred at
room temperature under N2 atmosphere for 24 h until the
reactants were consumed. Then the reaction mixture was
quenched by adding 1 N NaOH, and the product was extracted
with CH2Cl2. The CH2Cl2 extract was washed with brine and
dried over MgSO4. The solvent was evaporated and purified by
HPLC to give 13 (61 mg, 78%). 1H NMR (300 MHz, CD3OD)
δ 8.24 (s, 1H), 7.70−7.68 (m, 1H), 7.58−7.47 (m, 5H), 7.39−
7.31 (m, 3H), 7.12 (d, J = 7.0 Hz, 2H), 7.02−6.89 (m, 6H), 8.18
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-{3-
[(4-{[(4-{[(R)-4-(dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitrophenyl)sulfonyl]carbamoyl}phenyl)-
ethynyl]phenyl}-N-[3-(4-methylpiperazin-1-yl)propyl]-
1H-pyrrole-2-carboxamide (9). CuI (6.9 mg, 0.036 mmol)
and Pd(PPh3)4 (13.9 mg, 0.012 mmol) were added to a solu-
tion of 26 (83 mg, 0.12 mmol), 30 (66 mg, 0.12 mmol), and
Et3N (50 μL, 0.36 mmol) in DMF (5 mL) at room temperature
under nitrogen. The mixture was stirred at 40 °C for 3 h and
then filtered through Celite, washed with 50 mL of CH2Cl2,
and concentrated under reduced pressure. The residue was re-
solved in MeOH (5 mL) and treated with 0.2 mL of HCl
solution (4 M in dioxane) for 10 min, and then the solvent was
removed in vacuum. Purification of the residue by HPLC
afforded 9 (97 mg, 75%). 1H NMR (300 MHz, CD3OD) δ 8.70
(d, J = 1.9 Hz, 1H), 7.94 (d, J = 7.4 Hz, 1H), 7.83 (d, J = 8.3
Hz, 2H), 7.58 (d, J = 8.2 Hz, 2H), 7.48 (d, J = 7.7 Hz, 1H),
7.38−7.34 (m, 2H), 7.24−7.01 (m, 12H), 4.37−4.29 (m, 2H),
4.19−4.17 (m, 1H), 3.56−3.39 (m, 8H), 3.27−3.13 (m, 9H),
2.87−2.76 (m, 11H), 2.31−2.22 (m, 2H), 2.04−2.01 (m, 1H),
1.75−1.70 (m, 3H); 13C NMR (75 MHz, CD3OD) δ 166.9,
165.4, 148.6, 136.8, 136.2, 135.5, 134.8, 134.7, 132.9, 132.8,
132.6, 132.2, 131.5, 130.6, 130.1, 129.9, 129.5, 129.4, 129.3,
4676
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Journal of Medicinal Chemistry
Article
127.9, 126.9, 126.8, 125.0, 124.7, 123.9, 123.5, 93.4, 89.6, 70.2,
67.3, 55.9, 55.5, 52.6, 52.5, 50.4, 46.1, 43.6, 39.4, 37.7, 36.4,
30.1, 25.7; ESI MS m/z 1075.8 (M + H)+.
The resulting mixture was stirred at room temperature overnight
and then was diluted with water (10 mL) and extracted with
CH2Cl2 (3 × 20 mL). The combined organic layer was washed
with brine, dried over Na2SO4, and concentrated under reduced
pressure. The residue was resolved in MeOH (5 mL) and
treated with HCl solution (4 M in dioxane, 0.2 mL) for 10 min,
and then the solvent was removed in vacuum. Purification of
the residue by HPLC afforded 8 (97 mg, 75%). 1H NMR (300
MHz, CD3OD) δ 8.67 (s, 1H), 8.38 (d, J = 2.2 Hz, 1H), 7.78−
7.70 (m, 4H), 7.62 (dd, J = 2.2, 9.2 Hz, 1H), 7.47 (t, J = 7.0 Hz,
1H), 7.24−7.21 (m, 3H), 7.12−7.01 (m, 7H), 6.94−6.88
(m, 4H), 4.35−4.28 (m, 2H), 4.07−4.06 (m, 1H), 3.54−3.34
(m, 9H), 3.20−3.11 (m, 8H), 2.84−2.80 (m, 11H), 2.19−2.15
(m, 3H), 1.71−1.66 (m, 3H); 13C NMR (75 MHz, CD3OD)
δ 165.4, 148.9, 148.1, 139.3, 138.3, 138.0, 136.1, 134.7, 134.3,
132.9, 132.4, 132.2, 131.5, 131.0, 130.8, 130.0, 129.4,
128.1, 127.8, 127.7, 127.4, 127.1, 124.8, 124.5, 123.6, 123.4,
121.9, 120.1, 119.9, 116.4, 70.2, 67.3, 55.9, 55.5, 52.4, 50.3,
46.1, 43.5, 39.4, 37.7, 36.4, 30.1, 25.7; ESI MS m/z 1091.4
(M + H)+.
(R)-4-{[4-(Dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-N-(4-ethynylphenyl)-3-nitrobenzenesulfona-
mide (31). Compound 31 was prepared by a similar procedure
to that used for compound 30 in 80% yield. 1H NMR (300 MHz,
CD3OD) δ 8.43 (s, 1H), 7.64 (d, J = 8.8 Hz, 1H), 7.38 (d, J =
8.2 Hz, 2H), 7.21−7.14 (m, 4H), 7.10−6.93 (m, 4H), 4.13−
4.12 (m, 1H), 3.44−3.37 (m, 2H), 3.26−3.14 (m, 3H), 2.88 (s,
6H), 2.30−2.21 (m, 2H); 13C NMR (75 MHz, CD3OD) δ
146.7, 138.0, 134.7, 132.9, 132.7, 130.9, 130.1, 128.7, 126.6,
126.5, 125.9, 119.6, 118.3, 114.9, 82.4, 77.3, 54.5, 51.0, 42.1,
38.0, 28.7; ESI MS m/z 526.0 (M + H)+.
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-(3-
{[4-(4-{[(R)-4-(dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitrophenylsulfonamido)phenyl]ethynyl}-
phenyl)-N-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyr-
role-2-carboxamide (10). Compound 10 was prepared from
1
31 by a similar procedure as that for 9 in 79% yield. H NMR
Ethyl 4-(4-Chlorophenyl)-3-(4-iodophenyl)-1H-pyr-
role-2-carboxylate. Compound 33 was prepared by a similar
procedure as was used to prepare 24 in 58% yield in two steps.
