Design, synthesis, and evaluation of a potent, cell-permeable, conformationally constrained second mitochondria derived activator of caspase (Smac) mimetic

Article abstract of DOI:10.1021/jm061108d

A potent, cell-permeable, conformationally constrained second mitochondria derived activator of caspase mimetic (SM-131, 2) has been designed, synthesized, and evaluated. Compound 2 binds to X-linked inhibitors of apoptosis proteins (XIAP) with a K_i<

Full text of DOI:10.1021/jm061108d

7916
J. Med. Chem. 2006, 49, 7916-7920
Design, Synthesis, and Evaluation of a Potent, Cell-Permeable, Conformationally Constrained
Second Mitochondria Derived Activator of Caspase (Smac) Mimetic
Haiying Sun,† Zaneta Nikolovska-Coleska,† Jianfeng Lu,† Su Qiu,† Chao-Yie Yang,† Wei Gao,† Jennifer Meagher,‡
Jeanne Stuckey,‡ and Shaomeng Wang*,†
ComprehensiVe Cancer Center, Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry, and Life Sciences Institute,
UniVersity of Michigan, 1500 E. Medical Center DriVe, Ann Arbor, Michigan 48109
ReceiVed September 24, 2006
A potent, cell-permeable, conformationally constrained second mitochondria derived activator of caspase
mimetic (SM-131, 2) has been designed, synthesized, and evaluated. Compound 2 binds to X-linked inhibitors
of apoptosis proteins (XIAP) with a Ki of 61 nM in a competitive binding assay and directly antagonizes
the XIAP inhibition of caspase-9 activity in a cell-free functional assay. Compound 2 achieves an IC50 of
100 nM in inhibition of cell growth and effectively induces cell death in the MDA-MB-231 human breast
cancer cell line.
Introduction
BIR3.14,15 The binding site in the BIR3 domain of XIAP where
Smac and caspase-9 bind is ideally suited for the design of
small-molecule Smac mimetics that antagonize XIAP.8 Because
XIAP is widely expressed in human cancers, Smac mimetics
may hold great promise for the treatment of many types of
human cancers by promoting apoptosis in cancer cells.18-24
Apoptosis, or programmed cell death, is a critical cell process
in normal development and homeostasis of multicellular organ-
isms.1 Apoptosis dysfunction is a hallmark of human cancers
and plays a major role in the failure of current anticancer drugs.2
Design and development of small molecules that target critical
apoptosis regulators to induce cancer cells to undergo apoptosis
is an attractive therapeutic approach for the development of new
classes of anticancer agents.3,4
Design and Synthesis
Our laboratory has previously reported the structure-based
design and synthesis of a series of conformationally constrained
Smac mimetics.22,23 Among them, 1 is the most potent Smac
mimetic and binds to a recombinant XIAP BIR3 protein with a
Ki of 25 nM in our competitive, fluorescence-polarization-based
(FP-based) binding assay.22 However, 1 has very weak activity
in cell-based assays,22 a major shortcoming that must be
overcome for the development of our Smac mimetics as new
anticancer drugs. Herein, we report the design, synthesis, and
biochemical and biological evaluation of a potent Smac mimetic
based on 1 but with dramatically improved cellular activity. In
addition, we show that our potent Smac mimetics function as
direct antagonists of XIAP BIR3 protein in a cell-free functional
assay.
Analysis of the chemical structure of 1 showed that it contains
a primary amino group that is positively charged at physiological
pHs, which may account for its poor cell permeability.
Consistent with this observation, peptide-based Smac mimetics
in which this free amino group is methylated are found to have
potent cellular activity.24 We have therefore focused our
modifications on the primary amino group in 1 to improve its
cell permeability.
