Structure-based discovery of BM-957 as a potent small-molecule inhibitor of Bcl-2 and Bcl-xL capable of achieving complete tumor regression

Article abstract of DOI:10.1021/jm3010306

Bcl-2 and Bcl-xL antiapoptotic proteins are attractive cancer therapeutic targets. We have previously reported the design of 4,5-diphenyl-1H-pyrrole-3- carboxylic acids as a class of potent Bcl-2/Bcl-xL inhibitors. In the present study, we report our structure-based optimization for this class of compounds based upon the crystal structure of Bcl-xL complexed with a potent lead compound. Our efforts accumulated into the design of compound 30 (BM-957), which binds to Bcl-2 and Bcl-xL with K_i < 1 nM and has low nanomolar IC₅₀ values in cell growth inhibition in cancer cell lines. Significantly, compound 30 achieves rapid, complete, and durable tumor regression in the H146 small-cell lung cancer xenograft model at a well-tolerated dose schedule.

Full text of DOI:10.1021/jm3010306

Article
pubs.acs.org/jmc
Structure-Based Discovery of BM-957 as a Potent Small-Molecule
Inhibitor of Bcl‑2 and Bcl-xL Capable of Achieving Complete Tumor
Regression
Jianfang Chen,†,§ Haibin Zhou,†,§ Angelo Aguilar,†,§ Liu Liu,†,§ Longchuan Bai,†,§ Donna McEachern,†
Chao-Yie Yang,† Jennifer L. Meagher,‡ 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
ABSTRACT: Bcl-2 and Bcl-xL antiapoptotic proteins are attractive
cancer therapeutic targets. We have previously reported the design of
4,5-diphenyl-1H-pyrrole-3-carboxylic acids as a class of potent Bcl-2/Bcl-
xL inhibitors. In the present study, we report our structure-based
optimization for this class of compounds based upon the crystal
structure of Bcl-xL complexed with a potent lead compound. Our efforts
accumulated into the design of compound 30 (BM-957), which binds to
Bcl-2 and Bcl-xL with Ki < 1 nM and has low nanomolar IC50 values in cell growth inhibition in cancer cell lines. Significantly,
compound 30 achieves rapid, complete, and durable tumor regression in the H146 small-cell lung cancer xenograft model at a
well-tolerated dose schedule.
surface binding groove in Bcl-2 and Bcl-xL will inhibit their
antiapoptotic function and promote apoptosis in tumor cells with
high expression of these proteins. In the past 10 years, a number
of classes of nonpeptide, small-molecule inhibitors have been
designed to target the BH3 binding grooves in Bcl-2 and Bcl-xL
proteins.15−36 ABT-737 (1) and its analogue ABT-263 (2)
(Figure 1), from Abbott Laboratories, represent two of the most
potent small-molecule inhibitors of Bcl-2 and Bcl-xL proteins
reported to date. Both compounds bind to Bcl-2 and Bcl-xL with
very high affinities (Ki < 1 nM) and efficiently induce tumor
regression in multiple xenograft tumor models.24−32 In phase I/
II clinical trials, ABT-263 shows evidence of promising antitumor
activity in patients in chronic lymphocytic leukemia37 but has a
very limited single-agent activity in patients with small cell lung
cancer.38
Our laboratory has recently reported the design of compound
3 as a potent Bcl-2 and Bcl-xL inhibitor.39 Compound 3 binds to
Bcl-2 and Bcl-xL with Ki values of <1 nM and potently inhibits
cancer cell growth with IC50 values of approximately 10 nM in
multiple cancer cell lines. Compound 3 is also capable of
achieving a strong in vivo antitumor activity in the H146
xenograft model in mice at a well-tolerated dose schedule.
Although compound 3 shows strong in vivo antitumor activity
and in fact induces tumor regression during the treatment in the
H146 xenograft tumor model, it fails to achieve durable tumor
regression. After the treatment was stopped, tumors quickly
regrew, suggesting that further optimization is needed toward
our goal of obtaining a highly optimized Bcl-2/Bcl-xL inhibitor
for clinical development. In the present study, we have
performed further structure-based optimization for this class of
INTRODUCTION
■
Apoptosis is a tightly regulated cellular process to eliminate
damaged and unwanted cells and plays a critical role in normal
development and homeostasis of multicellular organisms.
Evasion of apoptosis is a hallmark of human cancer,1−3 and
targeting key apoptotic regulators with the goal of restoring
apoptosis in tumor cells is a promising cancer therapeutic
strategy.2,4
The B-cell lymphoma 2 (Bcl-2) family proteins are key
regulators of apoptosis and consist of both proapoptotic and
antiapoptotic members.5−7 Proapoptotic Bcl-2 proteins are
structurally subdivided into two groups: those like Bax, Bak,
and Bok, which contain three Bcl-2 homology (BH) domains
(BH1-BH3), and Bad, Bid, Bim, Bik, Puma, Noxa, and others
that contain only the BH3 domain.8 Although the precise
mechanism of the Bcl-2 and Bcl-xL prosurvival function is not
completely understood, it is clear that these proteins inhibit
apoptosis by directly binding to and sequestering their prodeath
counterparts such as Bim, Bid, and Bad.9 Bcl-2 and Bcl-xL are
frequently overexpressed in cancer cell lines and human cancer
tissues. Such overexpression helps cancer cells suppress a variety
of apoptotic stimuli including those associated with cancer
chemotherapeutic agents and confers on tumor cells resistance to
current therapeutic agents.10,11 Thus, inhibition of these
antiapoptotic Bcl-2 family members offers an attractive approach
for the development of new cancer therapeutics.
Experimentally determined three-dimensional structures of
Bcl-2 and its closely related homologous protein Bcl-xL showed
that the BH1, BH2, and BH3 domains in these proteins form a
well-defined hydrophobic surface binding groove (the BH3
binding groove), which interacts with BH3-only proapoptotic
proteins such as Bad, Bid, and Bim.12−14 It has been proposed
that small-molecule inhibitors designed to bind to the BH3
Received: July 13, 2012
© XXXX American Chemical Society
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Figure 1. Chemical structures of previously reported representative potent Bcl-2/Bcl-xL inhibitors.
Figure 2. (A−D). Co-crystal structure of compound 4 (BM-903) complexed with Bcl-xL. Compound 4 is colored in yellow, and the surface
representation of Bcl-xL is shown. Residues interact with the nitro-phenyl, and the thio-phenyl groups on the compounds are labeled. Hydrogen bonds
are depicted in dash cyan lines. The modifications are highlighted in (B−D) with a red cycle.
compounds based upon a cocrystal structure (Figure 2) of Bcl-xL
complexed with compound 4 (BM-903), an analogue of
compound 3. Our efforts accumulated into the design of a
new, highly potent Bcl-2/Bcl-xL inhibitor, which is capable of
achieving complete and durable tumor regression in the H146
xenograft tumor model.
designed and synthesized compound 5 (BM-916, Table 1) as a
less potent but more soluble compound and used it as the
template for further modifications of the nitro group. Compound
5 has a Ki value of 31.3 nM to Bcl-2 and 37.7 nM to Bcl-xL,
respectively (Table 1).
To determine the contribution of the nitro group, we first
synthesized compound 6 in which the nitro group is replaced
with a hydrogen atom. Compound 6 is 18 and 30 times less
potent than 5 in its binding affinities to Bcl-2 and Bcl-xL,
respectively, confirming the importance of the nitro group.
Because the nitro group inserts into a small hydrophobic
pocket in Bcl-xL in the crystal structure of 4 complexed with Bcl-
xL (Figure 2), we designed and synthesized a series of analogues
of 5 by replacing the nitro group with a small hydrophobic group.
These include 7−9, in which the nitro group is replaced with a
halogen atom (F, Cl, and Br), 10−13, in which the nitro group is
RESULTS AND DISCUSSION
■
Analysis of the cocrystal structure of compound 4 complexed
with Bcl-xL showed that the nitro group in 4 binds to a
hydrophobic pocket, which can accommodate a larger group
than the nitro group (Figure 2B). We have therefore modified
the nitro group to determine structure−activity relationship at
this site. Because 4 binds to Bcl-2 and Bcl-xL with very high
affinities (Ki value <1 nM to both Bcl-2 and Bcl-xL), exceeding
the lower limits of our binding assays for these two proteins, we
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replaced with a small alkyl group, 14−17 with a small alkyloxyl
group, and 18−21 with an alkylsulfonyl group. The binding data
(Table 1) show that, with the exception of compounds 9 and 18,
in which the nitro group is replaced by Br and trifluorome-
thylsulfonyl, respectively, these analogues bind to Bcl-2 and Bcl-
xL 5−20 times less potently than 5. While 9 is still slightly less
potent than 5, 18 is as potent as 5 in binding to both Bcl-2 and
Bcl-xL.
Having identified trifluoromethylsulfonyl as a suitable
replacement group for the nitro group for effective interaction
with both Bcl-2 and Bcl-xL, we next synthesized 22, in which the
nitro group in 4 is replaced with trifluoromethylsulfonyl.
