Development of dimeric modulators for anti-apoptotic Bcl-2 proteins

Article abstract of DOI:10.1016/j.bmcl.2007.10.088

Bcl-2 family proteins can be classified into two subfamilies-anti-apoptotic members and pro-apoptotic members. Mechanistically, these two subfamilies can antagonize each other through heterodimerization while homodimerization has been proposed for each su

Full text of DOI:10.1016/j.bmcl.2007.10.088

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 236–240
Development of dimeric modulators for anti-apoptotic
Bcl-2 proteins
Liangyou Wang,a Fansen Kong,a Candis L. Kokoski,b
David W. Andrewsb and Chengguo Xinga,*
aDepartment of Medicinal Chemistry, University of Minnesota Minneapolis, MN 55455, USA
bDepartment of Biochemistry and Biomedical Sciences, McMaster University, Ont., Canada L8S 4L8
Received 26 September 2007; revised 20 October 2007; accepted 25 October 2007
Available online 30 October 2007
Abstract—Bcl-2 family proteins can be classified into two subfamilies—anti-apoptotic members and pro-apoptotic members. Mech-
anistically, these two subfamilies can antagonize each other through heterodimerization while homodimerization has been proposed
for each subfamily to carry out their corresponding anti-apoptotic or pro-apoptotic functions. To date, many small-molecule antag-
onists against anti-apoptotic Bcl-2 proteins have been developed, which are monomeric modulators. In this study, a series of BH3I-1
based dimeric modulators were developed with structure–activity relationship explored. Dimeric modulators compared to the mono-
meric antagonists have enhanced binding activity against anti-apoptotic Bcl-2 proteins. In addition, the acidic functional group was
demonstrated to be critical for the binding interaction of the small-molecule antagonists with anti-apoptotic Bcl-2 proteins. Finally,
the representative dimeric modulator revealed enhanced activity in inducing cytochrome c release from mitochondria compared to
its monomeric counterpart. Taken together, dimerization of monomeric modulators is one practical approach to enhance the bio-
activity of Bcl-2 antagonists.
Ó 2007 Elsevier Ltd. All rights reserved.
Drug resistance is a major challenge in cancer chemo-
therapy.1 The over-expression of anti-apoptotic Bcl-2
proteins which protects cells from apoptosis is one
mechanism for tumors to acquire drug resistance.2–4
Inhibiting anti-apoptotic Bcl-2 proteins, therefore, is
one promising strategy to nullify drug resistance in can-
cer treatment, which has been proved valid through
anti-sense approach.5 Mechanistically, anti-apoptotic
Bcl-2 proteins protect cells from apoptosis by antagoniz-
ing pro-apoptotic Bcl-2 proteins through dimeriza-
tion.6,7 Structural studies of a complex of Bcl-XL
protein and a pro-apoptotic peptide (Bak BH3) have re-
vealed a hydrophobic cleft on Bcl-XL protein as the
binding pocket for the pro-apoptotic peptide8 and mol-
ecules binding to that hydrophobic cleft may overcome
the protective effect of the Bcl-XL protein.9 These obser-
vations have stimulated the recent discovery of small
molecule based antagonists—a few of which are cur-
rently at various stages of clinical evaluation, such as
gossypol, apogossypol, TW-37, and ABT-737.10–14
As Bcl-2 proteins form homo/hetero oligomers for their
physiological functions, multivalent modulators may
potentially possess unique functions that are not present
in the monomeric modulators.15 In addition, it is well
established that multivalency of ligands could enhance
potency compared to the monomeric ones.16,17 To test
these concepts in the Bcl-2 protein family, we developed
a series of dimeric analogs of 3e based on BH3I-1, a
small-molecule Bcl-2 antagonist developed by Degterev
et al.18 Several dimeric modulators demonstrated en-
hanced potency to inhibit the binding of a BH3-domain
Bak peptide binding to the anti-apoptotic Bcl-2 proteins
while dimerization through the carboxylic acid group on
3e resulted in substantial loss of the activity. Further
SAR studies confirmed that the carboxylic acid was crit-
ical for the antagonism of the small molecule against the
Bcl-2 proteins. The enhanced potency of the dimeric
modulators was further confirmed by its ability to in-
duce cytochrome c release from isolated mitochondria.