Our modeled structure of 1 complexed with XIAP BIR3
showed that the primary amino group forms an extensive
hydrogen bond network with a number of negatively charged
residues in XIAP, including Asp309 and Glu314 residues.22
Indeed, blocking of this amino group with an acetyl group in
Smac-based peptides made the resulting peptides inactive in
binding to XIAP BIR3,21 indicating the critical importance of
the hydrogen-bonding network for binding between Smac
mimetics and XIAP BIR3. Accordingly, we have designed a
series of analogues of 1, in which the primary amino group has
been replaced with a methylated secondary or tertiary amino
group or with a hydroxyl group (Figure 1). Under physiological
conditions, the methylated secondary or tertiary amino group
Inhibitors of apoptosis proteins (IAPs) are a class of key
apoptosis inhibitors.5,6 Among all the IAP members, the
X-linked IAP (XIAP) has attracted the greatest attention because
of its potent biological functions and its role in cancer.5-9 XIAP
has been found to be widely expressed in many human cancer
cell lines and tumor tissues,7 and such overexpression confers
apoptosis resistance on cancer cells to various anticancer
agents.8,9 Accordingly, targeting XIAP is a promising therapeutic
strategy for the design of a new class of anticancer therapy.7-9
Smac/DIABLO (second mitochondria derived activator of
caspase or direct IAP-binding protein with low pI) is a potent
proapoptotic protein.5,6 It promotes apoptosis, at least in part,
by directly interacting with XIAP and other IAP members and
inhibiting their antiapoptotic function.10-15 XIAP contains three
baculoviral IAP repeat (BIR) domains.5,6 Its BIR2 domain,
together with the linker before the BIR2 domain, binds to
effector caspases-3 and -7 and inhibits their activity, and its
BIR3 domain binds to and inhibits an initiator caspase-9.5,6
Biological and structural studies have demonstrated that Smac
binds to the BIR3 domain of XIAP, where caspase-9 binds.13-17
The binding of Smac protein to XIAP blocks the XIAP-
caspase-9 interaction and relieves the inhibition of XIAP to
caspase-9.16,17 This leads to an increased activity of caspase-9
and other caspases in cells and promotes apoptosis.
High-resolution X-ray crystal and NMR solution experiments
have shown that the interaction between Smac and the BIR3
domain of XIAP is mediated by the N-terminal four residues
(AVPI) in Smac and a well-defined surface groove in XIAP
* 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.
‡ Life Sciences Institute.
10.1021/jm061108d CCC: $33.50 © 2006 American Chemical Society
Published on Web 11/23/2006

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7917
Figure 1. Chemical structures of designed Smac mimetics.
Scheme 1. Synthesis of New Analogues of Compound 1a
Figure 3. Evaluation of Smac mimetics in cell-free caspase-9
functional assay.
1000 times less potent than 1. These binding data indicate that
primary and secondary amino groups are clearly preferred for
Smac mimetics to interact with XIAP BIR3 at this site and to
achieve high affinity, whereas a tertiary amino group or a
hydroxyl group is highly detrimental to binding.
a Reagents and conditions: (a) aminodiphenylmethane, EDC, HOBt, N,N-
diisopropylethylamine, CH2Cl2, 0 °C- to room temp, 92%; (b) (i) 4 N
HCl in 1,4-dioxane, MeOH; (ii) (S)-N-methyl-N-Boc-aminobutyric acid,
EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, room temp; (iii) 4 N HCl
in 1,4-dioxane, MeOH, 79% over three steps; (c) (i) 4 N HCl in 1,4-dioxane,
MeOH; (ii) L-lactic acid, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2,
room temp; 82% over rwo steps; (d) formaldehyde (37.4% solution in water),
NaBH3CN, MeOH, 78%.
