Pyrogallol-based molecules as potent inhibitors of the antiapoptotic Bcl-2 proteins

Article abstract of DOI:10.1021/jm061400l

We report herein a new class of small-molecule inhibitors of antiapoptotic Bcl-2 proteins. The most potent compound, 7, binds to Bcl-2, Bcl-xL, and Mcl-1 proteins with K_i of 110, 638, and 150 nM, respectively. Compound 7 is highly effective in

Full text of DOI:10.1021/jm061400l

© Copyright 2007 by the American Chemical Society
Volume 50, Number 8
April 19, 2007
Letters
Bak, and Bax.4-7 The antiapoptotic proteins in the Bcl-2 family
are overexpressed in many cancer cell lines and human cancer
tissues. This overexpression protects cancer cells from the
induction of apoptosis by current anticancer therapies and plays
a role in the failure of conventional anticancer drugs.4-7
Consequently, these antideath Bcl-2 proteins are considered to
be promising molecular targets for the design of novel anticancer
drugs.
Pyrogallol-Based Molecules as Potent Inhibitors
of the Antiapoptotic Bcl-2 Proteins
Guozhi Tang,† Chao-Yie Yang,†
Zaneta Nikolovska-Coleska,† Jie Guo,† Su Qiu,†
Renxiao Wang,† Wei Gao,† Guoping Wang,†
Jeanne Stuckey,‡ Krzysztof Krajewski,# Sheng Jiang,#
Peter P. Roller,# and Shaomeng Wang*,†
Although the precise mechanism by which Bcl-2 proteins
regulate apoptosis in cells is still under intense investigation,8
it is very clear that these antiapoptotic Bcl-2 proteins effectively
inhibit apoptosis, at least in part, by directly binding to
proapoptotic Bcl-2 proteins such as Bim, Bid, Bak, and Bax
and blocking their proapoptotic activity. Experimentally deter-
mined three-dimensional structures of Bcl-2, Bcl-xL, and Mcl-1
by NMR or X-ray crystallography showed that the BH1 (Bcl-2
homology domain 1), BH2, and BH3 domains in these proteins
form a well-defined, hydrophobic surface binding groove,
known as the BH3 binding groove, into which Bad, Bid, and
Bim bind.9-12 Hence, small molecules that are designed to target
the BH3 binding groove in these antiapoptotic Bcl-2 proteins
are predicted to promote apoptosis in cancer cells by antagoniz-
ing their antiapoptotic function. Design of nonpeptidic, cell-
permeable, small-molecule inhibitors that bind to the BH3
binding groove in these antideath Bcl-2 proteins is being
intensely pursued as a new anticancer therapeutic strategy.13-22
Departments of Internal Medicine, Pharmacology, and
Medicinal Chemistry, ComprehensiVe Cancer Center, and
Life Sciences Institute, UniVersity of Michigan, 1500 East Medical
Center DriVe, Ann Arbor, Michigan 48109, and Laboratory of
Medicinal Chemistry, National Cancer InstitutesFrederick,
National Institutes of Health, Frederick, Maryland 21702
ReceiVed December 6, 2006
Abstract: We report herein a new class of small-molecule inhibitors
of antiapoptotic Bcl-2 proteins. The most potent compound, 7, binds
to Bcl-2, Bcl-xL, and Mcl-1 proteins with Ki of 110, 638, and 150 nM,
respectively. Compound 7 is highly effective in induction of cell death
in breast cancer cells with high levels of Bcl-2, Bcl-xL, and Mcl-1
proteins and represents a promising lead compound for the design of
new anticancer drugs.
Apoptosis, or programmed cell death, is a critical cell process
in normal development and homeostasis of multicellular organ-
isms. Inappropriate regulation of apoptosis has been implicated
in many human diseases, including cancer.1-3 Targeting critical
apoptosis regulators is an attractive therapeutic approach for
the development of new classes of therapies for the treatment
of cancer and other human diseases.1
Design of nonpeptidic, small-molecule inhibitors to target
protein-protein interactions (PPIs) is considered one of the most
challenge tasks in modern drug discovery and medicinal
chemistry. Nevertheless, significant progress has been made in
the past few years in the design of small-molecule inhibitors to
target the Bcl-2 PPIs,13-22 and a number of classes of potent
small-molecule inhibitors, shown in Figure 1, have been
reported. Among them, 1 binds to Bcl-2, Bcl-xL, and Bcl-w
proteins with a very high affinity but does not bind to Mcl-1.18
Compound 2, a natural product isolated from cotton seeds,
concurrently targets Bcl-2, Bcl-xL, and Mcl-1 proteins with
similar affinities21 and is currently in clinical trials as an orally
administered agent for the treatment of multiple forms of human
cancer.22 Using a structure-based strategy, we have recently
reported the design of 3 as a new class of potent, cell-permeable
small-molecule inhibitor of Bcl-2, Bcl-xL, and Mcl-1 proteins.21
The Bcl-2a family proteins are a class of central arbiters of
apoptosis and consist of antiapoptotic members such as Bcl-2,
Bcl-xL, and Mcl-1 and proapoptotic members such as Bim, Bid,
* To whom correspondence should be addressed. Phone: 734-615-0362.
Fax: 734-647-9647. E-mail: shaomeng@umich.edu.
† Departments of Internal Medicine, Pharmacology, and Medicinal
Chemistry, Comprehensive Cancer Center, University of Michigan.
‡ Life Sciences Institute, University of Michigan.
# National Cancer InstitutesFrederick
a Abbreviations: Bcl-2, B-cell leukemia/lymphoma 2; Mcl-1, (myeloid
cell leukemia sequence 1; Bid, BH3 interacting domain death agonist; Bim,
Bcl-2 interacting mediator of cell death; Bax, Bcl-2-associated X protein;
Bak, Bcl-2 homologous antagonist/killer.
10.1021/jm061400l CCC: $37.00 © 2007 American Chemical Society
Published on Web 03/23/2007

