Structure-based design of flavonoid compounds as a new class of small-molecule inhibitors of the anti-apoptotic Bcl-2 proteins

Article abstract of DOI:10.1021/jm070383c

Structure-based strategy was employed to design flavonoid compounds to mimic the Bim BH3 peptide as a new class of inhibitors of the anti-apoptotic Bcl-2 proteins. The most potent compound, 4 (BI-33), binds to Bcl-2 and Mcl-1 with K_i values of

Full text of DOI:10.1021/jm070383c

J. Med. Chem. 2007, 50, 3163-3166
3163
Structure-Based Design of Flavonoid Compounds
As a New Class of Small-Molecule Inhibitors of
the Anti-apoptotic Bcl-2 Proteins
Guozhi Tang,† Ke Ding,† Zaneta Nikolovska-Coleska,†
Chao-Yie Yang,† Su Qiu,† Sanjeev Shangary,†
Renxiao Wang,† Jie Guo,† Wei Gao,† Jennifer Meagher,‡
Jeanne Stuckey,‡ Krzysztof Krajewski,§ Sheng Jiang,§
Peter P. Roller,§ 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, and Laboratory of
Medicinal Chemistry, National Cancer Institute-Frederick, National
Institutes of Health, Frederick, Maryland 21702
ReceiVed April 1, 2007
Figure 1. Structure-based design of flavonoid compounds as a new
class of small-molecule inhibitors to target the antiapoptotic Bcl-2
proteins.
Abstract: Structure-based strategy was employed to design flavonoid
compounds to mimic the Bim BH3 peptide as a new class of inhibitors
of the anti-apoptotic Bcl-2 proteins. The most potent compound, 4 (BI-
33), binds to Bcl-2 and Mcl-1 with Ki values of 17 and 18 nM,
respectively. Compound 4 inhibits cell growth in the MDA-MB-231
breast cancer cell line with an IC50 value of 110 nM and effectively
induces apoptosis.
The Bcl-2 family proteins are central regulators of apoptosis
and comprise anti-apoptotic proteins such as Bcl-2, Bcl-xL, and
Mcl-1 and pro-apoptotic proteins such as Bak, Bax, Bim, Bid,
and Bad.1-4 Although the precise molecular mechanism by
which these Bcl-2 proteins regulate apoptosis is still under
intensive investigation,5,6 it is very clear that the anti-apoptotic
proteins and the pro-apoptotic proteins modulate their opposing
functions through heterodimerization. Experimental three-
dimensional structures of Bcl-2, Bcl-xL, and Mcl-1 show that
these proteins form a well-defined, hydrophobic surface binding
groove, known as the Bcl-2 homology domain 3 (BH3) binding
groove, into which these pro-apoptotic proteins bind.7-11 It has
been hypothesized that nonpeptide, small-molecule inhibitors
that bind in the BH3 binding groove in Bcl-2, Bcl-xL, and Mcl-1
can block the heterodimerization between the anti-apoptotic and
pro-apopototic Bcl-2 members.12-19 Because cancer cells often
express high levels of one or more of these anti-apoptotic Bcl-2
proteins, such small-molecule inhibitors can induce apoptosis
on their own and/or sensitize cancer cells for apoptosis induction
by antagonism of these anti-apoptotic Bcl-2 proteins.2 Design
of inhibitors of Bcl-2, Bcl-xL, and Mcl-1 is being intensely
pursued as a novel strategy for the development of new
anticancer drugs.12-19 The development of potent, druglike,
nonpeptide small-molecule inhibitors to block these Bcl-2
protein-protein interactions remains one of the most challenging
tasks in modern drug discovery and medicinal chemistry.
In this report, we wish to present our structure-based design
of a potent, cell-permeable, non-peptidic small-molecule that
mimics the key binding residues in the Bim BH3 peptide and
binds to Bcl-2 and Mcl-1 proteins with high affinities.