Our Smac mimetics are designed to target XIAP BIR3, which
is known to bind to caspase-9 and effectively inhibits caspase
activity. We next sought to evaluate if these Smac mimetics
function as direct antagonists of XIAP in a functional assay
and if their potencies correlate with their binding affinities to
XIAP BIR3. For this purpose, we established a cell-free
functional assay using recombinant XIAP BIR3 protein and cell
lysates (Supporting Information) and tested our Smac mimetics
for their ability to directly overcome the inhibitory effect of
XIAP BIR3 protein to caspase-9 activity in this functional assay
(Figure 3). In this assay, addition of dATP and cytochrome c
into cell lysates activates caspase-9 (Figure 3 and Support
Information). As expected, recombinant XIAP BIR3 protein
effectively inhibits the activity of caspase-9 (Figure 3). Con-
sistent with their high binding affinities, 1 and 2 are potent and
effective in relieving the inhibition of XIAP BIR3 to caspase-9
activity. At 3 and 10 µM respectively, 1 and 2 can restore the
activity of caspase-9 by 40% and 75%, respectively, in the
presence of 0.5 µM XIAP BIR3 protein. In comparison, 3 with
a low binding affinity to XIAP BIR3 is much less effective in
this functional assay than 1 and 2. At 10 µM, 3 has a minimal
effect, and at 100 µM it restores only 15% of the activity of
caspase-9. Thus, these functional data show that our designed
Smac mimetics act as direct antagonists of XIAP BIR3 protein
and their potencies in this functional assay nicely correlate with
their binding affinities to XIAP BIR3.
Figure 2. Competitive binding curves of designed Smac mimetics to
recombinant XIAP BIR3 protein as determined using a fluorescence-
polarization-based assay.
or the hydroxyl group should still be capable of forming one or
more hydrogen bonds with XIAP BIR3.
Compounds 2-4 were synthesized using the procedures
outlined in Scheme 1. Briefly, the key intermediate 5 was
synthesized using the same procedure in our previous publica-
tion.22 Condensation of 5 with aminodiphenylmethane gave 6.
Removal of the Boc protecting group in 6 followed by
condensation of the resulting ammonium salt with (S)-N-methyl-
N-Boc-aminobutyric acid afforded an amide. Removal of the
Boc protecting group in this amide by treatment with HCl in
methanol yielded 2. Condensation of the ammonium salt derived
from 6 by deprotection of the Boc protecting group with L-lactic
acid furnished 4. Condensation of 1 with formaldehyde followed
by reduction of the resulted enamine with NaBH3CN gave 3.
Our major goal for the design and synthesis of these new
Smac mimetics is to improve their cellular activity. For this
purpose, we have evaluated them for their activity in a number
of cell-based assays.
These compounds were evaluated for their activity in inhibit-
ing cell growth in the MDA-MB-231 human breast cancer cell
line using the WST-based assay (Support Information). The
results are shown in Figure 4. As can be seen, 1 has an IC50 of
50 µM in inhibition of cell growth. In comparison, 2 achieves
an IC50 of 100 nM and is 500 times more potent than 1.
Although 3 has a much weaker binding affinity than 1, it is
more potent than 1 with an IC50 of 2.9 µM, indicating its
superior cell permeability. Compound 4 with a hydroxyl group
has an IC50 of 65 µM, consistent with its very weak binding
affinity to XIAP BIR3.
Results and Discussion
Compounds 2-4, together with 1, were first evaluated for
their binding affinities to recombinant XIAP BIR3 protein using
our FP-based competitive.25 The results are shown in Figure 2.
Compound 2 binds to XIAP BIR3 with a Ki of 61 nM in this
competitive binding assay, while 3 and 4 have Ki of 14.4 and
29.9 µM, respectively. In comparison, 1 has a Ki of 25 nM.
Thus, 2 with a secondary amino group is 2 times less potent in
our binding assay than 1 with a primary amino group.
Compound 3, with a tertiary amino group, is more than 500
times less potent than 1. Compound 4 with a hydroxyl group is
XIAP functions as a potent inhibitor of cell death, and Smac
mimetics may effectively induce cell death in certain cancer

7918 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26
Brief Articles
1 and 4 at 10 µM have a minimal effect in induction of caspase-9
processing and cleavage of caspase-3. Compound 3 at 1 and
10 µM clearly induces cleavage of caspase-3, although to lower
degree than 2. These data are highly consistent with the activity
of these compounds in inhibition of cell growth and induction
of cell death (Figures 4 and 5).