Compound 22 binds to Bcl-2 and Bcl-xL with high affinities
(Ki = 2.1 nM for Bcl-2 and <1 nM for Bcl-xL). Similarly to 3 and
4, compound 22 has no appreciable binding to Mcl-1 at
concentrations as high as 5 μM, indicating that it is a potent and
specific Bcl-2/Bcl-xL inhibitor.
has IC50 values of 21 and 22 nM, respectively, in these two cancer
cell lines, compound 31 has IC50 values of 9 and 13 nM,
respectively. However, compounds 32 and 33 are >10 times less
potent than 30 and 31.
Further in Vitro Evaluations of Potent Bcl-2 and Bcl-xL
Inhibitors. We next tested compounds 28, 30, and 31 for their
ability to induce cell death in the H146 cancer cell line, in direct
comparison to compounds 1 and 2. The results are shown in
Figure 3.
We next evaluated 22 for its activity in inhibition of cell growth
in the H1417 and H146 small-cell lung cancer cell lines, which
are sensitive to potent and specific Bcl-2/Bcl-xL inhibitors such
as compounds 1−4.24,27,39 Compound 22 potently inhibits cell
growth in the H1417 and H146 cancer cell lines, with IC50 values
of 151 and 98 nM, respectively. The binding and cellular data
thus indicated that 22 is a promising lead compound for further
modifications.
Figure 3. Induction of cell death by compounds 1, 2, 28, 30, and 31 in
the H146 cell line. Cells were treated with different concentrations of the
compounds for 24 h. Cell viability was determined using a trypan blue
exclusion assay.
The cocrystal structure (Figure 2C) also showed that the
dimethylamino group of compound 4 forms hydrogen bonds
with side chains of E96 and Y195 residues of Bcl-xL. We next
modified the dimethylamino group in 22 using different sized
nitrogen containing rings with or without a hydroxyl group.
Compound 23 with morpholinyl shows weaker binding affinity
than 22 to Bcl-2, but all other compounds have similar potencies
to both Bcl-2 and Bcl-xL as compared to 22. Consistent with its
weaker binding affinity to Bcl-2, compound 23 is 3−5 times less
potent than 22 in inhibition of cell growth in both H146 and
H1417 cancer cell lines. All other compounds show similar
potencies, with IC50 values of 100−200 nM in inhibition of cell
growth in the H1417 and H146 cell lines.
In our subsequent in vivo evaluation, we found that compound
28 is effective in inhibition of tumor growth in the H146 tumor
xenograft model at a well-tolerated dose-schedule (see below).
Therefore, we focused subsequent modifications on 28.
The cocrystal structure (Figure 2D) shows that the N-methyl
group in the core structure of compound 4 inserts into the well-
defined hydrophobic pocket formed by L112, V126, and F146 in
Bcl-xL, and there is more space available in this pocket to
accommodate a larger hydrophobic group than methyl. We
therefore probed this hydrophobic binding pocket by replacing
the methyl in compound 28 with ethyl, propyl, isopropyl, and
butyl groups (Table 3). Compound 30 with ethyl and compound
31 with isopropyl bind to both Bcl-2 and Bcl-xL with very high
affinities. While 30 and 31 bind to Bcl-2 with IC50 values of 5.4
and 4.0 nM, respectively (Ki values = 1.2 and 0.8 nM,
respectively), they bind to Bcl-xL with IC50 values of 6.0 and
3.9 nM, respectively (Ki values <1 nM). However, compound 32
with propyl and compound 33 with butyl have lower affinities to
Bcl-2 than 30 and 31. Interestingly, these four compounds have
similar high binding affinities to Bcl-xL. Similar to 22,
compounds 28−33 have no appreciable binding to Mcl-1 at
concentrations as high as 5 μM.
All these compounds induce cell death in a dose-dependent
manner but have different potencies. While 28 is somewhat less
potent than 1 and 2, 30 and 31 are several times more potent
than 1 and 2. For example, 30 and 31 at 10 nM with 24 h
treatment induces >50% of the H146 cells to undergo cell death,
whereas 1 and 2 at 30−100 nM have a similar effect.
We further tested compounds 2, 28, and 30 in the H146 cell
line for their ability to induce cleavage of poly(ADP-ribose)
polymerase (PARP) and caspase-3, two key biochemical markers
of apoptosis. The results are shown in Figure 4. Compound 30 at
10 nM, 28 at 100 nM, and 2 at 30 nM all induce clear cleavage of
PARP and activation of caspase-3 and have similar effects. Hence,
the potencies for these three compounds in induction of cleavage
Figure 4. Western blot analysis of biochemical markers of apoptosis in
H146 cancer cells treated with compounds 2, 28, and 30. H146 cancer
cells were treated with indicated concentrations of each compound for
24 h. PARP, cleaved PARP (Cl PARP), caspase-3, and cleaved caspase-3
(Cl Cas-3) were probed with specific antibodies. GAPDH was used as
the loading control.
Testing of compounds 30−33 in the H1417 and H146 cell
lines showed that these compounds are very potent in inhibition
of cell growth and are 5−10 times more potent than 28. While 30
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Figure 5. Induction of cleavage of PARP and caspase-3 in H146 xenograft tumors by compounds 28 and 30. SCID mice bearing H146 xenograft tumors
(100−200 mm3) were treated with vehicle control, single dose of 28 (50 mg/kg, iv) or 30 (25 mg/kg, iv). Mice were sacrificed at indicated time-point
and tumors were removed for Western blot analysis of cleavage of PARP and caspase-3. Cl PARP, cleaved PARP; Cl Caspase-3, cleaved Caspase-3.
Figure 6. Antitumor activity of compounds 28 and 30 in H146 xenograft tumors model in two separate experiments. SCID mice bearing xenograft
tumors (∼100 mm3) were treated with vehicle control, compound 28 at 50 mg/kg (iv) or compound 30 at 25 mg/kg, (iv), daily, 5 days a week for two
weeks.
of PARP and activation of caspase-3 in the H146 cells are
consistent with their potencies in induction of cell death.
In Vivo Evaluation of Compounds 28, 30, and 31. We
determined the maximum tolerated dose (MTD) for compounds
28, 30, and 31 in severe combined immunodeficiency (SCID)
mice. It was found that 28 at 50 mg/kg, 30 at 25 mg/kg, and 31 at
10 mg/kg, daily intravenous (iv) dosing 5 days a week for 2 weeks
were well tolerated in SCID mice and the animals had less than
10% weight loss. Higher doses of these compounds (75 mg/kg
for 28, 50 mg/kg for 30, and 25 mg/kg for 31) caused more than
10% of weight loss. These experiments established the MTDs for
these three compounds in SCID mice for our subsequent
pharmacodynamics (PD) and efficacy experiments. On the basis
of the toxicity data, we decided to further evaluate compounds 28
and 30 for their in vivo antitumor activity.
We tested compounds 28 and 30 for their ability to induce
cleavage of PARP and activation of caspase-3 in the H146
xenograft tumor tissue in mice at their respective MTD in a
pharmacodynamics experiment. Mice bearing H146 tumors were
given a single iv dose of 28 at 50 mg/kg or 30 at 25 mg/kg. The
mice were then sacrificed at 3, 6, and, 24 h time points, and
tumors were harvested for analysis. Western blotting analysis
showed that while both 28 and 30 effectively induce robust
cleavage of PARP in H146 tumor tissues at 3 and 6 h time points,
30 is much more effective than 28 in induction of cleavage of
caspase-3 (Figure 5). These data suggested that while both
compounds induce apoptosis in tumor tissues, 30 is clearly more
effective than 28.
regression. Of seven mice treated with 30, all mice were still
tumor-free at day 47 (15 days post-treatment) and five (71%)
remained tumor-free on day 58 (28 days post-treatment). Similar
to the data obtained from our MTD experiment, both
compounds 28 and 30 are well tolerated in tumor-bearing
animals. All treated animals experienced less than 10% weight
loss compared to the vehicle control, and all regained their
weight quickly after the treatments were finished. This in vivo
experiment thus established that 30 achieves complete and
durable tumor regression in the H146 xenograft tumor model
and is more efficacious than 28.
Synthesis of Designed Bcl-2 Family Protein Inhibitors.
The general synthetic route to compounds in Tables 1 and 2 is
shown in Scheme 1. Compound 34, which was prepared
according to a previously reported method,39 was coupled with 3-
(4-methyl-piperazin-1-yl)-propylamine to give phenylnitro 35.
Reduction of the phenylnitro under hydrogen atmosphere in the
presence of Pd/C yielded the corresponding aniline, which was
subjected to different m-substituted benzenesulfonyl chloride in
pyridine to generate compounds in Table 1.
Using a converged synthetic strategy, compounds in Table 2
were prepared by coupling amine 37a−f with highly activated
fluoro containing intermediate 38. Amine 37a−f were obtained
by treatment of 36 with different amines, followed by removal of
the Fmoc group with Et2NH and reduction with borane.
Phenylnitro 34 was reduced by hydrogenation and then treated
w i t h c o m m e r c i a l l y a v a i l a b l e 4 - fl u o r o - 3 -
(trifluoromethylsulfonyl)benzenesulfonyl chloride to yield 38.
