Bivalent Smac mimetics with a diazabicyclic core as highly potent antagonists of XIAP and cIAP1/2 and novel anticancer agents

Article abstract of DOI:10.1021/jm201072x

Nonpeptidic, bivalent Smac mimetics designed based upon monovalent Smac mimetics with a diazabicyclic core structure bind to XIAP, cIAP1, and cIAP2 with low to subnanomolar affinities and are highly effective in antagonizing XIAP in cell-free functional a

Full text of DOI:10.1021/jm201072x

Article
pubs.acs.org/jmc
Bivalent Smac Mimetics with a Diazabicyclic Core as Highly Potent
Antagonists of XIAP and cIAP1/2 and Novel Anticancer Agents
Yuefeng Peng,‡,† Haiying Sun,† Jianfeng Lu, Liu Liu, Qian Cai,§ Rong Shen,∥ Chao-Yie Yang, Han Yi,
and Shaomeng Wang*
Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology, and Medicinal Chemistry,
University of Michigan, 1500 E. Medical Center Drive, Ann Arbor, Michigan 48109, United States
ABSTRACT: Nonpeptidic, bivalent Smac mimetics designed
based upon monovalent Smac mimetics with a diazabicyclic
core structure bind to XIAP, cIAP1, and cIAP2 with low to
subnanomolar affinities and are highly effective in antagonizing
XIAP in cell-free functional assays. They efficiently induce the
degradation of cIAP1 and cIAP2 in cancer cells at concentra-
tions as low as 1 nM, activate caspase-3 and -8, and cleave
PARP at 3−10 nM. The most potent compounds in the series
have IC50 of 3−5 nM in inhibition of cell growth in both MDA-MB-231and SK-OV-3 cell lines and are promising lead
compounds for the development of a new class of cancer therapy.
cIAP2, and XIAP.14,15 The interaction between Smac and
INTRODUCTION
Apoptosis, or programmed cell death, is a cell process critical
■
IAPs is mediated by the N-terminal AVPI tetrapeptide motif
in Smac and one or more BIR domains in these IAP proteins.16,17
for homeostasis, normal development, host defense, and
Smac is a homodimer that binds to both the BIR2 and BIR3
suppression of oncogenesis. Faulty regulation of apoptosis has
been implicated in many human diseases,1 including cancer,2,3
domains in XIAP and antagonizes the inhibition of XIAP to
caspase-3/-7 and caspase-9.18 In comparison, Smac binds to
and it is now recognized that resistance to apoptosis is a
only the BIR3 domain in cIAP1 and cIAP219 and induces the
proteins’ rapid degradation in cells.20 Through two distinct
mechanisms, Smac is a very efficient antagonist of these
three IAP proteins.
hallmark of cancer.4 As a consequence, targeting of key
apoptosis regulators has emerged as an attractive strategy for
the development of new approaches to human cancer
treatment.1
Although their roles are not limited to regulation of apoptosis,7,8
inhibitors of apoptotic proteins (IAP) are a class of key
apoptosis regulators and are characterized by the presence of
one or more BIR (baculoviral IAP repeat) domains.5,6 Among
the IAPs, cellular IAP1 (cIAP1) and cIAP2 play a key role in
the regulation of death-receptor mediated apoptosis, whereas
X-linked IAP (XIAP) inhibits both death-receptor mediated
and mitochondria mediated apoptosis by binding to and
inhibiting caspase-3/7 and caspase-9, three cysteine proteases
critical for execution of apoptosis.5 These IAP proteins are
highly overexpressed both in cancer cell lines and in human
tumor tissues and have low expression in normal cells and
tissues.9 Extensive studies have demonstrated that over-
expression of IAP proteins makes cancer cells resistant to
apoptosis induction by a variety of anticancer drugs.10−12
Hence, targeting one or more of these IAP proteins is thought
to be a novel and promising therapeutic strategy for the
treatment of human cancer.10−13
The crystal and NMR structures of XIAP BIR3 complexed
with Smac protein or Smac peptide show that the AVPI
tetrapeptide motif in Smac binds to a well-defined surface
groove in XIAP, and this interaction represents an attractive site
for the design of small-molecule XIAP inhibitors.16−18 By use of
AVPI tetrapeptide as the lead structure, several classes of
small-molecule Smac mimetics have been designed as
antagonists of XIAP and cIAP1/2.21−38 Two different
types of Smac mimetics have been designed.21−23 The first
type, designed to mimic a single AVPI binding motif, is
called monovalent Smac mimetics.21−23 The second type,
the bivalent Smac mimetics, consists of two AVPI mimetics,
tethered through a linker, to mimic the dimeric form of Smac
proteins.21−23 One key advantage for monovalent Smac
mimetics as potential drugs is that they can achieve oral
bioavailability, but a drawback is that they only have modest
potency in antagonizing full-length XIAP in functional
assays. A major advantage of bivalent Smac mimetics is
that they are much more potent antagonists of XIAP than
monovalent Smac mimetics by concurrently targeting both
BIR2 and BIR3 domains in XIAP.30 Bivalent Smac mimetics
RESULTS AND DISCUSSION
■
Smac/DIABLO (second mitochondria-derived activator of
caspases or direct IAP binding protein with low pI) is a
protein released from mitochondria in response to apoptotic
stimuli and functions as an endogenous inhibitor of cIAP1,
Received: August 10, 2011
Published: December 7, 2011
© 2011 American Chemical Society
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Figure 1. Chemical structures of representative Smac mimetics reported by our laboratory. Compounds 1−5 are monovalent Smac mimetics, and
compounds 6 and 7 are bivalent Smac mimetics.