1724 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
Letters
Figure 1. Representative small-molecule inhibitors of Bcl-2 proteins.
Figure 2. Structure-based design of novel small-molecule inhibitors
of Bcl-2 proteins.
Figure 3. Competitive binding curves of designed small-molecule
inhibitors to Bcl-2 as determined using a fluorescence polarization based
binding assay. Recombinant human Bcl-2 protein and fluorescently
tagged Bid BH3 peptide were employed in this competitive binding
assay.
Although Bcl-2 and Bcl-xL proteins have been the primary
focus for the design of small-molecule inhibitors to target these
proteins,13-22 recent studies have demonstrated that the Mcl-1
protein plays a crucial role in protecting cancer cells from
induction of apoptosis by a variety of anticancer agents.
Compound 1 had potent activity only in cancer cells with low
levels of Mcl-1 protein but a much weaker activity in cancer
cells with high levels of Mcl-1.23 Knocking down Mcl-1 using
siRNA in cancer cells greatly sensitizes the activity of 1. Hence,
small-molecule inhibitors that target not only Bcl-2 and
Bcl-xL but also Mcl-1 could be highly effective in induction
of cell death in cancer cells with high levels of these proteins.
Herein, we report the structure-based design, synthesis, and
initial evaluations of pyrogallol-based compounds as novel
small-molecule inhibitors of Bcl-2, Bcl-xL, and Mcl-1.
Figure 4. Predicted binding models of designed small-molecule
inhibitors 4-7 to Bcl-2. For inhibitors, carbon atoms are shown in
yellow, oxygen atoms in red, nitrogen atoms in blue, and sulfur atoms
in yellow. For protein, carbon atoms are shown in yellow, oxygen atoms
in red, nitrogen atoms in blue. Side chains of crucial residues L94,
I97, D99, and F101 in the Bim BH3 peptide are displayed with carbon
atoms shown in green and oxygen atoms in red.
Bim peptide. The predicted binding model for 4 in complex
with Bcl-2 forms the structural basis for our current design and
optimization.
In our previous study,21 4 was designed as an initial lead
starting from 2 (Figure 2). In our fluorescence polarization (FP)
based binding assay, 4 binds to Bcl-2 with a Ki of 31.9 µM
(Figure 3). Analysis of its binding model to Bcl-2 (Figure 4
and Supporting Information) showed that two of the hydroxyl
groups in its phenyl ring form hydrogen bonds with R146 and
N143 in Bcl-2, mimicking the key residue D99 in the Bim BH3
peptide. Its phenyl ring mimics I97, and its isopropyl group
partially fills the hydrophobic pocket occupied by F101 in the
On the basis of the binding model for 4, we designed 5 (Fig-
ure 2), in which a fused, conformationally constrained bicyclic
system was used to replace the phenyl ring in 4. Our modeling
showed that 5 maintains all the hydrogen bonding observed for
4 binding to Bcl-2 but has enhanced hydrophobic contacts
mediated by the tetrahydroisoquinoline ring (Figure 4). Model-
ing predicted that 5 may bind to Bcl-2 with a higher affinity
than 4 (Supporting Information). Compound 5 was synthesized
(Supporting Information) and was determined to bind to Bcl-2