The general synthetic route for compounds in Table 3 is
shown in Scheme 2. Briefly, condensation of 39 with primary
amines resulted in pyrrole 40a−b. Phenylnitro 41a−b were
prepared by Ullman coupling of 1-(p-nitrophenyl)piperazine
with 40a−b, followed by hydrolysis of these ethyl esters, which
yielded free carboxylic acid 42a−b. Reduction of the nitro group
On the basis of the encouraging in vivo pharmacodynamic
data, we next evaluated 28 and 30 for their antitumor efficacy in
the H146 xenograft tumor model (Figure 6). Our data showed
that although compound 28 at 50 mg/kg effectively inhibits
tumor growth, it fails to induce tumor regression. In contrast,
compound 30 at 25 mg/kg achieved rapid and complete tumor
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Table 1. Structure−Activity Relationships of the Nitro Group Replacements
of 42a−b and 42c−d,39 followed by coupling of 4-fluoro-3-
(trifluoromethylsulfonyl)benzene-1-sulfonyl chloride, yielded
sulfonanilide 43a−d, treatment of which with 37e in the
presence of DIPEA in DMF gave compounds in Table 3.
the H146 cell line. In vivo, compound 30 achieves complete and
durable tumor regression in H146 xenograft tumors and is thus
more efficacious than compound 3 in the same tumor model.
The efficacy data thus suggest that compound 30 has a superior
pharmacokinetic property to compound 3. Taken together,
compound 30 represents a promising Bcl-2/Bcl-xL inhibitor for
extensive evaluations as a new anticancer agent.
SUMMARY
■
We have performed further structure-based optimization for a
new class of Bcl-2/Bcl-xL inhibitors containing 4,5-diphenyl-1H-
pyrrole-3-carboxylic acid as the core structure. Our efforts
accumulated into the design of compound 30 (BM-957), which
binds to Bcl-2 and Bcl-xL with the Ki values <1 nM and shows
potent activity in cell growth inhibition in cancer cell lines, with
IC50 values of ∼20 nM against the H1147 and H146 small-cell
lung cancer cell lines. Compound 30 induces robust cleavage of
PARP and caspase-3 in 24 h at concentrations as low 10 nM in
EXPERIMENTAL SECTION
■
Chemistry: General Methods. Unless otherwise noted, all
reactions were performed under nitrogen atmosphere in dry solvents
under anhydrous conditions and reagents were used as supplied without
further purification. 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
E
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Table 2. Structure−Activity Relationships of the Tail Soluble Group
Scheme 1. Synthesis of Target Compounds in Tables 1 and 2
Reagents and conditions: (a) 3-(4-methyl-piperazin-1-yl)-propylamine, EDCI, DMAP, CH2Cl2, room temp; (b) (i) Pd/C, H2, MeOH, room temp,
(ii) sulfonyl chlorides, Py, 0 °C; (c) (i) amines, EDCI, HOBT, CH2Cl2, room temp, (ii) Et2NH, CH3CN, room temp; (iii) BH3, THF, room temp;
(d) (i) Pd/C, H2, MeOH, room temp, (ii) 4-fluoro-3-(trifluoromethylsulfonyl)benzene-1-sulfonyl chloride, Py, 0 °C; (e) DIPEA, DMF, room temp.
C18 reverse phase semipreparative HPLC column with solvent A (0.1%
of TFA in H2O) and solvent B (0.1% of TFA in CH3CN) as eluents. All
the target compounds have purities of >95% based upon UPLC analysis.
Compounds 1 (ABT-737)24 and 2 (ABT-263) were purchased from
SellechBio.com, and their purity was confirmed to be >95% based upon
UPLC analysis.
5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-nitrophenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-
carboxamide (35). A solution of 34 (1.06 g, 2.0 mmol), 3-(4-
methylpiperazin-1-yl)propan-1-amine (472 mg, 3.0 mmol), EDCI (768
mg, 4.0 mmol), and DMAP (244 mg, 2.0 mmol) in CH2Cl2 (10 mL) was
stirred for 3 h until no 34 was observed by TLC. The mixture was diluted
F
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Table 3. Modifications of the N-Methyl Site in Compound 28
Scheme 2. Synthesis of Compounds 30−33 in Table 3
Reagents and conditions: (a) amines, MeOH, room temp; (b) 1-(4-nitrophenyl)piperazine, CuI, L-proline, K2CO3, 100 °C; (c) excess NaOH, 1:1:1
Dioxane, EtOH, H2O, reflux; (d) (i) Pd/C, H2, MeOH, room temp; (ii) 4-fluoro-3-(trifluoromethylsulfonyl)benzene-1-sulfonyl chloride, Py, 0 °C;
(e) 37e, DIPEA, DMF, room temp.
with EtOAc (200 mL), washed sequentially with 1 M HCl (50 mL),
H2O (50 mL), and brine (20 mL), dried (Na2SO4), filtered, and
concentrated. The concentrate was flash chromatographed on silica gel
with 5% MeOH/CH2Cl2 to provide 1.15 g (86%) of 35. 1H NMR (300
MHz, CDCl3): δ 8.14 (d, J = 10.8 Hz, 2H), 7.27−7.05 (m, 5H), 6.89−
6.68 (m, 5H), 3.53−3.47 (m, 4H), 3.42 (s, 3H), 3.26−3.19 (m, 6H),
2.61 (s, 3H), 2.45−2.09 (m, 8H), 2.24 (s, 3H), 2.11 (t, J = 6.4, 2H),
1.50−1.41 (m, 2H). 13C NMR (75 MHz, CDCl3): δ 165.99, 154.57,
150.26, 138.54, 135.84, 133.68, 133.31, 132.28, 130.58, 129.41, 129.17,
128.42, 125.88, 122.37, 121.04, 118.58, 114.83, 114.33, 112.67, 55.90,
55.07, 53.48, 53.03, 48.39, 46.72, 46.00, 37.63, 31.58, 26.47, 11.40. MS
(ESI): m/z 670.92 (M + H)+.
General Procedure I. 5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-
m e t h y l p i p e r a z i n - 1 - y l ) p r o p y l ) - 4 - ( 3 - ( 4 - ( 4 - ( 3 -
nitrophenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-
3-carboxamide (5). To a solution of 35 (100 mg, 0.15 mmol) in 15 mL
of methanol was added 10% wt Pd/C (10 mg, 0.1 equiv m/m). The
solution was stirred under hydrogen atmosphere for about 20 min at
room temperature until no 35 was observed by TLC. The reaction
mixture was filtered, and the filtrate was concentrated in vacuum. The
residue was used for next step directly without purification. To the
solution of this aniline in pyridine, 3-nitrobenzene-1-sulfonyl chloride
(40 mg, 0.18 mmol) was added at 0 °C. The mixture was stirred at 0 °C
to room temperature for 1 h until no aniline was observed by TLC. H2O
(10 mL) was added, and the mixture was extracted with EtOAc (2 × 30
mL). The EtOAc solution was washed with brine (50 mL), dried over
Na2SO4, and concentrated in vacuo. The concentrate was purified by
HPLC to give the pure product 5 (as the trifluoroacetate salt, 89 mg),
yield 72%, over two steps. The gradient ran from 80% of solvent A and
20% of solvent B to 40% of solvent A and 60% of solvent B in 40 min. 1H
NMR (300 MHz, CD3OD): δ 8.44−8.39 (m, 2H), 8.09−8.06 (m, 1H),
7.75 (t, J = 8.0 Hz, 1H), 7.32−7.22 (m, 3H), 7.14−7.04 (m, 7H), 6.86 (t,
J = 2.2 Hz, 2H), 3.51 (br, 4H), 3.42−3.34 (m, 17H), 2.97 (t, J = 7.3 Hz,
2H), 2.92 (s, 3H), 2.45 (s, 3H), 1.87−1.83 (m, 2H). MS (ESI): m/z
826.00 (M + H)+.
G
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5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-(phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1H-pyrrole-3-carboxamide (6). 6 was prepared from 35 and
7H), 6.87−6.85 (m, 2H), 3.52−3.25 (m, 21H), 2.98 (t, J = 7.3 Hz, 2H),
2.90 (s, 3H), 2.41 (s, 3H), 1.89−1.79 (m, 2H). MS (ESI): m/z 864.92
(M + H)+.
1
benzenesulfonyl chloride according to general procedure I. H NMR
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-methoxyphenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (15). 15 was prepared
from 35 and 3-methoxybenzene-1-sulfonyl chloride according to
general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.35−7.00 (m,
14H), 6.88−6.84 (m, 2H), 3.71 (s, 3H), 3.54−3.24 (m, 21H), 3.01 (t, J =
6.7 Hz, 2H), 2.91 (s, 3H), 2.40 (s, 3H), 1.89−1.82 (m, 2H). MS (ESI):
m/z 811.00 (M + H)+.
(300 MHz, CD3OD): δ 7.76−7.74 (m, 2H), 7.60−7.45 (m, 3H), 7.34−
7.23 (m, 3H), 7.16−7.04 (m, 7H), 6.90−6.86 (m, 2H), 3.52−3.9 (m,
21H), 3.00 (t, J = 7.4 Hz, 2H), 2.93 (s, 3H), 2.47 (s, 3H), 1.89−1.84 (m,
2H). MS (ESI): m/z 780.75 (M + H)+.