1H NMR (300 MHz, CCl3D) δ 9.52 (br s, 1H), 7.56 (d, J = 8.2
Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 7.09 (d, J = 3.0 Hz, 1H),
7.04−7.01 (m, 4H), 4.23 (q, J = 7.1 Hz, 2H), 1.20 (t, J = 7.1
Hz, 3H); 13C NMR (75 MHz, CCl3D) δ 161.0, 136.8, 133.7,
132.7, 132.2, 129.6, 128.5, 127.9, 125.5, 120.4, 120.2, 92.9,
60.5, 14.1.
(300 MHz, CD3OD) δ 8.39 (d, J = 2.2 Hz, 1H), 7.63 (dd, J =
2.2, 9.0 Hz, 1H), 7.40−7.37 (m, 3H), 7.32−7.27 (m, 2H),
7.15−7.10 (m, 8H), 7.01−6.89 (m, 6H), 4.37−4.21 (m, 2H),
4.09−4.06 (m, 1H), 3.53−3.49 (m, 1H), 3.45−3.43 (m, 2H),
3.33−3.31 (m, 5H), 3.24−3.08 (m, 9H), 2.82 (s, 9H), 2.72−
2.68 (m, 2H), 2.24−1.98 (m, 3H), 1.79−1.63 (m, 3H); 13C
NMR (75 MHz, CD3OD) δ 165.4, 148.1, 139.4, 136.6, 136.2,
134.8, 134.4, 134.3, 133.7, 132.7, 132.2, 132.0, 131.5, 131.2,
130.5, 130.1, 129.8, 129.3, 128.0, 127.9, 127.4, 126.7, 125.2,
124.7, 124.6, 123.5, 121.1, 120.1, 116.3, 90.2, 90.0, 70.2, 67.3,
55.9, 55.4, 52.7, 52.3, 50.5, 46.1, 43.6, 43.5, 39.4, 37.6, 36.4,
30.1, 25.7; ESI MS m/z 1048.4 (M + H)+.
(S)-Ethyl 4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-di-
oxolan-4-yl)ethyl]-3-(4-iodophenyl)-1H-pyrrole-2-car-
boxylate (34). Compound 34 was prepared from 33 by a
1
(S)-3-(3-Azidophenyl)-4-(4-chlorophenyl)-1-[2-(2,2-di-
methyl-1,3-dioxolan-4-yl)ethyl]-N-[3-(4-methylpipera-
zin-1-yl)propyl]-1H-pyrrole-2-carboxamide (32). A mix-
ture of 26 (300 mg, 0.43 mmol), sodium azide (42 mg, 0.65
mmol), CuI (8.3 mg, 0.043 mmol), L-proline (10 mg, 0.087
mmol), and NaOH (3.5 mg, 0.087 mmol) in DMSO (4 mL) in
a sealed tube was heated to 70 °C under N2. After the reaction
was completed, the cooled mixture was partitioned between
CH2Cl2 and H2O. The organic layer was separated, and the
aqueous layer was extracted twice with CH2Cl2. The combined
organic layers were washed with brine, dried over Mg2SO4, and
concentrated in vacuo. The residue was purified by flash
chromatography on silica gel to afford 32 (134 mg, 51%).1H
NMR (300 MHz, CCl3D) δ 7.35−7.29 (m, 1H), 7.11 (d, J =
8.3 Hz, 2H), 6.98 (d, J = 7.7 Hz, 2H), 6.93−6.87 (m, 4H), 5.54
(br s, 1H), 4.47−4.43 (m, 1H), 4.38−4.29 (m, 1H), 4.10−4.08
(m, 1H), 4.03−3.00 (m, 1H), 3.53 (t, J = 7.3 Hz, 1H), 3.21−
3.15 (m, 2H), 2.36−1.96 (m, 15H), 1.42−1.40 (m, 5H), 1.32
(s, 3H); 13C NMR (75 MHz, CCl3D) δ 161.5, 140.6, 136.8,
132.8, 131.8, 130.2, 129.1, 128.4, 127.2, 124.1, 123.8, 122.2,
121.2, 118.1, 108.9, 73.2, 69.1, 55.7, 54.9, 52.7, 46.3, 45.8, 37.8,
35.7, 27.0, 26.1, 25.6; ESI MS m/z 606.8 (M + H)+.
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-(3-{4-
[4-(4-{[(R)-4-(dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitrophenylsulfonamido)phenyl]-1H-1,2,3-tri-
azol-1-yl}phenyl)-N-[3-(4-methylpiperazin-1-yl)propyl]-
1H-pyrrole-2-carboxamide (8). Compounds 32 (90 mg,
0.15 mmol) and 31 (78 mg, 0.15 mmol) were suspended in a
1:2 mixture of H2O and t-BuOH (9 mL), and then a solution of
CuSO4·5H2O (3.7 mg, 0.015 mmol) and (+)-sodium L-ascor-
bate (8.8 mg, 0.045 mmol) in H2O (1 mL) was added.
procedure similar to that used for 25 in 90% yield. H NMR
(300 MHz, CCl3D) δ 7.62 (d, J = 8.4 Hz, 2H), 7.16 (d, J = 8.6
Hz, 2H), 7.05 (s, 1H), 6.98−6.93 (m, 4H), 4.62−4.55 (m, 1H),
4.46−4.36 (m, 1H), 4.15−4.03 (m, 4H), 3.63−3.57 (m, 1H),
2.21−2.12 (m, 1H), 2.08−1.94 (m, 1H), 1.47 (s, 3H), 1.38 (s,
3H), 0.99 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CCl3D) δ
161.2, 136.7, 135.4, 132.6, 131.9, 130.0, 129.3, 128.4, 126.4,
123.0, 119.9, 109.1, 92.3, 73.1, 69.0, 60.0, 46.8, 35.5, 27.0, 25.6,
13.7.