The cytochrome c release study also suggested that the
dimeric modulator functionally mimics the monomeric
antagonist.
Keywords: Bcl-2; Apoptosis; BH3I-1; Dimeric; Modulator; Mono-
meric; Antagonist; Potency.
The preparation of the dimeric modulators (3e-D1–D4)
followed the general synthetic route outlined in Scheme
*
Corresponding author. Tel.: +1 612 626 5675; e-mail: xingx009@
umn.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.10.088

L. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 236–240
237
O
O
OR
RO
S
S
O
N
N
a
O
b
S
S
OR
S
O
O
OR
N
O
NH2
S
O
O
3e-D1: R = H
3e ethyl ester dimer: R = Et
Scheme 1. General synthetic route for the syntheses of the bottom-linked dimeric modulators. Reagents and conditions: (a) CS2, NaOH;
ClCH2COONa; 5.5 M HCl, reflux, overnight; (b) 4,40-[methylenebis(oxy)]bis benzaldehyde (0.45 equivalent), NH4OAc in Toluene, reflux, 16 h. (For
the syntheses of 3e-D2, D3, and D4, 4,40-ethanediyldioxydibenzaldehyde, 4,40-(3-oxapentanediyldioxy)dibenzaldehyde, and 4,40-(3,6-diox-
aoctanediyldioxy)dibenzaldehyde were used, respectively, instead of 4,40-[methylenebis(oxy)]bis benzaldehyde).
Table 1. The inhibitory potency of BH3I-1 based modulators against
Bak peptide binding to anti-apoptotic Bcl-2 proteins
of Bak peptide binding to anti-apoptotic Bcl-2, Bcl-
XL, and Bcl-w. All of the four dimeric modulators dem-
Ki (lM)a
onstrated improved potency to prevent the binding of
Bak peptide to the three anti-apoptotic Bcl-2 proteins
tested (Table 1). The best candidate, 3e-D2, demon-
strated 5- to 16-fold increase of potency compared to
the monomeric 3e. The optimal linker between the
monomers is a four-atom ethylene glycolic liner—in-
crease or decrease of linker length would result in de-
creased activity.
Bcl-2
Bcl-XL
Bcl-w
3e
3e-D1
14.2
4.3
5.25
2.4
21.5
2.3
3e-D2
3e-D3
1.7
4.3
0.97
2.3
1.3
2.2
3e-D4
3e-D5
21.2
243
>1000
161
226
260
8.7
13.7
101
99
134
104
112
58.4
176
3e-D6
3e-D7
Delighted by these results, we further explored the other
dimerization of the monomeric 3e. 3e-D5–D7 were pre-
pared by using L-phenylalanine based rhodanine, which
first reacted with various ethylene glycol to form the di-
meric rhodanines in 34–81% yield (Supplementary
Materials). The dimeric rhodanines were then con-
densed with 5-bromobenzaldehyde (2.2 equivalent) for
3e-D5–D7 (82–95%). These three dimers, however, all
demonstrated substantial decrease of potency to prevent
Bak peptide binding to anti-apoptotic Bcl-2 proteins
(Table 1). One potential cause for the loss of activity
would be due to the mask of the carboxylic acid func-
tional group on 3e. To test this hypothesis, we synthe-
sized 3e ethyl ester and 3e ethyl ester dimer by
following the same procedure for the preparation of
3e19 and 3e-D1 except that the ethyl ester of L-phenylal-
anine was used instead of L-phenylalanine. As hypothe-
sized, 3e ethyl ester and 3e ethyl ester dimer all
demonstrated substantially weakened ability to prevent
88.3
39.2
116
3e-Dimer ethyl ester
3e Ethyl ester
a Values (Ki) are given as an average of three experiments with tripli-
cate performed in each individual experiment.
1 (The preparation of the dimeric modulators 3e-D5–D7
followed a similar synthetic route detailed in Supple-
mentary Materials.).19 Briefly, the syntheses of 3e-D1–
D4 started with L-phenylalanine, which first reacted with
carbon disulfide and cyclized upon chloroacetate treat-
ment to form the rhodanine compound in 45% yield.