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-fluorophenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (7). 7 was prepared from
35 and 3-fluorobenzene-1-sulfonyl chloride according to general
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-ethoxyphenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (16). 16 was prepared
from 35 and 3-ethoxybenzene-1-sulfonyl chloride according to general
1
procedure I. H NMR (300 MHz, CD3OD): δ 7.50−7.36 (m, 3H),
7.26−7.18 (m, 4H), 7.13−6.95 (m, 7H), 6.87−6.81 (m, 2H), 3.52−3.22
(m, 21H), 2.99 (t, J = 7.0 Hz, 2H), 2.88 (s, 3H), 2.37 (s, 3H), 1.82−1.75
(m, 2H). MS (ESI): m/z 799.00 (M + H)+.
1
procedure I. H NMR (300 MHz, CD3OD): δ 7.37−7.02 (m, 14H),
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-chlorophenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (8). 8 was prepared from
35 and 3-chlorobenzene-1-sulfonyl chloride according to general
6.88−6.86 (m, 2H), 4.01−3.94 (m, 2H), 3.54−3.28 (m, 21H), 3.00 (t, J
= 7.1 Hz, 2H), 2.93 (s, 3H), 2.44 (s, 3H), 1.92−1.82 (m, 2H), 1.34 (t, J =
7.0 Hz, 3H). MS (ESI): m/z 825.00 (M + H)+.
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-isopropoxyphenylsulfonamido)-
phenyl)piperazin-1-yl) phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (17). 17 was prepared
from 35 and 3-isopropoxylbenzene-1-sulfonyl chloride according to
general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.42−6.95 (m,
14H), 6.81−6.78 (m, 2H), 4.59−4.51 (m, 1H), 3.47 (s, 3H), 3.38−3.10
(m, 18H), 2.85 (s, 3H), 2.75 (t, J = 7.3 Hz, 2H), 2.50 (s, 3H), 1.77−1.75
(m, 2H), 1.28 (d, J = 6.0 Hz, 6H). MS (ESI): m/z 839.08 (M + H)+.
5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-(3-(trifluoromethylsulfonyl)phenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (18). 18
was prepared from 35 and 3-(trifluoromethylsulfonyl)benzene-1-
1
procedure I. H NMR (300 MHz, CD3OD): δ 7.67−7.40 (m, 4H),
7.30−7.20 (m, 3H), 7.11−7.00 (m, 7H), 6.87−6.84 (m, 2H), 3.52−3.26
(m, 21H), 2.98 (t, J = 7.4 Hz, 2H), 2.90 (s, 3H), 2.42 (s, 3H), 1.87−1.82
(m, 2H). MS (ESI): m/z 815.42 (M + H)+.
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-bromophenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (9). 9 was prepared from
35 and 3-bromobenzene-1-sulfonyl chloride according to general
1
procedure I. H NMR (300 MHz, CD3OD): δ 7.81−7.64 (m, 3H),
7.38−7.21 (m, 4H), 7.11−7.00 (m, 7H), 6.88−6.82 (m, 2H), 3.53−3.26
(m, 21H), 2.99 (t, J = 6.9 Hz, 2H), 2.91 (s, 3H), 2.42 (s, 3H), 1.87−1.82
(m, 2H). MS (ESI): m/z 859.92 (M + H)+.
1
sulfonyl chloride according to general procedure I. H NMR (300
5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-(3-(trifluoromethyl)phenylsulfonamido)phenyl)-
piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (10). 10 was pre-
pared from 35 and 3-trifluoromethylbenzene-1-sulfonyl chloride
MHz, CD3OD): δ 8.29 (t, J = 6.4 Hz, 2H), 8.22 (s, 1H), 7.96 (t, J = 7.9
Hz, 1H), 7.37−7.15 (m, 5H), 7.04−7.00 (m, 5H), 6.83−6.79 (m, 2H),
3.46 (s, 3H), 3.40−3.22 (m, 18H), 2.88 (s, 3H), 2.82 (t, J = 7.3 Hz, 2H),
2.50 (s, 3H), 1.83−1.79 (m, 2H). MS (ESI): m/z 913.25 (M + H)+.
5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-(3-(methylsulfonyl)phenylsulfonamido)phenyl)-
piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (19). 19 was pre-
pared from 35 and 3-(methylsulfonyl)benzene-1-sulfonyl chloride
1
according to general procedure I. H NMR (300 MHz, CD3OD): δ
7.94−7.86 (m, 3H), 7.68 (t, J = 8.0 Hz, 1H), 7.32−7.01 (m, 10H), 6.88−
6.86 (m, 2H), 3.52−3.28 (m, 21H), 2.98 (t, J = 7.5 Hz, 2H), 2.91 (s,
3H), 2.44 (s, 3H), 1.91−1.81 (m, 2H). MS (ESI): m/z 849.08 (M + H)+.
5 - ( 4 - C h l o r o p h e n y l ) - 1 , 2 - d i m e t h y l - 4 - ( 3 - ( 4 - ( 4 - ( 3 -
methylphenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-N-(3-(4-
methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (11). 11
was prepared from 35 and 3-methylbenzene-1-sulfonyl chloride
1
according to general procedure I. H NMR (300 MHz, CD3OD): δ
8.12−8.02 (m, 3H), 7.74 (t, J = 7.8 Hz, 1H), 7.31−7.23 (m, 3H), 7.12−
7.02 (m, 7H), 6.93−6.91 (m, 2H), 3.56−3.36 (m, 21H), 3.06−3.01 (m,
5H), 2.93 (s, 3H), 2.42 (s, 3H), 1.92−1.83 (m, 2H). MS (ESI): m/z
859.00 (M + H)+.
1
according to general procedure I. H NMR (300 MHz, CD3OD): δ
7.56−7.51 (m, 2H), 7.38−7.21 (m, 5H), 7.13−7.00 (m, 7H), 6.87−6.84
(m, 2H), 3.52−3.28 (m, 21H), 2.98 (t, J = 7.1 Hz, 2H), 2.91 (s, 3H),
2.44 (s, 3H), 2.33 (s, 3H), 1.90−1.81 (m, 2H). MS (ESI): m/z 795.00
(M + H)+.
5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-ethylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-(3-(methylsulfonyl)phenylsulfonamido)phenyl)-
piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (20). 20 was pre-
pared from 35 and 3-(ethylsulfonyl)benzene-1-sulfonyl chloride
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-ethylphenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (12). 12 was prepared
from 35 and 3-ethylbenzene-1-sulfonyl chloride according to general
1
according to general procedure I. H NMR (300 MHz, CD3OD): δ
8.10−8.03 (m, 3H), 7.78 (t, J = 7.9 Hz, 1H), 7.34−7.24 (m, 3H), 7.16−
7.05 (m, 7H), 6.92−6.89 (m, 2H), 3.54−3.29 (m, 21H), 3.14 (dd, J =
14.8, 7.41 Hz, 2H), 3.01 (t, J = 7.2 Hz, 2H), 2.94 (s, 3H), 2.46 (s, 3H),
1.90−1.85 (m, 2H), 1.09 (t, J = 7.4 Hz, 3H). MS (ESI): m/z 873.00 (M
+ H)+.
5-(4-Chlorophenyl)-4-(3-(4-(4-(3-(cyclopropylsulfonyl)-
phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-N-
(3-(4-methylpiperazin-1-yl)propyl)-1H-pyrrole-3-carboxamide (21).
21 was prepared from 35 and 3-(cyclopropyl)benzene-1-sulfonyl
chloride according to general procedure I. 1H NMR (300 MHz,
CD3OD): δ 8.09−8.02 (m, 3H), 7.80 (t, J = 7.8 Hz, 1H), 7.65−6.71 (m,
11H), 6.25 (s, 1H), 5.89 (d, J = 1.7 Hz, 1H), 3.46 (s, 3H), 3.37−3.23 (m,
17H), 2.92 (br, 1H), 2.79 (s, 3H), 2.60 (t, J = 3.9 Hz, 2H), 2.50 (s, 3H),
1.83 (s, 2H), 1.70−1.68 (m, 2H), 1.32 (s, 2H). MS (ESI): m/z 885.17
(M + H)+.
5 - ( 4 - C h l o r o p h e n y l ) - 4 - ( 3 - ( 4 - ( 4 - ( 4 - fl u o r o - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (38). 38 was
prepared from 34 and 4-fluoro-3-(trifluoromethylsulfonyl)benzene-1-
sulfonyl chloride according to general procedure I. 1H NMR (300 MHz,
CDCl3): δ 8.29 (dd, J = 6.0, 2.0 Hz, 1H), 8.08−8.04 (m, 1H), 7.35−6.62
1
procedure I. H NMR (300 MHz, CD3OD): δ 7.58−7.34 (m, 6H),
7.24−7.16 (m, 3H), 7.06−6.94 (m, 5H), 6.80−6.75 (m, 2H), 3.46 (s,
3H), 3.30−3.05 (m, 18H), 2.83 (s, 3H), 2.71−2.64 (m, 4H), 2.50 (s,
3H), 1.77−1.73 (m, 2H), 1.20 (t, J = 7.6, 3H). MS (ESI): m/z 809.00
(M + H)+.
