Potent bivalent smac mimetics: Effect of the linker on binding to inhibitor of apoptosis proteins (IAPs) and anticancer activity

Article abstract of DOI:10.1021/jm101651b

Figure Presented. We have synthesized and evaluated a series of nonpeptidic, bivalent Smac mimetics as antagonists of the inhibitor of apoptosis proteins and new anticancer agents. All these bivalent Smac mimetics bind to full-length XIAP with low nanomolar affinities and function as ultrapotent antagonists of XIAP. While these Smac mimetics bind to cIAP1/2 with similar low nanomolar affinities, their potencies to induce degradation of cIAP1/2 proteins in cells differ by more than 100-fold. The most potent bivalent Smac mimetics inhibit cell growth with IC₅₀ from 1 to 3 nM in the MDA-MB-231 breast cancer cell line and are 100 times more potent than the least potent compounds. Determination of intracellular concentrations for several representative compounds showed that the linkers in these bivalent Smac mimetics significantly affect their intracellular concentrations and hence the overall cellular activity. Compound 27 completely inhibits tumor growth in the MDA-MB-231 xenografts while causing no signs of toxicity in the animals.

Full text of DOI:10.1021/jm101651b

NIH Public Access
Author Manuscript
J Med Chem. Author manuscript; available in PMC 2012 May 12.
Published in final edited form as:
J Med Chem. 2011 May 12; 54(9): 3306–3318. doi:10.1021/jm101651b.
Potent Bivalent Smac Mimetics: Effect of the Linker on Binding
to Inhibitor of Apoptosis Proteins (IAPs) and Anticancer Activity
+
+
+
+
Δ
Haiying Sun , Liu Liu , Jianfeng Lu , Longchuan Bai , Xiaoqin Li , Zaneta Nikolovska-
+
+
+
+
+
Δ
Coleska , Donna McEachern , Chao-Yie Yang , Su Qiu , Han Yi , Duxin Sun , and
Shaomeng Wang
*
+ Comprehensive Cancer Center and Departments of Internal Medicine, Pharmacology and
Medicinal Chemistry, University of Michigan, Ann Arbor, MI 48109, USA
Δ Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, Ann
Arbor, MI 48109, USA
Abstract
We have synthesized and evaluated a series of non-peptidic, bivalent Smac mimetics as
antagonists of the inhibitor of apoptosis proteins and new anticancer agents. All these bivalent
Smac mimetics bind to full-length XIAP with low nanomolar affinities and function as ultra-
potent antagonists of XIAP. While these Smac mimetics bind to cIAP1/2 with similar low
nanomolar affinities, their potencies to induce degradation of cIAP1/2 proteins in cells differ by
more than 100-fold. The most potent bivalent Smac mimetics inhibit cell growth with IC values
50
from 1–3 nM in the MDA-MB-231 breast cancer cell line and are 100-times more potent than the
least potent compounds. Determination of intracellular concentrations for several representative
compounds showed that the linkers in these bivalent Smac mimetics significantly affect their
intracellular concentrations, hence the overall cellular activity. Compound 27 completely inhibits
tumor growth in the MDA-MB-231 xenografts, while causing no signs of toxicity in the animals.
Introduction
Apoptosis is a critical cell suicide process by which damaged or unwanted cells are
removed. It plays an important role in homeostasis, normal development, host defense and
1
suppression of oncogenesis. Dysfunction of apoptosis machinery is a hallmark of cancer
and defects in the apoptosis machinery confer on cancer cells resistance to current anticancer
2
therapies, making them less effective and leading to their ultimate failure. Targeting key
apoptosis regulators with the goal of promoting apoptosis in tumor cells is therefore being
3
pursued as a new therapeutic strategy for human cancer.
The inhibitor of apoptosis proteins (IAPs) are a class of key apoptosis regulators and are
4–7
characterized by the presence of one or more baculoviral IAP repeat (BIR) domains.
Among a total of 8 mammalian IAPs, X-linked IAP (XIAP) inhibits apoptosis by directly
4–7
binding to and effectively inhibiting three caspases, caspase-3, -7, and -9. The third BIR
domain (BIR3) of XIAP binds to the processed caspase-9 and inhibits its activity, and the
BIR2 domain of XIAP, together with the linker preceding it, binds to and inhibits both
caspase-3 and caspase-7. Hence, XIAP plays a central role in the inhibition of apoptosis by
inhibiting these three caspases. Two other IAPs, cIAP1 and cIAP2 were originally identified
4
through their interaction with tumor necrosis factor associated factor 2 (TRAF2). This
interaction leads to their recruitment to TNF receptor 1- and 2-associated complexes, where
*To whom correspondence should be addressed. Phone: (734)615-0362. Fax: (734)6479647. shaomeng@umich.edu.

Sun et al.
Page 2
4
they suppress caspase-8 activation and death-receptor-mediated apoptosis. Furthermore,
although these IAPs were initially characterized for their role in apoptosis regulation, they
also modulate many other cellular processes, such as inflammation, proliferation, mitosis
8–10
and metastasis,
which are frequently deregulated in cancer and contribute directly or
indirectly to tumor initiation, maintenance and/or progression. Accordingly, these IAP
11–13
proteins are very attractive cancer therapeutic targets.
The second mitochondria derived activator of caspases (Smac) or direct IAP binding protein
with low pI (DIABLO) has been identified as an endogenous antagonist of IAP
14,15
proteins.
Once released from mitochondria into the cytosol, Smac is processed by
proteases to remove the first 55 N-terminal residues, exposing an Ala-Val-Pro-Ile (AVPI)
14,15
tetrapeptide binding motif.
Smac forms a homodimer and promotes apoptosis by
7
directly interacting with and antagonizing XIAP and cIAP1 and cIAP2. In its homodimer
form, Smac protein binds concurrently to both the BIR2 and BIR3 domains of XIAP using
two AVPI binding motifs and nullifies the inhibition of XIAP to caspase-9 and
caspase-3/7. Smac binds to the BIR3 domain, but not to other BIR domains of cIAP1 and
cIAP2, via a single AVPI binding motif. By antagonizing these multiple IAP proteins,
7,
19
Smac efficiently promotes apoptosis. There have been intense research efforts in recent
years in the design and development of small-molecule Smac mimetics as a new class of
20–35
anticancer drugs.
Two different types of Smac mimetics have been designed;
monovalent Smac mimetics possess one AVPI mimic and and bivalent Smac mimetics
20,21
contain two AVPI mimics tethered with a linker.
Representatives of previously reported
monovalent and bivalent Smac mimetics are shown in Figure 1 and Figure 2, respectively.
Although Smac mimetics were initially designed primarily based upon the interaction
between Smac and XIAP proteins, recent studies have shown that Smac mimetics induce
36–39
rapid degradation of cIAP proteins in cells.
One major difference between bivalent and
monovalent Smac mimetics is their ability to antagonize XIAP. While monovalent Smac
mimetics can potently antagonize the inhibition of XIAP BIR3 protein to the activity of
caspase 9, they are much less effective in antagonizing the inhibition of caspase-9 and 3 by
39
XIAP protein containing both BIR2 and BIR3 domains. In comparison, bivalent Smac
mimetics function as ultra-potent antagonists of XIAP protein containing both BIR2 and
28,39
BIR3 domains through currently binding to both BIR domains.
Both monovalent and
bivalent Smac mimetics are effective in killing cancer cells in a subset of human cancer cell
36–39
lines in a TNFα-dependent manner,
but bivalent Smac mimetics are much more potent
39
than their corresponding monovalent Smac mimetic analogues. One major advantage for
monovalent Smac mimetics, however, is their much favorable pharmacokinetic properties;
properly designed monovalent Smac mimetics can achieve excellent oral
20,21
bioavailability.
To date, three monovalent Smac mimetics and two bivalent Smac
20
mimetics have been advanced into early clinical development for cancer treatment. Of
which, an orally active monovalent Smac mimetic, SM-406/AT-406 (compound 9 in Figure
23
1) from our group, is currently in Phase I clinical trials. The chemical structures of the
other four clinical stage compounds have not been disclosed.
Starting from a non-peptide, monovalent Smac mimetic, 15, we have designed and
35,39
synthesized a bivalent Smac mimetic 16 (Figure 3).
We have shown that 16 is >100-
times more potent in binding to XIAP containing both BIR2 and BIR3 domains, and 10-
35,39
times more potent in binding to cIAP1 BIR3 protein and cIAP2 BIR3 protein, than 15.
