Design, synthesis, and activity evaluation of selective inhibitors of anti-apoptotic Bcl-2 proteins: The effects on the selectivity of the P1 pockets in the active sites

Article abstract of DOI:10.1016/j.bmcl.2016.09.061

The anti-apoptotic Bcl-2 proteins are attractive targets for anti-cancer drug development, and the discovery of their selective inhibitors has become a research focus. In this Letter, obvious differences in the P1 pocket of the active site between Bcl-2, Bcl-x_L, and Mcl-1 proteins were proposed by the structural comparison of these proteins. As a result, the groups in their inhibitors binding to the P1 pockets may have significant effect on the selectivity for these proteins. Based on this hypothesis, five types of derivatives of the lead compound B-1 were designed, and several highly selective inhibitors of Bcl-x_L(E-1) or Mcl-1 proteins (G) were found. The selective inhibitors of Mcl-1 protein found in this Letter provide new structural types for the development of novel antitumor agents.

Full text of DOI:10.1016/j.bmcl.2016.09.061

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, and activity evaluation of selective inhibitors of
anti-apoptotic Bcl-2 proteins: The effects on the selectivity of the P1
pockets in the active sites
Mingping Wang a,b, Wei Tian b, Chongqing Wang b, Shihai Lu b, Chao Yang b, Juan Wang c, Yunlong Song b,
Youjun Zhou b, Ju Zhu b, , Zhiyu Li a, , Canhui Zheng b,
⇑
⇑
⇑
a Jiangsu Key Laboratory of Drug Design and Optimization, China Pharmaceutical University, 24 Tongjiaxiang, Nanjing 210009, China
b School of Pharmacy, Second Military Medical University, Shanghai 200433, China
c Shanghai Institute of Pharmaceutical Industry, Shanghai 200437, China
a r t i c l e i n f o
a b s t r a c t
Article history:
The anti-apoptotic Bcl-2 proteins are attractive targets for anti-cancer drug development, and the discov-
ery of their selective inhibitors has become a research focus. In this Letter, obvious differences in the P1
pocket of the active site between Bcl-2, Bcl-xL, and Mcl-1 proteins were proposed by the structural com-
parison of these proteins. As a result, the groups in their inhibitors binding to the P1 pockets may have
significant effect on the selectivity for these proteins. Based on this hypothesis, five types of derivatives of
the lead compound B-1 were designed, and several highly selective inhibitors of Bcl-xL (E-1) or Mcl-1
proteins (G) were found. The selective inhibitors of Mcl-1 protein found in this Letter provide new struc-
tural types for the development of novel antitumor agents.
Received 1 March 2016
Revised 20 August 2016
Accepted 26 September 2016
Available online xxxx
Keywords:
Anti-apoptotic Bcl-2 proteins
Selective inhibitors
Mcl-1 protein
Ó 2016 Elsevier Ltd. All rights reserved.
Anti-tumor
Apoptosis is a process of natural, programmed cell death, which
plays a key role in ontogenesis and homeostasis in multicellular
organisms.1,2 The Bcl-2 protein family plays a key regulatory role
in this process. It contains both anti-apoptotic (e.g., Bcl-2, Bcl-xL,
Mcl-1, Bcl-w, A1) and pro-apoptotic members.1,3 The overexpres-
sion of the anti-apoptotic members of the Bcl-2 family, which is
found in a wide variety of human cancers, is associated with
tumour progression and resistance to anti-cancer therapy.3–5 This
is why the anti-apoptotic Bcl-2 proteins have become attractive
targets for anti-cancer drug development. In recent years, a series
of small-molecule inhibitors of anti-apoptotic Bcl-2 proteins with
different structure types have been reported, which show anti-
cancer effects.4–16 At present, three small-molecular inhibitors,
AT-101,17 ABT-263,18,19 and GX15-07020,21 are in clinical research
(Fig. 1). ABT-199 (venetoclax)22,23 has been approved recently by
FDA for the treatment of patients with chronic lymphocytic leuke-
mia (CLL) who have a chromosomal abnormality called 17p
deletion.
anti-apoptotic Bcl-2 proteins or directly activate Bax-like pro-
apoptotic Bcl-2 proteins (Bax, Bak, Bok), which can lead to cyto-
chrome C release from mitochondria to activate apoptosis.4,5,24,25
Different anti-apoptotic Bcl-2 proteins show diverse selectivity
and preference for binding BH3-only pro-apoptotic Bcl-2 proteins.