4-(3-(4-(4-(3-tert-Butylphenylsulfonamido)phenyl)piperazin-1-
yl)phenyl)-5-(4-chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpipera-
zin-1-yl)propyl)-1H-pyrrole-3-carboxamide (13). 13 was prepared
from 35 and 3-tert-butylbenzene-1-sulfonyl chloride according to
general procedure I. 1H NMR (300 MHz, CD3OD): δ 7.58−7.50 (m,
3H), 7.36−7.18 (m, 4H), 7.07−7.01 (m, 7H), 6.87−6.84 (m, 2H),
3.53−3.22 (m, 21H), 2.99 (t, J = 7.2 Hz, 2H), 2.88 (s, 3H), 2.38 (s, 3H),
1.85−1.81 (m, 2H), 1.17 (s, 9H). MS (ESI): m/z 837.00 (M + H)+.
5-(4-Chlorophenyl)-1,2-dimethyl-N-(3-(4-methylpiperazin-1-yl)-
propyl)-4-(3-(4-(4-(3-(trifluoromethoxy)phenylsulfonamido)-
phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxamide (14). 14
was prepared from 35 and 3-trifluoromethoxylbenzene-1-sulfonyl
chloride according to general procedure I. 1H NMR (300 MHz,
CD3OD): δ 7.70−7.43 (m, 4H), 7.29−7.20 (m, 3H), 7.10−7.01 (m,
H
dx.doi.org/10.1021/jm3010306 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(m, 13H), 3.40 (s, 3H), 3.13−3.07 (m, 8H), 2.60 (s, 3H). MS (ESI): m/
z 791.64 (M + H)+.
ution of 38 (79 mg, 0.1 mmol) and (R)-N1,N1-dimethyl-4-(phenylthio)-
butane-1,3-diamine (44 mg, 0.2 mmol) in DMF (1 mL) was added
DIPEA (0.5 mL). The solution was stirred for 4 h at room temperature
until no 38 was observed by TLC. The reaction mixture was
concentrated in vacuo, and the residue was purified by HPLC to give
the pure product 22 (trifluoroacetic acid salt, 81 mg, yield 81%). The
gradient ran from 60% of solvent A and 40% of solvent B to 20% of
General Procedure II. (R)-4-(Azetidin-1-yl)-1-(phenylthio)butan-
2-amine (37a). A solution of 36 (1.0 g 2.3 mmol), azetidine (394 mg,
6.9 mmol), EDCI (662 mg, 3.5 mmol), and HOBt (466 mg, 3.5 mmol)
in DCM (12 mL) was stirred for 4 h at room temperature until no 36
was observed by TLC and then diluted with DCM (100 mL), washed
sequentially with 1 M HCl (50 mL), H2O (50 mL), and brine (20 mL),
dried (MgSO4), filtered, and concentrated. The concentrate was flash
chromatographed on silica gel with 50% EtOAc/hexanes to provide (R)-
(9H-fluoren-9-yl)methyl 4-(azetidin-1-yl)-4-oxo-1-(phenylthio)butan-
2-ylcarbamate, 957 mg (88%). The amide was dissolved in MeCN (20
mL) and treated with Et2NH (2.1 mL, 20 mmol). The solution was
stirred for 2 h at room temperature until no starting material was
observed by TLC and concentrated. The concentrate was flash
chromatographed on silica gel with 5% MeOH/CH2Cl2 to provide
(R)-3-amino-1-(azetidin-1-yl)-4-(phenylthio)butan-1-one, 497 mg
(98%). A mixture of this free amine and 1 M BH3 in THF (5 mL)
was stirred for 16 h at room temperature, treated with MeOH (1.5 mL),
and concentrated HCl (0.5 mL), stirred at 80 °C for 3 h, cooled to room
temperature, adjusted to pH 10 with 4 M Na2CO3, diluted with CH2Cl2
(150 mL), washed with H2O (50 mL) and brine (10 mL), dried
(MgSO4), filtered, and concentrated. The concentrate was flash
chromatographed on silica gel with 15% MeOH/CH2Cl2 to provide
37a, 445 mg (95%). 1H NMR (300 MHz, D2O), δ 7.53 (d, J = 7.2 Hz,
2H), 7.49−7.38 (m, 3H), 4.32−4.18 (m, 2H), 4.03−3.82 (m, 2H),
3.54−3.15 (m, 5H), 2.61−2.51 (m, 1H), 2.44−2.39 (m, 1H), 2.07−1.90
(m, 2H). 13C NMR (75 MHz, D2O): δ 132.99, 131.13, 129.78, 128.10,
54.91, 54.77, 50.64, 48.53, 35.92, 26.45, 15.82. MS (ESI): m/z 237.66
(M + H)+.
(R)-1-(Phenylthio)-4-(pyrrolidin-1-yl)butan-2-amine (37b). 37b
was prepared from 36 and pyrrolidine according to general procedure
II. 1H NMR (300 MHz, D2O): δ 7.55 (d, J = 7.2 Hz, 2H), 7.46−7.37 (m,
3H), 3.54−3.48 (m, 3H), 3.39−3.05 (m, 4H), 3.02−2.89 (m, 1H),
2.87−2.68 (m, 1H), 2.25−1.94 (m, 6H). 13C NMR (75 MHz, D2O): δ
133.02, 131.13, 129.78, 128.09, 54.38, 54.08, 50.74, 48.77, 35.85, 27.70,
22.58. MS (ESI): m/z 251.26 (M + H)+.
(R)-1-(Phenylthio)-4-(piperidin-1-yl)butan-2-amine (37c). 37c was
prepared from 36 and piperidine according to general procedure II. 1H
NMR (300 MHz, D2O): δ 7.59 (d, J = 7.3 Hz, 2H), 7.50−7.39 (m, 3H),
3.50−3.00 (m, 7H), 2.90−2.72 (m, 2H), 2.30−2.07 (m, 2H), 2.01−1.58
(m, 5H), 1.54−1.35 (m, 1H). 13C NMR (75 MHz, D2O): δ 133.02,
131.20, 129.78, 128.11, 53.52, 53.19, 52.68, 48.88, 35.88, 25.89, 22.74,
20.93. MS (ESI): m/z 265.38 (M + H)+.
1
solvent A and 80% of solvent B in 40 min. H NMR (300 MHz,
CD3OD): δ 7.84 (d, J = 2.0 Hz, 1H), 7.70 (dd, J = 9.1, 2.2 Hz, 1H),
7.26−6.96 (m, 16H), 6.86−6.78 (m, 2H), 3.98−3.91 (m, 1H), 3.42−
3.31 (m, 11H), 3.20−3.02 (m, 4H), 2.80 (s, 6H), 2.57 (s, 3H), 2.25−
1.98 (m, 2H). MS (ESI): m/z 996.30 (M + H)+.
(R)-5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(4-(4-morpholino-
1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)-
phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-
carboxylic Acid (23). 23 was prepared from 38 and (R)-4-morpholino-
1
1-(phenylthio)butan-2-amine according to general procedure III. H
NMR (300 MHz, CD3OD): δ 7.93 (d, J = 2.1 Hz, 1H), 7.74 (dd, J = 9.2,
2.22 Hz, 1H), 7.44−6.89 (m, 17H), 6.83 (d, J = 9.4 Hz, 1H), 4.03−3.98
(m, 3H), 3.78−3.71 (m, 2H), 3.56−3.36 (m, 11H), 3.25−3.09 (m, 8H),
2.64 (s, 3H), 2.32−2.09 (m, 2H). MS (ESI): m/z 1037.80 (M + H)+.
(R)-4-(3-(4-(4-(4-(4-(Azetidin-1-yl)-1-(phenylthio)butan-2-ylami-
no)-3-(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)-
piperazin-1-yl)phenyl)-5-(4-chlorophenyl)-1,2-dimethyl-1H-pyrrole-
3-carboxylic Acid (24). 24 was prepared from 38 and 37a according to
general procedure III. 1H NMR (300 MHz, CD3OD): δ 7.90 (d, J = 2.0
Hz, 1H), 7.60 (dd, J = 9.1, 2.0 Hz, 1H), 7.31−7.26 (m, 4H), 7.24−6.96
(m, 13H), 6.88−6.82 (m, 1H), 4.25−4.15 (m, 2H), 4.03−3.97 (m, 3H),
3.43 (s, 3H), 3.40−3.11 (m, 12H), 2.67 (s, 3H), 2.54−2.35 (m, 2H),
2.07−1.85 (m, 2H). MS (ESI): m/z 1007.08 (M + H)+.