Table 1. Binding Affinities of Newly Designed Smac Mimetics to XIAP L-BIR2-BIR3, cIAP1 BIR3, cIAP2 BIR3, XIAP BIR3,
a
and XIAP BIR3 Proteins, As Determined in Fluorescence-Polarization Assays
XIAP L-BIR2-BIR3
cIAP1 BIR3
cIAP2 BIR3
XIAP BIR3
XIAP BIR2
compd
IC50 (nM)
Ki (nM)
IC50 (nM)
Ki (nM)
IC50 (nM)
Ki (nM)
IC50 (nM)
Ki (nM)
IC50 (μM)
Ki (μM)
1
1240 42
169 28
7.5 0.8
6.4 2.7
13.7 3.8
7.6 0.3
7.8 2.0
10.0 1.3
17.7 1.7
11.5 3.2
6.3 1.3
24.0 13
408 14
46.2 8.3
7.6 0.1
4.6 0.7
2.8 0.8
6.1 0.7
4.5 0.4
8.6 1.6
12.9 1.9
12.4 2.6
17.4 1.9
8.5 2.2
21.2 1.7
6.7 1.2
1.1 0.1
0.5 0.1
74.7 12.1
14.5 2.5
8.5 4.2
8.2 1.9
9.9 0.6
8.2 1.0
18.3 2.9
3.0 0.7
2.0 1.0
636 51
208 18
191 15
18.9 4.0
14.3 1.1
8.2 1.5
5.5 0.5
4
51
2
2
3
2
2
2
3
2
1
5
9
70
45
39
6
2
3
b
6
0.2
b
153
5
b
7
1
<0.5
2
0.4
134 11
188 37
145 19
161 23
217 11
289 39
311 43
203 22
715 114
b
8
1
0.7 0.1
0.4 0.1
1.2 0.3
2.0 0.4
1.9 0.5
2.9 0.4
1.2 0.4
3.6 0.3
1.7 0.2
1.3 0.3
3.3 0.7
6.4 1.1
6.9 1.2
63 13
b
b
b
b
b
b
9
0.1
0.4
0.3
0.3
0.6
0.2
48
54
73
7
8
4
10
11
12
13
14
15
15
27
29
52
18
43
3
4
4
6
2
5
4.3 0.5
9.4 0.1
1.1 0.2
3.4 0.1
98 13
14
4.0 0.5
11
2
106 15
68
8
b
2
1
246 39
a
b
Compounds 1, 4, 6, and 7 were included as control compounds for comparison. Exceeded lower assay limits: Ki values are estimated.
are typically 2−3 orders of magnitude more potent than their
monovalent Smac mimetic counterparts in induction of
apoptosis in cancer cells.21
Currently, three monovalent and two bivalent Smac mimetics
have been advanced into clinical trials for the treatment of
human cancer.21 A number of representative monovalent and
bivalent Smac mimetics designed by our laboratory are shown
in Figure 1. Compound 5 (AT-406), an orally active Smac
mimetic, is currently in phase I clinical trials for the treatment
of solid tumors and leukemia.37
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Figure 2. Predicted binding models of compound 4 in complex with XIAP (A, B) BIR3 and (C, D) BIR2. The crystal structure of Smac AVPI
peptide (green color) in complex with XIAP BIR3 and the predicted binding model of the same peptide with BIR2 are superimposed in (A) and
(C), respectively. Key residues around the binding site are shown and labeled. The dimer linkage sites in compound 4 are circled in yellow.
Figure 3. Chemical structures of designed new bivalent Smac mimetics based upon the core structure of monovalent Smac mimetics 4 and 5.
Since bivalent Smac mimetics are much more potent than
monovalent Smac mimetics in targeting XIAP and cIAP1/2 and
in induction of apoptosis of cancer cells in vitro and in vivo and
in inhibition of tumor growth, we have pursued the design and
development of such compounds for cancer treatment.21 In
earlier studies,30,31,38 we reported the design of a series of
bivalent Smac mimetics, exemplified by compounds 6 and 7,
based upon the core structure of monovalent Smac mimetic 1.
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In the present study, we report the design, synthesis, and
evaluation of a new class of bivalent Smac mimetics containing
a diazabicyclic core structure contained in monovalent Smac
mimetics 4−6, which have a favorable pharmacokimetic (PK)
and toxicity profile. For example, compound 5 (SM-406/AT-406)
demonstrates an excellent PK and toxicity profile in rodents
and non-rodents and is now in clinical trials for cancer
treatment.37 Therefore, we have sought to design new classes of
bivalent Smac mimetics based upon the core structure in
compounds 4−6.