(S)-Ethyl 4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-di-
oxolan-4-yl)ethyl]-3-{4-[4-(4-nitrophenyl)piperazin-1-
yl]phenyl}-1H-pyrrole-2-carboxylate (35). Compound 34
(380 mg, 0.66 mmol), 1-(4-nitrophenyl)piperazine (271 mg,
1.31 mmol), CuI (12 mg, 0.066 mmol), L-proline (15 mg,
0.13 mmol), and K2CO3 (181 mg, 1.31 mmol) were dissolved
in 5 mL of DMSO. This mixture was heated to 80 °C for 2 h
under nitrogen. After the solution was cooled, saturated ammonium
chloride solution was added and the mixture was extracted with
CH2Cl2. The combined organic layers were washed with brine,
dried over sodium sulfate, and concentrated. Purification of
the residue by flash chromatography on silica gel afforded 35
(354 mg, 82%). 1H NMR (300 MHz, CCl3D) δ 8.15 (d, J = 8.9
Hz, 2H), 7.15−7.11 (m, 4H), 7.05−7.00 (m, 3H), 6.90−6.87
(m, 4H), 4.63−4.54 (m, 1H), 4.45−4.36 (m, 1H), 4.12−4.04
(m, 4H), 3.62−3.57 (m, 5H), 3.39−3.37 (m, 4H), 2.21−2.15
(m, 1H), 2.09−2.00 (m, 1H), 1.47 (s, 3H), 1.38 (s, 3H), 1.02
(t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CCl3D) δ 161.5,
154.7, 149.4, 138.6, 133.2, 131.5, 131.1, 129.3, 128.3, 127.5,
126.4, 126.0, 123.1, 120.0, 115.3, 112.7. 109.1, 73.2, 69.1, 59.8,
48.9, 47.1, 46.9, 35.6, 27.0, 25.6, 13.8; ESI MS m/z 659.8
(M + H)+.
4677
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Journal of Medicinal Chemistry
Article
(S)-4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-dioxo-
lan-4-yl)ethyl]-N-[3-(4-methylpiperazin-1-yl)propyl]-3-
{4-[4-(4-nitrophenyl)piperazin-1-yl]phenyl}-1H-pyrrole-
2-carboxamide (36). Compound 36 was prepared from 35 in
85% yield in two steps by a procedure similar to that used for
109.1, 73.2, 69.1, 59.8, 46.8, 43.0, 42.5, 35.5, 27.0, 25.6, 13.7;
ESI MS m/z 633.4 (M + H)+.
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-{3-
[(2-{[4-(4-{[(R)-4-(dimethylamino)-1-(phenylthio)but-2-
yl]amino}-3-nitrophenylsulfonamido)phenyl]amino}-
ethyl)amino]phenyl}-N-[3-(4-methylpiperazin-1-yl)-
propyl]-1H-pyrrole-2-carboxamide (11). Compound 11
was prepared from 37 in 40% yield over six steps by a procedure
1
26. H NMR (300 MHz, CCl3D) δ 8.14 (d, J = 8.9 Hz, 2H),
7.17−7.10 (m, 4H), 6.89−6.86 (m, 7H), 5.61 (t, J = 5.3 Hz,
1H), 4.58−4.49 (m, 1H), 4.44−4.35 (m, 1H), 4.13−4.09 (m,
1H), 4.05−4.01 (m, 1H), 3.62−3.53 (m, 5H), 3.41−3.40 (m,
4H), 3.18 (q, J = 6.2 Hz, 2H), 2.35−1.98 (m, 15H), 1.48−1.40
(m, 5H), 1.35 (s, 3H); 13C NMR (75 MHz, CCl3D) δ 161.8,
154.5, 149.8, 138.7, 133.4, 131.7, 131.4, 129.1, 128.3, 126.0,
125.8, 125.0, 124.2, 123.5, 122.3, 116.0, 112.8, 108.9, 73.4, 69.1,
55.8, 55.0, 53.0, 48.2, 47.0, 46.5, 45.9, 37.5, 35.7, 27.0, 26.4,
25.7; ESI MS m/z 770.4 (M + H)+.
1
similar to that used for compound 12. H NMR (300 MHz,
CD3OD) δ 8.35 (d, J = 1.9 Hz, 1H), 7.65 (d, J = 7.4 Hz, 1H),
7.33−6.94 (m, 14H), 6.78−6.61 (m, 5H), 4.45−4.31 (m, 2H),
4.15−4.12 (m, 1H), 3.70−3.39 (m, 8H), 3.28−3.17 (m, 13H),
2.90−2.88 (m, 9H), 2.69−2.64 (m, 2H), 2.30−2.05 (m, 3H), 1.85−
1.82 (m, 1H), 1.70−1.68 (m, 2H); 13C NMR (75 MHz, CD3OD)
165.2, 147.9, 137.6, 136.2, 135.0, 134.5, 132.5, 132.3, 131.6, 130.9,
130.3, 130.1, 129.2, 128.02, 127.96, 127.7, 125.9, 125.1, 125.0, 123.4,
116.2, 70.2, 67.3, 55.9, 55.4, 53.0, 52.3, 50.7, 46.4, 43.7, 43.5, 39.6,
37.6, 36.4, 30.1, 25.8; ESI MS m/z 1081.6 (M + H)+.
4-(4-Chlorophenyl)-1-[(S)-3,4-dihydroxybutyl]-3-(4-{4-
[4-(4-{[(R)-4-(dimethylamino)-1-(phenylthio)but-2-yl]-
amino}-3-nitrophenylsulfonamido)phenyl]piperazin-1-
yl}phenyl)-N-[3-(4-methylpiperazin-1-yl)propyl]-1H-pyr-
role-2-carboxamide (12). Pd−C (10%; 20 mg) was added to
a solution of compound 36 (124 mg, 0.16 mmol) in CH2Cl2
(3 mL) and MeOH (3 mL). The solution was stirred under
1 atm of H2 at room temperature for 0.5 h before it was filtered
through Celite and concentrated. The resulting aniline was
used in the next step without purification. To this aniline in
pyridine (5 mL) was added 4-fluoro-3-nitrobenzene-1-sulfonyl
chloride (39 mg, 0.16 mmol) at 0 °C. The mixture was stirred
at 0 °C for 30 min. The pyridine was removed under vacuum and
the residue was purified by flash chromatography on silica gel to
give (S)-4-(4-chlorophenyl)-1-[2-(2,2-dimethyl-1,3-dioxolan-4-yl)-
ethyl]-3-{4-[(2-{[4-(4-fluoro-3-nitrophenylsulfonamido)phenyl]-
amino}ethyl)amino]phenyl}-N-[3-(4-methylpiperazin-1-yl)-
propyl]-1H-pyrrole-2-carboxamide. N,N-Diisopropylethylamine
(DIEA; 56 μL, 0.32 mmol) was added to a solution of this
sulfonamide and (R)-N1,N1-dimethyl-4-(phenylthio)butane-
1,3-diamine (39 mg, 0.16 mmol) in DMF. The solution was
stirred overnight and concentrated. The residue was dissolved
in MeOH (5 mL) and treated with HCl solution (4 M in
dioxane, 0.2 mL) for 10 min, and then the solvent was removed
in vacuum. Purification of the residue by HPLC afforded 12
Ethyl 4-(4-Chlorophenyl)-3-(3-iodophenyl)-1-methyl-
1H-pyrrole-2-carboxylate (38). K2CO3 (1.83 g, 13.3 mmol)
and MeI (1.89 g, 13.3 mmol) were added to a solution of 24 (3.0 g,
6.6 mmol) in DMF (40 mL). The reaction mixture was stirred for
4 h under nitrogen at room temperature. Then the reaction was
diluted with water (40 mL) and extracted with EtOAc (2 × 60 mL).