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8 1725
Scheme 1. Synthesis of Designed Inhibitor 7a
Figure 5. Binding curve of 7 to Mcl-1 protein in competition with a
biotinylated Bim BH3 peptide as determined using an ELISA assay.
a Reagents and conditions: (a) ClSO3H, room temp, 91%; (b) 3-phenyl-
propylamine, NEt3, CH2Cl2, 97%; (c) (i) MeI, K2CO3, acetone, reflux,
overnight; (ii) KOH, MeOH, 60 °C, 3 h, 92%; (d) substituted tetrahydroiso-
quinoline, EDAC, HOBt, DiPEA, CH2Cl2, 85%; (e) BBr3 (15 equiv),
CH2Cl2, -78 to -20 °C, 70%.
with a Ki of 3.1 µM (Figure 3). Compound 5 is thus 10 times
more potent than 4, supporting our modeling prediction.
Comparison of the predicted binding models for 5 and the
Bim BH3 peptide suggested that the hydrophobic pocket
occupied by I94 in the Bim peptide is not utilized by 5 (Figure
4). Taking advantage of this key hydrophobic interaction should
further improve the binding affinity. We have thus designed 6
(Figure 2), in which a phenethyl group is attached to the
7-position of the fused bicyclic system through a sulfonamide
linker. This facilitates the synthesis and enhances the solubility
of the resulting compound. Modeling predicted that the phen-
ethyl group in 6 is inserted into the hydrophobic pocket in Bcl-2
occupied by the I94 residue in the Bim peptide (Figure 4).
Compound 6 was synthesized (Supporting Information) and
determined to bind to Bcl-2 with a Ki of 590 nM (Figure 3). It
is thus 5 times more potent than 5, providing further support
for our design strategy.
Analysis of the predicted binding model for 6 in complex
with Bcl-2 showed that its isopropyl group inserts into the
hydrophobic pocket occupied by F101 (Figure 4). It is clear,
however, that this hydrophobic pocket can accommodate a larger
hydrophobic group. Although one could simply replace the
isopropyl group with a larger hydrophobic group, the resulting
compounds would become too hydrophobic and insoluble. To
address this problem, we replaced the isopropyl group with
N-methyl-N-(3-phenylpropyl)sulfamoyl, as in 7. Modeling
predicted that the phenylpropyl group in 7 should significantly
improve the hydrophobic interactions with Bcl-2 compared to
the isopropyl group in 6. Importantly, the use of a sulfonamide
as the linker leads to a compound with a much improved solu-
bility. Interestingly, our modeling also suggested that the sul-
fonyl group in 7 forms a hydrogen bond with Y108 (Figure 4).
Compound 7 was synthesized (Scheme 1) and was found to
bind to Bcl-2 with a Ki of 110 nM (Figure 3). It is therefore 5
times more potent than 6 and 290 times more potent than the
initial lead compound 4.
Figure 6. Induction of cell death in the MDA-MB-231 (2LMP) human
breast cancer cell line by designed small-molecule inhibitors. Cells were
treated with each inhibitor for 96 h, and cell viability was determined
using Trypan blue exclusion assay.
To further confirm the binding affinity of 7 to Mcl-1, we
have developed and validated an ELISA assay using recombi-
nant Mcl-1 protein and a biotinylated Bim peptide (Supporting
Information). In this assay, 7 has an IC50 of 39 ( 10 nM to
