Design, synthesis, and activity evaluation of broad-spectrum small-molecule inhibitors of anti-apoptotic Bcl-2 family proteins: Characteristics of broad-spectrum protein binding and its effects on anti-tumor activity

Article abstract of DOI:10.1016/j.bmcl.2011.11.101

On the basis of the comparison of the structure of the Bim BH3: Bcl-x _L complex and that of the ABT-737: Bcl-x_L complex, a series of class A compounds were designed. These compounds had the basic skeleton of ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim BH3's broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B compounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most of these class B compounds showed better binding affinity to the target proteins than the class A compounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple tumor cell lines known to express Bcl-x _L, Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spectrum binding to Bcl-x_L, Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum properties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth of many tumor cells. Finally, our study provides a series of lead compounds that merit further research into anti-cancer therapeutics.

Full text of DOI:10.1016/j.bmcl.2011.11.101

Bioorganic & Medicinal Chemistry Letters 22 (2012) 39–44
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and activity evaluation of broad-spectrum small-molecule
inhibitors of anti-apoptotic Bcl-2 family proteins: Characteristics of
broad-spectrum protein binding and its effects on anti-tumor activity
Can-Hui Zheng a, , Hui Yang a, , Meng Zhang a, Shi-Hai Lu a, Duo Shi a, Juan Wang b, Xiu-Hua Chen b,
a,
Xiao-Hui Ren a, Jia Liu a, Jia-Guo Lv a, Ju Zhu a, , You-Jun Zhou
⇑
⇑
a School of Pharmacy, Second Military Medical University, Shanghai 200433, China
b Shanghai Institute of Pharmaceutical Industry, Shanghai 200040, China
a r t i c l e i n f o
a b s t r a c t
Article history:
On the basis of the comparison of the structure of the Bim BH3: Bcl-xL complex and that of the ABT-737:
Bcl-xL complex, a series of class A compounds were designed. These compounds had the basic skeleton of
ABT-737 and the h2 residues of Bim BH3. These residues had shown themselves to be relevant to Bim
BH3’s broad-spectrum binding properties in saturation mutagenesis assays. Unlike ABT-737, which is a
selective inhibitor of anti-apoptotic members of the Bcl-2 protein family, the class A compounds showed
broad-spectrum binding activity to target proteins similar to those of Bim BH3 peptide. Then class B com-
pounds were synthesized by modifying the structure of the most effective class A compound, A-4. Most
of these class B compounds showed better binding affinity to the target proteins than the class A com-
pounds had. They also showed themselves more effective than ABT-737 at inhibiting growth in multiple
tumor cell lines known to express Bcl-xL, Bcl-2, and Mcl-1 proteins at high levels. Compounds B-11 and
B-12 had the strongest anti-tumor activity of any compounds we produced. This study suggests that it is
feasible to design small-molecule inhibitors based on the structure of Bim BH3, which shows broad-spec-
trum binding to Bcl-xL, Bcl-2, and Mcl-1 proteins. Our results also suggest that the broad-spectrum prop-
erties of small-molecule inhibitors binding to target proteins play a critical role in inhibiting the growth
of many tumor cells. Finally, our study provides a series of lead compounds that merit further research
into anti-cancer therapeutics.
Received 22 September 2011
Revised 2 November 2011
Accepted 24 November 2011
Available online 1 December 2011
Keywords:
Anti-apoptotic Bcl-2 family proteins
Inhibitor
Broad-spectrum
Anti-tumor agent
Ó 2011 Elsevier Ltd. All rights reserved.