Figure 2. (a) Predicted binding models of Bcl-2 in complex with (a)
compound 1; (b) mBim BH3 peptide; and (c) designed compounds 2
and (d) 4. Bcl-2 is shown in surface representation where carbon,
oxygen, nitrogen, and sulfur atoms are colored in gray, red, blue, and
orange, respectively. The carbon and oxygen atoms in compounds 1,
2, and 4 are shown in yellow and red, respectively. The mBim BH3
peptide was shown in a light blue helix. Hydrogen bonds are depicted
in dotted lines in cyan. Bim peptide residues are labeled in italic.
Mcl-1 with Ki values of 320, 480, and 180 nM, respectively,
determined by competitive fluorescence polarization-based
(FP-based) binding assays.18 Compound 1, currently in clinical
trials as a single, oral agent for the treatment of human cancers,
has demonstrated antitumor activity and manageable toxicity.21
It, therefore, is a promising lead compound for the design of
potent, nonpeptidic small-molecule inhibitors targeting the anti-
apoptotic Bcl-2 proteins.
Based upon our predicted binding model (Figure 2a), 1 forms
a hydrogen-bonding network with residues Arg146 and Asn143
in Bcl-2 through the aldehyde group and its adjacent hydroxyl
group on one of the naphthalene rings. This mimics the
hydrogen-bonding network formed by Asp99 and Asn102 in
Bim and Arg146 and Asn143 in Bcl-2 (Figure 2b). The
hydrophobic isopropyl group on the same naphthalene ring
inserts into a hydrophobic pocket in Bcl-2, in part mimicking
the Phe101 in the Bim peptide. The other naphthalene ring
interacts with Bcl-2 primarily through hydrophobic contacts,
mimicking Ile97 in the Bim peptide. Thus, this predicted binding
model provides a structural basis for the design of novel small-
molecule inhibitors of Bcl-2.
Through structure-based database screening, we discovered
previously18,20 that 1, a natural product isolated from seeds and
roots of the cotton plant, is a fairly potent inhibitor of Bcl-2,
Bcl-xL, and Mcl-1. Compound 1 binds to Bcl-2, Bcl-xL, and
* To whom correspondence should be addressed. Phone: 734-615-0362.
Fax: 734-647-9647. E-mail: shaomeng@umich.edu.
† Comprehensive Cancer Center and Departments of Internal Medicine,
Pharmacology, and Medicinal Chemistry, University of Michigan.
‡ Life Sciences Institute, University of Michigan.
§ National Cancer Institutes of Health.
10.1021/jm070383c CCC: $37.00 © 2007 American Chemical Society
Published on Web 06/07/2007

3164 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
Scheme 1. Synthesis of Designed Compounds 2, 4, 5, and 6a
Letters
Figure 4. Competitive binding curves of small-molecule inhibitors to
Mcl-1 as determined in our fluorescence-polarization-based Mcl-1
binding assay using a fluorescently tagged Bim peptide as the tracer
(Supporting Information).
with Bcl-2. This led to the design of template 3 (Figure 1), which
contains two identical isoflavonoid moieties of 2 tethered
through a linker.
a Reagents and conditions: (a) isobutyric chloride, BF3‚Et2O, Cl(CH2)2Cl,
reflux, 85%; (b) Et3SiH, TFA, 95%; (c) AcCl, BF3·Et2O, Cl(CH2)2Cl, reflux,
87%; (d) Ac2O, pyridine; (e) NaH, DMF; (f) HCl, 82% for 3 steps; (g) I2,
CF3CO2Ag, CH2Cl2, 0 °C, 94%; (h) PhB(OH)2, Pd2(dpf)2Cl2‚CH2Cl2,
Na2CO3, EtOH, DMF, H2O, 60 °C, 92%; (i) BBr3, CH2Cl2, -78∼0 °C; (j)
1,4-phenylenebisboronic acid (0.5 equiv), Pd2(dpf)2Cl2‚CH2Cl2, Na2CO3,
EtOH, DMF, H2O, 90 °C, 96%.