In summary, our chemical modifications on the primary
amino group in 1 yielded a series of new analogues. Of these,
2 binds to XIAP BIR3 with a Ki of 61 nM, similar to that of 1.
Consistent with their modes of action, 1 and 2 function as potent
and direct antagonists of XIAP BIR3 in promoting the activity
of caspase-9 in a cell-free functional assay. However, 2 is 500
times more potent than 1 in inhibition of cell growth in the
MDA-MB-231 human breast cell line. Compound 2 is highly
effective in induction of cell death and activates caspase-9 and
-3 in the MDA-MB-231 cancer cells. Significantly, 2 shows
minimal toxicity to normal cells (data not shown). Collectively,
these data demonstrate that 2, which we named as SM-131
(Smac mimetic 131), is a potent, cell-permeable, and promising
Smac mimetic. Compound 2 is an excellent pharmacological
tool to elucidate the role of XIAP and other IAP proteins in
human cancer and other diseases and warrants extensive
evaluation in vitro and in vivo as a potential new anticancer
therapy.
Figure 4. Inhibition of cell growth by Smac mimetics in the MDA-
MB-231 human breast cancer cell line. Cells were treated for 4 days,
and cell growth inhibition was determined using the WST-based assay.
Figure 5. Induction of cell death in the MDA-MB-231 breast cancer
cells by Smac mimetics. Cells were treated for 4 days, and cell viability
was determined using the Trypan blue exclusion assay.
Experimental Section
I. Chemistry. General Methods. 1H NMR spectra were recorded
at 300 MHz and 13C NMR spectra at 75 MHz on a Bruker
ADVANCE300 spectrometer. 1H chemical shifts are reported with
TMS (0.00 ppm) or DHO (4.70 ppm) as internal standards. 13C
chemical shifts are reported with CDCl3 (77.00 ppm) or 1,4-dioxane
(67.16 ppm) as internal standards.
All starting materials, solvents, and silica gel were purchased
from Aldrich, Fisher, or Lancaster and were used without further
purification. The final products were purified by a C18 reversed-
phase semipreparative HPLC column with solvent A (0.1% of TFA
in water) and solvent B (0.1% of TFA in CH3CN) as eluents.
(3S,6S,10S)-[3-(Benzhydrylcarbamoyl)-5-oxooctahydropyrrolo-
[1,2-a]azepin-6-yl]carbamic Acid tert-Butyl Ester (6). To a
mixture of 5 (320 mg, 1 mmol), EDC (230 mg, 1.2 mmol), HOBt
(160 mg, 1.2 mmol), and aminodiphenylmethane (185 mg, 1 mmol)
in 5 mL of CH2Cl2 was added 0.6 mL of N,N-diisopropylethylamine
at 0 °C. The mixture was warmed to room temperature and stirred
overnight. The solvent was evaporated and the residue was purified
by chromatography on silica gel to give 6 (440 mg, yield 92%).
1H NMR (300 M Hz, CDCl3) δ 7.99 (brd, J ) 8.7 Hz, 1H), 7.37-
7.19 (m, 10H), 6.22 (d, J ) 8.7 Hz, 1H), 5.96 (d, J ) 6.1 Hz, 1H),
4.80 (d, J ) 7.4 Hz, 1H), 4.28 (m, 1H), 3.75 (m, 1H), 2.43 (m,
1H), 2.24 (m, 1H), 1.91-1.60 (m, 6H), 1.46 (s, 9H), 1.25 (m, 1H),
1.03 (m, 1H); 13C NMR (75 MHz,CDCl3) δ 173.27, 169.66, 155.05,
141.98, 141.20, 128.62, 128.47, 127.34, 127.23, 127.03, 61.00,
60.36, 59.40, 56.69, 54.06, 35.01, 33.41, 31.88, 28.32, 27.41, 25.21.