(R)-5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(4-(1-(phenyl-
t h i o ) - 4 - ( p y r r o l i d i n - 1 - y l ) b u t a n - 2 - y l a m i n o ) - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1H-pyrrole-3-carboxylic Acid (25). 25 was prepared from 38
1
and 37b according to general procedure III. H NMR (300 MHz,
CD3OD): δ 7.95 (d, J = 1.9 Hz, 1H), 7.74 (dd, J = 9.1, 2.1 Hz, 1H),
7.30−7.01 (m, 14H), 6.84 (d, J = 9.2 Hz, 1H), 6.58−6.42 (m, 3H), 3.99
(br, 1H), 3.80−3.53 (m, 2H), 3.46 (s, 3H), 3.44−2.95 (m, 14H), 2.61 (s,
3H), 2.29−1.68 (m, 6H). MS (ESI): m/z 1021.75 (M + H)+.
(R)-5-(4-Chlorophenyl)-1,2-dimethyl-4-(3-(4-(4-(4-(1-(phenyl-
t h i o ) - 4 - ( p i p e r i d i n - 1 - y l ) b u t a n - 2 - y l a m i n o ) - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1H-pyrrole-3-carboxylic Acid (26). 26 was prepared from 38
1
and 37c according to general procedure III. H NMR (300 MHz,
CD3OD): δ 8.36 (d, J = 2.2 Hz, 1H), 7.61 (dd, J = 9.2, 2.2 Hz, 1H),
7.20−6.92 (m, 15H), 6.53−6.38 (m, 3H), 4.12 (br, 1H), 3.42 (s, 3H),
3.36−3.17 (m, 11H), 2.88 (s, 5H), 2.61 (s, 3H), 2.29−2.19 (m, 8H). MS
(ESI): m/z 1035.92 (M + H)+.
(R)-1-(3-Amino-4-(phenylthio)butyl)azetidin-3-ol (37d). 37d was
prepared from 36 and azetidin-3-ol according to general procedure II.
1H NMR (300 MHz, D2O): δ 7.55 (d, J = 7.1 Hz, 2H), 7.50−7.39 (m,
3H), 4.72−4.63 (m, 1H), 4.51−3.69 (m, 4H), 3.51−3.43 (m, 1H),
3.38−3.20 (m, 4H), 2.08−190 (m, 2H). 13C NMR (75 MHz, D2O): δ
132.92, 131.13, 129.78, 128.15, 63.27, 62.52, 58.73, 50.71, 48.44, 35.93,
26.66. MS (ESI): m/z 253.60 (M + H)+.
(R)-1-(3-Amino-4-(phenylthio)butyl)piperidin-4-ol (37e). 37e was
prepared from 36 and piperidin-4-ol according to general procedure II.
1H NMR (300 MHz, D2O): δ 7.51 (d, J = 3.5 Hz, 2H), 7.41−7.33 (m,
3H), 4.09−3.77 (m, 1H), 3.46−2.69 (m, 9H), 2.12−2.08 (m, 3H), 1.87
(br, 2H), 1.69−1.57 (m, 1H). 13C NMR (75 MHz, D2O): δ 132.99,
131.18, 129.76, 128.04, 64.59, 52.58, 51.38, 48.87, 47.89, 35.88, 30.88,
28.85, 26.16. MS (ESI): m/z 281.46 (M + H)+.
(R)-1-(3-Amino-4-(phenylthio)butyl)-4-methylpiperidin-4-ol (37f).
37f was prepared from 36 and 4-methylpiperidin-4-ol according to
general procedure II. 1H NMR (300 MHz, D2O): δ 7.44 (d, J = 6.8 Hz,
2H), 7.40−7.34 (m, 3H), 3.55−3.47 (m, 1H), 3.39−2.81 (m, 8H),
2.67−2.04 (m, 2H), 1.81−1.76 (m, 4H), 1.27 (s, 3H). 13C NMR (75
MHz, D2O): δ 133.09, 131.26, 129.81, 128.15, 65.02, 52.64, 49.10,
48.96, 48.56, 35.84, 34.62, 28.45, 25.88. MS (ESI): m/z 295.36 (M +
H)+.
(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(3-hydroxyazetidin-1-yl)-
1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)-
phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-1H-
pyrrole-3-carboxylic Acid (27). 27 was prepared from 38 and 37d
1
according to general procedure III. H NMR (300 MHz, CD3OD): δ
7.89 (d, J = 1.9 Hz, 1H), 7.75 (dd, J = 9.0, 2.0 Hz, 1H), 7.36−7.09 (m,
13H), 7.01−6.66 (m, 5H), 4.03 (br, 1H), 3.81−3.50 (m, 2H), 3.44 (s,
3H), 3.40−2.89 (m, 15H), 2.62 (s, 3H), 2.28−1.73 (m, 2H). MS (ESI):
m/z 1024.00 (M + H)+.
(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-
1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)-
phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1,2-dimethyl-1H-
pyrrole-3-carboxylic Acid (28). 28 was prepared from 38 and 37e
according to general procedure III. 1H NMR (300 MHz, CD3OD): δ
7.86 (s, 1H), 7.71 (d, J = 9.2 Hz, 1H), 7.27−7.25 (m, 4H), 7.20−6.79
(m, 14H), 4.03−3.75 (m, 2H), 3.49−3.31 (m, 13H), 3.14−2.89 (m,
6H), 2.58 (s, 3H), 2.26−1.88 (m, 5H), 1.67−1.63 (m, 1H). 13C NMR
(75 MHz, CD3OD): δ 167.60, 151.31, 146.91, 144.09, 137.99, 137.13,
136.10, 134.72, 133.49, 133.40, 132.64, 131.05, 130.84, 130.39, 128.98,
128.37, 128.14, 127.35, 126.82, 123.13, 122.17, 121.99, 117.62, 116.34,
113.93, 110.08, 108.62, 64.34, 59.71, 53.60, 52.62, 51.06, 50.69, 48.51,
37.97, 31.40, 30.84, 29.54, 28.24, 27.94, 10.71. MS (ESI): m/z 1051.30
(M + H)+.
General Procedure III. (R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-
(dimethylamino)-1-(phenylthio)butan-2-ylamino)-3-
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (22). To a sol-
I
dx.doi.org/10.1021/jm3010306 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxy-4-methylpi-
p e r i d i n - 1 - y l ) - 1 - ( p h e n y l t h i o ) b u t a n - 2 - y l a m i n o ) - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1,2-dimethyl-1H-pyrrole-3-carboxylic Acid (29). 29 was
prepared from 38 and 37f according to general procedure III. 1H
NMR (300 MHz, CD3OD): δ 7.79 (d, J = 1.6 Hz, 1H), 7.66 (dd, J = 9.1,
1.7 Hz, 1H), 7.20−6.75 (m, 18H), 4.03−3.88 (m, 1H), 3.45−3.33 (m,
10H), 3.25−2.95 (m, 9H), 2.51 (s, 3H), 2.22−1.93 (m, 2H), 1.80−1.66
(m, 4H), 0.97 (s, 3H). MS (ESI) m/z 1066.16 (M + H)+.
and the solution was refluxed for 20 h until no 41a was observed by TLC.
After cooling, the reaction was neutralized with 1 M HCl and the
compound was extracted with EtOAc. The EtOAc solution was washed
with brine, dried (Na2SO4), and concentrated in vacuo to produce 900
mg of compound 42a as a white solid (95% yield, used for next steps
directly without purification). 1H NMR (300 MHz, CDCl3): δ 8.13 (d, J
= 9.3 Hz, 2H), 7.26 (d, J = 8.2 Hz, 2H), 7.09−7.03 (m, 3H), 6.84−6.64
(m, 5H), 3.74 (t, J = 7.5 Hz, 2H), 3.48 (t, J = 4.5 Hz, 4H), 3.15 (t, J = 5.2
Hz, 4H), 2.62 (s, 3H), 1.57−1.50 (m, 2H), 0.76 (t, J = 7.3 Hz, 3H). 13C
NMR (75 MHz, CDCl3): δ 169.73, 154.75, 149.50, 138.56, 137.41,
135.73, 133.77, 132.67, 130.70, 130.50, 128.51, 128.14, 125.93, 124.55,
123.26, 119.87, 114.30, 112.67, 109.84, 48.92, 46.79, 45.94, 23.98, 12.09,
11.04. MS (ESI): m/z 559.17 (M + H)+.
1-Butyl-5-(4-chlorophenyl)-2-methyl-4-(3-(4-(4-nitrophenyl)-
piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylic Acid (42b). 42b was
prepared from 41b according to general procedure VI. 1H NMR (300
MHz, CDCl3): δ 8.11 (d, J = 9.4 Hz, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.08
(d, J = 8.4 Hz, 2H), 7.03 (t, J = 7.7 Hz, 1H), 6.82−6.63 (m, 5H), 3.78 (t, J
= 8.0 Hz, 2H), 3.44 (t, J = 4.8 Hz, 4H), 3.13 (t, J = 5.4 Hz, 4H), 2.61 (s,
3H), 1.54−1.44 (m, 2H), 1.20−1.10 (m, 2H), 0.79 (t, J = 7.2 Hz, 3H).