For the design of bivalent Smac mimetics, three key issues
need to be considered. The first is the identification of a
suitable monovalent Smac mimetic, which can bind to both the
BIR2 and BIR3 domains of XIAP with good affinities. The
second issue is the identification of a suitable tethering site, and
the third is the determination of optimal length and properties
of the linker used to tether two monovalent mimetics. In our
previous study,38 we have shown that the linker has a major
effect on the overall cellular activity of the designed bivalent
Smac mimetics by modulation of their cell permeability,
although it has a minimal effect on the biochemical binding
to XIAP, cIAP1, and cIAP2.
In our binding assays, compound 4 binds to XIAP BIR3 with
Ki = 51 nM and to XIAP BIR2 with Ki = 5.5 μM (Table 1).
Since compound 4 has good affinities to both BIR3 and BIR2
domains in XIAP, it represents an excellent monovalent lead
compound for the design of new bivalent Smac mimetics with
the objective to currently target both BIR2 and BIR3 domains
in XIAP. To identify suitable sites for tethering, we modeled 4
in a complex with the BIR2 and BIR3 domains of XIAP (Figure
2). These models revealed that the amide group in the eight-
membered ring being exposed to solvent (Figure 2) is a suitable
site for tethering. Accordingly, we designed a series of bivalent
Smac mimetics (8−15) by linking two molecules of compound
4 through this site (Figure 3). The synthesis of these
compounds is shown in Scheme 1.
evaluated their binding affinities to XIAP protein containing
only the BIR3 domain or BIR2 domain (Table 1). Our data
showed that compounds 8−12 bind to XIAP BIR3 protein with
IC50 of 145−289 nM and Ki of 48−98 nM, very similar to those
for compounds 6 and 7. Compounds 11 and 13 bind to XIAP
BIR2 protein with Ki of 1.1 and 3.4 μM, respectively (Table 1).
Therefore, these new bivalent Smac mimetics bind to XIAP
protein containing both BIR2 and BIR3 domains with affinities
>20 times higher than to XIAP BIR3 protein and >100 times
higher than to XIAP BIR2 proteins. Hence, consistent with our
previous study for compound 6,30 these data suggest that this
new class of bivalent Smac mimetics achieves a much higher
affinity to XIAP BIR2-BIR3 proteins than to XIAP BIR2 and
BIR3 proteins by concurrently binding to both BIR domains in
XIAP. Furthermore, the very similar high binding affinities to
XIAP BIR2-BIR3 protein between these new bivalent Smac
mimetics suggest that the region between BIR2 and BIR3
domains in XIAP is flexible and the protein can readily adopt a
conformation for concurrently and efficiently binding to both
of the AVPI mimetics in these bivalent Smac mimetics.
Compounds 8−12 also bind to cIAP1 BIR3 protein with
high affinities, with IC50 of 4.5−12.9 nM and Ki of 0.4−2.0 nM
(Table 1). They also have high affinities for cIAP2 BIR3 protein
with IC50 of 8.2−29 nM and Ki of 1.3−6.9 nM, a 4-fold
difference (Table 1). Compound 9 with a four-carbon linker
appears to have the highest binding affinities to these three IAP
proteins. These data show that the length of the linker in this
class of bivalent Smac mimetics has only a modest effect on
binding affinities to all three IAP proteins. Furthermore, the
binding affinities of these new, bivalent Smac mimetics to
cIAP1 and cIAP2 proteins are also similar to those of the
corresponding monovalent Smac mimetic 4 (Table 1).
To investigate if the nature of the linker has a significant
effect on binding to the three IAP proteins, we synthesized
compounds 13, 14, and 15. The linker in 13 has a length
similar to that in 12 but is less flexible because of the presence
of the phenyl group in its linker. Compound 13 binds to XIAP
BIR2-BIR3, cIAP1, and cIAP2 proteins with Ki values of 2.0,
2.9, and 14 nM, respectively, very similar to those for 12.
Hence, we concluded that the conformational restriction by the
phenyl group has no significant effect on binding affinities to
these IAP proteins. Compound 14, in which an oxygen atom is
inserted into the linker in compound 11, has Ki values of 1.0,
1.2, and 4.0 nM to XIAP, cIAP1, and cIAP2, respectively, and
thus is slightly more potent than 11. Compound 15, in which a
phenyl ring was used in the linker, has Ki values of 5.0, 3.6, and
11 nM to XIAP, cIAP1, and cIAP2, respectively. Compounds
15 and 9 have similar linker lengths, but 15 is several times less
potent than 9 in binding to the three IAP proteins. Since the
linker in 15 is much more conformationally rigid than the linker
in 9, the binding data indicate that a short, rigid linker is not
optimal for binding to these IAP proteins in this class of
compounds.
XIAP functions as a potent inhibitor of caspase-3 and
caspase-9,18 and dimeric Smac protein antagonizes XIAP by
binding concurrently to both the BIR2 and BIR3 domains.30
We thus evaluated the functional antagonism of these bivalent
Smac mimetics in in vitro functional assays. Since XIAP binds
to and antagonizes caspase-3 using its BIR2 domain, together
with the immediate linker preceding BIR2, and binds to and
antagonizes caspase-9 through its BIR3 domain, we used an
XIAP construct containing linker-BIR2-BIR3 domain (residues
120−356) in our functional assays. In the caspase-9 functional
a
Scheme 1. Synthesis of Bivalent Smac Mimetics 8−15
a
Reagents and conditions: (i) diacyl chloride, N,N-diisopropylethyl-
amine, CH2Cl2; (ii) 4 N HCl, 1,4-dioxane, methanol.