Compound 16 is capable of effectively inducing apoptosis and inhibiting cell growth in a
subset of human cancer cell lines at concentrations as low as 1–10 nM and is 100-times
39
more potent than 15. Furthermore, 16 also strongly induces apoptosis in MDA-MB-231
xenograft tumor tissues and achieves tumor regression at 5 mg/kg in the MDA-MB-231
39
xenograft model in mice. These in vitro and in vivo data identify compound 16 as a
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Sun et al.
Page 3
promising lead for further structure-activity relationship studies, with the ultimate goal of
developing a potent bivalent Smac mimetic for the treatment of human cancer.
We report here the design, synthesis and evaluation of a series of analogues of 16, as well as
several control compounds (Figure 4). One main objective in the present study was to
investigate the effect of the linker on binding affinities to XIAP and cIAP1/2 proteins and
their anticancer activity. Our study has led to the identification of several highly potent
bivalent Smac mimetics and yielded new structure-activity insights into the design of potent
bivalent Smac mimetics as a new class of anticancer drugs.
Chemistry
The synthesis of the newly designed compounds 18–30 is similar to that of 16 and is shown
35
in Scheme 1. Briefly, the key intermediate 31 was synthesized using a method we
35
published previously. Cycloaddition of 31 with corresponding bis-azide in the presence of
CuSO and (+)-sodium-L-ascorbate afforded a series of bis-triazoles, removal of the Boc
4
protecting groups, which gave compounds 18–29. Cycloaddition of 31 with excess 1,4-
bis-4-azidobutylbenzene, catalyzed by CuSO and (+)-sodium-L-ascorbate, furnished 32,
4
35
which was reacted with 33 to afford a bis-triazole. Removal of the Boc protecting group
from this bis-triazole gave 30.
Results and Discussion
Compound 16 is a bivalent Smac mimetic containing two monovalent IAP binding motifs,
tethered together through a flexible linker.
In order to explore the influence of the linker region on the activity of bivalent Smac
mimetics, we designed a series of new analogues (compounds 18–29 in Figure 4) with
linkers of various lengths, flexibility and hydrophobicity. A previously synthesized
compound (17), in which both N-methylalanine residues were replaced with N-
acetyltryptophan groups, and compound 30, in which one N-methylalanine residue in 16 has
been replaced with N-acetyltryptophan, were employed as control compounds. Compounds
15–30 were tested in fluorescence-polarization (FP) based binding assays for their binding
affinities to XIAP, cIAP1 and cIAP2 and the results are summarized in Table 1.
We had shown previously that 16 binds to XIAP BIR3 protein and XIAP protein containing
35
both BIR2-BIR3 domains with different affinities. To evaluate the affinities to XIAP we
employed two recombinant XIAP proteins: XIAP BIR3 protein (residues 241–356), which
possesses only the BIR3 domain of XIAP, and XIAP L-BIR2-BIR3 (residues 120–356)
which contains both BIR2 and BIR3 domains of XIAP and the linker preceding BIR2.
Compounds 18–22, which differ from 16 only in the length of the linker, bind to XIAP L-
BIR2-BIR3 protein with very high affinities, achieving IC values of 6–17 nM with
50
calculated K values of 1.5–5.0 nM. These compounds also bind to XIAP BIR3 protein with
i
high affinities and have IC values of 177–613 nM with calculated K values = 55–185 nM.
50
i
Comparison of their K values for their binding to these two XIAP proteins showed that each
i
of these bivalent Smac mimetics binds to XIAP L-BIR2-BIR3 with an affinity 20–40 times
higher than to XIAP BIR3. Notwithstanding the significant differences in the linker lengths
in these compounds, the most potent compounds, 16 and 21, are only three times more
potent than the least potent compound (22) in their binding affinities to XIAP L-BIR2-BIR3
35
protein. Our previous study showed that 16 has a higher affinity for XIAP L-BIR2-BIR3
protein than for XIAP BIR3 protein because it concurrently targets both the BIR2 and BIR3
domains. Hence, the binding data for these new analogues to the two XIAP protein
constructs also suggest that they concurrently interact with both BIR2 and BIR3 domains in
XIAP in the presence of the XIAP protein containing both BIR domains.
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Sun et al.
Page 4
We also employed two cIAP1 proteins containing BIR3-only domain or both BIR2 and
BIR3 domains to determine if these bivalent Smac mimetics can interact concurrently with
both BIR2 and BIR3 domains in cIAP1. Bivalent Smac mimetics 16 and 18–22 bind to
cIAP1 BIR3 and cIAP1 BIR2-BIR3 proteins with similar high affinities and have K = 1–3
i
nM to both cIAP1 protein constructs (Table 1). We conclude that in contrast to XIAP, only
the BIR3 domain in cIAP1 is involved in the binding to these bivalent Smac mimetics.
Compounds 16 and 18–22 also bind to cIAP2 BIR3 protein with very high affinities and
have K = 1–6 nM. Taken together, these binding data show that these new bivalent Smac
i
mimetics have very high affinities to XIAP and cIAP1/2 proteins and the length of linkers in
these bivalent Smac mimetics has only a modest effect on these binding affinities.
We designed 23 and 24, in which the phenyl group in the linker of 16 is replaced with a
more flexible (CH ) or (CH ) to investigate the influence of linker rigidity on the binding
2 2
2 4
affinities of bivalent Smac mimetics. Compounds 23 and 24 have potent binding affinities to
all of these IAP proteins which are similar to that of 16, indicating that the rigidity of the
linker lacks significant influence on the binding affinities to these IAP proteins.
The linkers in 16–24 are very hydrophobic and in order to explore the influence that polarity
and hydrophobicity have on binding affinities to any of these three IAP proteins, we
designed compounds 25–29 in which the phenyl group in the linker of 16 is replaced with a
more hydrophilic triazole (25), urea (26), or the entire linker in 16 is replaced with alkyl
chains containing one or more oxygen atoms (27–29). All of these compounds have binding
affinities to these three IAPs similar to that of 16, indicating that hydrophobicity and polarity
in the linker have little influence on the binding affinities.
Finally, focusing on the involvement of the two AVPI mimetics in these bivalent Smac
mimetics for binding to XIAP, we designed compound 30, in which one N-methylalanine
residue in 16 was replaced with N-acetyltryptophan to disrupt the interaction of one AVPI
mimetic to these IAP proteins. Compound 30 is 4 times less potent than 16 in binding to
XIAP BIR3, but more than 40 times less potent than 16 in binding to XIAP linker-BIR2-
28
BIR3. These data are consistent with our previous study using compound 16 that showed
both of the two IAP binding motifs to be involved in the binding to full length XIAP.
These binding data show that in bivalent Smac mimetics, both AVPI motifs are involved in
binding to XIAP protein containing both BIR2 and BIR3 domains. The length,
conformational rigidity, and hydrophobicity of the linker tethered to the two AVPI mimetics
all appear to have only a modest effect on their binding affinities to the XIAP protein
containing both BIR2 and BIR3 domains. These data are, however, consistent with the fact
that the BIR2 and BIR3 domains in XIAP are connected by a 25-residue segment apparently
40
lacking any significant secondary structure, which would allow XIAP to efficiently
interact with bivalent Smac mimetics with linkers of different length, rigidity and
hydrophobicity. In comparison, our binding data indicate that only the BIR3 domain in
cIAP1 is involved in the binding to these bivalent Smac mimetics.
4,7
Because XIAP functions as a potent inhibitor of caspase-9 and caspase-3/-7, we evaluated
several representative new analogues (18–22, 24, 29 and 30), together with compounds 15,
16 and 17, in cell-free functional assays for their functional antagonism against XIAP
(Figure 5). In the caspase-9 functional assay, the XIAP linker-BIR2-BIR3 protein dose-
dependently inhibits the activity of caspase-9, achieving 80% inhibition at 500 nM. Bivalent
Smac mimetics 16, 18–21, 24 and 29 have similar potencies and can restore 60–80% of
caspase-9 activity at concentrations of 1.5 μM. Interestingly, compound 22 with the longest
linker shows much less activity than 16, restoring only 25% of the caspase-9 activity at 1.5
μM concentration. The monovalent Smac mimetic 15 is approximately equipotent with 30,
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Sun et al.
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in which one side has been disabled, but both compounds are much less potent than 16, 18–
21, 24 and 29. The inactive control 17, at a concentration as high as 100 μM, fails to restore
any caspase-9 activity. These results show that both AVPI mimetics in these bivalent Smac
mimetics are important to the antagonism of XIAP linker-BIR2-BIR3 proteins in this
caspase-9 functional assay.