(3S,6S,10S)-6-((S)-2-Methylaminobutyrylamino)-5-oxooctahy-
dropyrrolo[1,2-a]azepine-3-carboxylic Acid Benzhydrylamide
(2). To a solution of compound 6 (95 mg, 0.2 mmol) in 3 mL of
MeOH was added 1 mL of a solution of 4 N HCl in 1,4-dioxane.
The solution was stirred at room temperature overnight and then
evaporated. The residue was suspended in 5 mL of CH2Cl2. To
this mixture were added L-N-Boc-N-methyl-2-aminobutyric acid (50
mg, 0.25 mmol), EDC (58 mg, 0.3 mmol), HOBt (40 mg, 0.3
mmol), and N,N-diisopropylethylamine (0.2 mL). After the mixture
was stirred at room temperature overnight, the solvent was
evaporated and the residue was purified by chromatography to give
an amide. To a solution of this amide in 3 mL of methanol was
added 0.5 mL of 4 N HCl in 1,4-dioxane. The solution was stirred
at room temperature overnight and then evaporated to give crude
2. The crude product was purified by HPLC on a C18 reversed-
phase column to give the pure compound as a salt with trifluoacetic
Figure 6. Western blot analysis of activation of caspase-9 and -3 in
the MDA-MB-231 breast cancer cells induced by Smac mimetics. Cells
were treated for 24 h by Smac mimetics, and procaspase-9 and cleaved
caspase-3 were examined using specific monoclonal antibodies.
cells where XIAP acts as the final defensive mechanism to
protect cells from undergoing cell death. We have tested these
compounds for their ability to induce cell death in the MDA-
MB-231 cell line using the Trypan blue exclusion assay (Figure
5 and Support Information). Our data showed that 2 effectively
induces cell death in MDA-MB-231 cells in a dose-dependent
manner. Significant cell death induction was observed at
concentrations as low as 10 nM. When cells were treated with
100 nM and 1 µM 2 for 4 days, 40% and 80% of cells underwent
cell death. These data showed that 2 is a highly effective and
potent inducer of cell death in the MDA-MB-231 cancer cells.
Consistent with their activity in inhibition of cell growth, 1, 3,
and 4 are much less potent than 2 in induction of cell death in
MDA-MB-231 cells, and 3 is clearly more potent than 1 and 4.
Hence, their activities in induction of cell death for 1-4 correlate
with their potencies in inhibition of cell growth.
Potent and cell-permeable Smac mimetics are predicted to
activate caspase-9 and -3 by overcoming the inhibition of XIAP
in cancer cells. We have therefore evaluated 1-4 for their ability
to activate caspase-9 and -3 in the MDA-MB-231 cells using
Western blot analysis. The results are shown in Figure 6.
As can be seen, 2, effectively and in a dose-dependent
manner, induces a decrease of procaspase-9 protein, indicative
of processing and activation of procaspase-9. Induction of
caspase-9 processing is evident using 0.1 µM 2 and becomes
more pronounced at 1 and 10 µM. In addition, 2 at concentra-
tions as low as 0.1 µM also causes significant caspase-3
cleavage, indicative of activation of caspase-3. In comparison,

Brief Articles
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 26 7919
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acid (95 mg, yield 79%). The gradient ran from 75% of solvent A
and 25% of solvent B to 60% of solvent A and 40% of solvent B
in 30 min. The purity was confirmed by analytical HPLC to be
over 98%. 1H NMR (300 M Hz, D2O) δ 7.34-7.19 (m, 10H), 5.96
(s, 1H), 4.52-4.46 (m, 2H), 3.91 (m, 1H), 3.73 (m, 1H), 2.56 (s,
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tahydropyrrolo[1,2-a]azepine-3-carboxylic Acid Benzhydryla-
mide (3). To a solution of 1 (salt with HCl, 100 mg, 0.2 mmol) in
5 mL of methanol was added 1 mL of formaldehyde solution
(37.4% in water). The solution was cooled to 0 °C and stirred for
10 min. Then NaBH3CN (80 mg, 1 mmol) was added. After the
mixture was stirred for 1 h at 0 °C, 10 mL of water was added to
quench the reaction. The mixture was extracted by CH2Cl2 three
times. The combined organic layers were dried over Na2SO4 and
then evaporated. The residue was purified by chromatography to
give 3. To a solution of 3 in CH2Cl2 was added 3 equiv of
trifluoroacetic acid. After removal of solvent, the residue was
purified by HPLC to give the pure product (salt with TFA, 94 mg,
yield 78%). The gradient ran from 75% of solvent A and 25% of
solvent B to 60% of solvent A and 40% of solvent B in 30 min.