Compounds 8−12 were designed to have a linear, flexible
alkane linker with different lengths, from 2 carbon atoms (8) to
10 carbon atoms (12), in order to investigate the influence of
the linker length on binding affinities to IAP proteins and
cellular activities. In our fluorescence-polarization-based (FP-
based) binding assay,38 compounds 8−12 have similar high
affinities to XIAP protein containing both BIR2 and BIR3
domains, with IC50 ranging from 7.6 to 17.7 nM and calculated
Ki of 2−3 nM (Table 1). Their binding affinities to XIAP BIR2-
BIR3 protein exceed the lower limits of the assay, and so their
Ki values are underestimated. Our previous study has shown
that the bivalent Smac mimetic 6 achieves a much higher
affinity to XIAP containing both BIR2 and BIR3 domains than
its corresponding monovalent counterparts by concurrently
binding to both BIR domains.30 To investigate this aspect, we
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assay (Figure 4), XIAP protein dose-dependently inhibits the
activity of caspase-9, achieving 80% of inhibition at 500 nM.
Smac mimetics can effectively inhibit cell growth in a subset
of human cancer cell lines, such as the MDA-MB-231 breast
cancer and SK-OV-3 ovarian cancer cell lines.31 We tested these
new bivalent Smac mimetics in cell growth inhibition assays
against both the MDA-MB-231 and SK-OV-3 cancer cell lines,
including 4 and 6 as controls.
In the MDA-MB-231 cell line, these new bivalent Smac
mimetics inhibited cell growth with IC50 of 3.4−50.8 nM
(Figure 6). Compounds 13 and 14 are the most potent
Figure 4. Smac mimetics antagonize XIAP L-BIR2-BIR3 in an in vitro
caspase-9 functional assay. A 500 nM XIAP L-BIR2-BIR3 protein
achieves 80% inhibition of caspase-9 activity, and Smac mimetics dose-
dependently restore the activity of caspase-9. Caspase-9 activity was
determined using the Z-LEHD fluorescent substrate at the 1 h time-
point and was normalized to the control.
Consistent with their high binding affinities to XIAP,
compounds 8−15 can dose-dependently antagonize XIAP to
restore the activity of caspase-9, with IC50 of 0.5−1.0 μM, and
have potencies very similar to that of our previously reported
bivalent Smac mimetic 6. The monovalent Smac mimetics 3
and 4 can also antagonize XIAP in the caspase-9 functional
assay but is less potent than the bivalent Smac mimetics.
In the caspase-3 functional assay (Figure 5), XIAP protein at
20 nM can inhibit 90% of the enzymatic activity of caspase-3.
Figure 6. Inhibition of cell growth by Smac mimetics in the MDA-
MB-231 human breast cancer cell line. Cells were seeded in 96-well
flat-bottom cell culture plates at a density of (3−4) × 1000 cells/well
and grown overnight, then incubated with Smac mimetics for 4 days.
Cell growth was determined using a WST-based assay. Compounds 4
and 6 were included as control compounds.
compounds and have IC50 values of 3.4 and 4.7 nM,
respectively, and are slightly more potent than compound 6.
Compound 15 is the least potent among these new bivalent
Smac mimetics and has an IC50 of 50.8 nM. Since compounds
10 and 13 essentially have the same binding affinities to XIAP
and cIAP1/2 proteins (Table 1), their 5-fold difference in their
IC50 values in inhibition of cell growth is probably due to their
different cell permeability, as we have demonstrated in our
previous study.38 Hence, the linker has a significant effect on
the cellular growth inhibitory activity of these bivalent Smac
mimetics. Of note, the monovalent control compound 4 has an
IC50 of 115 nM and is thus >20 times less potent than
compounds 13 and 14.
In the SK-OV-3 cell line, these new bivalent Smac mimetics
also effectively inhibit cell growth, with IC50 of 3.1−176 nM
(Figure 7). Compounds 11 and 14 are the most potent with
IC50 values of 3.1 and 4.7 nM, respectively. Compound 15 is
also the least potent against the SK-OV-3 cell line. Compounds
11 and 14 are several times more potent than the bivalent
control compound 6 and 50 times more potent than the
monovalent control 4 in the SK-OV-3 cell line in the cell
growth assay.
Mechanistic studies have shown that upon binding to cIAP1/
2 in cells, Smac mimetics induce rapid degradation of cIAP1/
2.39,40 Upon cIAP1/2 degradation, Smac mimetics then induce
tumor necrosis factor α (TNFα) dependent apoptosis in cancer
cells that produce and secrete TNFα.39,40 Degradation of
cIAP1/2 is an essential and key early event of apoptosis
induction by Smac mimetics.39,40 To explore their cellular
mechanism of action, we performed Western blot analysis of
MDA-MB-231 cells treated for 24 h with bivalent mimetics 11
and 13 or the monovalent mimetic 4, and the results are shown
in Figure 8. Consistent with our previous results,31 these
compounds are potent and effective in induction of cIAP1
Figure 5. Smac mimetics antagonize XIAP L-BIR2-BIR3 in an in vitro
caspase-3 functional assay. Recombinant XIAP L-BIR2-BIR3 protein at
20 nM inhibits caspase-3 activity by 90%. Smac mimetics dose-
dependently reactivate caspase-3 activity. Caspase-3 activity was
determined using the Ac-DEVD-AFC fluorescent substrate at the 1
h time-point and was normalized to the control.