Modeling showed that a phenyl group has the appropriate
geometry to tether these two flavonoid moieties together through
its 1,4 positions to mimic Ile90, Leu94, Ile97, and Phe101 in
the Bim peptide (Figure 2d), and this led to the design of
compound 4 (Figure 1). In our model, compound 4 also forms
an extensive hydrogen-bonding network with Arg146 and
Asn143 in Bcl-2 through one of the flavonoid moieties, thus
mimicking the interactions of Asp99 in Bim with Bcl-2 (Figure
2). In addition, an additional hydrogen bond is formed between
one hydroxyl group on the other flavonoid moiety and Val133
in Bcl-2. The model also showed that compound 4 mimics these
key residues in the Bim BH3 and predicted that compound 4
should achieve a higher affinity to Bcl-2 than the initial lead
compound 2. Indeed, compound 4 was determined to bind to
Bcl-2 with a Ki value of 17 nM (Figure 3) in our FP-based
binding assay, which is 43 times more potent than 2. Of note,
4 is also 10 times more potent than 1.
Figure 3. Competitive binding curves of small-molecule inhibitors to
Bcl-2 as determined in our fluorescence-polarization-based Bcl-2
binding assay using a fluorescently tagged Bim peptide as the tracer
(Supporting Information).
Our modeling suggested that one-half of compound 1 forms
an extensive hydrogen-bonding network and also has hydro-
phobic interactions with Bcl-2. We searched for structures that
would mimic the interactions mediated by the half of compound
1 with Bcl-2. Among a number of templates we have investi-
gated, compound 2 was predicted by modeling to mimic one-
half of compound 1 closely in its interaction with Bcl-2 (Figure
2c).
Compound 2 was synthesized (Scheme 1) and was found to
bind to Bcl-2 with a Ki value of 730 nM (Figure 3) in our FP-
based binding assay (Supporting Information). Although it is
four times less potent than 1, it has a significant affinity for
Bcl-2. Compound 2 contains a flavonoid core structure found
in many natural products, has well balanced hydrophobic and
hydrophilic properties and is thus a promising new template
for further optimization.
Our modeling predicted that the phenyl group in compound
4 functions as a linker and the hydroxyl groups in 4 play a
critical role through formation of an extensive hydrogen-bonding
network with Bcl-2. To test these predictions, we designed and
synthesized compounds 5 and 6 (Figure 1 and Scheme 1) and
evaluated their binding to Bcl-2. Compound 5 binds to Bcl-2
with a Ki value of 340 nM and is only twice as potent as
compound 2, indicating that the additional phenyl ring in
compound 5 primarily functions as a tethering group. As
predicted, compound 6 with all these six hydroxyl groups
methylated shows no appreciable binding to Bcl-2 at concentra-
tions as high as 50 µM.
We found with an FP-based competitive binding assay that
compound 4 binds to Bcl-xL with a Ki value of 1.2 µM. Hence,
compound 4 binds to Bcl-2 with an affinity 70 times more potent
than to Bcl-xL.
Based upon our modeling (Figure 2b), the Bim BH3 peptide
interacts primarily with Bcl-2 through 4 hydrophobic residues
(Ile90, Leu94, Ile97, and Phe101) and one negatively charged
Asp99 residue. Modeling also shows that compound 2 binds to
Bcl-2 by mimicking the Phe101, Ile97, and Asp99 in the Bim
peptide, but lacks the interactions of Ile90 and Leu94 in the
Bim peptide with Bcl-2. This suggests that capture of these
additional hydrophobic interactions should significantly improve
the binding affinity of compound 2. Our modeling indicates that
the isobutyl and the methyl groups in compound 2 can mimic
Ile90 and Leu94 residues in the Bim peptide, two hydrophobic
residues separated by a single helical turn in the Bim BH3
peptide. We have thus investigated if the basic core structure
of compound 2 can be used as a general helical mimic to capture
the interactions by Ile90 and Leu94 residues in the Bim peptide
Although Bcl-2 and Bcl-xL have been the primary focus for
the design of small-molecule inhibitors that target the anti-
apoptotic Bcl-2 proteins, recent studies have demonstrated that
Mcl-1 plays a critical role in apoptosis resistance of cancer cells
and Mcl-1 has become a highly attractive target for anticancer
drug design.22 We have therefore evaluated the binding affinities
of our designed compounds to Mcl-1. As can be seen in Figure
4, compound 4 binds to Mcl-1 with a Ki value of 18 nM. In
comparison, compounds 1, 2, and 5 bind to Mcl-1 with Ki values
of 280, 1690, and 400 nM, respectively, while compound 6
shows no appreciable binding at concentrations as high as 50
µM.