The rhodanine compound (2.2 equivalent) was then con-
densed with various dimeric aldehydes in refluxing tolu-
ene to afford compound 3e-D1–D4 in 76–92% yield.
These four dimeric analogs were evaluated in the anti-
apoptotic Bcl-2 protein binding assays.19 The binding
was assessed for their activity to prevent the binding
HO2C
NO
2 H
N
N
O
N
H
N
O
H
OH
OH
O
S
OH
H
S
S
S
HO
HO
O
O
OH
H
O
N
N
Br
BH3I-1
Gossypol
OH
OH
OH
OH
HO
OH
HO
HO
H
Cl
N
OH
O
S
O
ABT-737
O
TW-37
apogossypol
Figure 1. Structures of some reported small-molecule antagonists against anti-apoptotic Bcl-2 proteins (the acidic protons are highlighted).

238
L. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 236–240
O
O
O
O
OH
S
O
OH
N
S
OH
HO
O
S
S
HO
S
N
N
N
S
N
S
S
S
O
O
O
S
O
O
O
O
Br
3e
O
n
3e-D2: n = 1
3e-D3: n = 2
3e-D4: n = 3
3e-D1
O
O
O
O
O
O
O
O
S
OEt
EtO
n
S
S
N
N
S
O
S
N
O
N
O
N
S
S
S
S
O
S
O
O
O
Br
Br
Br
3e-D5: n = 1
3e-D6: n = 2
3e-D7: n = 3
3e ethyl ester dimer
3e ethyl ester
Figure 2. Structures of BH3I-1 based monomeric modulators and dimeric modulators against anti-apoptotic Bcl-2 proteins.
cytochrome c release
Bak peptide binding to the anti-apoptotic Bcl-2 proteins
relative to 3e (>5-fold loss) and 3e-1D (>15- fold loss),
respectively. 3e Ethyl ester dimer still revealed better po-
tency than the monomeric 3e ethyl ester. These SAR
studies demonstrate the carboxylic acid functional
group on 3e is critical for its interaction with anti-apop-
totic Bcl-2 proteins. In fact, quite a few of the reported
small-molecule antagonists against anti-apoptotic Bcl-2
proteins have acidic functional groups, such as gossypol,
apogossypol, ABT-737, TW-37, and the recent flavo-
noid compound developed by Wang et al. (The acidic
protons are highlighted in Fig. 1.) Wang et al. also ob-
served that masking the acidic functional group resulted
in dramatic loss of the activity against anti-apoptotic
Bcl-2 proteins.20,21 Our study herein, in combination
with the other studies, suggests that an acidic functional
group may be optimal for small-molecule antagonists
against anti-apoptotic Bcl-2 proteins (Fig. 2).
110
100
90
80
70
60
50
40
30
20
10
0
3e
3e-D2
10-1.0 10-0.5 100.0 100.5 101.0 101.5 102.0
Concentration (μM)
3e
3e-D2
IC50 SE(μM)
69.1 3.4
6.1 3.0
Figure 3. The potency of 3e and 3e-D2 in releasing cytochrome c from
isolated mitochondria.
unambiguously identify the target of the compounds
on mitochondria.
To explore whether the monomeric modulator and di-
meric modulator may function differently, we evaluated
3e and 3e-D2 for their ability to induce cytochrome c re-
lease from mitochondria, a key feature for mitochon-
drial-mediated apoptotic cascade. Both candidates
induced cytochrome c release from the isolated mito-
chondria (Fig. 3). The respective concentration to in-
duce 50% cytochrome c release for 3e was about
69 lM while that for 3e-D2 was ꢀ6 lM, correlating with
their potency to antagonize the anti-apoptotic Bcl-2 pro-
teins. However, further investigations are required to
In summary, dimerization of monomeric antagonist 3e
could enhance the potency to bind the anti-apoptotic
Bcl-2 proteins. The dimeric modulator also had en-
hanced activity to induce cytochrome c release from
mitochondria. These studies demonstrated that the
dimerization is an effective approach to enhance the po-
tency of Bcl-2 antagonists. In addition, our SAR studies
in combination with others suggest that an acidic func-
tional group is critical for the antagonism of the small
molecules against anti-apoptotic Bcl-2 proteins. Our

bottom flask equipped with a magnetic stirrer, L
-phenyl-
L. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 236–240
239
current studies also suggest that the dimeric modulators
have the same function as the monomeric antagonists.