The purity was confirmed by analytical HPLC to be over 98%. 1H
NMR (300 M Hz, D2O) δ 7.30-7.14 (m, 10H), 5.89 (s, 1H), 4.48-
4.36 (m, 2H), 3.79 (m, 1H), 3.66 (m, 1H), 2.76 (s, 3H), 2.71 (s,
3H), 2.10-1.38 (m, 12H), 0.84 (t, J ) 7.3 Hz, 3H); 13C NMR (75
MHz, D2O) δ 173.46, 172.35, 167.65, 141.61, 141.45, 129.51,
129.43, 128.34, 127.88, 127.70, 70.00, 62.59, 60.13, 58.19, 54.46,
43.06, 41.08, 33.45, 33.07, 28.34, 27.55, 22.26, 8.84.
(3S,6S,10S)-6-((S)-2-Hydroxypropionylamino)-5-oxooctahy-
dropyrrolo[1,2-a]azepine-3-carboxylic Acid Benzhydrylamide
(4). To a solution of 6 (110 mg, 0.23 mmol) in 3 mL of MeOH
was added 1 mL of a solution of 4 N HCl in 1,4-dioxane. The
solution was stirred at room temperature overnight and then
evaporated. The residue was suspended in 5 mL of CH2Cl2. To
this mixture were added L-lactic acid (22 mg, 0.23 mmol), EDC
(58 mg, 0.3 mmol), HOBt (40 mg, 0.3 mmol), and N,N-diisopro-
pylethylamine aminodiphenylmethane (0.2 mL). After the mixture
was stirred at room temperature overnight, the solvent was
evaporated and the residue was purified by chromatography to give
4 (84 mg, yield 82%). Compound 4 was purified by HPLC again.
The gradient ran from 70% of solvent A and 30% of solvent B to
50% of solvent A and 50% of solvent B. The purity was confirmed
by analytical HPLC to be over 98%. 1H NMR (300 M Hz, CDCl3)
δ 7.86 (d, J ) 8.7 Hz, 1H), 7.65 (d, J ) 6.8 Hz, 1H), 7.45-7.15
(m, 10H), 6.22 (d, J ) 8.7 Hz, 1H), 4.75 (d, J ) 6.4 Hz, 1H), 4.53
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2.23 (m, 1H), 2.15-1.58 (m, 6H), 1.43 (d, J ) 6.8 Hz, 3H), 1.26
(m, 1H), 1.13 (m, 1H); 13C NMR (75 MHz, CDCl3) δ 173.77,
172.83, 169.63, 141.90, 141.17, 128.65, 128.51, 127.40, 127.32,
127.28, 127.13, 68.14, 61.10, 59.41, 56.74, 52.59, 34.83, 33.34,
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Acknowledgment. We are grateful for financial support from
the National Cancer Institute, National Institutes of Health
(Grant R01CA109025), the Breast Cancer Research Foundation,
the Susan G. Komen Foundation, the Prostate Cancer Founda-
tion, the University of Michigan Cancer Center (Core Grant
P30CA046592), and Ascenta Therapeutics Inc.
Supporting Information Available: Information on the binding
assay, cell-free functional assay, cell growth assay, cell death
induction, and Western blot analysis. This material is available free
of charge via the Internet at http://pubs.acs.org.
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