Bad and Bmf are selective for the Bcl-2, Bcl-xL, and Bcl-w proteins,
while Noxa is selective for the Mcl-1 and A1 protein, and Bim, Bid
and Puma are able to bind all anti-apoptotic proteins well.16,25–28
These observations suggest that apoptosis can be regulated by dif-
ferent anti-apoptotic Bcl-2 proteins by diverse ways. And they may
have different physiological effect.5,24 Furthermore, human cancers
may have different expression spectrum of anti-apoptotic Bcl-2
proteins.5,29,30
To explore the exact role of each anti-apoptotic Bcl-2 protein, its
selective inhibitors can be utilized as good molecular tools.24 In
addition, these selective inhibitors have the opportunity to be
developed to anticancer drug candidates with fewer adverse side
effects than general inhibitors. Therefore, the discovery of these
selective inhibitors has become a research focus. However, the
structural similarity of anti-apoptotic Bcl-2 proteins makes the
design of its selective inhibitors challenging. Presently, not many
structure types of selective inhibitors have been reported. ABT-
263 was reported to have high selectivity for Bcl-2 and Bcl-xL pro-
teins.10 ABT-199,11 WEHI-53912 and A-121047713,31 were reported
Apoptosis can be triggered by BH3-only pro-apoptotic Bcl-2
proteins (Bad, Bim, Bmf, Bik, Hrk, Bid, Puma, Noxa), which are
activated upon cellular stresses. These proteins antagonize
⇑
Corresponding authors. Tel.: +86 021 81871240.
E-mail addresses: zhuju@smmu.edu.cn (J. Zhu), zhiyuli@cpu.edu.cn (Z. Li),
canhuizheng@smmu.edu.cn (C. Zheng).
http://dx.doi.org/10.1016/j.bmcl.2016.09.061
0960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
Please cite this article in press as: Wang, M.; et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.09.061

2
M. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
OH
O
O
three-dimensional pharmacophore for Bcl-2 inhibitors we con-
O
O
OH
S
SO2CF3
NH
N
structed, hydrophobic centers H1, H2 and H3 were three points
bound in L, P1 and P2, respectively. They were found to serve as
the linear molecular scaffold that satisfies the overall geometric
and steric requirements for inhibitor binding.16,32
H
H
H
HO
O
N
OH
N
HO
HO
S
N
O
At present, several high-resolution three-dimensional struc-
tures of Bcl-2, Bcl-xL, and Mcl-1 proteins in different states were
reported, which offer good templates for determining the struc-
tural difference in their active sites. The structures of these three
proteins which are free (PDB entry: 1GJH, 1LXL, and 1WSX) or
complexed with a same substrate (BH3 of Bax) (PDB entry:
2XAO, 3PL7 and 3PK1) were superimposed and compared
(Fig. 2).34,35 Obvious difference in the opening of the P1 pocket
could be observed because of the difference of the residues in the
active site among these proteins (Fig. S1). The size order of free
proteins was: Bcl-xL < Bcl-2 < Mcl-1, that of complexed proteins
was: Mcl-1 < Bcl-xL < Bcl-2.
The different opening size order of free proteins and complexed
proteins also suggested that there was difference in the flexibility
when binding to substrates of the P1 pocket in the active site.
Therefore, the root mean square deviation (RMSD) values of the
changes between active sites of Bcl-2, Bcl-xL, and Mcl-1 proteins
in free and complexed states were calculated (Table 1). The order
of the value of the changes between active sites in whole was:
Mcl-1 < Bcl-2 < Bcl-xL. In addition, the RMSD values of the changes
between each domain of the active site were also calculated
AT-101
ABT-263
Cl
O
HN
O
S O
N
N
N
NO2
HN
O
N
H
O
Cl
HN
N
GX15-070
ABT-199
O
HN
O
O
N
N
O
HO
N
S
OH
O
O
HN
N
S
N
N
N
N
N
S
O
N
O
O
A-1210477
WEHI-539
NH2
Figure 1. Representative small-molecule inhibitors of anti-apoptotic Bcl-2
proteins.
to have high selectivity for Bcl-2, Bcl-xL and Mcl-1 proteins, respec-
tively (Fig. 1).