13C NMR (75 MHz, CDCl3): δ 170.66, 154.77, 149.44, 138.47, 137.36,
General Procedure IV. Ethyl 5-(4-Chlorophenyl)-4-(3-iodophen-
yl)-2-methyl-1-propyl-1H-pyrrole-3-carboxylate (40a). To the sol-
ution of 39 (2.0 g, 4.1 mmol) in MeOH (20 mL) was added
propylamine (1.0 mL, 12.3 mmol), and the solution was stirred for 6 h at
room temperature until no starting material was observed by TLC. The
solution was acidified with 1 M HCl (50 mL) and extracted with EtOAc
(2 × 50 mL). The combined EtOAc solutions were washed with brine
(50 mL), dried (Na2SO4), and concentrated in vacuo. The concentrate
was flash chromatographed on silica gel with 10% EtOAc/hexane to
provide 1.89 g of 40a (91%). 1H NMR (300 MHz, CDCl3): δ 7.52 (t, J =
1.6 Hz, 1H), 7.46−7.42 (m, 1H), 7.28−7.24 (m, 2H), 7.08−6.94 (m,
3H), 6.84 (t, J = 7.8 Hz, 1H), 4.08 (dd, J = 14.3, 7.1 Hz, 2H), 3.73 (t, J =
7.7 Hz, 2H), 2.61 (s, 3H), 1.58−1.46 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H),
0.75 (t, J = 7.4 Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.60, 139.89,
138.20, 136.00, 134.64, 133.88, 132.60, 130.30, 130.17, 129.77, 128.79,
128.56, 122.57, 111.21, 92.91, 59.30, 45.83, 24.01, 14.03, 11.69, 11.07.
Ethyl 1-Butyl-5-(4-chlorophenyl)-4-(3-iodophenyl)-2-methyl-1H-
pyrrole-3-carboxylate (40b). 40b was prepared from 39 and butyl-
amine according to general procedure IV. 1H NMR (300 MHz, CDCl3):
δ 7.52 (s, 1H), 7.43 (d, J = 7.9 Hz, 1H), 7.26 (d, J = 8.4 Hz, 2H), 7.06 (d,
J = 8.4 Hz, 2H), 6.95 (d, J = 8.1 Hz, 1H), 6.84 (t, J = 7.7 Hz, 1H), 4.07
(dd, J = 14.4, 7.3 Hz, 2H), 3.76 (t, J = 7.6 Hz, 2H), 2.62 (s, 3H), 1.53−
1.43 (m, 2H), 1.22−1.09 (m, 2H), 1.04 (t, J = 7.1 Hz, 3H), 0.78 (t, J = 7.3
Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.60, 139.90, 138.21, 135.96,
134.63, 133.88, 132.62, 130.29, 130.14, 129.78, 128.79, 128.55, 122.56,
111.21, 92.92, 59.30, 44.05, 32.77, 19.79, 14.04, 13.55, 11.70.
135.79, 133.74, 132.74, 130.75, 130.52, 128.49, 128.03, 125.92, 124.68,
123.29, 119.99, 114.22, 112.65, 109.95, 48.94, 46.75, 44.15, 32.73, 19.78,
13.51, 12.13. MS (ESI): m/z 573.80 (M + H)+.
5 - ( 4 - C h l o r o p h e n y l ) - 4 - ( 3 - ( 4 - ( 4 - ( 4 - fl u o r o - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-2-methyl-1-propyl-1H-pyrrole-3-carboxylic Acid (43a). 43a
was prepared from 42a according to general procedure I. 1H NMR (300
MHz, CDCl3): δ 8.31 (dd, J = 5.9, 1.8 Hz, 1H), 8.09−8.04 (m, 1H), 7.37
(t, J = 8.9 Hz, 1H), 7.25−6.64 (m, 12H), 3.75 (t, J = 7.4 Hz, 2H), 3.18−
3.11 (m, 8H), 2.64 (s, 3H), 1.58−1.51 (m, 2H), 0.77 (t, J = 7.3 Hz, 3H).
MS (ESI): m/z 819.00 (M + H)+.
1-Butyl-5-(4-chlorophenyl)-4-(3-(4-(4-(4-fluoro-3-
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (43b). 43b was
General Procedure V. Ethyl 5-(4-Chlorophenyl)-2-methyl-4-(3-
(4-(4-nitrophenyl)piperazin-1-yl)phenyl)-1-propyl-1H-pyrrole-3-car-
boxylate (41a). Ester 40a (1.52 g, 3.0 mmol), 1-(4-nitrophenyl)-
piperazine (1.87 g, 9.0 mmol), CuI (57 mg, 0.3 mmol), L-proline (173
mg, 1.5 mmol), and K2CO3 (1.24 g, 9.0 mmol) were dissolved in 30 mL
of DMSO. This solution was stirred for 8 h under a nitrogen atmosphere
at 80 °C until no 40a was observed by TLC. The reaction mixture was
cooled, saturated NH4Cl solution (50 mL) was added, and the solution
extracted with EtOAc (2 × 50 mL). The combined EtOAc solutions
were washed with brine (50 mL), dried (Na2SO4), and concentrated in
vacuo. The concentrate was flash chromatographed on silica gel with
1
prepared from 42b according to general procedure I. H NMR (300
MHz, CDCl3): δ 8.31 (dd, J = 5.9, 2.2 Hz, 1H), 8.09−8.04 (m, 1H), 7.37
(t, J = 8.8 Hz, 1H), 7.26−6.64 (m, 12H), 3.78 (t, J = 8.0 Hz, 2H), 3.21−
3.08 (m, 8H), 2.64 (s, 3H), 1.55−1.45 (m, 2H), 1.23−1.10 (m, 2H),
0.79 (t, J = 7.2 Hz, 3H). MS (ESI): m/z 833.52 (M + H)+.
5-(4-Chlorophenyl)-1-ethyl-4-(3-(4-(4-(4-fluoro-3-
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (43c). 43c was
prepared from 42c39 according to general procedure I. 1H NMR (300
MHz, CDCl3): δ 8.32 (dd, J = 6.0, 2.0 Hz, 1H), 8.08−8.03 (m, 1H), 7.35
(t, J = 8.9 Hz, 1H), 7.26−6.64 (m, 12H), 3.83 (dd, J = 14.0, 6.9 Hz, 2H),
3.17−3.08 (m, 8H), 2.64 (s, 3H), 1.63 (t, J = 7.1 Hz, 3H). MS (ESI): m/
z 805.66 (M + H)+.
1
40% EtOAc/hexane to provide 1.44 g of 41a (82%). H NMR (300
MHz, CDCl3): δ 8.14−8.09 (m, 2H), 7.26−7.06 (m, 5H), 6.86−6.62
(m, 5H), 4.09 (dd, J = 14.2, 7.08 Hz, 2H), 3.75 (t, J = 7.7 Hz, 2H), 3.48
(t, J = 4.9 Hz, 4H), 3.15 (t, J = 5.3 Hz, 4H), 2.62 (s, 3H), 1.61−1.48 (m,
2H), 1.05 (t, J = 7.1 Hz, 3H), 0.76 (t, J = 7.4 Hz, 3H). 13C NMR (75
MHz, CDCl3): δ 165.88, 154.75, 149.55, 138.55, 136.55, 135.45, 133.55,
132.68, 131.00, 129.94, 128.43, 127.88, 125.93, 124.32, 123.39, 119.32,
114.08, 112.69, 111.39, 59.26, 49.01, 46.90, 45.86, 24.06, 14.06, 11.7,
11.08. MS (ESI): m/z 587.50 (M + H)+.
Ethyl 1-Butyl-5-(4-chlorophenyl)-2-methyl-4-(3-(4-(4-
nitrophenyl)piperazin-1-yl)phenyl)-1H-pyrrole-3-carboxylate (41b).
41b was prepared from 40b according to general procedure V. 1H NMR
(300 MHz, CDCl3): δ 8.11 (d, J = 9.3 Hz, 2H), 7.26−7.06 (m, 5H),
6.84−6.63 (m, 5H), 4.09 (dd, J = 14.3, 7.1 Hz, 2H), 3.79 (t, J = 7.6 Hz,
2H), 3.48 (t, J = 4.7 Hz, 4H), 3.14 (t, J = 5.3 Hz, 4H), 2.62 (s, 3H), 1.55−
1.45 (m, 2H), 1.22−1.10 (m, 2H), 1.05 (t, J = 7.1 Hz, 3H), 0.79 (t, J = 7.3
Hz, 3H). 13C NMR (75 MHz, CDCl3): δ 165.88, 154.75, 149.54, 138.52,
136.55, 135.41, 133.54, 132.71, 130.98, 129.91, 128.42, 127.88, 125.93,
124.31, 123.38, 119.33, 114.07, 112.69, 111.38, 59.26, 49.00, 46.89,
44.06, 32.82, 19.79, 14.07, 13.55, 11.74. MS (ESI): m/z 601.82 (M +
H)+.
5 - ( 4 - C h l o r o p h e n y l ) - 4 - ( 3 - ( 4 - ( 4 - ( 4 - fl u o r o - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-1-isopropyl-2-methyl-1H-pyrrole-3-carboxylic Acid (43d).