In the caspase-3/7 functional assay, 20 nM of XIAP protein containing linker-BIR2-BIR3
domains inhibits 90% of the enzymatic activity of caspase-3/7, and bivalent Smac mimetics
can dose dependently restore this activity (Figure 6). Most of these bivalent Smac mimetics
show activity comparable to that of 16 in this assay. At a 60 nM concentration, the bivalent
Smac mimetics 16, 18–21, 24 and 29 for example, can restore 55–80% of the activity of
caspase-3/7. However, the monovalent compound 15 at 60 μM, at 1,000 times higher
concentration, restores only 40% of the caspase-3/7 activity. Compound 30 is only several
times less potent than the most potent bivalent Smac mimetics. It is interesting that the
inactive control compound 17 shows a comparable potency to 15 in this caspase-3/7
functional assay. Since the BIR2 domain, together with the preceding linker, binds to and
inhibits caspase-3/7, such functional data suggest that IAP binding motifs in 17 still can
interact with the BIR2 domain of XIAP, although this compound binds to XIAP BIR3
protein with a very low affinity (Table 1). These assay results thus show that bivalent Smac
mimetics are highly potent antagonists of XIAP linker-BIR2-BIR3 protein, much more
potent than their corresponding monovalent Smac mimetics in both caspase-9 and
caspase-3/7 functional assays.
Compound 16 was shown to inhibit cell growth effectively and to induce apoptosis in
multiple human cancer cell lines, including the MDA-MB-231 breast cancer cell line.
35,39
Accordingly, the new Smac mimetics were evaluated for their ability to inhibit cell growth
in this cell line and the data are summarized in Table 1. It was found that although the linker
length in these bivalent Smac mimetics has little influence on the binding affinities to XIAP
and cIAP1/2, it has a dramatic effect on the compounds’ ability to inhibit cell growth. While
the bivalent compound 18, with the shortest linker, has IC = 159 nM and a potency similar
50
to monovalent compound 15, the analogues with longer linkers in general show cellular
activities that increase as the linker length is extended. Compound 21 is the most potent in
the series, with IC = 1.6 nM, equipotent with 16 and 400 times more potent than 15.
50
Compound 22 with the longest linker is slightly less potent than 21, suggesting that the
length of the linker in compound 21 is optimal, further extension failing to improve the
cellular activity.
The hydrophobicity of the linker also has significant influence on the cellular activity. While
23 and 24 have linkers of length comparable to that of 16 and are as potent as 16 in this
cellular assay, compounds 27–29, which contain one to three oxygen atoms in their linker
region, have diminished cellular activity. While 27 has IC = 19.6 nM, and is thus 6 times
50
less potent than 16, 28 has an IC = 175 nM, 53 times less potent than 16. Compound 29,
50
whose linker contains 3 oxygen atoms, has IC = 225 nM, and is 68 times less potent than
50
16. Insertion of other polar and hydrophilic groups into the linker also decreases the cellular
activity. Compounds 25 and 26, with a triazole- or a urea-containing linker, have IC = 107
50
and 263 nM respectively in this assay, and thus are 32 and 80 times less potent than 16.
Compound 30 is 203 times less potent than 16 and has the same potency as 15, indicating
the two active IAP binding motifs are required for achieving ultra-potent cellular activity.
These data show clearly that the linkers in these bivalent Smac mimetics have a major effect
on the compounds’ ability to inhibit cell growth.
Previous studies have shown that potent Smac mimetics can induce rapid degradation of
both cIAP1 and cIAP2 and that degradation of these cIAP proteins is a prerequisite to
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Sun et al.
Page 6
36–39
initiation of apoptosis by Smac mimetics in cancer cells.
To explore the mechanism of
action of the bivalent Smac mimetics, we performed western blot analysis of the cIAP
proteins in MDA-MB-231 cells treated with compounds 16 and 18–21. The results are
shown in Figure 7. Although these bivalent Smac mimetics bind to cIAP1 and cIAP2 with
comparable binding affinities in biochemical assays, they have different potencies in
induction of cIAP1/2 degradation, as well as caspase-3 processing and poly(ADPribose)
polymerase (PARP) cleavage, two biochemical markers of apoptosis. While 19 at 30 nM has
little effect on cIAP1 degradation, 20 at a concentration of 10 nM induces clear cIAP1
degradation, whereas the highly potent compounds 16 and 21 induce robust cIAP1
degradation at 1 nM. Similar results have been obtained with respect to their potencies in
induction of cIAP2 degradation (Figure 5). At a concentration of 30 nM, both compounds 18
and 19 fail to induce caspase-3 processing and PARP cleavage. In comparison, compound
20 starts to cause PARP cleavage at 3 nM and caspase-3 processing at 10 nM, while
compounds 16 and 21 induce robust caspase-3 processing and PARP cleavage at 3 nM
concentrations. Thus the western blot analysis shows that the potency of these bivalent Smac
mimetics in inhibition of cell growth correlates well with their ability to induce degradation
of cIAP1 and cIAP2, PARP cleavage and caspase-3 processing in cancer cells.
Hence, although the linker in the bivalent Smac mimetics has only modest effect on the
binding affinities to XIAP and cIAP1/2, it has a significant influence on the cellular activity
in inhibition of cell growth, as well as in induction of cIAP1/2 degradation and cleavage of
PARP and caspase-3. While the linker determines the distance between the two IAP binding
motifs, it also affects the overall hydrophobic properties of a bivalent Smac mimetic.
Therefore, we hypothesized that the linker in these bivalent Smac mimetics may have a
major effect on their cell permeability and thus the intracellular concentrations of the
compounds.
To test this hypothesis, we developed an assay to determine the intracellular concentrations
of some representative compounds. In this assay, a compound was incubated with MDA-
MB-231 cells at different concentrations for 6 hours or less, i.e. before significant cell death
occurs. After incubation, the cell culture medium was discarded and the remaining cells
were washed promptly and extensively to minimize both the nonspecific adsorption of the
compound on outer cell walls and leakage of the compound from the cells during washing.
The cells were then lysed and resuspended in water. Concentrations of the compound in the
resuspended cell lysates were determined by a sensitive LC-MS/MS technique. Because the
exact volume of the cells was unknown, the concentrations of each compound determined in
this way are not the actual intracellular concentrations of the compound, but the
concentrations of different compounds determined in this way will be a measure of their
actual intracellular concentrations.
We evaluated the assay conditions using compounds 16 and 18, which possess significantly
different linker lengths and show different cellular activities, and the results are shown in
Figure 8. With a very short incubation time of 15, 30, or 60 seconds, approximately 50 and
10 nM of compounds 16 and 18 were detected in the resuspended cell lysates, but no
significant changes were observed for the concentrations of both compounds. The
concentrations for both compounds detected with incubation time less than 60 seconds were
assumed to represent nonspecific binding, but when the incubation time was extended to one
hour, concentrations in cell lysates for compounds 16 and 18 were found to have increased
by factors of 5 and 2, respectively, over that observed with incubation time below one
minute. Increasing incubation times to 3 and 6 hours did not significantly alter the cell lysate
concentrations for both compounds, suggesting that for both compounds, equilibrium
between the intra- and extracellular concentrations has been reached after one hour’s
incubation (Figure 8A). It is also clear that compound 16 has a much higher concentration
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Sun et al.
Page 7
than compound 18 in resuspended cell lysates. Compound concentrations obtained were
normalized to the total protein amount in the cell lysates to compensate for potentially
significant differences in cell numbers in each culture dish. The ratios between these two
compounds after normalization (Figure 8B) with incubation times of 1, 3 and 6 hr are
consistent with those obtained using concentrations in resuspended cell lysates.
Using these established assay conditions, we next evaluated the intracellular concentrations
for three additional representative compounds (19, 20 and 29), together with compounds 16
and 18. These compounds were incubated with the MDA-MB-231 cells for 1 minute and 3
hours, respectively, and the concentrations in resuspended cell lysates in 100 μl of water for
each compound were determined using LC-MS/MS. The results are provided in Table 2.
After subtraction of the concentration after 1 minute’s incubation from that after 3 hr
incubation, the concentrations in resuspended cell lysates for these compounds are used to
assess their relative intracellular concentrations (Table 2). The data indicated that 16 has the
highest intracellular concentration, whereas 29 has the lowest concentration among these
compounds. Significantly, the calculated concentrations of these compounds in resuspended
cell lysates correlate well with their potencies to induce the degradation of cIAP1/2, as well
as their ability to cleave caspase-3 and PARP. These data show that the major difference in
the overall cellular activity of these bivalent Smac mimetics, including degradation of
cIAP1/2, induction of apoptosis and cell growth inhibition, is most likely due to their
different cell permeabilities rather than their different binding affinities to IAP proteins.