(Table 1). In Bcl-2 and Bcl-xL proteins, the
a3 domain appeared
most flexible among the active site. While the
a4 domain appeared
Determining the structural difference in the active site between
anti-apoptotic Bcl-2 proteins is useful to rationally design of its
selective inhibitors. In this Letter, important differences were pro-
posed by the structural comparison of Bcl-2, Bcl-xL, and Mcl-1 pro-
teins, three most studied anti-apoptotic Bcl-2 proteins. Based on
this hypothesis, several types of derivatives of the lead compound
were designed. Among them, several selective inhibitors of Bcl-xL
protein or Mcl-1 protein were found. These results validated our
hypothesis and provided several lead compounds that merit fur-
ther research into anti-cancer therapeutics.
most flexible in Mcl-1 protein. This conclusion is consistent with
the finding of the molecular dynamic simulations to investigate
the conformational flexibility of Bcl-xL and Mcl-1 proteins.36
On basis of above analysis, there were obvious differences in the
P1 pocket of the active site between Bcl-2, Bcl-xL, and Mcl-1 pro-
teins. As a result, the groups in their inhibitors binding to the P1
pocket may have significant effect on the selectivity for these pro-
teins. In order to validate the hypothesis, a broad-spectrum inhibi-
tor B-1 we found, with a benzamide group binding to the P1 pocket
predictively, was chosen as the lead compound (Fig. 3).16 Then five
types of derivatives C–F were designed by changing the benzamide
group to other groups with diverse lengths and volumes, using dif-
ferent carboxylic acids from our in-house library (Fig. 3).
All of the anti-apoptotic Bcl-2 proteins have a similar active site,
which is a narrow and long groove on the protein surface. The bot-
tom of it is bound by
a5, and on the side, it is bound by a2, a3, a4,
a
7 and 8. The active site comprises three main areas: the P1 and
a
The synthetic routes of target compounds were shown in
Scheme 1. The key intermediates 3 and 6 were synthesized
by known procedures.16 The target compounds C and G were
P2 are two big and deep pockets in the active site, and the L1 is a
narrow and shallow channel linking P1 and P2 (Fig. 2).9,32,33 In the
Figure 2. Comparison between the activity sites of Bcl-2, Bcl-xL and Mcl-1 proteins. The PDB entries of the structures of free Bcl-2, Bcl-xL and Mcl-1 proteins (A) are 1GJH,
1LXL, and 1WSX. Those of Bcl-2, Bcl-xL and Mcl-1 proteins complexed with Bax BH3 (B) are 2XAO, 3PL7 and 3PK1. They are rendered as ribbons with different colors. The
three main areas of the active site are labeled as P1, L and P2.
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M. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
3
Table 1
RMSD values of the changes between the activity sites of Bcl-2, Bcl-xL and Mcl-1 proteins in different statuses
Proteins
Structuresa (PDB entry)
RMSD of activity sitesb (C-
a)
Whole
a2
a
3
a4
a5
a7
a8
Bcl-2
Bcl-xL
Mcl-1
1GJH, 2XAO
1LXL, 3PL7
1WSX, 3PK1
1.96
2.24
1.75
1.48
1.56
1.48
4.11
4.15
1.87
1.32
1.77
2.12
0.77
1.31
0.90
1.50
1.61
1.42
1.09
1.08
0.90
a
The structures of free Bcl-2, Bcl-xL and Mcl-1 proteins and those complexed with Bax BH3.
The range of the activity site of Bcl-2 protein is V92-V159 and H186-G203. That of Bcl-xL protein is A85-V152 and E179-G196. That of Mcl-1 protein is K208-K276 and
b
R303-H320.
O
The selective inhibitor of Bcl-xL protein can be used as the
molecular tool to explore the role of anti-apoptotic Bcl-2 proteins.