The combined organics were washed with water (60 mL) and brine
(60 mL) and then dried over Na2SO4. After evaporation of the
solvent, the residue was purified by flash chromatography on silica
1
gel to provide 38 (2.88 g, 93%). H NMR (300 MHz, CCl3D) δ
7.64 (t, J = 1.6 Hz, 1H), 7.60 (dt, J = 1.6, 7.8 Hz, 1H), 7.15−7.12
(m, 2H), 7.07−7.04 (m, 1H), 7.00−6.91 (m, 4H), 4.06 (q, J =
7.1 Hz, 2H), 3.97 (s, 3H), 1.00 (t, J = 7.1 Hz, 3H); 13C NMR (75
MHz, CCl3D) δ 161.6, 139.8, 138.2, 135.7, 132.8, 132.0, 129.42,
129.39, 129.0, 128.5, 127.0, 122.9, 121.0, 93.4, 60.1, 37.8, 14.0.
Ethyl 4-(4-Chlorophenyl)-1-methyl-3-{3-[4-(4-
nitrophenyl)piperazin-1-yl]phenyl}-1H-pyrrole-2-car-
boxylate (39). Compound 39 was prepared in 83% yield by a
1
procedure similar to that used for compound 35. H NMR (300
MHz, CCl3D) δ 8.12 (d, J = 9.1 Hz, 2H), 7.26−7.21 (m, 1H), 7.13
(d, J = 8.3 Hz, 2H), 7.03 (d, J = 8.3 Hz, 2H), 6.96 (s, 1H), 6.90−6.79
(m, 5H), 4.09 (q, J = 7.1 Hz, 2H), 3.99 (s, 3H), 3.54−3.51 (m, 4H),
3.27−3.24 (m, 4H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz,
CCl3D) δ 161.7, 154.7, 150.1, 138.5, 136.7, 133.2, 131.5, 131.0, 129.2,
128.4, 128.2, 126.6, 125.9, 123.1, 122.7, 121.0, 119.0, 114.9, 112.7,
59.8, 49.0, 46.9, 37.6, 13.8; ESI MS m/z 545.8 (M + H)+.
1
(94 mg, 51% in four steps). H NMR (300 MHz, CD3OD) δ
8.36 (s, 1H), 7.66 (d, J = 9.1 Hz, 1H), 7.22−7.04 (m, 18H),
6.97 (d, J = 9.2 Hz, 1H), 4.43−4.31 (m, 2H), 4.15−4.13 (m,
1H), 3.59−3.37 (m, 17H), 3.26−3.21 (m, 8H), 2.90−2.82 (m,
11H), 2.30−2.05 (m, 3H), 1.83−1.74 (m, 3H); 13C NMR (75
MHz, CD3OD) δ 165.6, 147.9, 136.2, 135.1, 134.4, 132.7,
132.5, 132.3, 131.6, 130.5, 130.1, 129.2, 128.0, 127.9, 127.7,
126.3, 125.9, 125.1, 124.2, 123.7, 119.2, 117.9, 116.2, 70.3, 67.3,
55.9, 55.5, 52.5, 52.4, 51.4, 50.6, 50.4, 46.3, 43.6, 43.5, 39.5,
37.5, 36.5, 30.1, 25.7; ESI MS m/z 1107.7 (M + H)+.
(S)-Ethyl 4-(4-Chlorophenyl)-1-[2-(2,2-dimethyl-1,3-di-
oxolan-4-yl)ethyl]-3-[3-({2-[(4-nitrophenyl)amino]ethyl}-
amino)phenyl]-1H-pyrrole-2-carboxylate (37). Com-
pound 37 was prepared in 78% yield from 26 by a procedure
similar to that used for 35. 1H NMR (300 MHz, CCl3D) δ 8.07
(d, J = 8.6 Hz, 2H), 7.15−7.13 (m, 3H), 7.05−7.02 (m, 3H),
6.66 (d, J = 7.5 Hz, 1H), 6.59 (d, J = 8.5 Hz, 1H), 6.52 (d, J =
8.8 Hz, 2H), 6.46 (s, 1H), 4.58−4.56 (m, 1H), 4.45−4.35 (m,
1H), 4.11−3.99 (m, 4H), 3.59 (t, J = 6.9 Hz, 1H), 3.37 (br s,
4H), 2.18−2.16 (m, 1H), 2.06−2.01 (m, 1H), 1.47 (s, 3H),
1.38 (s, 3H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz,
CCl3D) δ 161.5, 153.3, 146.9, 138.1, 136.9, 133.1, 131.6, 129.2,
128.6, 128.2, 126.4, 126.2, 122.8, 121.1, 120.0, 115.3, 111.9,
(R)-4-(4-Chlorophenyl)-3-(3-{4-[4-(4-{[4-(dimethylamino)-
1-(phenylthio)but-2-yl]amino}-3-nitrophenylsulfonamido)-
phenyl]piperazin-1-yl}phenyl)-1-methyl-1H-pyrrole-2-car-
boxylic acid (20). Compound 20 was prepared from 38 in 49%
1
yield in four steps by a procedure similar to that used for 12. H
NMR (300 MHz, CD3OD) δ 8.29 (d, J = 2.2 Hz, 1H), 7.58 (dd,
J = 2.3, 9.2 Hz, 1H), 7.27 (t, J = 7.8 Hz, 1H), 7.15−6.89 (m, 18H),
4.08−4.05 (m, 1H), 3.93 (s, 3H), 3.67−3.30 (m, 9H), 3.20−3.14
(m, 3H), 2.82 (s, 6H), 2.23−2.14 (m, 2H); 13C NMR (75 MHz,
CD3OD) δ 164.3, 147.9, 138.9, 136.2, 134.8, 134.4, 132.8, 132.6,
132.2, 131.7, 131.6, 130.6, 130.1, 130.0, 129.2, 128.7, 128.3, 128.0,
127.9, 127.6, 124.1, 124.0, 122.4, 122.0, 119.1, 117.8, 116.2, 55.9,
52.8, 52.4, 50.6, 43.5, 39.5, 38.1, 30.1; ESI MS m/z 895.1 (M + H)+.