Apoptosis, the process of natural, programmed cell death (PCD),
is stimulated by a diverse range of cell signals. It plays a key role in
ontogenesis and homeostasis in multicellular organisms.1 The Bcl-
2 protein family, which contains both anti-apoptotic (e.g., Bcl-xL,
Bcl-2, Bcl-w, Mcl-1, A1) and pro-apoptotic members,2,3 includes
many central regulators of these apoptotic signaling pathways.4
The pro-apoptotic members of this protein family can be either
Bax-like (Bax, Bak, Bok) or BH3-only (Bad, Bim, Bmf, Bik, Hrk,
Bid, Puma, Noxa).5 The anti-apoptotic members of the Bcl-2 family,
such as Bcl-xL and Bcl-2, are over-expressed in many types of can-
cer and contribute to tumor initiation, progression, and resistance
to conventional anti-cancer therapy.2–4,6,7 This is why the anti-
apoptotic members of the Bcl-2 protein family have become attrac-
tive targets for anti-cancer drug development.
shown that anti-apoptotic Bcl-2 proteins exert their functionally
anti-apoptotic effects by antagonizing the pro-apoptotic members.8
This antagonistic function is mediated by constructive binding of a
hydrophobic groove on the surface of anti-apoptotic proteins with
the BH3 region in pro-apoptotic molecules.9,10 One new strategy
for the design of anti-tumordrugs takes this into account by employ-
ing non-peptide small-molecule inhibitors that bind to the hydro-
phobic groove and inhibit its function. In recent years, series of
small-molecule inhibitors with different structures have been re-
ported.11–28 At present, three small-molecular inhibitors, ABT-263,
AT-101, and GX15-070, have been investigated as oral anti-cancer
medications for use in clinical research (Fig. 1).29–31 The analog com-
pounds of ABT-263, including ABT-737, have high affinity to Bcl-2
family proteins (nM level). However, they also have a narrow anti-
tumor spectrum with limited clinical effects and only work on cer-
tain types of hematological malignancies, when used alone.22,32–35
This could be explained by the fact that these compounds have high
affinity only to the Bcl-xL and Bcl-2 proteins and poor affinity to Mcl-
1 protein.33 This hypothesis has been supported by study results
showing that blocking Mcl-1 protein in cells by a variety of methods,
such as siRNA, can restore cells’ sensitivity to ABT-737.33,35 In
Although further studies into the mechanisms of the regulation
of apoptosis by the Bcl-2 protein family are needed, it has been
⇑
Corresponding authors. Tel.: +86 21 81871238 (J.Z.); +86 021 81871231 (Y.-J.Z.).
E-mail addresses: zhuju@smmu.edu.cn (J. Zhu), zhuoyoujun2006@yahoo.com.cn
(Y.-J. Zhou).
These authors contributed equally to this work.
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.11.101

40
C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 39–44
Figure 1. Representative small-molecule inhibitors of anti-apoptotic Bcl-2 proteins.
addition, it has recently been shown that homologous Mcl-1 protein,
in addition to the well-known Bcl-xL and Bcl-2 proteins, is an impor-
tant survival factor in many tumors.36–38 Over-expression of Mcl-1
has been shown to affect cancer cells’ resistance to anti-cancer
drugs.38,39 For these reasons, broad-spectrum small-molecule inhib-
itors targeting anti-apoptotic members of the Bcl-2 protein family
may be ideal anti-cancer drugs.
binding abilities. By comparing the structure of the Bim BH3:
Bcl-xL complex to that of the ABT-737: Bcl-xL complex, we found
that the chlorinated biphenyls and thiophenyl at the end of ABT-
737 bound to the same two sites on the active cavity to which
hydrophobic residues h2 and h4 bound (Fig. 2A). This prompted
us to determine whether it would be possible to replicate the
broad-spectrum binding properties of Bim BH3 by designing a ser-
ies of new class A compounds (Fig. 3). These compounds would
have the basic skeleton of ABT-737 but the chlorinated biphenyls
would be replaced with the h2 residues, which had shown them-
selves relevant to Bim BH3’s broad-spectrum binding properties
in saturation mutagenesis assays. The binding modes of represen-
tative A-4 compounds (Table 1) docking with the protein target
(Fig. 2B) showed that the amino acids within this structure had
good overlap with the h2 residues in Bim BH3. These were
expected to simulate the interactions between the h2 residues
and the protein target.54–56
Global topological structures, especially the binding groove, are
quite similar among anti-apoptotic members of the Bcl-2 family,
but different protein sequences do produce a few significant differ-
ences.9,10,22,40–45 Before binding to ligands, the binding groove of
the Mcl-1 protein is in a more open, ready-to-bind conformation,
than that of Bcl-xL, Bcl-2, or other proteins.44,46,47 Additionally,
the binding groove of the Mcl-1 protein appears less flexible when
binding to different substrates than the grooves of Bcl-xL, Bcl-2,
and other proteins do.45,48,49 These structural differences may
explain why different anti-apoptotic Bcl-2 proteins show different
selectivities and preferences for binding different substrates. For
example, these proteins show their selectivity when they bind to
the physiological substrates of BH3-only proteins. Bad BH3 is
selective for the Bcl-xL and Bcl-2 proteins, while Noxa BH3 is
selective for the Mcl-1 protein, and Bim BH3 is able to bind all
pro-survival proteins well.50,51 When we design broad-spectrum
small-molecule inhibitors, we may be inspired by studies on Bim
protein, which can combine with a broad range of anti-apoptotic
Bcl-2 proteins.