We have previously shown that compound 16 was very effective in inhibition of tumor
39
growth in the MDA-MB-231 xenograft model. To further investigate the antitumor
activity for this class of compounds, we have evaluated compound 27 for its antitumor
activity in the MDA-MB-231 xenograft model based upon its excellent solubility and good
in vitro activity. The results are shown in Figure 9. Our data showed that 27 inhibits tumor
growth in a dose-dependent manner and can completely inhibit tumor growth at 5 mg/kg,
while causing minimal weight loss or other signs of toxicity in SCID mice. The antitumor
activity is statistically significant (p =0.03 and 0.0005 for compound 27 at 1 and 5 mg/kg
versus the control at the end of the treatment, respectively). The antitumor activity for 27 is
also long lasting. On day 50, tumors treated with 27 at 5 mg/kg have an average size of 269
3
mm , whereas tumors treated with vehicle control have grown to an average size of 1136
3
mm . In comparison, while taxotere has a similar antitumor activity in this model, it causes
significant weight loss during the treatment. Hence, compound 27 is very effective in
inhibiting tumor growth of the MDA-MB-231 xenografts at well-tolerated dose-schedules.
Summary
A series of bivalent Smac mimetics with linkers of various lengths and different
hydrophobicities were synthesized and evaluated. These compounds bind to XIAP and
cIAP1/2 with very high affinities. They are highly potent XIAP antagonists and efficiently
induce cIAP1 and cIAP2 degradation. Several of these new bivalent Smac mimetics, such as
compounds 21, 22 and 24, are most potent in inhibition of cell growth in the MDA-MB-231
cell line with IC values of 1–3 nM. While the linker has no significant influence on the
50
binding affinities of these bivalent Smac mimetics to XIAP and cIAP1/2, it can dramatically
affect their cell permeability, and hence their overall cellular activity. Compound in vivo
evaluation showed that compound 27 is capable of completely inhibiting tumor growth in
the MDA-MB-231 xenograft model. Additional in vivo studies are underway with the goal
to identify the most promising compounds for advanced preclinical development and the
results will be reported in due course.
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Experimental Section
I. Chemistry
1
13
General Methods. H NMR spectra were acquired at 300 MHz and C spectra at 75
1
MHz. H chemical shifts are reported with CDCl (7.27 ppm) or HDO (4.70 ppm) as
3
13
internal standards. C chemical shifts are reported relative to CDCl (77.00 ppm) or 1,4-
3
dioxane (67.16 ppm) as internal standards. The final products were purified by C reverse
18
phase semi-preparative HPLC column with solvent A (0.1% of TFA in H O) and solvent B
2
(0.1% of TFA in CH CN) as eluents. Purity for all the final compounds was determined by
3
reverse phase analytical HPLC to be over 95%.
Synthesis of bivalent Smac mimetics. General procedure—A mixture of CuSO
4
(0.1 eq) and (+)-sodium l-ascorbate (0.3 eq) in H O (5 mL per mmol of 31) was added to a
2
solution of compound 31 (1 eq); then a bis-azide (0.5 eq) in tert-butyl alcohol (10 mL per
mmol of 31) was also added. The mixture was stirred at room temperature overnight and
then extracted with CH Cl (3 × 30 mL). The combined organic layer was washed with
2
2
brine, dried over Na SO , and evaporated to afford a residue which was purified by
2
4
chromatography to give a bis-triazole. HCl (4N in 1,4-dioxane, 2 mL per mmol of bis-
triazole) was added to a solution of this bis-triazole in MeOH (5 mL per mmol of bis-
triazole). The solution was stirred at room temperature overnight and then concentrated to
furnish a crude product which was purified by C18 reversed phase semipreparative HPLC to
give a bivalent Smac mimetic.
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(1,4-
phenylenebis(methylene))bis(1H-1,2,3-triazole-4,1-
diyl))bis(phenylmethylene))bis(6-((S)-2-(methylamino)propanamido)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide) (18)—Yield 62% over two
1
steps. Purity was determined by reverse phase analytical HPLC to be over 98%. H NMR
(D O): δ 7.55 (s, 2H), 7.20-7.02 (m, 10H), 6.82 (brs, 4H), 5.95 (s, 2H), 5.12 (brs, 4H), 4.65
2
13
(m, 2H), 4.20 (m, 2H), 4.12 (m, 2H), 3.80 (m, 2H), 2.52 (s, 6H), 2.05-1.15 (m, 30H);
C
NMR (D O): δ 173.04, 172.16, 169.49, 148.55, 139.08, 136.43, 135.38, 129.27, 128.82,
2
128.51, 127.40, 123.90, 61.92, 60.92, 53.62, 51.05, 50.39, 35.87, 33.05, 32.28, 31.32, 27.66,
+
25.06, 21.91, 15.64; ESI MS: m/z 1037.6 (M + H) ; Anal. (C H N O 2.2CF COOH): C,
56 72 14
6
3
H, N.
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(1,4-phenylenebis(ethane-2,1-
diyl))bis(1H-1,2,3-triazole-4,1-diyl))bis(phenylmethylene))bis(6-((S)-2-
(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide) (19)—Yield 64% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 98%. H NMR (D O): δ 7.28-7.15 (m, 6H), 7.12-7.02 (m, 6H),
2
6.50 (brs, 4H), 5.95 (s, 2H), 4.65 (m, 2H), 4.35 (m, 4H), 4.28 (m, 2H), 4.18 (m, 2H), 3.82
13
(m, 2H), 2.85 (m, 4H), 2.55 (s, 6H), 2.20-1.20 (m, 30H); C NMR (D O): δ 172.64, 171.79,
2
169.10, 147.24, 138.78, 135.59, 128.76, 128.09, 127.00, 123.79, 61.55, 60.58, 56.78, 51.66,
50.66, 49.75, 35.57, 35.39, 32.65, 31.93, 30.90, 27.34, 24.68, 21.52, 15.23; ESI MS: m/z
+
1065.6 (M + H) ; Anal. (C H N O 2.6CF COOH): C, H, N.
58 76 14
6
3
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(1,4-
phenylenebis(propane-3,1-diyl))bis(1H-1,2,3-triazole-4,1-
diyl))bis(phenylmethylene))bis(6-((S)-2-(methylamino)propanamido)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide) (20)—Yield 59% over two
1
steps. Purity was determined by reverse phase analytical HPLC to be over 98%. H NMR
(D O): δ 7.52 (s, 2H), 7.22-7.05 (m, 10H), 6.55 (s, 4H), 6.02 (s, 2H), 4.65 (m, 2H), 4.32 (m,
2
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13
2H), 4.15 (m, 2H), 4.01 (m, 4H), 3.85 (m, 2H), 2.58 (s, 6H), 2.25-1.20 (m, 38H); C NMR
(D O): δ 174.82, 172.49, 171.74, 147.88, 139.03, 138.15, 128.87, 128.25, 128.08, 127.04,
2
124.34, 61.53, 60.53, 56.81, 50.65, 49.99, 49.62, 35.57, 32.71, 31.94, 31.28, 30.93, 30.68,
+
27.34, 24.71, 21.55, 15.26; ESI MS: m/z 1093.6 (M + H) ; Anal. (C H N O
60 80 14
6
2.3CF COOH): C, H, N.
3
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(1,4-phenylenebis(hexane-6,1-
diyl))bis(1H-1,2,3-triazole-4,1-diyl))bis(phenylmethylene))bis(6-((S)-2-
(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide) (21)—Yield 64% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 98%. H NMR (300 MHz, D O): δ 7.55 (s, 2H), 7.40-7.02 (m,
2
10H), 6.70 (s, 4H), 6.15 (s, 2H), 4.70 (m, 2H), 4.35 (m, 2H), 4.15 (m, 2H), 4.10-3.90 (m,
4H), 3.85 (m, 2H), 2.60 (s, 6H), 2.30-1.05 (m, 0.90-0.70 (m, 8H); ESI MS: m/z 1177.7 (M +
+
H) .
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(1,4-phenylenebis(octane-8,1-
diyl))bis(1H-1,2,3-triazole-4,1-diyl))bis(phenylmethylene))bis(6-((S)-2-
(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide) (22)—Yield 66% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 98%. H NMR (300 MHz, D O): δ 7.55 (s, 2H), 7.25-6.95 (m,
2
10H), 6.62 (s, 4H), 6.05 (s, 2H), 4.65 (m, 2H), 4.30 (m, 2H), 4.12 (m, 2H), 3.92 (m, 4H),
13
3.80 (m, 2H), 2.52 (s, 6H), 2.25-1.05 (m, 34H), 1.02-0.72 (m, 24H); C NMR (D O): δ
2
171.91, 171.42, 168.93, 148.11, 139.59, 139.36, 128.66, 127.95, 127.04, 122.65, 61.32,
60.34, 56.79, 50.50, 49.81, 35.13, 32.83, 31.99, 31.33, 29.79, 19.16, 28.83, 27.20, 26.13,
+
24.88, 21.52, 15.28; ESI MS: m/z 1233.8 (M + H) ; Anal. (C H N O 2.9CF COOH):
70 100 14
6
3
C, H, N.