(R)-4-(4-Chlorophenyl)-3-(3-{4-[4-(4-{[4-(dimethylamino)-
1-(phenylthio)but-2-yl]amino}-3-nitrophenylsulfonamido)-
phenyl]piperazin-1-yl}phenyl)-1-methyl-N-[3-(4-methylpi-
perazin-1-yl)propyl]-1H-pyrrole-2-carboxamide (16). A
mixture of 20 (76 mg, 0.085 mmol), 1-(3-aminopropyl)-4-
methylpiperazine (20 mg, 0.127 mmol), EDCI (24.5 mg,
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Journal of Medicinal Chemistry
Article
0.127 mmol), HOBt (16.4 mg, 0.127 mol), and DIEA (44 μL,
0.25 mmol) in DCM (5 mL) was stirred for 8 h and then con-
centrated. The residue was purified by HPLC to provide 16
(74 mg, 84%). 1H NMR (300 MHz, CD3OD) δ 8.30 (d, J = 2.2
Hz, 1H), 7.59 (dd, J = 2.2, 9.1 Hz, 1H), 7.28 (t, J = 7.9 Hz,
1H), 7.16−6.89 (m, 16H), 6.82−6.77 (m, 2H), 4.09−4.06 (m,
1H), 3.82 (s, 3H), 3.33−3.28 (m, 6H), 3.25−3.09 (m, 16H),
2.83 (s, 9H), 2.62−2.57 (m, 2H), 2.24−2.14 (m, 2H), 1.65−
1.61 (m, 2H); 13C NMR (75 MHz, CD3OD) δ 165.1, 151.6,
147.9, 137.4, 136.2, 135.0, 134.4, 132.5, 132.2, 131.6, 130.7,
130.4, 130.1, 129.2, 128.0, 127.9, 127.6, 126.7, 126.4, 125.8,
124.2, 123.3, 120.1, 118.9, 116.9, 116.2, 55.9, 55.4, 52.9. 52.4,
51.1, 50.6, 50.5, 43.6, 43.5, 39.5, 37.5, 36.3, 30.1, 25.9; ESI MS
m/z 1033.7 (M + H)+.
134.4, 133.3, 132.7, 132.3, 131.6, 130.5, 130.1, 130.0, 129.2,
128.6, 128.0, 127.9, 127.6, 124.0, 123.9, 122.0, 121.9, 119.4,
117.7, 116.2, 60.9, 55.9, 52.44, 52.37, 51.0, 43.5, 39.5, 37.8, 30.1,
14.2; ESI MS m/z 922.8 (M + H)+.
Ethyl 5-Bromo-4-(4-chlorophenyl)-3-(3-iodophenyl)-
1-methyl-1H-pyrrole-2-carboxylate (40). N-Bromosuccini-
mide (0.59 g, 3.3 mmol) was added in portions to a solution of
24 (1.5 g, 3.3 mmol) in DMF (20 mL) and the resulting mix-
ture was stirred at room temperature for 2 h. The reaction mix-
ture was diluted with H2O (30 mL) and extracted into EtOAc
(2 × 30 mL). The combined organics were washed with water
(30 mL) and brine (30 mL) and dried over Na2SO4. After
removal of the solvent under vacuum, a solution of the residue
in DMF (20 mL) was added to K2CO3 (0.92 g, 6.6 mmol) and
MeI (0.94 g, 6.6 mmol). This reaction mixture was stirred for
4 h under nitrogen at room temperature. The reaction was
diluted with water (20 mL) and extracted into EtOAc (2 ×
30 mL). The combined organics were washed with water
(30 mL) and then brine (30 mL) and dried over Na2SO4. After
evaporation of the solvent, the residue was purified by flash
chromatography on silica gel to provide 40 (1.3 g, 71% yield
(R)-4-(4-Chlorophenyl)-3-(3-{4-[4-(4-{[4-(dimethylamino)-
1-(phenylthio)but-2-yl]amino}-3-nitrophenylsulfonamido)-
phenyl]piperazin-1-yl}phenyl)-N,1-dimethyl-1H-pyrrole-2-
carboxamide (17). A mixture of 20 (42 mg, 0.047 mmol), 2 M
methylamine in THF (47 μM, 0.094 mmol), EDCI (18.0 mg,
0.094 mmol), HOBt (12.1 mg, 0.094 mol), and DIEA (25 μL,
0.14 mmol) in DCM (3 mL) was stirred for 8 h and then con-
centrated. The residue was purified by HPLC to provide 17
1
over two steps). H NMR (300 MHz, CCl3D) δ 7.61 (s, 1H),
1
(36.6 mg, 86%). H NMR (300 MHz, CD3OD) δ 8.32 (d, J =
7.56 (d, J = 7.5 Hz, 1H), 7.21 (d, J = 8.4 Hz, 2H), 7.05 (d, J =
8.4 Hz, 2H), 6.99−6.89 (m, 2H), 4.09 (q, J = 7.1 Hz, 2H), 4.05
(s, 3H), 1.01 (t, J = 7.1 Hz, 3H); 13C NMR (75 MHz, CCl3D)
δ 160.9, 139.7, 137.2, 135.7, 132.8, 131.7, 131.5, 130.0, 129.7,
129.1, 128.2, 123.6, 121.7, 111.7, 93.1, 60.3, 35.6, 13.8.
1.2 Hz, 1H), 7.58 (d, J = 9.1 Hz, 1H), 7.28 (t, J = 7.9 Hz, 1H),
7.17−6.99 (m, 15H), 6.90 (d, J = 9.3 Hz, 1H), 6.86 (s, 1H),
6.80 (d, J = 7.5 Hz, 1H), 4.09−4.07 (m, 1H), 3.80 (s, 3H),
3.45−3.33 (m, 9H), 3.21−3.08 (m, 3H), 2.84 (s, 6H), 3.05 (s,
3H), 2.25−2.10 (m, 2H); 13C NMR (75 MHz, CD3OD) δ
165.4, 149.9, 148.0, 147.2, 137.4, 136.2, 135.1, 134.4, 133.6,
132.6, 132.3, 131.6, 130.7, 130.6, 130.1, 129.2, 128.0, 127.9,
127.6, 126.9, 125.9, 125.8, 125.6, 124.0, 123.1, 120.8, 119.5,
117.4, 116.2, 55.9, 52.4, 51.4, 43.5, 39.6, 36.2, 30.1, 26.4; ESI
MS m/z 907.6 (M + H)+.