In order to validate the hypothesis, we used the approach
shown in Scheme 1 to synthesize compounds A-(1–5), which con-
tained Leu, Ile, Met, Phe, and Trp amino acids, respectively. As key
intermediates of our designed compounds, 7 were synthesized by
the known procedure.22 Compound 1 was treated with phosphorus
tribromide and provided 2 by substitution reaction. Compound 2, a
halide, formed a temporary intermediate with 4-nitrophthalimide
potassium salt. Then hydrazine hydrate was added and hydrolyzed
to form 3. Ethyl 4-aminobenzoate reacted with different acid, EDCI
and DMAP to form different Ester 9A. Then sodium hydroxide solu-
tion was added for hydrolysis to produce another key intermediate,
10A. Compounds 7 and 10A reacted to form target compounds A.
Using an FP-based binding assay (Table 1), we found that the
class A compounds, like Bim BH3 peptide, could broadly bind to
Bcl-xL, Bcl-2, and Mcl-1 proteins, although the binding affinity to
Structural analysis of Bim BH3: Bcl-xL complexes shows that
four hydrophobic residues (h1–h4) on one face of the
a-helix of
the Bim protein BH3 domain insert themselves into the hydropho-
bic surface groove of the Bcl-xL protein (Fig. 2A).42,52 The saturation
mutagenesis analysis of the two most critical residues at positions
h2 and h4 suggests that the residue at the h2 position plays a more
important role than the one at the h4 position in the broad-spec-
trum binding properties of Bim protein when binding to various
anti-apoptotic Bcl-2 proteins.53 After Leu is substituted with large
hydrophobic amino acids such as Ile, Met, Phe, or Trp, at the h2
residue position in Bim BH3, the molecules will retain their
broad-spectrum binding properties. These amino acids bind to
the active cavity mainly through the hydrophobic activity of their
side chains, whose size is just adequate for three protein active
cavities. This might be the reason why they have broad-spectrum
the target proteins was only up to the
l
M level.57,58 Their low
binding affinities are partly induced by the absence of the substi-
tuted ethylamine chain, appended to the thiophenyl group, which
are different from ABT-737. In addition, we synthesized compound
A-6, containing a Val amino acid. It has been reported that Bim BH3
peptides with Val amino acids at the h2 residue have relatively
lower affinity to Bcl-xL and Bcl-2 proteins than to Mcl-1.53 The
results of the binding assay used in this study showed compound
A-6 have a similar binding property. These data suggest that it is

C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 39–44
41
feasible to design small-molecule inhibitors with broad-spectrum
binding affinity through the above approach.
We also evaluated the ability of these compounds and the con-
trol compound, ABT-737, to inhibit cell growth in the Raji human
Burkitt lymphoma cell line and the MDA-MB-435 human breast
carcinoma cell line, both of which express Bcl-xL, Bcl-2, and Mcl-
1 proteins at high levels (Table 1).59–61 Consistent with the
reported results, ABT-737 showed low inhibitory activity in these
two cell lines, both of which have high levels of expression of the
Mcl-1 protein.33 Compounds A-(1–6) demonstrated a certain de-
gree of inhibitory activity to the same two tumor cell lines, but this
inhibitory activity was also very low. One possible reason for this
may be that the binding affinities between compounds A-(1–6)
and the three proteins are very low. This hypothesis was confirmed
by the fact that compound A-6 showed the least ability to inhibit
tumor cell growth and had the lowest affinity to Bcl-xL and Bcl-2
proteins. Our purpose from this point on was to modify the struc-
ture of the class A compounds to increase their biological activity.