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(decane-1,10-diyl)bis(1H-1,2,3-
triazole-4,1-diyl))bis(phenylmethylene))bis(6-((S)-2-
(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide) (23)—Yield 69% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 95%. H NMR (300 MHz, D O): δ 7.67 (s, 2H), 7.26-7.10 (m,
2
10H), 6.07 (s, 2H), 4.73 (m, 2H), 4.28 (m, 2H), 4.25-4.10 (m, 6H), 3.83 (m, 2H), 2.54 (s,
13
6H), 2.20-1.92 (m, 4H), 1.86-1.30 (m, 30H), 0.98-0.80 (m, 12H); C NMR (75 MHz, D O):
2
δ 177.43, 173.05, 172.22, 148.26, 139.29, 129.28, 128.50, 127.43, 123.81, 62.00, 60.99,
57.20, 51.08, 50.76, 50.40, 36.00, 33.06, 32.36, 31.32, 29.52, 28.62, 28.26, 27.77, 25.76,
+
25.10, 21.93, 15.65; ESI MS: m/z 1073.7 (M + H) .
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(dodecane-1,12-
diyl)bis(1H-1,2,3-triazole-4,1-diyl))bis(phenylmethylene))bis(6-((S)-2-
(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide) (24)—Yield 65% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 95%. H NMR (300 MHz, D O): δ 7.60 (s, 2H), 7.30-7.10 (m,
2
10H), 6.20 (s, 2H), 4.80 (m, 2H), 4.65 (m, 2H), 4.55-4.20 (m, 6H), 3.75 (m, 2H), 2.45 (s,
6H), 2.25-1.45 (m, 28H), 1.38 (d, J = 7.2 Hz, 6H), 1.10-0.80 (m, 16H); ESI MS: m/z 1101.7
+
(M + H) .
(3S,6S,10aS)-6-((S)-2-(methylamino)propanamido)-N-((1-(4-(4-(4-(4-((S)-((3S,6S,
10aS)-6-((S)-2-(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-
a]azocine-3-carboxamido)(phenyl)methyl)-1H-1,2,3-triazol-1-yl)butyl)-1H-1,2,3-
triazol-1-yl)butyl)-1H-1,2,3-triazol-4-yl)(phenyl)methyl)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide (25)—Yield 51% over two
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Page 10
1
steps. Purity was determined by reverse phase analytical HPLC to be over 95%. H NMR
(300 MHz, D O): δ 7.87 (s, 1H), 7.71 (s, 2H), 7.30-7.11 (m, 10H), 6.05 (s, 2H), 4.64 (m,
2
2H), 4.35-4.16 (m, 10H), 3.81 (m, 2H), 2.60 (t, J = 6.2 Hz, 2H), 2.54 (s, 6H), 2.10 (m, 2H),
13
1.98 (m, 2H), 1.75-1.42 (m, 34H); C NMR (75 HMz, D O): δ 175.85, 174.79, 172.08,
2
150.63, 147.37, 141.60, 131.85, 131.09, 129.93, 128.50, 126.79, 64.57, 63.53, 59.73, 54.54,
53.66, 52.90, 52.59, 38.49, 35.57, 34.90, 33.93, 31.29, 30.32, 28.96, 28.65, 27.63, 27.38,
+
25.24, 24.49, 18.22; ESI MS: m/z 1112.7 (M + H) .
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-
((carbonylbis(azanediyl))bis(butane-4,1-diyl))bis(1H-1,2,3-triazole-4,1-
diyl))bis(phenylmethylene))bis(6-((S)-2-(methylamino)propanamido)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide) (26)—Yield 42% over two
1
steps. Purity was determined by reverse phase analytical HPLC to be over 95%. H NMR
(300 MHz, D O): δ 7.75 (s, 2H), 7.40-7.20 (m, 10H), 6.16 (s, 2H), 4.74 (m, 2H), 4.36 (m,
2
2H), 4.32-4.20 (m, 6H), 3.89 (m, 2H), 2.95 (t, J = 6.6 Hz, 4H), 2.64 (s, 6H), 2.32-1.20 (m,
+
38H); ESI MS: m/z 1103.7 (M + H) .
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-(oxybis(pentane-5,1-
diyl))bis(1H-1,2,3-triazole-4,1-diyl))bis(phenylmethylene))bis(6-((S)-2-
(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-a]azocine-3-
carboxamide) (27)—Yield 68% over two steps. Purity was determined by reverse phase
1
analytical HPLC to be over 95%. H NMR (300 MHz, D O): δ 7.73 (s, 2H), 7.20-7.02 (m,
2
10H), 6.05 (s, 2H), 4.65 (m, 2H), 4.30 (m, 2H), 4.22-4.08 (m, 6H), 3.84 (m, 2H), 3.08 (m,
13
4H), 2.52 (s, 6H), 2.25-0.90 (m, 42H); C NMR (75 MHz, D O): δ 173.02, 172.13, 169.49,
2
147.91, 139.07, 129.31, 128.55, 127.41, 124.14, 70.25, 66.87, 61.93, 60.90, 57.18, 50.97,
50.22, 36.01, 33.08, 32.37, 31.40, 29.33, 28.23, 27.74, 25.11, 22.62, 21.97, 15.70; ESI MS:
+
m/z 1089.7 (M + H) .
(S,3S,3′S,6S,6′S,10aS,10a′S)-N,N′-((1S,1′S)-(1,1′-((butane-1,4-
diylbis(oxy))bis(ethane-2,1-diyl))bis(1H-1,2,3-triazole-4,1-
diyl))bis(phenylmethylene))bis(6-((S)-2-(methylamino)propanamido)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide) (28)—Yield 63% over two
1
steps. Purity was determined by reverse phase analytical HPLC to be over 95%. H NMR
(300 MHz, D O): δ 7.77 (s, 2H), 7.22-7.08 (m, 10H), 6.05 (s, 2H), 4.65 (m, 2H), 4.37-4.22
2
(m, 6H), 4.16 (m, 2H), 3.82 (m, 2H), 3.48 (m, 4H), 3.08 (m, 4H), 2.52 (s, 6H), 2.16-1.42 (m,
13
30H), 1.01 (m, 4H); C NMR (75 HMz, D O): δ 173.11, 172.16, 169.49, 148.01, 139.10,
2
129.32, 128.56, 127.38, 124.62, 70.59, 68.39, 66.87, 61.96, 60.95, 57.19, 50.73, 50.24,
35.98, 33.09, 32.36, 31.40, 27.77, 25.43, 25.12, 21.96, 15.69; ESI MS: m/z 1077.6 (M + H) .
+
(3S,6S,10aS)-6-((S)-2-(methylamino)propanamido)-N-((1-(2-(2-(2-(2-(4-((S)-((3S,
6S,10aS)-6-((S)-2-(methylamino)propanamido)-5-oxodecahydropyrrolo[1,2-
a]azocine-3-carboxamido)(phenyl)methyl)-1H-1,2,3-triazol-1-
yl)ethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-yl)(phenyl)methyl)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide (29)—Yield 67% over two
1
steps. Purity was determined by reverse phase analytical HPLC to be over 95%. H NMR
(300 MHz, D O): δ 7.58 (s, 2H), 7.29-7.13 (m, 10H), 6.08 (s, 2H), 4.70 (m, 2H), 4.38 (m,
2
4H), 4.27 (m, 2H), 4.22 (m, 2H), 3.85 (m, 2H), 3.73 (m, 4H), 3.32 (m, 4H), 3.25 (m, 4H),
13
2.58 (s, 6H), 2.25-1.48 (m, 22H), 1.40 (d, J = 7.0 Hz, 6H), 1.39 (m, 2H); C NMR (75
HMz, D O): δ 173.36, 172.32, 169.56, 148.12, 139.21, 129.32, 128.54, 127.40, 124.51,
2
69.98, 69.75, 68.97, 62.07, 61.07, 57.20, 51.13, 50.46, 50.41, 35.94, 33.01, 32.35, 31.31,
+
27.81, 25.07, 21.92, 15.63; ESI MS: m/z 1093.7 (M + H) .