All of the bivalent Smac mimetics, including compound 6, show
very similar potencies in this assay and have EC50 of 8−20 nM.
However, the monovalent Smac mimetics 3 and 4 have weak
potencies with EC50 values of 9.2 and 7.5 μM, respectively, and
are thus >500 times less potent than the bivalent Smac
mimetics. Our functional data thus show that these new
bivalent Smac mimetics are highly potent in antagonizing XIAP
to restore the activity of caspase-9 and caspase-3 and are >500
times more potent than monovalent Smac mimetics in the
caspase-3 functional assay.
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Figure 7. Inhibition of cell growth by Smac mimetics in the SK-OV-3
cancer cell line. Cells were seeded in 96-well flat-bottom cell culture
plates at a density of (3−4) × 1000 cells/well and grown overnight,
then incubated with Smac mimetics for 4 days. Cell growth was
determined using a WST-based assay. Compounds 4 and 6 were
included as control compounds.
Figure 9. Western blot analysis of degradation of cIAP1 and cIAP2,
cleavage of caspase-8, caspase-3, and PARP in the SK-OV-3 cell line
treated with compounds 11, 13, and 4 for 24 h. cIAP-1, cIAP1, cIAP2,
caspase-8, and caspase-3 were probed by specific antibodies and
GAPDH was used as the loading control.
better anticancer activity in cell-based assays than monovalent
Smac mimetics. The data for this new class of bivalent Smac
mimetics are also consistent with our previous observations
using a different class of bivalent compound 6 and its correspond-
ing monovalent 1.31
CONCLUSION
■
We have designed, synthesized, and evaluated a new class of
bivalent Smac mimetics based upon a class of conformationally
constrained monovalent Smac mimetics containing a diazabi-
cyclic core structure. These new bivalent Smac mimetics (8−15)
bind to XIAP, cIAP1, and cIAP2 with low nanomolar to subnano-
molar affinities and function as highly potent antagonists of XIAP
in functional assays. These compounds effectively induce
degradation of cIAP1 and cIAP2 at concentrations as low as
1 nM in MDA-MB-231 and SK-OV-3 cancer cells and result in
manifest cleavage of caspases and PARP at 3−10 nM. Consistent
with the high affinities against these IAP proteins, the most potent
of these compounds have IC50 of 3−5 nM in inhibition of cell
growth in both the MDA-MB-231and SK-OV-3 cell lines. Further
evaluation and optimization of these compounds can lead to the
development of a new class of anticancer drugs.
Figure 8. Western blot analysis of degradation of cIAP1 and cIAP2,
cleavage of caspase-8, caspase-3, and PARP in the MDA-MB-231 cell
line treated with compounds 11, 13, and 4 for 24 h. cIAP1, cIAP2,
PARP, caspase-8, and caspase-3 were probed by specific antibodies and
GAPDH was used as the loading control.
degradation. Degradation of the cIAP1 protein was essentially
complete in the MDA-MB-231 cells treated with 1 nM bivalent
compounds 11 and 13 or 10 nM monovalent compound 4.
These compounds also effectively induced cIAP2 degradation
in a dose-dependent manner, and significant cIAP2 degradation
was observed with 1−3 nM compounds 11 and 13 and 10−30
nM compound 4. Thus, although compounds 4, 11, and 13
have binding affinities comparable to those of cIAP1/2 in our
biochemical assays (Table 1), bivalent Smac mimetics 11 and
13 are approximately 10 times more potent than monovalent
Smac mimetic 4 in induction of cIAP1/2 degradation in cells.
Compounds 11 and 13 also induced robust cleavage of
casapse-8 and -3 and poly (ADP-ribose) polymerase (PARP),
three key biochemical markers of apoptosis, at concentrations
as low as 3 nM. Although the monovalent compound 4 can
efficiently induce cIAP1 degradation at concentrations as low as
10 nM, it has a minimal effect on cleavage of caspase-8, -3, and
PARP at concentrations as high as 300 nM. Similar results were
obtained with these compounds in the SK-OV-3 cell line
(Figure 9). Since bivalent Smac mimetics 11 and 13 are much
more potent antagonists of XIAP than monovalent Smac
mimetic 4, our data further suggest that the ability of bivalent
Smac mimetics to concurrently target not only cIAP1/2 but
also XIAP with very high affinities is responsible for their much
EXPERIMENTAL SECTION
■
I. Chemistry. General Methods. 1H NMR spectra were acquired
1
at 300 MHz. H chemical shifts are reported with CDCl3 (7.27 ppm)
or HDO (4.70 ppm) as internal standard. The final products were
purified by C18 reverse phase semipreparative HPLC column with
solvent A (0.1% of TFA in H2O) and solvent B (0.1% of TFA in
CH3CN) as eluents. Purity for all the tested compounds was measured
by reverse phase analytical HPLC and found to be >95%.