Ethyl 5-Bromo-4-(4-chlorophenyl)-1-methyl-3-{3-[4-
(4-nitrophenyl)piperazin-1-yl]phenyl}-1H-pyrrole-2-car-
boxylate (41). Compound 41 was prepared in 74% yield by a
1
procedure similar to that used for compound 35. H NMR
(300 MHz, CCl3D) δ 8.15 (d, J = 9.3 Hz, 2H), 7.21−7.14 (m,
3H), 7.07 (d, J = 8.5 Hz, 2H), 6.87−6.81 (m, 3H), 6.72 (d, J =
7.6 Hz, 1H), 6.63 (s, 1H), 4.09 (q, J = 7.1 Hz, 2H), 4.04 (s,
3H), 3.53−3.49 (m, 4H), 3.20−3.17 (m, 4H), 0.99 (t, J = 7.1
Hz, 3H); 13C NMR (75 MHz, CCl3D) δ 161.20, 154.7, 149.8,
138.6, 135.6, 132.5, 132.1, 131.9, 131.7, 128.2, 128.1, 125.9,
123.6, 123.1, 121.7, 119.1, 114.9, 112.7, 111.3, 60.1, 48.9, 46.9,
35.5, 13.8; ESI MS m/z 623.3 (M + H)+.
Ethyl 4-(4-Chlorophenyl)-1-methyl-3-{3-[4-(4-
nitrophenyl)piperazin-1-yl]phenyl}-5-[(trimethylsilyl)-
ethynyl]-1H-pyrrole-2-carboxylate (42). CuI (37 mg, 0.19
mmol) and Pd(PPh3)4 (111 mg, 0.096 mmol) were added to a
solution of 41 (600 mg, 0.96 mmol), ethynyltrimethylsilane
(473 mg, 4.8 mmol), and Et3N (0.4 mL, 2.9 mmol) in DMF
(20 mL) in a sealed tube at room temperature under nitrogen.
After being stirred at 80 °C for 6 h, the reaction mixture was
cooled and filtered through Celite, washed with 50 mL of
CH2Cl2, and concentrated under reduced pressure. Purification
of the residue by flash chromatography on silica gel afforded 42
(160 mg, 26%). 1H NMR (300 MHz, CCl3D) δ 8.15 (d, J = 9.3
Hz, 2H), 7.23−7.14 (m, 5H), 6.87−6.84 (m, 3H), 6.74 (d, J =
7.6 Hz, 1H), 6.69 (s, 1H), 4.09 (q, J = 7.1 Hz, 2H), 4.06 (s,
3H), 3.54−3.51 (m, 4H), 3.24−3.21 (m, 4H), 0.99 (t, J = 7.1 Hz,
3H), 0.26 (s, 9H); 13C NMR (75 MHz, CCl3D) δ 161.2, 154.7,
150.0, 138.7, 135.9, 132.1, 130.9, 130.4, 128.4, 127.8, 127.5, 125.9,
123.2, 121.7, 119.7, 119.1, 114.9, 112.7, 103.9, 95.3, 60.1, 49.0, 46.9,
34.9, 13.7, −0.26; ESI MS m/z 641.9 (M + H)+.
(R)-N-[4-(4-{3-[4-(4-Chlorophenyl)-1-methyl-1H-pyr-
rol-3-yl]phenyl}piperazin-1-yl)phenyl]-4-{[4-(dimethyla-
mino)-1-(phenylthio)but-2-yl]amino}-3-nitrobenzene-
sulfonamide (18). TFA (0.5 mL) was added to a solution of
20 (37 mg, 0.041 mmol) in DCM (2 mL). The solution was stirred
for 15 min and then evaporated. The residue was purified by
1
HPLC to afford compound 18 (28 mg, 80%). H NMR (300
MHz, CD3OD) δ 8.30 (d, J = 2.2 Hz, 1H), 7.59 (dd, J = 2.2, 9.2
Hz, 1H), 7.25 (m, 1H), 7.18−6.89 (m, 17H), 6.80 (dd, J = 2.3,
9.7 Hz, 2H), 4.10−4.06 (m, 1H), 3.67 (s, 3H), 3.35−3.30 (m,
9H), 3.20−3.14 (m, 3H), 2.82 (s, 6H), 2.23−2.14 (m, 2H); 13C
NMR (75 MHz, CD3OD) δ 148.0, 147.9, 139.3, 136.3, 136.2,
134.4, 133.0, 132.34, 132.25, 131.6, 131.0, 130.8, 130.1, 129.3,
128.0, 127.9, 127.6, 126.0, 124.1, 123.6, 123.4, 123.2, 123.1,
123.0, 119.7, 119.2, 116.8, 116.2, 55.9, 53.0, 52.4, 50.5, 43.5,
39.5, 36.4, 30.1; ESI MS m/z 851.6 (M + H)+.
(R)-Ethyl 4-(4-Chlorophenyl)-3-(3-{4-[4-(4-{[4-(dime-
thylamino)-1-(phenylthio)but-2-yl]-amino}-3-
nitrophenylsulfonamido)phenyl]piperazin-1-yl}phenyl)-
1-methyl-1H-pyrrole-2-carboxylate (19). A mixture of 20
(37 mg, 0.041 mmol), EtOH (1 mL), N,N′-diisopropylcarbo-
diimide (16 mg, 0.124 equiv), and 4-(dimethylamino)pyridine
(1.0 mg, 0.008 mmol) in THF (3 mL) was stirred for 8 h and
then concentrated. The residue was purified by HPLC to provide
19 (29 mg, 76%). 1H NMR (300 MHz, CD3OD), δ 8.31 (d, J =
2.1 Hz, 1H), 7.60 (dd, J = 2.1, 9.1 Hz, 1H), 7.31 (t, J = 7.8 Hz,
1H), 7.17−6.90 (m, 18H), 4.11−4.07 (m, 1H), 3.99 (q, J = 7.1
Hz, 2H), 3.93 (s, 3H), 3.39−3.31 (m, 9H), 3.21−3.14 (m, 3H),
4-(4-Chlorophenyl)-5-ethynyl-1-methyl-3-{3-[4-(4-
nitrophenyl)piperazin-1-yl]phenyl}-1H-pyrrole-2-car-
boxylic acid (43). KOH (56 mg, 1.0 mmol) was added to a
solution of 42 (160 mg, 0.25 mmol) in a mixture of dioxane/
EtOH/H2O (1:1:1, 10 mL) and the solution was refluxed for
2.83 (s, 6H), 2.27−2.11 (m, 2H), 0.93 (t, J = 7.1 Hz, 3H); 13
NMR (75 MHz, CD3OD) δ 162.9, 148.0, 139.1, 136.2, 134.6,
C
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2 h. After being cooled, the reaction was neutralized with 1 M
HCl and extracted with EtOAc. The EtOAc solution was washed
with brine, dried over Na2SO4, and concentrated in vacuum.