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Page 11
tert-butyl ((S)-1-(((3S,6S,10aS)-3-(((R)-(1-(4-(4-(4-
azidobutyl)phenyl)butyl)-1H-1,2,3-triazol-4-yl)(phenyl)methyl)carbamoyl)-5-
oxodecahydropyrrolo[1,2-a]azocin-6-yl)amino)-1-oxopropan-2-yl)
(methyl)carbamate (32)—CuSO (10 mg, 0.04 mmol) and (+)-sodium l-ascorbate (20
4
mg, 0.1 mmol) in water (3mL) was added to a solution of compound 31 (32 mg, 0.061
mmol) and 1,4-bis-(4-azidobutyl)benzene (60 mg, 0.22 mmol) in tert-butyl alcohol (5 mL).
The mixture was stirred at room temperature overnight and then extracted with CH Cl (3 ×
2
2
10 mL). After the combined organic layer was washed with brine, dried over Na SO , and
2
4
1
evaporated, the residue was purified by chromatography to give 32 (30 mg, yield 62%). H
NMR (CDCl ): δ 8.01 (brd, J = 8.3 Hz, 1H), 7.40-7.20 (m, 6H), 7.15-7.02 (m, 4H), 6.90 (m,
3
1H), 6.29 (d, J = 8.3 Hz, 1H), 4.84 (m, 1H), 4.68 (m, 1H), 4.52 (brm, 1H), 4.31 (t, J = 7.1
Hz, 2H), 4.14 (m, 1H), 3.29 (t, J = 6.5 Hz, 2H), 2.81 (s, 3H), 2.62 (t, J = 7.2 Hz, 4H), 2.55
13
(m, 1H), 2.20-1.20 (m, 2H); C NMR (CDCl ): δ 171.41, 170.53, 169.68, 148.06, 140.58,
3
139.50, 138.89, 128.60, 128.44, 128.38, 127.68, 127.39, 121.51, 59.94, 59.22, 51.33, 50.22,
50.01, 36.37, 35.97, 34.92, 34.69, 32.01, 30.13, 29.74, 28.48, 28.44, 28.41, 28.21, 24.93,
+
24.50, 23.13, 13.83; ESI MS: 797.5 (M + H) .
(3S,6S,10aS)-6-((R)-2-acetamido-3-(1H-indol-3-yl)propanamido)-N-((S)-(1-(4-(4-
(4-(4-((S)-((3S,6S,10aS)-6-((R)-2-(methylamino)propanamido)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamido)(phenyl)methyl)-1H-1,2,3-
triazol-1-yl)butyl)phenyl)butyl)-1H-1,2,3-triazol-4-yl)(phenyl)methyl)-5-
oxodecahydropyrrolo[1,2-a]azocine-3-carboxamide (30)—CuSO (10 mg, 0.04
4
mmol) and (+)-sodium l-ascorbate (20 mg, 0.1 mmol) in H O (5 mL) was added to a
2
solution of compound 33 (15 mg, 0.026 mmol) and 32 (21 mg, 0.026 mmol) in tert-butyl
alcohol (10 mL). The mixture was stirred at room temperature overnight and then extracted
with CH Cl (3 × 30 mL). The combined organic layer was washed with brine, dried over
2
2
Na SO , and evaporated. The residue was purified by chromatography to give a bis-triazole.
2
4
To a solution of this bis-triazole in MeOH (5 mL) was added HCl (4N in 1,4-dioxane, 1
mL). The solution was stirred at room temperature overnight and then concentrated to
furnish a crude product which was purified by C18 reversed phase semipreparative HPLC to
1
give compound 30 (21.4 mg, yield 65%). H NMR (D O-CD OD 1:1): δ 8.90 (m, 1H), 7.72
2
3
(s, 2H), 7.60 (m, 1H), 7.50-7.30 (m, 11H), 7.25-6.95 (m, 7H), 6.30-6.25 (m, 2H), 4.70 (m,
1H), 4.65 (m, 1H), 4.52 (m, 2H), 4.50-4.45 (m, 4H), 4.45-4.30 (m, 3H), 3.85 (m, 1H), 3.30
(m, 1H), 3.20 (m, 1H), 2.70 (m, 3H), 2.65 (m, 4H), 2.30-1.40 (m, 38H); ESI MS: m/z 1264.7
+
(M + H) ; Anal. (C H N O 1.7CF COOH): C, H, N.
71 89 15
7
3
II. Fluorescence polarization based assays for XIAP, cIAP-1 and cIAP-2 proteins
A set of sensitive and quantitative fluorescence polarization (FP)-based assays were used to
determine the binding affinities of the designed Smac mimetics to XIAP BIR3, XIAP linker-
BIR2-BIR3, cIAP-1 BIR3, cIAP-1 BIR2-BIR3 and cIAP-2 BIR3 proteins.
Protein expression and purification—Human XIAP BIR3 (residues 241–356) and
linker-BIR2-BIR3 (residues 120–356) were cloned into a pET28 vector (Novagen)
containing an N-terminal 6xHis tag. Protein was produced in E. coli BL21(DE3) cells grown
at 37°C in 2xYT containing kanamycin to an OD of 0.6.
600
Protein expression was induced by IPTG (0.4 mM) at 27°C for 4 hours. Cells were lysed by
sonication in buffer containing Tris pH 7.5 (50 mM), NaCl (200 mM), ZnAc (50 μM), 0.1%
βME and Leupectin/Aprotin protease inhibitors. Protein was purified from the soluble
fraction using Ni-NTA resin (QIAGEN) followed by gel filtration on a Superdex 75 column
in Tris pH 7.5 (20 mM), NaCl (200 mM), ZnAc (50 μM), and dithiothreital (DTT, 1 mM).
After purification, DTT was added to a final concentration of 10 mM. Human cIAP-1 BIR3
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Sun et al.
Page 12
(residues 253–363), cIAP-1 BIR2-BIR3 (residues 139–363) and cIAP2 BIR3 (residues 238–
349) were cloned into pHis-TEV vector, produced and purified using the same method as for
the XIAP protein.
FP-based binding assays—A fluorescently labeled Smac mimetic (Smac-2F) was used
in FP assays to determine the binding affinities of our Smac mimetics to XIAP BIR3,
33
cIAP-1 BIR3, cIAP-2 BIR3, and cIAP-1 BIR2-BIR3 proteins. The K values of Smac-2F
d
to XIAP BIR3, cIAP-1 BIR3, cIAP-2 BIR3, and cIAP-1 BIR2-BIR3 were determined by
monitoring the total fluorescence polarization of mixtures composed with fluorescent tracer
at a fixed concentration and proteins with increasing concentrations up to full saturation.
Fluorescence polarization values were measured using the Infinite M-1000 plate reader
(Tecan U.S., Research Triangle Park, NC) in Microfluor 2 96-well, black, round-bottom
plates (Thermo Scientific). To each well, SMAC-2F (2nM, 1nM, 1nM, and 1nM for
experiments with XIAP BIR3, cIAP-1BIR3, cIAP-2 BIR3, and cIAP-1 BIR2-BIR3,
respectively) and increasing concentrations of protein were added to a final volume of 125
μl in the assay buffer (100mM potassium phosphate, pH 7.5, 100 μg/ml bovine γ-globulin,
0.02% sodium azide, Invitrogen, with 4% DMSO). Plates were incubated at room
temperature for 2–3 hours and mixed with gentle shaking to assure equilibrium. The
polarization values in millipolarization units (mP) were measured at an excitation
wavelength of 485 nm and an emission wavelength of 530 nm. Equilibrium dissociation
constants (K ) were then calculated by fitting the sigmoidal dose-dependent FP increases as
d
a function of protein concentrations using Graphpad Prism 5.0 software (Graphpad
Software, San Diego, CA).
The K values of inhibitors were determined through an inhibitor dose-dependent
i
competitive binding experiment in which serial dilutions of inhibitor competed against fixed
concentration of the fluorescent tracer for binding to a fixed concentration of the protein
(typically 2 to 3 times the K values determined above). Mixtures of 5 μl of the tested
d
compounds in DMSO and 120 μl of preincubated protein/tracer complex in the assay buffer
(100mM potassium phosphate, pH 7.5, 100 μg/ml bovine γ-globulin, 0.02% sodium azide,
Invitrogen) were added into assay plates and incubated at room temperature for 3 h with
gentle shaking. Final concentrations of proteins and tracers were 10nM and 2nM, 3nM and
1nM, 5nM and 1nM, and 6nM and 1nM for assays for XIAP BIR3, cIAP-1 BIR3, cIAP-2
BIR3, and cIAP-1 BIR2-BIR3, respectively. Negative controls containing protein/tracer
complex only (equivalent to 0% inhibition), and positive controls containing only free
tracers (equivalent to 100% inhibition), were included in each assay plate. FP values were
measured as described above. IC values were determined by nonlinear regression fitting of
50
the competition curves. The K values of competitive inhibitors were calculated using the
i
41
derived equation described previously , based upon the measured IC values, the K
50
d
values of the tracer to different proteins, and the concentrations of the proteins and tracers in
the competitive assays.