We selected A-4 as a leading compound because it showed good
affinity to all three proteins (Bcl-xL, Bcl-2, and Mcl-1) and was suit-
able for structural derivatization. According to binding models of
compound A-4 with its protein targets, the acetyl amino group of
Phe did not interact with the active cavity, so we removed that part
of the amino acid during structural modification. We designed the
class B compounds (Fig. 3) taking into account both the binding
model and the requirements of structural derivatization. The bind-
ing models of representative compound B-1 (Table 2) docking with
its target protein showed that the introduced benzene ring
behaved like the side chain of the amino acids in class A com-
pounds, which had very good overlap with the h2 residues in the
Bim BH3 peptide. We also introduced a halogen bond and different
hydrophobic groups at different positions on the benzene ring to
examine their impact on the biological activity of derivatives.
We synthesized a series of compounds B-(1–12) (Table 2) using
methods similar to those used with compounds A-(1–6) shown in
Scheme 1. Using an FP-based binding assay, we determined the
binding affinity of some representative compounds to Bcl-xL, Bcl-
2, and Mcl-1 proteins (Table 2). The results showed that these
compounds maintained broad-spectrum binding affinity to the
Bcl-xL, Bcl-2, and Mcl-1 proteins. Their binding affinity to the target
proteins was one to five times higher than that of the leading com-
pound A-4. Then we systematically evaluated the cell growth inhib-
itory activities of all class the B compounds in Raji human Burkitt’s
lymphoma, MDA-MB-435 human breast carcinoma and HCT-116
human colon cancer cell lines, all of which have high levels of
expression of Bcl-xL, Bcl-2, and Mcl-1 proteins (Table 2).59–63 The re-
sults showed a clear linear relationship between the anti-tumor
activity of class B compounds and their affinity to the target pro-
teins. Although the affinity of the class B compounds to Bcl-xL and
Bcl-2 proteins was still significantly lower than that of ABT-737,
most of the compounds showed more pronounced growth inhibi-
tory activity than ABT-737 in the three tumor cell lines studied.
These results provide further evidence that the broad-spectrum
properties of small-molecule inhibitors binding to Bcl-xL, Bcl-2,
Figure 2. (A) Overlay of the structures of Bcl-xL–Bim complex (PDB ID: 3FDL) and
Bcl-xL–ABT-737 complex (PDB ID: 2YXJ). (B) Binding modes of compound A-4 to
Bcl-xL. (C) Binding modes of compound B-1 to Bcl-xL.
Figure 3. Design of broad-spectrum anti-apoptotic-Bcl-2-protein inhibitors.

42
C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 39–44
Table 1
The structure and biological activity of class A compounds
No.
h2 residue
Compound structure
FP (Ki,
l
M)
Cytotoxicity (IR in 100 lg/ml)
R1
Bcl-xL
5.82
Bcl-2
Mcl-1
1.80
Raji
MDA-MB-435
O
S O
A-1
Leu
3.78
58.27%
25.61%
O
H
HN
O
N
R1
NO2
A-2
A-3
Ile
4.84
3.91
3.38
3.52
2.29
3.11
40.85%
40.12%
13.48%
16.01%
NHCOCH3
S
NH
Met
S
A-4
A-5
Phe
Trp
3.42
6.45
3.39
3.11
4.03
26.79%
31.78%
36.57%
9.36%
0.83
9.49
HN
A-6
Val
/
16.44
3.27
8.85%
6.33%
ABT-737
/
/
<0.001
<0.001
>100
42.52%
55.43%
Scheme 1. Synthesis of target compounds. Reagents and conditions: (a) PBr3, rt; (b) (i) 4-nitrophthalimide potassium salt/DMF, reflux, (ii) NH2NH2ÁH2O/CH3OH, reflux; (c)
ClHO3S, 80 °C, reflux; (d) NH4OH, À78 to À65 °C, rt; (e) 3/DIEA/DMSO, rt; (f) C9H11NO2/DMAP/EDCI, rt; (g) NaOH/THF/CH3OH, rt; (h) 7/DMAP/EDCI/CH2Cl2, rt.
and Mcl-1 proteins play a critical role in growth inhibitory activity
in many tumor cells. By comparing the binding affinity to target
proteins and the growth inhibitory activity in tumor cells of
B-(2–4), B-(5–7), and B-(8–10), we found that the position of the

C.-H. Zheng et al. / Bioorg. Med. Chem. Lett. 22 (2012) 39–44
43
Table 2
The structure and biological activity of class B and C compounds
No.