General Synthesis of Bivalent Smac Mimetics. N,N-Diisopro-
pylethylamine (3 equiv) was added to a solution of compound 16
(1 equiv) and a corresponding diacyl chloride (0.55 equiv) in CH2Cl2.
The solution was stirred at room temperature overnight and then
concentrated. The residue was purified by chromatography to give a
diamide. To a solution of this diamide in methanol was added HCl
solution (4 N in 1,4-dioxane, 3 mL/mmol). The solution was stirred at
room temperature overnight and then concentrated to yield crude
product that was purified on C18 reverse phase semipreparative HPLC
column to afford pure Smac mimetic as a salt with TFA.
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(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-Succinylbis(N-benzhydryl-
5-((S)-2-(methylamino)propanamido)-6-oxodecahydropyrrolo-
[1,2-a][1,5]diazocine-8-carboxamide) (8). Yield 59% over two
steps. 1H NMR (300 MHz, CD3OD): 7.42−7.20 (m, 20H), 6.17 (s, 2H),
5.15 (m, 2H), 4.58 (m, 2H), 4.50−4.20 (m, 4H), 4.10−3.70 (m, 6H),
3.70−3.35 (m, 4H), 2.70 (m, 6H), 2.55−1.70 (m, 8H), 1.55 (d, J =
7.5 Hz, 6H). ESI MS: m/z 1037.6 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-Adipoylbis(N-benzhydryl-
5-((S)-2-(methylamino)propanamido)-6-oxodecahydropyrrolo-
[1,2-a][1,5]diazocine-8-carboxamide) (9). Yield 62% over two
steps. 1H NMR (300 MHz, CD3OD): 7.40−7.20 (m, 20H), 6.20 (s, 2H),
4.80 (m, 1H), 4.55 (m, 2H), 4.25 (m, 2H), 4.05−3.70 (m, 6H), 3.70−
3.30 (m, 4H), 2.70 (s, 6H), 2.69−2.30 (m, 6H), 2.25−1.75 (m, 10H),
1.75−1.55 (m, 4H), 1.55−1.48 (m, 6H). ESI MS: m/z 1065.6 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-Octanedioylbis(N-benz-
h y d r y l - 5 - ( (S ) - 2 - ( m e t hy l a m i n o ) p r o p a n am i d o ) - 6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide)
(10). Yield 61% over two steps. 1H NMR (300 MHz, CD3OD):
7.45−7.20 (m, 20H), 6.15 (s, 2H), 4.80 (m, 2H), 4.55 (m, 2H), 4.25
(m, 2H), 4.05−3.80 (m, 6H), 3.80−3.30 (m, 4H), 2.70 (s, 6H), 2.70−
2.25 (m, 6H), 2.20−1.75 (m, 10H), 1.75−1.50 (m, 10H), 1.50−1.30
(m, 4H). ESI MS: m/z 1093.6 (M + H)+.
simulation was used to predict the binding between AVPI and
designed compounds in the docking simulations.
We used the AMBER program suite (version 8)42 to perform the
molecular dynamics (MD) simulations. The AMBER force field
(ff99)42,43 was used for the natural amino acids in the complex, and
the TIP3P model44 was used for water molecules. There is one Zn2+
ion covalently bound to C200, C203, H220, and C227 in the XIAP
BIR2 domain. This Zn2+ ion, while important for structural integrity,
has no direct interaction with the ligands. We used parameters
developed by Ryde45 for the Zn2+ ion and its coordination with the
neighboring four residues to model this chelating structure in our
simulation. All the MD simulations were carried out at NTP. The
SHAKE algorithm46 was used to fix the bonds involving hydrogen.
The PME method47 was used to account for long-range electrostatic
interactions and the nonbonded cutoff distance was set at 10 Å. The
time step was 2 fs, and the neighboring pairs list was updated after
every 20 steps. For the refinement of the structure between designed
compounds and the proteins, the protocol is as follows: A 500-step
minimization of the solvated system was performed followed by 6 ps
of MD simulation to gradually heat the system from 0 to 298 K. The
system was then equilibrated by another 34 ps simulation at 298 K.
Finally, the 1 ns production simulation was run and the snapshots of
conformations (typically 2000), evenly spaced in time, were collected
for structural analysis.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-Decanedioylbis(N-benz-
h y d r y l - 5 - ( (S ) - 2 - ( m e t hy l a m i n o ) p r o p a n am i d o ) - 6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide)
Binding poses of designed compounds with XIAP BIR2 and BIR3
were predicted using the GOLD program (version 3.1.1).48,49 The
center of the binding site was set at T308 for XIAP BIR3 and at K208
for XIAP BIR2, respectively. The radius for the binding sites was
defined as 13 Å, large enough to cover the binding pocket. For each
genetic algorithm (GA) run, a maximum number of 200 000
operations were performed on a population of 5 islands of 100
individuals. Operator weights for crossover, mutation, and migration
were set to 95, 95, and 10, respectively. The docking simulations were
terminated after 20 runs for each compound. GoldScore implemented
in Gold 3.1.1 was used as the fitness function to evaluate the docked
conformations. The highest ranked conformation from each of the
20 runs was saved for further analysis. The top ranked conformation
from the 20 runs was taken as the predicted binding mode.