42
The FP-based assay for XIAP linker-BIR2-BIR3 protein has been described in detail. In
this assay, a bivalent fluorescently tagged peptidic Smac mimetic (Smac-1F) was used as the
42
fluorescent tracer in this FP-based binding assay.
III. Caspase-9 and Caspase-3 activity assays
For the caspase-9 activity assay, the enzymatic activity of active recombinant caspase-9
(Enzo Life Sciences) was evaluated by the caspase-Glo 9 Assay kit from Promega. 2.5 μL of
a solution of the compound in caspase assay buffer (CAB, 50 mM of HEPES, 100 mM of
NaCl, 1 mM of EDTA with 0.1% of CHAPS and 10% of Glycerol, pH 7.4) containing 20%
DMSO was mixed with 7.5 μL of XIAP protein containing linker-BIR2-BIR3 and pre-
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incubated for 15 minutes, followed by addition of 2.5 μL of active caspase-9 solution in
CAB. This mixture was incubated at room temperature for 15 minutes. Luminogenic Z-
LEHD substrate was added with 1:1 ratio to give final concentrations of XIAP and
Caspase-9 of 500 nM and 2.5 unit/reaction (according to the manufacturer’s instructions),
respectively. This mixture was incubated at room temperature without light for 1 h, and
luminescence from the substrate cleavage was then determined by Tecan Infinite M-1000
multimode plate reader.
For caspase-3 activity assay, the enzymatic activity of Caspase-3 was determined using the
Caspase-3 Fluorescent Assay kit (BD Biosciences). 5 μL of a solution of the compound in
CAB with 20% DMSO was pre-incubated with 15 μL of XIAP linker-BIR2-BIR3 protein
for 15 minutes followed by addition of 5 μL of active Caspase-3 solution and the mixture
was incubated at room temperature for 15 minutes. Fluorescent Ac-DEVD-AFC substrate
was added at 1:1 ratio to give final concentrations of XIAP, Caspase-3, and Ac-DEVD-AFC
at 20 nM, 40ng/mL, and 125ng/mL, respectively. Fluorescence from the cleavage of
substrate was measured by Tecan Infinite M-1000 multimode plate reader using an
excitation wavelength of 400 nm and an emission wavelength of 505 nm. The reaction was
monitored for 1–2 h.
IV. Cell growth inhibition assay
The MDA-MB-231 cell line was purchased from the American Type Culture Collection.
3
Cells were seeded in 96-well flat bottom cell culture plates at a density of 3–4×10 cells/
well and grown overnight, then incubated with a compound at different concentrations. The
rate of cell growth inhibition after treatment with different concentrations of a compound
was determined by assaying with (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-
disulfophenyl)-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. The concentrations of the
compounds that inhibited cell growth by 50% (IC ) was calculated by comparing
50
absorbance in the untreated cells and the treated cells.
V. 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 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 cIAP-1 and
cIAP-2 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.
VI. Determination of intracellular concentrations of Smac mimetics
6
MDA-MB-231 cells were cultured in 100 mm cell culture dishes at a density of 10–15×10
cells/dish and incubated with 300 nM of a compound at 37°C for 5s, 30s, 60s, 1 h, 3 h, or 6
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Sun et al.
Page 14
h. After incubation, culture medium with a compound was aspirated and the adherently
growing cells were washed with cold PBS (10mL × 3, 10 seconds/wash). Cells were then
scraped directly into 2 mL of pure methanol. After methanol was removed by evaporation,
cell pellets were reconstituted in 100 μL of deionized water. To complete the cell lyses, cell
suspensions were sonicated in a water bath for 10 minutes followed by centrifuge at 14,000
RPM for 5 minutes. Supernatant aliquot (20 μL) was mixed with 60 μL of acetonitrile
(containing internal standard at 300nM) to precipitate proteins. Supernatant (5 μL) was
injected for LC-MS/MS analysis after centrifuging at 14,000 RPM for 5 min. Total protein
concentrations of the supernatant were determined by Micro BCA protein assay kit from
Pierce. Compound concentrations determined by LC-MS/MS were normalized to the total
protein concentrations to compensate the cell number difference of each cell dish.
Quantitative LC-MS/MS analysis was conducted using an Agilent 1200 HPLC system
coupled to an API 3200 mass spectrometer (Applied Biosystems, MDS Sciex Toronto,
Canada) equipped with an API electrospray ionization (ESI) source.
Aliquots (5 μL) were injected onto a reversed-phase column [5 cm × 2.1 mm I.D., packed
with 3.5 μm Zorbax Bonus-RP (Agilent)]. The mobile phase consisted of 0.1% formic acid
in water (A) and 0.1% formic acid in acetonitrile (B). The mobile phase A was held at 10%
for 0.5 min, increased from 10% to 98% over 0.1 min, held at 98% for an additional 4 min,
and then immediately stepped back down to 10% for re-equilibration. The mobile phase was
eluted at 0.4 mL/min.
VII. In vivo antitumor efficacy study
Female severe combined immunodeficiency (SCID) mice were injected subcutaneously with
6
5 × 10 MDA-MB-231 cells in 50% Matrigel per mouse. Treatment started when the tumors
3
reached an average volume of 150 mm on day 26. Mice were treated with vehicle (9 mice
per group), taxotere at 7.5mg/kg intravenously on treatment days 2 & 9 (8 per group),
compound 27 at 1 or 5mg/kg given intravenously on days 1–5 & 8–12 with 8 mice per
group. Tumor sizes and animal weights were measured 3 times a week during the treatment
and twice a week after the treatment. Data are presented as mean tumor volumes ± SEM.
Statistical analyses were performed by two-way ANOVA and unpaired two-tailed t test,
using Prism (version 4.0, GraphPad, La Jolla, CA). P < 0.05 was considered statistically
significant. The efficacy experiment was performed under the guidelines of the University of
Michigan Committee for Use and Care of Animals.
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 secretary assistance.
Abbreviations
IAP
Inhibitor of apoptotic protein
X-linked IAP
XIAP
cIAP
Smac
BIR
cellular IAP
second mitochondria derived activator of caspases
baculoviral IAP repeat
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Page 15
PARP
FP
poly(ADPribose) polymerase
fluorescence polarization
mP
millipolarization units
TRAF1
tumor necrosis factor associated factor 2
References
1. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell. 2000; 100:57–70. [PubMed: 10647931]
2. Lowe SW, Lin AW. Apoptosis in cancer. Carcinogenesis. 2000; 21:485–495. [PubMed: 10688869]
3. Nicholson DW. From bench to clinic with apoptosis-based therapeutic agents. Nature. 2000;
407:810–816. [PubMed: 11048733]
4. Salvesen GS, Duckett CS. Apoptosis: IAP proteins: blocking the road to death’s door. Nat Rev Mol
Cell Biol. 2002; 3:401–410. [PubMed: 12042762]
5. Deveraux QL, Reed JC. IAP family proteins—suppressors of apoptosis. Genes Dev. 1999; 13:239–
252. [PubMed: 9990849]
6. Holcik M, Gibson H, Korneluk RG. XIAP: Apoptotic brake and promising therapeutic target.
Apoptosis. 2001; 6:253–261. [PubMed: 11445667]