However, they may not ideal to be developed as anticancer agents,
O
O
HN
O
S O
O
S
O
HN
O
H
N
NO2
NH
NO2
n
because they can induce
a rapid, concentration-dependent
S
S
NH
D
C
decrease in the number of circulating platelets.11 However, it has
been shown that Mcl-1 protein, in addition to the well-known
Bcl-2 protein, is also an important factor in tumor initiation, pro-
gression,30,37,38 and resistance to anti-cancer drugs.39,40 Therefore,
only compounds F and G with good binding affinity to Mcl-1 pro-
tein were further evaluated in the human cancer cell growth inhi-
bitory activity test (Table 3). ABT-737, an analogue of ABT-263,
which is a weak inhibitor of Mcl-1 protein but potent inhibitor of
Bcl-2 protein, was chosen as the control. On the U266 human
myeloma,42 MCF-7 human breast cancer,43 SKOV3 human ovar-
ian,44 NCI-H23 human lung cancer cell lines with high levels of
expression of the Mcl-1 protein,37 these compounds displayed
more pronounced growth inhibitory activity than ABT-737. How-
ever, on the HL-60 human leukemia cell line without high levels
P2
P1
L
O
O
O
O
H
HN
O
S O
HN
O
S
O
N
N
H
NO2
NH
NO2
NH
R1
S
S
B-1
E
O
O
O
O
HN
O
S
HN
O
S
O
H
N
NO2
NO2
NH
S
NH
S
F
G
Figure 3. Design of target compounds. On the structure of lead compound B-1, the
areas of the active site which its groups were binded to are shown in orange, and
the benzamide group binding to the P1 pocket is shown in blue. On the other
structures, the groups replacing the benzamide group of lead compound B-1 are
also shown in blue.
b
a
Br
NH2
OH
c
S
S
S
2
3
1
SO2NH2
SO2NH2
synthesized from the intermediate 6 and different carboxylic acids
by condensation reactions under the condition of EDCI and DMAP,
with 74% and 75% yields, respectively. While the target compounds
D, E and F were synthesized by another route. Firstly, ethyl
4-aminobenzoate (7) and different carboxylic acids reacted to gen-
erate different amides 8 with 70–80% yields. Then these intermedi-
ates hydrolyzed under the condition of sodium hydroxide alcohol
aqueous solution to form corresponding carboxylic acids 9 with
85–95% yields. Finally, these carboxylic acids condensed with the
intermediate 6 to gain target compounds with 70–80% yields.
Binding affinities of all of target compounds to Bcl-2, Bcl-xL and
Mcl-1 proteins were evaluated by a fluorescence polarization (FP)-
based method (Table 2). Based on the results, some of compounds
C-E showed a certain degree of selectivity for the Bcl-xL protein rel-
ative to Bcl-2 protein (selectivity index > 10). Among them, com-
pounds E-1 was found having high selectivity with selectivity
index of 35. Additionally, some of them were found having binding
affinities to Bcl-xL protein better than that of the lead compound B-
1, such as E-7 with Ki values of 70 nM. The results also showed that
compounds F and G displayed selectivity for the Mcl-1 protein. The
compound G had high selectivity with selectivity index of 70 and
good binding affinity with Ki value of 60 nM. These data suggest
that the inhibitors of anti-apoptotic Bcl-2 proteins with diverse
groups binding to the P1 pocket may indeed have obviously differ-
ent binding selectivity for these proteins. The inhibitors with this
group of substituted cinnamamides would show good binding
affinity and selectivity for Bcl-xL protein. While the inhibitors with
this group of substituted (tetrahydronaphthalen-2-yl)vinyls would
display such binding characteristic for Mcl-1 protein.