Purification of the residue by flash chromatography on silica gel
afforded 43 (116 mg, 86%). 1H NMR (300 MHz, CCl3D) δ 8.15
(d, J = 9.1 Hz, 2H), 7.22−7.10 (m, 5H), 6.86−6.83 (m, 3H),
6.76−6.73 (m, 2H), 4.06 (s, 3H), 3.51−3.49 (m, 5H), 3.22−3.19
(m, 4H); 13C NMR (75 MHz, CCl3D) δ 154.7, 149.9, 138.7,
134.9, 132.5, 132.1, 131.6, 131.0, 128.6, 128.1, 126.0, 123.1,
120.1, 119.4, 115.2, 112.7, 86.1, 74.2, 48.8, 46.8, 35.5; ESI MS
m/z 541.8 (M + H)+.
were determined by nonlinear regression fitting of the com-
petition curves. The Ki value of a compound to a protein was
calculated by use of the equation described previously,26 based
upon the measured IC50 value, the Kd value of the probe to the
protein, and the concentration of the protein and probe in the
competitive assays. Ki values were also calculated from an
equation published in the literature.27 The values obtained from
both equations were found to be in excellent agreement.
Molecular Modeling. Crystal structures of Bcl-xL with 118
(PDB entry 2YXJ) and 4 were used to model the binding poses
of our designed compounds with Bcl-xL. In the binding models,
the structure of Bcl-xL in complex with 4 was superimposed on
that of Bcl-xL in complex with 1. The core scaffold of 4 then
replaced the 4′-chloro-2-methyl-1,1′-biphenyl group of 1 to
generate the initial binding model, which was then refined by a
1 ns molecular dynamics (MD) simulation. All the modifica-
tions of the ligands were performed by the Sybyl program.28
The charge and force field parameters of the compounds
were obtained with the most recent Antechamber module in
the Amber 10 program suite,29 where the charge models were
calculated from the Gaussian 98 program30 at the Hartree−
Fock level by use of the 6-31G** basis sets. Protocols for the
MD simulation are the following. The total charge of the sys-
tem was neutralized by first adding counterions. Then, the
system was solvated in a 10 Å cubic box of water by use of the
TIP3P water model.31 Two thousand steps of minimization
of the system were performed where the protein and the
modeled compound were constrained by a force constant of
50 kcal·mol−1·Å−2. After minimization, a 20 ps simulation was
used to gradually raise the temperature of the system to 298 K
while the whole system was constrained by a force constant of
10 kcal·mol−1·Å−2. Another 40 ps of equilibrium run was used
where only the backbone atoms of the protein and the ligand
atoms were constrained by a force constant of 2 kcal·mol−1·Å−2.
A final production run of 1 ns was performed with no con-
straints on any atoms of the complex structure. When con-
straints were applied, the initial complex structure was used as a
reference structure. All the MD simulations were at NTP. The
SHAKE algorithm32 was used to fix the bonds involving
hydrogen. The PME method33 was used and the nonbonded
cutoff distance was set at 10 Å. The time step was 2 fs, and the
neighboring pairs list was updated in every 20 steps. Final
conformations of Bcl-xL in complex with 6 and 7 are shown in
Figure S1 in Supporting Information.
(R)-4-(4-Chlorophenyl)-3-(3-[4-[4-(4-{[4-(dimethylamino)-
1-(phenylthio)but-2-yl]amino}-3-nitrophenylsulfonamido)-
phenyl]piperazin-1-yl}phenyl)-5-ethyl-1-methyl-1H-pyr-
role-2-carboxylic acid (21). Pd−C (10%; 15 mg) was added to
a solution of compound 43 (82 mg, 0.15 mmol) in a mixture of
CH2Cl2 (3 mL) and MeOH (3 mL). The mixture was stirred
under 1 atm of H2 at room temperature for 0.5 h before being
filtered through Celite and concentrated. The resulting aniline
was used in the next step without purification. To this aniline in
pyridine (5 mL) was added 4-fluoro-3-nitrobenzene-1-sulfonyl
chloride (36 mg, 0.15 mmol) at 0 °C. The mixture was stirred
at 0 °C for 30 min. The pyridine was removed under vacuum
and the residue was purified by flash chromatography on silica
gel to give 4-(4-chlorophenyl)-5-ethyl-3-(3-{4-[4-(4-fluoro-3-
nitrophenylsulfonamido)phenyl]-piperazin-1-yl}phenyl)-1-
methyl-1H-pyrrole-2-carboxylic acid. DIEA (53 μL, 0.30 mmol)
was added to a solution of this sulfonamide and (R)-N1,N1-
dimethyl-4-(phenylthio)butane-1,3-diamine (34 mg, 0.15
mmol) in DMF. The reaction mixture was stirred overnight
and concentrated. The residue was resolved in a mixture of
H2O and CH3CN (a trace of HCl was added to assist with
solubility), and purification by HPLC with H2O and CH3CN
(without TFA) as eluents afforded 21 (41 mg, 29% in four
1
steps). H NMR (300 MHz, CD3OD) δ 8.34 (d, J = 1.9 Hz,
1H), 7.60 (dd, J = 1.9, 9.1 Hz, 1H), 7.20−6.93 (m, 16H), 6.83−
6.81 (m, 2H), 4.13−4.11 (m, 1H), 3.92 (s, 3H), 3.41−3.35 (m,
2H), 3.24−3.21 (m, 10H), 2.86 (s, 6H), 2.66 (q, J = 7.5 Hz,
2H), 2.35−2.13 (m, 2H), 1.16 (t, J = 7.5 Hz, 3H); 13C NMR
(75 MHz, CD3OD) δ 164.8, 147.9, 140.3, 138.3, 136.2, 135.6,
134.5, 133.3, 133.1, 132.3, 131.6, 130.1, 129.2, 129.0, 128.0,
127.9, 127.7, 124.5, 123.0, 122.1, 120.3, 118.5, 116.8, 116.2,
56.0, 52.4, 52.1, 50.3, 43.5, 39.6, 33.6, 30.1, 18.9, 14.5; ESI MS
m/z 922.5 (M + H)+.
Fluorescence Polarization-Based Binding Assays. De-
tails of the expression and purification of Bcl-2, Bcl-xL, and
Mcl-1 proteins and determination of Kd values of fluorescent
probes to proteins are provided in the Supporting Information.