Compound structure
FP (Ki,
lM)
Cytotoxicity (IC50
Raji
, lM)
R2
Bcl-xL
Bcl-2
Mcl-1 HCT116
MDA-MB-435
B-1
B-2
B-3
B-4
B-5
B-6
B-7
B-8
B-9
/
2.51
1.71
2.08
1.94
/
/
/
/
/
1.67
1.09
1.40
0.93
/
/
/
/
/
4.16
3.02
3.09
2.54
/
/
/
/
/
136.45
39.99
>168.17 (100
78.86
>163.64 (100
33.14
43.38
160.73
169.30 (100
48.61
10.53
14.62
39.78
32.19
g/ml) 39.40
38.58
g/ml) 41.17
37.54
59.90
48.10
O
2-F
3-F
4-F
2-Cl
3-Cl
4-Cl
2-CH3
3-CH3
4-CH3
4-Br
O
HN
H
S O
l
59.50
57.53
69.88
46.67
49.89
47.56
>169.29 (100
52.97
39.48
N
O
NO2
l
S
NH
37.54
38.84
92.62
38.84
34.99
22.94
R2
lg/ml)
lg/ml)
B-10
B-11
B-12
/
/
/
1.50
1.27
0.76
0.80
4.10
3.11
4-OCH3
36.07
O
O
S N
HN
H
S
O
O
C-1
/
/
2.29
15.19
7.22
162.95
47.70
>163 (100
93.49
lg/ml) >163 (100 lg/ml)
O
NO2
S
NH
ABT-737
/
<0.001 <0.001 >100
>122.94 (100 lg/ml)
halogen placed on the benzene ring could influence their activity.
The relative activity of halogens at different positions was found
to be as follows: 4 > 3 > 2. We also found that the large size of the
replacement groups at position 4 had a profound effect on their
activity. Compounds B-11 and B-12, which had the largest halogens
at position 4, Br and OCH, respectively, showed the best activity.
According to the binding models of representative compound B-1
docking with its target protein (Fig. 2C), the introduction of halo-
gens or hydrophobic groups at position 4 of the benzene ring may
be more beneficial for increasing the hydrophobic activity than hal-
ogens placed at other positions, and the greater the size of the
groups, the stronger the hydrophobic activity.
In addition, we also used a method similar to that used to syn-
thesize compounds A-(1–6) (Scheme 1) to synthesize the class C
compounds, in which the carbonyl at benzene ring was replaced
with sulfonyl (Table 2). The class C compounds’ binding affinity
to Bcl-2 and Mcl-1 proteins decreased significantly, indicating that
the amide backbone connecting to the benzene ring in class B com-
pounds plays a critical role in maintaining their broad-spectrum
binding activity to target proteins. Our results showed that the
anti-tumor activity of class C compounds significantly decreased
alongside decreased affinity (Table 2). This confirmed the impor-
tance of broad-spectrum binding activity of inhibitors in curtailing
the growth of tumor cells.
inhibitors based on the structure of Bim BH3, which shows
broad-spectrum binding to Bcl-xL, Bcl-2, and Mcl-1 proteins. Our
results also suggest that the broad-spectrum properties of small-
molecule inhibitors binding to target proteins play a critical role
in inhibiting the growth of many tumor cells. Finally, our study
provides a series of lead compounds that merit further research
into anti-cancer therapeutics.
Acknowledgments
The work was supported by the National Natural Science Foun-
dation of China (Grant No. 30901859) and Shanghai Natural Sci-
ence Foundation (Grant No. 09ZR1438800).
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2011.11.101.
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