e
d
NO2
NO2
NO2
HN
F
4
F
S
5
6
O
S O
O
S O
HN
O
HN
O
O
NO2
NH
NO2
NH
S
S
C
G
e
O
Z
OC2H5
O
OC2H5
O
f
O
OH
H
N
H
N
H2N
Z
O
8
9
7
O
O
S
O
S
O
O
HN
O
H
N
HN
O
O
N
H
NO2
O
n
NO2
NH
R1
R1
n
1
2
S
NH
D-1
D-2
S
e
O
R1
/
HN
S
O
H
N
E-8 2'- Cl
E-9 3'- Cl
E-10
E-11
E-12
E-1
O
NO2
E-2 2'- Me
E-3
E-4
E-5
4'- Cl
2'- CF3
3'- CF3
3'- Me
4'- Me
2'- OMe
S
NH
F
E-13 4'- CF3
E-6 3'- OMe
E-7 4'- OMe
Scheme 1. Synthesis of target compounds. Reagents and conditions: (a) PBr3, rt,
98%; (b) (1) 4-nitrophthalimide potassium salt/DMF, reflux, (2) NH2NH2ÁH2O/CH3-
OH, reflux, 72%; (c) (1) ClHO3S, 80 °C, reflux, (2) NH4OH, À75 to À60 °C, rt, 60%; (d)
3/DIEA/DMSO, rt, 85%; (e) carboxylic acids/EDCI/DMAP/DCM, rt, 70–80%; (f) NaOH/
CH3OH/H2O/THF, rt, 85–90%.
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M. Wang et al. / Bioorg. Med. Chem. Lett. xxx (2016) xxx–xxx
Table 2
Binding affinities of target compounds to Bcl-2, Bcl-xL and Mcl-1 proteins
No.
Ki (FP,
lM)
Selectivity index
Bcl-2
Bcl-xL
Mcl-1
Bcl-2/Bcl-xL
Bcl-2/Mcl-1
C
3.4
0.33
0.45
0.32
0.14
0.21
0.16
0.13
0.09
0.11
0.07
0.12
0.29
0.21
0.17
0.29
0.51
>20
4.00
5.60
6.90
1.60
0.83
0.44
0.63
8.60
0.80
0.59
14.0
1.30
2.70
0.66
5.40
1.70
0.29
0.06
6.70
0.64
>20
10
9
14
35
8
11
12
8
17
19
5
4
5
10
9
4
1
1
1
3
2
4
2
<1
2
2
<1
1
<1
3
<1
1
11
70
<1
1
D-1
D-2
E-1
E-2
E-3
E-4
E-5
E-6
E-7
4.00
4.40
4.90
1.70
1.70
1.50
0.72
1.90
1.30
0.60
1.30
1.10
1.70
2.60
2.00
3.10
4.20
1.70
0.44
0.001
E-8
E-9
E-10
E-11
E-12
E-13
F
<1
<1
3
1
1
G
>20
B-1
AT-101
ABT-737
0.63
0.31
0.001
<1
Table 3
Inhibitory activities of target compounds against human cancer cell lines
No.
IC50 (lM)
HL-60
U266
MCF-7
SKOV3
NCI-H23
F
G
2.92 1.02
4.86 1.53
0.97 0.40
6.95 2.31
7.63 2.87
27.35 5.29
0.26 0.14
2.25 1.42
25.33 6.56
7.83 3.22
7.64 2.95
46.59 9.26
8.38 3.97
7.40 2.78
71.16 13.84
ABT-737
of expression of the Mcl-1 protein,41 compounds F and G didn’t
show better growth inhibitory activity than ABT-737. These results
were consistent with their binding affinities to Bcl-2, Bcl-xL and
Mcl-1 proteins.
References and notes
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In summary, it was proposed that there were obvious differ-
ences in the P1 pocket of the active site between Bcl-2, Bcl-xL,
and Mcl-1 proteins, by the structural comparison of these proteins.
As a result, the groups in their inhibitors binding to the P1 pocket
may have significant effect on the selectivity for these proteins.
Based on this hypothesis, five types of derivatives C-F were
designed by changing the corresponding group of the lead com-
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Mcl-1 proteins (such as G) were found according to the results of
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Acknowledgments
We thank the Shanghai Rising-Star Program of China
(16QA1404800), State Scholarship Fund of China, ‘Chen Guang’
project supported by the Shanghai Municipal Education Commis-
sion and the Shanghai Education Development Foundation of China
(12CG42), and the National Natural Science Foundation of China
(Grant No. 21572266) for financial support.
Supplementary data
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Supplementary data (materials and methods of computational
modeling, chemistry and biology experiments) associated with this
article can be found, in the online version, at http://dx.doi.org/10.
1016/j.bmcl.2016.09.061.
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