IC50 and Ki values to Bcl-2/Bcl-xL/Mcl-1 of our synthesized
compounds and reference compounds were determined in
competitive binding experiments, in which an inhibitor in serial
dilutions was allowed to compete with a fixed concentration of
a fluorescent probe for a fixed concentration of a protein. Mix-
tures of 5 μL of the tested compound in DMSO and 120 μL of
preincubated protein/probe complex in the assay buffer were
added to assay plates and incubated at room temperature for
2 h with gentle shaking. The final concentrations of the protein
and probe, respectively, were 1.5 nM and 1 nM for the Bcl-2
assay, 10 nM and 2 nM for the Bcl-xL assay, and 20 nM and
2 nM for the Mcl-1 assay. Controls containing protein/probe
complex only (equivalent to 0% inhibition) or free probe only
(equivalent to 100% inhibition), were included in each assay
plate. FP values were measured as described above. IC50 values
Cell Growth Assay. The effect of compounds on cell
growth was evaluated by a WST-8 [2-(2-methoxy-4-nitrophenyl)-
3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monoso-
dium salt] assay (Dojindo Molecular Technologies, Gaithersburg,
Maryland). Human small-cell lung cancer cell lines H146 and
H1417 were purchased from the American Type Culture Collec-
tion (ATCC) and were maintained in RPMI-1640 medium
containing 10% fetal bovine serum (FBS). Cells were seeded in
96-well flat bottom cell culture plates at a density of 1 × 104
cells/well with various concentrations of compounds and in-
cubated for 4 days. At the end of incubation, WST-8 dye (20 μL)
was added to each well and incubated for an additional 1−2 h,
and then the absorbance was measured in a microplate reader
(Molecular Devices) at 450 nm. The concentration of com-
pounds that inhibited cell growth by 50% (IC50) was calculated
by comparing absorbance in the untreated cells and the cells
treated with the compounds by use of the GraphPad Prism soft-
ware (GraphPad Software, La Jolla, CA). At least three
4680
dx.doi.org/10.1021/jm300178u | J. Med. Chem. 2012, 55, 4664−4682

Journal of Medicinal Chemistry
Article
independent experiments were performed to obtain the standard
deviation for each compound in each cell line.
ASSOCIATED CONTENT
■
S
* Supporting Information
Cell Death Assay. Cell death assays were performed via
trypan blue staining. Cells were treated with the indicated
compounds. At the end of treatment, cells were collected and
stained with trypan blue. Cells that stained blue or mor-
phologically unhealthy cells were scored as dead cells. At least
100 cells were counted for each sample.
Additional text, one figure, and one table with experimental
procedures and spectral data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Accession Codes
†Coordinates for Bcl-xL complexed with 4 were deposited into
the Protein Data Bank under Accession Number 3SPF.
Western Blotting. Cells were lysed in radioimmunopreci-
pitation assay lysis buffer [phosphate-buffered saline containing
1% NP40, 0.5% sodium deoxycholate, and 0.1% sodium dodecyl
sulfate (SDS)] supplemented with 1 μmol/L phenylmethanesul-
fonyl fluoride and 1 protease inhibitor cocktail tablet per 10 mL on
ice for 20 min, and lysates were then cleared by centrifugation
before protein concentration determination by use of the Bio-
Rad protein assay kit according to the manufacturer’s instruc-
tions. Proteins were electrophoresed onto SDS-containing
4−20% polyacrylamide gels (Invitrogen) and transferred onto
poly(vinylidene difluoride) membranes. Following blocking in
5% milk, membranes were incubated with a specific primary
antibody, washed, and incubated with horseradish peroxidase-
linked secondary antibody (Amersham). The signals were visu-
alized with the chemiluminescent horseradish peroxidase anti-
body detection reagent (Denville Scientific). Rabbit antibodies
against PARP and caspase-3 were from Cell Signaling Tech-
nology, and rabbit anti-glyceraldehyde 3-phosphate dehydro-
genase (GAPDH) was from Santa Cruz Biotechnology.
Bcl-xL Crystallographic Studies. Expression and purifi-
cation of Bcl-xL ΔTM ΔLP is described in the Supporting
Information. Prior to crystallization, Bcl-xL was incubated with
a 5-fold molar excess of compound 4 in the presence of 4%
DMSO for 1 h at 4 °C and then concentrated to 7 mg/mL.
Crystals of Bcl-xL/4 were grown by vapor diffusion in a sitting
drop tray with 1.2 M sodium citrate and 25 mM
2-(cyclohexylamino)ethanesulfonic acid (CHES) buffer, pH
9.0, as the mother liquor. Crystals did not appear until after the
well was opened, perturbed with a loop, and resealed. In the
experiment, the crystallization drops contained equal volumes
of protein and well solution. Prior to data collection, crystals
were cryoprotected in well solution with increasing amounts of
glycerol, to a final concentration of 20%, and then flash-frozen
in liquid nitrogen.
X-ray data was collected at LS-CAT ID-21-F and G lines at
the Advanced Photon Source at Argonne National Lab. Data
were processed with HKL2000.34 The Bcl-xL/4 complex crys-
tallized in the P42212 space group and diffracted to 1.7 Å re-
solution. The structure contained one molecule in the asym-
metric unit. The structure of the complex was solved by
molecular replacement with Phaser35 by use of a structure of
Bcl-xL previously solved in our laboratory as a starting model.
Iterative rounds of refinement and model building were com-
pleted by use of Buster36 and Coot,37 respectively. The initial
Fo − Fc electron density map revealed the presence of the
compounds in the binding site of Bcl-xL. Only the three-ring
core of 4 was visible in the Fo − Fc electron density map con-
toured at 3σ. The PRODRG server38 was used to create the
starting coordinates and restraint files for the compounds. The
current Rfree/Rwork for the Bcl-xL/4 structure is 0.2036/0.1854. All
amino acids fall into the allowed regions of the Ramanchandran
plot with 98% in the preferred regions. Data collection and refine-
ment statistics are given in Table S1 in Supporting Information.
AUTHOR INFORMATION
■
Corresponding Author
*Telephone 734-615-0362; fax 734-647-9647; e-mail shaomeng@
umich.edu.
Author Contributions
§These authors contributed equally.
Notes
The authors declare the following competing financial interest(s):
Ascentage has licensed this class of Bcl-2/Bcl-xL inhibitors from the
University of Michigan. Dr. Shaomeng Wang is a co-founder for
Ascentage and owns stocks in Ascentage. Dr. Shaomeng Wang also
serves as a consultant for Ascentage.
ACKNOWLEDGMENTS
■
This research was supported in part by a grant from the National
Cancer Institute, National Institutes of Health (U19CA113317).
Use of the Advanced Photon Source was supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract DE-AC02-06CH11357. Use of the
LS-CAT Sector 21 was supported by the Michigan Economic
Development Corporation and the Michigan Technology Tri-
Corridor for the support of this research program (Grant
085P1000817).
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