alanine (1 mmol) was dissolved with sodium hydroxide
(80 mg, 2 mmol) in water (10 ml). Then, carbon disulfide
(60 ll, 1 mmol) was added to the reaction mixture, which
was stirred vigorously overnight. An aqueous solution of
ClCH2CO2Na (1 ml, 1 M, 1 mmol) was added and stirring
was continued at 23 °C for 3 h. Then hydrochloric acid
solution (3 ml, 5.5 N, 16.5 mmol) was added and the
reaction mixture was refluxed overnight. The reaction
mixture was neutralized with saturated NaHCO3 solution.
The solvent was removed under vacuum and the cyclized
product was purified by flash chromatography. In a
round-bottom flask equipped with a reflux condenser
and a magnetic stirrer, cyclized rhodanine intermediate
(1 mmol) in toluene (20 ml) was added. To this, 4,40-
[methylenebis(oxy)]bis benzaldehyde (0.45 mmol) was
added along with ammonium acetate (3 mmol). The
mixture was refluxed overnight and the solvent was
removed under vacuum. The final rhodanine candidate
was purified by flash chromatography. 3e-D1 (54%) yellow
powder. TLC (CH2Cl2/MeOH = 9:1), Rf = 0.18. M.p.
242–3 °C. IR (KBr, cmax, cmꢁ1): 3700–2200, 2923, 2852,
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.10.088.
References and notes
1. Reed, J. C.; Miyashita, T.; Takayama, S.; Wang, H. G.;
Sato, T.; Krajewski, S.; Aime-Sempe, C.; Bodrug, S.;
Kitada, S.; Hanada, M. J. Cell. Biochem. 1996, 60, 23.
2. Kiechle, F. L.; Zhang, X. Clin. Chim. Acta 2002, 326, 27.
3. Folkman, J. Semi. Cancer Biol. 2003, 13, 159.
4. Reinhold, W. C.; Kouros-Mehr, H.; Kohn, K. W.;
Maunakea, A. K.; Lababidi, S.; Roschke, A.; Stover, K.;
Alexander, J.; Pantazis, P.; Miller, L.; Liu, E.; Kirsch, I.
R.; Urasaki, Y.; Pommier, Y.; Weinstein, J. N. Cancer
Res. 2003, 63, 1000.
1695, 1590, 1506, 1344, 1273, 1239, 1218, 1170, 999 cmꢁ1
.
1H NMR (CDCl3): 7.60 (2H, s, 4-H, 40-H), 7.40 (4H, d,
J = 9 Hz, 10-H, 11-H, 100-H, 110-H), 7.30–7.10 (14H, m,
5-H, 6-H, 7-H, 8-H, 9-H, 12-H, 13-H, 50-H, 60-H, 70-H, 80-
H, 90-H, 120-H, 130-H), 6.10 (2H, t, J = 6.9 Hz, 2-H, 20-H),
5.81 (2H, s, 1-H), 3.62 (4H, d, J = 6.9 Hz, 3-H, 30-H) ESI-
MS (negative): m/z 446, 448 (MꢁꢁH); 402, 404 (MꢁꢁH–
CO2). ESI-MS (m/z, negative): 781 (Mꢁ1). The binding
assay of small-molecule antagonists to anti-apoptotic Bcl-
2 proteins was run essentially as described in Xing, C.;
Wang, L.; Tang, X.; Sham, Y. Bioorg. Med. Chem. 2007,
15, 2167. Human recombinant Bcl-2, Bcl-XL, and Bcl-w
were prepared. To determine the binding affinity of small
molecules for an anti-apoptotic Bcl-2 protein, a series of 3-
fold dilutions of small molecules were prepared in
dimethylsulfoxide, that is, 10 mM, 3.33 mM, 1.11 mM,
0.37 mM, 0.123 mM, 0.041 mM, 0.014 mM, and 0 mM.