Mcl-1 from three independent experiments (Figure 5). Hence,
the FP and the ELISA-based assay showed that 7 binds to Mcl-1
protein with high affinity.
Potent, cell-permeable, small-molecule inhibitors that concur-
rently target Bcl-2, Bcl-xL, and Mcl-1 proteins are predicted to
be highly effective in inducing cell death in cancer cells.23 We
evaluated these compounds for their ability to induce cell death
in the MDA-MB-231 (2LMP) human breast cancer cell line,
which has high levels of Bcl-2, Bcl-xL, and Mcl-1 proteins
(Supporting Information). Each inhibitor induces cancer cells
to undergo cell death in a dose-dependent manner (Figure 6).
Compound 7 effectively induces cell death at concentrations
as low as 10 nM and is much more potent than other
compounds. For example, 7 at 100 nM is as effective as 6 at
1000 nM and is much more effective than 4 and 5 at 1000 nM.
In direct comparison, 1, which does not bind to Mcl-1,18 is 100
times less effective than 7 in cell death induction in the MDA-
MB-231 cell line (Figure 6).
In summary, using a structure-based design strategy, we have
successfully designed a new class of potent small-molecule
inhibitors targeting Bcl-2, Bcl-xL, and Mcl-1 proteins. Our most
potent inhibitor, 7, which was named TM-1206, binds to
Bcl-2, Bcl-xL, and Mcl-1 proteins with Ki of 110, 639, and 150
nM, respectively. The binding of 7 to Bcl-2 and Mcl-1 proteins
has further been confirmed using ELISA-based assays. Com-
pound 7 is effective in induction of cell death in MDA-MB-
231 cancer cells with high levels of Bcl-2, Bcl-xL, and Mcl-1
proteins in a dose-dependent manner. Our present study indicates
that concurrently targeting Bcl-2, Bcl-xL, and Mcl-1 proteins
using a small-molecule inhibitor is potentially a promising
therapeutic strategy for the design of novel anticancer drugs.
Since FP-based assays can be influenced by autofluorescence
of the tested inhibitors, we have developed and validated an
enzyme-linked immunosorbent assay (ELISA) for Bcl-2 (Sup-
porting Information). Using this ELISA assay, we have deter-
mined the binding of our designed inhibitors to Bcl-2. The IC50
values for 5, 6, and 7 are 38.2 ( 6.2, 6.6 ( 2.7, and 0.33 (
0.05 µM, respectively, obtained from three independent experi-
ments. Hence, the relative binding affinities for these inhibitors,
obtained from the ELISA assay, are consistent with those
obtained from the FP-based assay.
Acknowledgment. We are grateful for the financial support
from the National Cancer Institute, National Institutes of Health
(Grant U19CA113317), the Department of Defense Breast
Cancer Program (Grant BC0009140), the Department of Defense
Prostate Cancer Program (Grant PC040537), the Prostate Cancer
Foundation, the Breast Cancer Research Foundation, the Susan
We next evaluated 7 for its binding affinities to Bcl-xL and
Mcl-1 proteins using our established FP-based binding assays
(Supporting Information). Compound 7 has Ki of 638 and 150
nM to Bcl-xL and Mcl-1 proteins, respectively.

1726 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 8
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