1
(11). Yield 64% over two steps. H NMR (300 MHz, CD3OD) δ
7.37−7.24 (m, 20H), 6.20 (s, 2H), 4.90 (m, 2H), 4.59 (m, 2H), 4.24
(m, 2H), 3.95 (m, 2H), 3.90−3.55 (m, 4H), 3.60−3.28 (m, 4H), 2.70
(s, 6H), 2.65−2.25 (m, 6H), 2.20−1.80 (m, 10H), 1.75−1.55 (m, 4H),
1.55 (d, J = 7.8 Hz, 6H), 1.45−1.25 (m, 8H). ESI MS: m/z 1121.7
(M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-Dodecanedioylbis(N-benz-
h y d r y l - 5 - ( (S ) - 2 - ( m e t hy l a m i n o ) p r o p a n am i d o ) - 6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide)
(12). Yield 63% over two steps. 1H NMR (300 MHz, CD3OD):
7.45−7.20 (m, 20H), 6.15 (s, 2H), 4.80 (m, 2H), 4.55 (m, 2H), 4.05−
3.70 (m, 6H), 3.70−3.30 (m, 4H), 2.70 (s, 6H), 2.68−2.25 (m, 8H),
2.25−1.75 (m, 10H), 1.75−1.50 (m, 10H), 1.45−1.25 (m, 10H). ESI
MS: m/z 1149.7 (M + H)+.
III. Fluorescence Polarization Based Assays for XIAP, cIAP1,
and cIAP2 Proteins. A set of sensitive and quantitative fluorescence
polarization (FP) based assays were used to determine the binding
affinities of Smac mimetics to XIAP linker-BIR2-BIR3, XIAP BIR3,
cIAP1 BIR3, and cIAP2 BIR3 proteins. The FP-based assay for XIAP
linker-BIR2-BIR3 was described in detail previously.30 The optimized
FP-based assays using a new tracer for XIAP BIR3, cIAP1 BIR3, and
cIAP2 BIRs proteins were published recently.37
IV. Caspase-9 and Caspase-3/7 Functional Assays. Cell-free
functional assays were employed to determine the functional
antagonism of our designed Smac mimetics. These assays have been
described previously in detail.38
V. Cell Growth Inhibition Assay. The MDA-MB-231 and SK-OV-3
cell lines were purchased from the American Type Culture Collection
(ATCC). Cells were seeded in 96-well flat bottom cell culture plates at
a density of 3−4 × 103 cells/well with compounds and incubated for 4
days. The rate of cell growth inhibition after treatment with different
concentrations of the inhibitors was determined by assaying with
(2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophen-
yl)-2H-tetrazolium monosodium salt (WST-8; Dojindo Molecular
Technologies Inc., Gaithersburg, Maryland). WST-8 was added to
each well to a final concentration of 10%, and then the plates were
incubated at 37 °C for 2−3 h. The absorbance of the samples was
measured at 450 nm using a TECAN ULTRA reader. Concentration
of the compounds that inhibited cell growth by 50% (IC50) was
calculated by comparing absorbance in the untreated cells with that in
the cells treated with the compounds.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-(5,5′-(1,4-Phenylene)bis-
(pentanoyl))bis(N-benzhydryl-5-((S)-2-(methylamino)-
propanamido)-6-oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-
carboxamide) (13). Yield 67% over two steps. 1H NMR (300 MHz,
CD3OD): 7.40−7.20 (m, 20H), 7.15−7.05 (m, 4H), 6.08 (s, 2H), 4.80
(m, 2H), 4.55 (m, 2H), 4.23 (m, 2H), 3.90 (m, 2H), 3.85 (m, 2H),
3.76 (m, 2H), 3.70−3.30 (m, 4H), 2.65 (s, 6H), 2.63−2.25 (m, 10H),
2.20−1.78 (m, 10H), 1.75−1.58 (m, 8H), 1.55 (d, J = 7.5 Hz, 6H).
ESI MS: m/z 1197.7 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-(5,5′-Oxybis(pentanoyl))-
bis(N-benzhydryl-5-((S)-2-(methylamino)propanamido)-6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide)
(14). Yield 58% over two steps. 1H NMR (300 MHz, CD3OD):
7.40−7.20 (m, 20H), 6.18 (s, 2H), 4.90 (m, 2H), 4.55 (m, 2H), 4.25
(m, 2H), 3.90 (m, 2H), 3.85−3.55 (m, 6H), 3.55−3.30 (m, 10H), 2.70
(s, 6H), 2.65−2.25 (m, 6H), 2.25−1.60 (m, 18H), 1.55 (d, J = 7.8 Hz,
6H). ESI MS: m/z 1137.6 (M + H)+.
(S,5S,5′S,8S,8′S,10aR,10a′R)-3,3′-Terephthaloylbis(N-benz-
h y d r y l - 5 - ( (S ) - 2 - ( m e t hy l a m i n o ) p r o p a n am i d o ) - 6-
oxodecahydropyrrolo[1,2-a][1,5]diazocine-8-carboxamide)
(15). Yield 53% over two steps. 1H NMR (300 MHz, CD3OD) 7.40−
7.14 (m, 24H), 6.15 (s, 2H), 5.05 (m, 2H), 4.58 (m, 2H), 4.44 (m, 2H),
3.90−3.75 (m, 6H), 3.50−3.43 (m, 4H), 2.51 (s, 6H), 2.30−1.84 (m, 12H),
1.47 (d, J = 6.9 Hz, 6H). ESI MS: m/ z 1085.7 (M + H)+.