To each well in a 96-well half-area black plate, 5 ll of the
small molecule stock solution was added. A solution
containing 50 nM Flu-Bak peptide and the anti-apoptotic
Bcl-2 protein to be tested in PBS buffer, pH 7.0, 1 mM
DTT was prepared and incubated at 23 °C for one hour.
The concentration of the anti-apoptotic Bcl-2 protein used
corresponds to the one that resulted in 60% of Flu-Bak
peptide complex with the anti-apoptotic Bcl-2 protein and
such a concentration was determined in the Flu-Bak and
Bcl-2 protein saturation binding experiment. The corre-
sponding concentrations of Bcl-2, Bcl-XL, and Bcl-w
needed to achieve 60% complex formation under the
assay conditions are 1000 nM, 880 nM, and 1200 nM,
respectively. To each well the Flu-Bak peptide and anti-
apoptotic Bcl-2 protein solution (45 ll) was added by
auto-injection at a rate of 200 ll/s. The sample was
incubated at 23 °C for one hour and shaken for two
seconds before fluorescence polarization (in mP unit) was
measured. Controls included dose–response measurements
in the absence of proteins to assess for any interactions
between the compounds and the Flu-Bak peptide. Even-
tual effects were taken into account by subtraction. The
inhibitory constant (Ki) was determined by fitting the FP
changes to the concentration of the small molecule
competing ligand by using an equation developed in
Nikolovska-Coleska, Z.; Wang, R.; Fang, X.; Pan, H.;
Tomita, Y.; Li, P.; Roller, P. P.; Krajewski, K.; Saito, N.
G.; Stuckey, J. A.; Wang, S. Anal. Biochem. 2004, 332,
261. For cytochrome c release, mitochondria were isolated
from mouse liver using differential centrifugation for
5. Chan, S. L.; Lee, M. C.; Tan, K. O.; Yang, L.; Lee, A. S.
Y.; Flotow, H.; Fu, N. Y.; Butler, M. S.; Soejarto, D. D.;
Buss, A. D.; Yu, V. C. J. Biol. Chem. 2003, 278, 20453.
6. Reed, J. C. Oncogene 1998, 17, 3225.
7. Adams, J. M.; Cory, S. Trends Biochem. Sci. 2001, 26, 61.
8. Sattler, M.; Liang, H.; Nettesheim, D. N.; Meadows, R.
P.; Harlan, J. E.; Eberstadt, M.; Yoon, H. S.; Shuker, S.
B.; Chang, B. S.; Minn, A. J.; Thompson, C. B.; Fesik, S.
W. Science 1997, 275, 983.
9. Holinger, E. P.; Chittenden, T.; Lutz, R. J. J. Biol. Chem.
1999, 274, 13298.
10. Kim, R.; Tanabe, K.; Emi, M.; Uchida, Y.; Toge, T.
Cancer 2005, 103, 2199.
11. Kitada, S.; Leone, M.; Sareth, S.; Zhai, D.; Reed, J. C.;
Pellecchia, M. J. Med. Chem. 2003, 46, 4259.
12. Oltersdorf, T.; Elmore, S. W.; Shoemaker, A. R.; Arm-
strong, R. C.; Augeri, D. J.; Belli, B. A.; Bruncko, M.;
Deckwerth, T. L.; Dinges, J.; Hajduk, P. J.; Joseph, M. K.;
Kitada, S.; Korsmeyer, S. J.; Kunzer, A. R.; Letai, A.; Li,
C.; Mitten, M. J.; Nettesheim, D. J.; Ng, S.; Nimmer, P.
M.; O’Connor, J. M.; Oleksijew, A.; Petros, A. M.; Reed,
J. C.; Shen, W.; Tahir, S. K.; Thompson, C. B.; Tomaselli,
K. J.; Wang, B.; Wendt, M. B.; Zhang, H.; Fesik, S. W.;
Rosenberg, S. H. Nature 2005, 435, 677.