43d was prepared from 42d39 according to general procedure I. H
1
NMR (300 MHz, CDCl3): δ 8.30 (dd, J = 5.9, 1.6 Hz, 1H), 8.10−8.07
(m, 1H), 7.35 (t, J = 8.9 Hz, 1H), 7.26−6.68 (m, 12H), 4.42−4.33 (m,
1H), 3.18−3.13 (m, 8H), 2.73 (s, 3H), 1.41 (d, J = 10.0 Hz, 6H). MS
(ESI): m/z 819.52 (M + H)+.
(R)-5-(4-Chlorophenyl)-1-ethyl-4-(3-(4-(4-(4-(4-(4-hydroxypiperi-
d i n - 1 - y l ) - 1 - ( p h e n y l t h i o ) b u t a n - 2 - y l a m i n o ) - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)
phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (30). 30 was prepared
1
from 43c and 37e according to general procedure III. H NMR (300
MHz, CD3OD): δ 7.86 (s, 1H), 7.71 (d, J = 9.1 Hz, 1H), 7.29−6.96 (m,
16H), 6.87−6.79 (m, 2H), 4.04−3.77 (m, 4H), 3.49−3.28 (m, 8H),
3.17−2.94 (m, 8H), 2.60 (s, 3H), 2.05−1.69 (m, 6H), 1.10 (t, J = 6.9 Hz,
3H). 13C NMR (75 MHz, CD3OD): δ 169.03, 152.67, 148.36, 145.43,
139.23, 137.46, 137.39, 136.08, 135.09, 134.77, 134.28, 132.37, 132.02,
131.76, 131.61, 130.35, 129.66, 129.54, 128.73, 128.19, 124.95, 124.51,
123.26, 118.97, 117.66, 115.29, 111.72, 109.98, 61.10, 53.98, 52.42,
52.05, 51.22, 50.93, 40.10, 39.34, 32.77, 30.90, 28.19, 27.32, 25.25, 25.06,
24.62, 24.57, 16.14, 13.88, 11.87. MS (ESI) m/z 1066.26 (M + H)+.
General Procedure VI. 5-(4-Chlorophenyl)-2-methyl-4-(3-(4-(4-
nitrophenyl)piperazin-1-yl)phenyl)-1-propyl-1H-pyrrole-3-carbox-
ylic Acid (42a). To a solution of 41a (1.00 g, 1.7 mmol) in 30 mL of
1:1:1 dioxane, EtOH, and H2O was added NaOH (680 mg, 17.0 mmol),
J
dx.doi.org/10.1021/jm3010306 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-
1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)-
phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-1-isopropyl-2-
methyl-1H-pyrrole-3-carboxylic Acid (31). 31 was prepared from 43d
ASSOCIATED CONTENT
Accession Codes
Coordinates for Bcl-xL complexed with 4 were deposited into the
Protein Data Bank with accession number 3SPF.
■
1
and 37e according to general procedure III. H NMR (300 MHz,
CD3OD): δ 7.84 (d, J = 1.8 Hz, 1H), 7.70 (dd, J = 9.2, 2.1 Hz, 1H),
7.26−6.95 (m, 17H), 6.80 (d, J = 9.2 Hz, 1H), 4,42−4.33 (m, 1H),
4.02−3.73 (m, 2H), 3.48−3.31 (m, 10H), 3.25−2.88 (m, 6H), 2.67 (s,
3H), 2.20−1.87 (m, 5H), 1.66−1.62 (m, 1H), 1.38 (d, J = 7.1 Hz, 6H).
MS (ESI) m/z 1080.30 (M + H)+.
AUTHOR INFORMATION
Corresponding Author
*Phone: 734-615-0362. Fax: 734-647-9647. E-mail: shaomeng@
umich.edu.
■
(R)-5-(4-Chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperidin-1-yl)-
1-(phenylthio)butan-2-ylamino)-3-(trifluoromethylsulfonyl)-
phenylsulfonamido)phenyl)piperazin-1-yl)phenyl)-2-methyl-1-
propyl-1H-pyrrole-3-carboxylic Acid (32). 32 was prepared from 43a
Author Contributions
§Equal contributions
Notes
1
and 37e according to general procedure III. H NMR (300 MHz,
The authors declare the following competing financial
interest(s): Dr. Wang is a co-founder of Ascentage, which has
licensed the Bcl-2/Bcl-xL inhibitors described in the manuscript.
Dr. Wang serves as an officer in Ascentage and owns stocks in
Ascentage.
CD3OD): δ 7.91 (d, J = 1.8 Hz, 1H), 7.78 (dd, J = 9.2, 2.2 Hz, 1H),
7.46−7.03 (m, 17H), 6.87 (d, J = 9.1 Hz, 1H), 4.02−4.00 (m, 2H), 3.86
(t, J = 7.6 Hz, 2H), 3.51−2.95 (m, 16H), 2.65 (s, 3H), 2.27−2.11 (m,
3H), 1.93 (br, 2H), 1.72−1.63 (m, 1H), 1.61−1.51 (m, 2H), 0.75 (t, J =
5.1 Hz, 3H). MS (ESI): m/z 1080.28 (M + H)+.
(R)-1-Butyl-5-(4-chlorophenyl)-4-(3-(4-(4-(4-(4-(4-hydroxypiperi-
d i n - 1 - y l ) - 1 - ( p h e n y l t h i o ) b u t a n - 2 - y l a m i n o ) - 3 -
(trifluoromethylsulfonyl)phenylsulfonamido)phenyl)piperazin-1-yl)-
phenyl)-2-methyl-1H-pyrrole-3-carboxylic Acid (33). 33 was prepared
from 43b and 37e according to general procedure III. 1H NMR (300 M
Hz, CD3OD): δ 7.91 (s, 1H), 7.73 (d, J = 8.6 Hz, 1H), 7.33−6.81 (m,
18H), 4.08−3.79 (m, 4H), 3.53−2.94 (m, 16), 2.63 (s, 3H), 2.33−2.19
(m, 1H), 2.17−2.04 (m, 2H), 1.98−1.87 (m, 2H), 1.75−1.58 (m, 1H),
1.54−1.44 (m, 2H), 1.23−1.11 (m, 2H), 0.78 (t, J = 7.3 Hz, 3H). MS
(ESI): m/z 1094.36 (M + H)+.
Fluorescence Polarization-Based Binding Assays for Bcl-2,
Bcl-xL, and Mcl-1 Proteins. The binding assays for Bcl-2, Bcl-xL and
Mcl-1 proteins were the same as those described in our previous
publications.39,40
Cell Death Assay. Cell death assays were performed using 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 the morphologically unhealthy cells were scored as
dead cells. At least 100 cells were counted for each sample.
Western Blotting. Cells were lysed using radioimmunoprecipita-
tion assay lysis buffer (PBS containing 1% NP40, 0.5% sodium
deoxycholate, and 0.1% SDS) supplemented with 1 μmol/L phenyl-
methylsulfonyl fluoride and one protease inhibitor cocktail tablet per 10
mL on ice for 20 min, and lysates were then cleared by centrifugation
before protein concentration determination using the Bio-Rad protein
assay kit according to the manufacturer’s instructions. Proteins were
electrophoresed onto 4−20% SDS-PAGE gels (Invitrogen) and
transferred onto polyvinylidenedifluoride membranes. Following block-
ing in 5% milk, membranes were incubated with a specific primary
antibody, washed, and incubated with horseradish peroxidase-linked
secondary antibody (Amersham). The signals were visualized with the
chemiluminescent horseradish peroxidase antibody detection reagent
(Denville Scientific). Rabbit antibodies against PARP and caspase-3 are
from Cell Signaling Technology. Rabbit anti-GAPDH is from Santa
Cruz Biotechnology.
Efficacy Studies. When tumors reached tumor volumes between 40
and 110 mm3, mice were randomized into different groups, eight mice
per group, with a mean tumor volume of 70 mm3. Mice were treated
with compound 28 at 50 mg/kg, or compound 30 at 25 mg/kg,
intravenously, daily, 5 days a week for 2 weeks, or vehicle control. Tumor
sizes and animal weights were measured 3 times a week during the
treatment and twice a week after the treatment. Data are presented as
mean tumor volumes SEM. Statistical analyses were performed by
two-way ANOVA and unpaired two-tailed t test, using Prism (version
4.0, GraphPad, La Jolla, CA). P < 0.05 was considered statistically
significant. The efficacy experiment was performed under the guidelines
of the University of Michigan Committee for Use and Care of Animals.
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 no. 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).
ABBREVIATIONS USED
■
Bcl-2, B-cell lymphoma 2; Bax, Bcl-2 related protein X; Bak, Bcl-2
antagonist/killer; Bok, Bcl-2-related ovarian killer protein; Bad,
Bcl-2 antagonist of cell death; Bid, BH3 interacting death
domain; Bim, Bcl-2 interacting mediator; Bik, bcl-2 interacting
killer; Puma, p53 upregulated modulator of apoptosis; Bcl-xL, B-
cell lymphoma x long; BH, Bcl homology; PARP, poly(ADP-
ribose) polymerase; SCID, severe combined immunodeficiency;
EDCI, N-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hy-
drochloride; HOBT, 1-hydroxybenzotriazole hydrate; DIPEA,
N,N-diisopropylethylamine; FPA, fluorescence polarization
assay
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