The recent publication of crystal structures11 of Mcl-1
provides an opportunity to examine computationally the docking
of 4 to Mcl-1 and thus to identify the structural basis for this

Letters
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14 3165
Figure 5. Predicted binding model of compound 4 with human Mcl-1
obtained after a 1 ns MD simulation. Residues colored in blue are the
superimposed human Bim BH3 peptide. The residue number of the
Bim BH3 peptide follows that in the mouse Bim BH3 (I90, L94, I97,
D99, and F101), which corresponds to I58, L62, I65, D67, and F69 in
the human Bim BH3 peptide.
Figure 7. Analysis of apoptosis induced by compounds 4 and 5 in
the MDA-MB-231 breast cancer cell line. Cells were treated with
compounds 4 and 5 for 3 days and apoptosis was analyzed using
Annexin-V and propium iodide (PI) double staining by flow cytometry.
Early apoptotic cells were defined as Annexin-V positive/PI-negative,
late apoptotic cells as Annexin-V/PI-double positive and necrotic cells
as Annexin V positive/PI positive.
Figure 6. Inhibition of cell growth by designed small-molecule BH3
mimetics in the MDA-MB-231 (2LMP) breast cancer cell line. Cells
were treated for 4 days and cell growth was determined using WST-
based assay.
high affinity binding. Our predicted binding model for 4 in the
complex with Mcl-1 shows that compound 4 mimics Ile90,
Leu94, Ile97, Phe101, and Asp99 in the Bim BH3 peptide
(Figure 5). The predicted binding models for compound 4 to
Mcl-1 and Bcl-2 are similar but not identical, likely due to
structural differences between these two proteins. From our
predicted binding models, it is also clear that compound 4 can
be further optimized to improve its binding to both Bcl-2 and
Mcl-1 proteins.
One major advantage of nonpeptide, small-molecule BH3
mimetics over peptide-based inhibitors is their superior cell
permeability. We have, therefore, tested our new nonpeptide
BH3 mimetics for their ability to inhibit cell growth in cancer
cell lines with high levels of Bcl-2, Bcl-xL, and Mcl-1 proteins.
The cell growth inhibition of these compounds in the MDA-
MB-231 (2LMP) breast cancer cell line is shown in Figure 6.
Consistent with its high binding affinity to Bcl-2 and Mcl-1,
compound 4 is a potent cell growth inhibitor, achieving an IC50
value of 110 nM in this assay. In direct comparison, compound
4 is 17 times more potent than 1 and 280 times more potent
than our initial lead compound 2 in inhibition of cell growth.
Significantly, the potencies of these inhibitors in cell growth
inhibition in the MDA-MB-231 breast cancer cell line positively
correlate with their binding affinities to Bcl-2 and Mcl-1
proteins. Furthermore, compound 4 was found to be at least 10
times less potent in normal primary prostate epithelial cells and
WI-38 normal fibroblasts with much lower levels of Bcl-2 and
Mcl-1 proteins. Hence, compound 4 displays a good selectivity
for cancer cells with high levels of Bcl-2 and Mcl-1 proteins
over normal cells.
Figure 8. Cell death induction by compounds 4 and 5 in the MDA-
MB-231 (2LMP) breast cancer cell line at different time points. Cells
were treated by compounds 4 and 5 for 24, 48, and 96 h and cell
viability was determined using trypan blue exclusion assay.
manner. Treatment of the MDA-MB-231 cells by 0.5 and 1 µM
of 4 for 3 days results in 24.5% and 64.4% of apoptotic cells
(early + late), as compared to 7.2% of apoptotic cells in an
untreated control. Compound 5 at 1 µM for 3 days has little
effect on apoptosis induction as compared to untreated control.
This is consistent with its much weaker binding affinity to both
Bcl-2 and Mcl-1 as compared to 4 and its modest activity in
inhibition of cell growth.