II. Molecular Modeling. The crystal structure of XIAP BIR3
complexed with Smac protein (PDB entry 1G73)16 was used to predict
the binding models of XIAP BIR3 bound to designed compounds. For
XIAP BIR2, the crystal structure of XIAP BIR2 complexed with
caspase 3 (PDB entry 1I3O)41 was used to predict the binding models
of XIAP BIR2 bound to designed compounds. This structure was
further refined through a 1 ns molecular dynamics (MD) simulation.
The final XIAP BIR2 conformation at the end of 1 ns of MD
VI. Western Blot Analysis. Cells were harvested and washed with
cold PBS. Cell pellets were lysed in double lysis buffer (DLB;
50 mmol/L Tris, 150 mmol/L sodium chloride, (1 mmol/L EDTA,
0.1% SDS, and 1% NP-40) in the presence of PMSF (1 mmol/L) and
protease inhibitor cocktail (Roche) for 10 min on ice, then centrifuged
112
dx.doi.org/10.1021/jm201072x | J. Med. Chem. 2012, 55, 106−114

Journal of Medicinal Chemistry
Article
at 13 000 rpm at 4 °C for 10 min. Protein concentrations were
determined using a Bio-Rad protein assay kit (Bio-Rad Laboratories).
Proteins were electrophoresed onto a 4−20% gradient SDS−PAGE
(Invitrogen) and then transferred to PVDF membranes. After blocking
in 5% milk, the membranes were incubated with a specific primary
antibody, washed, and incubated with horseradish peroxidase linked
secondary antibody (Amersham). The signals were visualized with a
chemiluminescent HRP antibody detection reagent (Denville
Scientific). When indicated, the blots were stripped and reprobed
with a different antibody. Primary antibody against cleaved caspase-3
was purchased from Stressgen Biotechnologies. Primary antibodies
against cIAP1 and cIAP2 were purchased from R&D systems. Primary
antibody against XIAP was purchased from BD Biosciences. Primary
antibodies against PARP and β-actin were purchased from Cell
Signaling Technology.
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AUTHOR INFORMATION
Corresponding Author
*Phone: (734) 615-0362. Fax: (734) 647-9647. E-mail:
shaomeng@umich.edu.
■
Present Addresses
‡Institute of Biomedical and Health Engineering, Shenzhen
Institutes of Advanced Technology, Chinese Academy of
Sciences, Shenzhen, Guangdong 518055, China.
§Key Laboratory of Regenerative Biology and Institute of
Chemical Biology, Guangzhou Institutes of Biomedicine and
Health, Chinese Academy of Sciences, Guangzhou 510530, China.
∥ZJU-ENS Joint Laboratory of Medicinal Chemistry, Zijingang
Campus, Zhejiang University, Hangzhou 310058, China.
Author Contributions
†These authors contribute equally.
ACKNOWLEDGMENTS
■
We are grateful for the financial support from the Breast Cancer
Research Foundation, the National Cancer Institute, NIH
(R01CA109025 and R01CA127551), the Susan G. Koman
Foundation, Ascenta Therapeutics, and University of Michigan
Cancer Center Core grant from the National Cancer Institute,
NIH (P30CA046592). The authors thank Dr. G.W.A. Milne for
his critical reading of the manuscript and many useful
suggestions and Ms. Karen Kreutzer for her excellent secretarial
assistance.
ABBREVIATIONS USED
■
IAP, inhibitor of apoptotic protein; cIAP, cellular inhibitor of
apoptotic protein; XIAP, X-linked inhibitor of apoptotic
protein; Smac/DIABLO, second mitochondria-derived activa-
tor of caspases or direct IAP binding protein with low pI; BIR,
baculoviral inhibitor of apoptotic protein repeat; FP,
fluorescence polarization; PK, pharmacokinetics; PARP, poly
(ADP-ribose) polymerase; TNFα, tumor necrosis factor α
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X.; Harran, P. G. A small molecule Smac mimic potentiates TRAIL-
and TNF alpha-mediated cell death. Science 2004, 305, 1471−1474.
(25) Oost, T. K.; Sun, C.; Armstrong, R. C.; Al-Assaad, A. S.; Betz, S.
F.; Deckwerth, T. L.; Ding, H.; Elmore, S. W.; Meadows, R. P.;
Olejniczak, E. T.; Oleksijew, A.; Oltersdorf, T.; Rosenberg, S. H.;
Shoemaker, A. R.; Tomaselli, K. J.; Zou, H.; Fesik, S. W. Discovery of
potent antagonists of the antiapoptotic protein XIAP for the treatment
of cancer. J. Med. Chem. 2004, 47, 4417−4426.
(26) Sun, H.; Nikolovska-Coleska, Z.; Yang, C.-Y.; Xu, L.; Liu, M.;
Tomita, Y.; Pan, H.; Yoshioka, Y.; Krajewski, K.; Roller, P. P.; Wang, S.
Structure-based design of potent, conformationally constrained smac
mimetics. J. Am. Chem. Soc. 2004, 126, 16686−16697.
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