13. Tanabe, K.; Kim, R.; Inoue, H.; Emi, M.; Uchida, Y.;
Toge, T. Int. J. Oncol. 2003, 22, 875.
14. Wang, J.; Liu, D.; Zhang, Z.; Shan, S.; Han, X.;
Srinivasula, S. M.; Croce, C. M.; Alnemri, E. S.; Huang,
Z. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 7124.
15. Clackson, T.; Yang, W.; Rozamus, L. W.; Hatada, M.;
Amara, J. F.; Rollins, C. T.; Stevenson, L. F.; Magari, S.
R.; Wood, S. A.; Courage, N. L.; Lu, X.; Cerasoli, F., Jr.;
Gilman, M.; Holt, D. A. Proc. Natl. Acad. Sci. U.S.A.
1998, 95, 10437.
16. Zhang, Z.; Merritt, E. A.; Ahn, M.; Roach, C.; Hou, Z.;
Verlinde, C. L. M. J.; Hol, W. G. J.; Fan, E. J. Am. Chem.
Soc. 2002, 124, 12991.
17. Green, N. S.; Palaninathan, S. K.; Sacchettini, J. C.; Kelly,
J. W. J. Am. Chem. Soc. 2003, 125, 13404.
18. Degterev, A.; Lugovskoy, A.; Cardone, M.; Mulley, B.;
Wagner, G.; Mitchison, T.; Yuan, J. Nat. Cell Biol. 2001,
3, 173.
19. The general experimental procedure for preparing the
BH3I-1 based antagonists is described below with the
synthesis of analog 3e-D1 as an example. In a round-

240
L. Wang et al. / Bioorg. Med. Chem. Lett. 18 (2008) 236–240
incubation with exogenous proteins as described by
samples contained an equal concentration of DMSO.
Mitochondria were incubated for 1 h at 30 °C, after which
the membranes were spun at 13,000g in a microcentrifuge
for 10 min to pellet mitochondria. Samples from the
supernatant and pellet were run on a 10% Tricene gel and
immunoblotted for cytochrome c using a sheep anti-
cytochrome c antibody (Ex Alpha Biologicals). Percent
cytochrome c release was determined using ImageQuant
5.2 (Molecular Dynamics).
Frezza, C.; Cipolat, S.; Scorrano, L. Nat. Protoc. 2007,
2, 287. Briefly, livers were removed from euthanized
animals and immersed in isolation buffer, IBc (10 mM
Tris/MOPS, 1 mM EGTA/Tris, 0.2 M sucrose, pH 7.4).
Samples were washed 5 times with IBc to remove excess
blood and minced on ice in IBc. The tissue was then
homogenized in a glass potter using a Teflon pestle for 4
strokes in fresh IBc. The homogenate was centrifuged at
600g for 10 min at 4 °C in a JA 25.4 rotor (Beckman). The
supernatant was then centrifuged at 7000g for 10 min to
pellet mitochondria. The pellet was washed in fresh IBc.
The final pellet was resuspended in a small amount of IBc.
Samples were quantified by Bradford and diluted to 1 mg/
ml in experimental buffer, EBc (125 mM KCl, 1 mM Tris/
MOPS, 0.1 mM EGTA/Tris, 0.1 mM KH2PO4). Isolated
mitochondria were incubated with increasing concentra-
tions of Monomer and Dimer solubilized in DMSO. All
20. Wang, G.; Nikolovska-Coleska, Z.; Yang, C. Y.; Wang,
R.; Tang, G.; Guo, J.; Shangary, S.; Qiu, S.; Gao, W.;
Yang, D.; Meagher, J.; Stuckey, J.; Krajewski, K.; Jiang,
S.; Roller, P. P.; Abaan, H. O.; Tomita, Y.; Wang, S. J.
Med. Chem. 2006, 49, 6139.
21. Tang, G.; Ding, K.; Nikolovska-Coleska, Z.; Yang, C. Y.;
Qiu, S.; Shangary, S.; Wang, R.; Guo, J.; Gao, W.;
Meagher, J.; Stuckey, J.; Krajewski, K.; Jiang, S.; Roller,
P. P.; Wang, S. J. Med. Chem. 2007, 50, 3163.