Finally, we tested compounds 4 and 5 for their ability to
induce cell death in the MDA-MB-231 cell line using the trypan
blue exclusion assay (Figure 8). As can be seen, compound 4
effectively induces cell death in a dose- and time-dependent
manner at concentrations as low as 100 nM and at the 24-hour
time point, whereas compound 5 is much less potent.
The synthesis for compounds 2, 4, 5, and 6 is outlined in
Scheme 1. Briefly, commercially available 3,4,5-trimethoxy-
phenol was subjected to Friedel-Crafts acylation under the
presence of BF3‚Et2O to give isobutyrophenone 7. Subsequent
reduction of the ketone group by triethylsilane in TFA afforded
phenol 8. A similar Friedel-Crafts reaction was carried out to
introduce the acetyl group. The obtained acetophenol 9 was
converted to flavonoid methyl ether 10 in three steps without
purification of intermediates. The product of O-acetylation of
9 by acetic anhydride in pyridine was dissolved in anhydrous
DMF and added to a suspension of NaH in DMF. The crude
product of the Baker-Venkataraman rearrangement23 readily
gave 10 upon acid treatment. Demethylation of 10 with excess
BBr3 afforded flavone 2. For the synthesis of designed
compound 4, compound 10 was treated with I2 in the presence
We evaluated compounds 4 and 5 for their ability to induce
apoptosis in the MDA-MB-231 cell line using Annexin-V and
propium iodide (PI) double staining by flow cytometry. The
results are shown in Figure 7. As can be seen, compound 4 is
very effective in induction of apoptosis in a dose-dependent

3166 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 14
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acetone. Isoflavone 5 was also synthesized in a similar manner
as 4, except that phenylboronic acid was used in the Suzuki
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In summary, we have designed and synthesized compound
4, which mimics the key binding residues in the Bim BH3
peptide in the interaction with Bcl-2. Compound 4, which was
named as BI-33, binds to Bcl-2 and Mcl-1 proteins with Ki
values of 17 nM and 18 nM, respectively, and has a relatively
weak affinity to Bcl-xL. Compound 4 is potent in inhibition of
cell growth in the MDA-MB-231 breast cancer cell line with
high levels of Bcl-2 and Mcl-1 proteins and effectively induces
apoptosis in a dose-dependent manner in the MDA-MB-231 cell
line. Of significance, compound 4 has a novel chemical scaffold
that is different from any known small-molecule inhibitors of
the anti-apoptotic Bcl-2 protein and represents a new class of
small-molecule inhibitors targeting the anti-apoptotic Bcl-2
proteins. Taken together, our data indicate that compound 4 is
a potent and cell-permeable small-molecule BH3 mimetic.
Extensive in Vitro and in ViVo studies are in progress to elucidate
further the molecular mechanism of action of compound 4 for
apoptosis induction and to ascertain its therapeutic potential,
and the results will be reported in due course. The study reported
here demonstrates that a structure-based strategy is effective
for the design of truly novel, potent, drug-like, nonpeptide,
small-molecule inhibitors that target protein-protein interac-
tions.
Acknowledgment. We are grateful for the financial support
from the National Cancer Institute, National Institutes of Health
(U19CA113317), the Department of Defense Breast Cancer
Program (BC0009140), the Department of Defense Prostate
Cancer Program (PC040537), the Prostate Cancer Foundation,
the Breast Cancer Research Foundation, the Susan G. Komen
Foundation, Ascenta Therapeutics, Inc., and the Intramural
Research Program of the National Institutes of Health from the
National Cancer Institute, Center for Cancer Research.
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G.; Guo, J.; Shangary, S.; Qiu, S.; Gao, W.; Yang, D.; Meagher, J.;
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Supporting Information Available: An experimental section
including information on the synthesis and chemical data for
compounds 2-6, molecular modeling methods and results for 2
and 4, the experimental procedure for the fluorescence polarization-
based binding assays for Bcl-2, Bcl-xL, and Mcl-1, details on the
cellular growth inhibition, and apoptosis assays. This material is
available free of charge via the Internet at http://pubs.acs.org.
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