7. Shiozaki EN, Shi Y. Caspases, IAPs and Smac/DIABLO: mechanisms from structural biology.
Trends Biochem Sci. 2004; 29:486–494. [PubMed: 15337122]
8. Srinivasula SM, Ashwell JD. IAPs: what’s in a name? Mol Cell. 2008; 30:123–135. [PubMed:
18439892]
9. Gyrd-Hansen M, Meier P. IAPs: from caspase inhibitors to modulators of NF-kappaB, inflammation
and cancer. Nat Rev Cancer. 2010; 10:561–574. [PubMed: 20651737]
10. Mehrotra S, Languino LR, Raskett CM, Mercurio AM, Dohi T, Altieri D. C IAP Regulation of
Metastasis. Cancer Cell. 2010; 17:53–64. [PubMed: 20129247]
11. LaCasse EC, Mahoney DJ, Cheung HH, Plenchette S, Baird S, Korneluk RG. IAP-targeted
therapies for cancer. Oncogene. 2008; 27:6252–6275. [PubMed: 18931692]
12. Vucic D, Fairbrother WJ. The inhibitor of apoptosis proteins as therapeutic targets in cancer. Clin
Cancer Res. 2007; 13:5995–6000. [PubMed: 17947460]
13. Fulda S. Inhibitor of apoptosis proteins as targets for anticancer therapy. Expert Rev Anticancer
Ther. 2007; 7:1255–1264. [PubMed: 17892425]
14. Du C, Fang M, Li Y, Li L, Wang X. Smac, a mitochondrial protein that promotes cytochrome c-
dependent caspase activation by eliminating IAP inhibition. Cell. 2000; 102:33–42. [PubMed:
10929711]
15. Verhagen AM, Ekert PG, Pakusch M, Silke J, Connolly LM, Reid GE, Moritz RL, Simpson RJ,
Vaux DL. Identification of DIABLO, a mammalian protein that promotes apoptosis by binding to
and antagonizing IAP proteins. Cell. 2000; 102:43–53. [PubMed: 10929712]
16. Wu G, Chai J, Suber TL, Wu JW, Du C, Wang X, Shi Y. Structural basis of IAP recognition by
Smac/DIABLO. Nature. 2000; 408:1008–1012. [PubMed: 11140638]
17. Liu Z, Sun C, Olejniczak ET, Meadows R, Betz SF, Oost T, Herrmann J, Wu JC, Fesik SW.
Structural basis for binding of Smac/DIABLO to the XIAP BIR3 domain. Nature. 2000;
408:1004–1008. [PubMed: 11140637]
18. Huang Y, Rich RL, Myszka DG, Wu H. Requirement of both the second and third BIR domains
for the relief of X-linked inhibitor of apoptosis protein (XIAP)-mediated caspase inhibition by
Smac. J Biol Chem. 2003; 278:49517–49522. [PubMed: 14512414]
19. Samuel T, Welsh K, Lober T, Togo SH, Zapata JM, Reed JC. Distinct BIR domains of cIAP1
mediate binding to and ubiquitination of tumor necrosis factor receptor-associated factor 2 and
second mitochondrial activator of caspases. J Biol Chem. 2006; 281:1080–1090. [PubMed:
16282325]
20. Wang S. Design of Small-Molecule Smac Mimetics as IAP Antagonists. Curr Top Microbiol
Immunol. 2010 Nov 12. [Epub ahead of print].
J Med Chem. Author manuscript; available in PMC 2012 May 12.

Sun et al.
Page 16
21. Sun H, Nikolovska-Coleska Z, Yang CY, Qian D, Lu J, Qiu S, Bai L, Peng Y, Cai Q, Wang S.
Design of small-molecule peptidic nonpeptidic Smac mimetics. Acc Chem Res. 2008; 41:1264–
1277. [PubMed: 18937395]
22. Mannhold R, Fulda S, Carosati E. IAP antagonists: promising candidates for cancer therapy. Drug
Discovery Today. 2010; 15:210–219. [PubMed: 20096368]
23. Cai Q, Haiying Sun H, Peng Y, Lu J, Nikolovska-Coleska Z, McEachern D, Liu L, Qiu S, Yang C-
Y, Miller R, Yi H, Zhang T, Sun D, Kang S, Guo M, Leopold L, Yang D, Wang S. A potent and
orally active antagonist (SM-406/AT-406) of multiple inhibitor of apoptosis proteins (IAPs) in
clinical development for cancer treatment. J Med Chem. 2011 (in press).
24. Sun H, Nikolovska-Coleska Z, Yang CY, Xu L, Liu M, Tomita Y, Pan H, Yoshioka Y, Krajewski
K, Roller PP, Wang S. Structure-Based Design of Potent, Conformationally Constrained Smac
Mimetics. J Am Chem Soc. 2004; 126:16686–16687. [PubMed: 15612682]
25. Sun H, Nikolovska-Coleska Z, Yang CY, Xu L, Tomita Y, Krajewski K, Roller PP, Wang S.
Structure-based design, synthesis, and evaluation of conformationally constrained mimetics of the
second mitochondria-derived activator of caspase that target the X-linked inhibitor of apoptosis
protein/caspase-9 interaction site. J Med Chem. 2004; 47:4147–4150. [PubMed: 15293984]
26. Li L, Thomas RM, Suzuki H, De Brabander JK, Wang X, Harran PG. A small molecule Smac
mimic potentiates TRAIL- and TNF alpha-mediated cell death. Science. 2004; 305:1471–1474.
[PubMed: 15353805]
27. Oost TK, Sun C, Armstrong RC, Al-Assaad AS, Betz SF, Deckwerth TL, Ding H, Elmore SW,
Meadows RP, Olejniczak ET, Oleksijew A, Oltersdorf T, Rosenberg SH, Shoemaker AR,
Tomaselli KJ, Zou H, Fesik SW. Discovery of potent antagonists of the antiapoptotic protein
XIAP for the treatment of cancer. J Med Chem. 2004; 47:4417–4426. [PubMed: 15317454]
28. Sun H, Nikolovska-Coleska Z, Lu J, Qiu S, Yang CY, Gao W, Meagher J, Stuckey J, Wang S.
Design, synthesis, and evaluation of a potent, cell-permeable, conformationally constrained second
mitochondria derived activator of caspase (Smac) mimetic. J Med Chem. 2006; 49:7916–7920.
[PubMed: 17181177]
29. Sun H, Stuckey JA, Nikolovska-Coleska Z, Qin D, Meagher JL, Qiu S, Lu J, Yang CY, Saito NG,
Wang S. Structure-based design, synthesis, evaluation, and crystallographic studies of
conformationally constrained Smac mimetics as inhibitors of the X-linked inhibitor of apoptosis
protein (XIAP). J Med Chem (Article). 2008; 51(22):7169–7180.
30. Peng Y, Sun H, Nikolovska-Coleska Z, Qiu S, Yang CY, Lu J, Cai Q, Yi H, Wang S. Design,
Synthesis and Evaluation of Potent and Orally Bioavailable Diazabicyclic Smac Mimetics. J Med
Chem. 2008; 51:8158–8162. [PubMed: 19049347]
31. Zhang B, Nikolovska-Coleska Z, Zhang Y, Bai L, Qiu S, Yang CY, Sun H, Wang S, Yikang Wu
Y. J Med Chem. 2008; 51:7352–7355. [PubMed: 19012392]
32. Sun W, Nikolovska-Coleska Z, Qin D, Sun H, Yang CY, Bai L, Qiu S, Ma D, Wang S. Design,
Synthesis and Evaluation of Potent, Non-Peptidic Smac Mimetics. J Med Chem. 2009; 52:593–
596. [PubMed: 19138149]
33. Sun H, Lu J, Liu L, Yi H, Qiu S, Yang CY, Deschamps JR, Wang S. Nonpeptidic and Potent
Small-Molecule Inhibitors of cIAP-1/2 and XIAP Proteins. J Med Chem. 2010; 53:6361–6367.
[PubMed: 20684551]
34. Zobel K, Wang L, Varfolomeev E, Franklin MC, Elliott LO, Wallweber HJ, Okawa DC, Flygare
JA, Vucic D, Fairbrother WJ, Deshayes K. Design, synthesis, and biological activity of a potent
Smac mimetic that sensitizes cancer cells to apoptosis by antagonizing IAPs. ACS Chem Biol.
2006; 1:525–533. [PubMed: 17168540]
35. Sun H, Nikolovska-Coleska Z, Lu J, Meagher JL, Yang CY, Qiu S, Tomita Y, Ueda Y, Jiang S,
Krajewski K, Roller PP, Stuckey JA, Wang S. Design synthesis and characterization of a potent
nonpeptide cell-permeable bivalent Smac mimetic that concurrently targets both the BIR2 BIR3
domains in XIAP. J Am Chem Soc. 2007; 129:15279–15294. [PubMed: 17999504]
36. Varfolomeev E, Blankenship JW, Wayson SM, Fedorova AV, Kayagaki N, Garg P, Zobel K,
Dynek JN, Elliott LO, Wallweber HJ, Flygare JA, Fairbrother WJ, Deshayes K, Dixit VM, Vucic
D. IAP antagonists induce autoubiquitination of c-IAPs, NF-kappaB activation, and TNFalpha-
dependent apoptosis. Cell. 2007; 131:669–681. [PubMed: 18022362]
J Med Chem. Author manuscript; available in PMC 2012 May 12.

Sun et al.
Page 17
37. Vince JE, Wong WW, Khan N, Feltham R, Chau D, Ahmed AU, Benetatos CA, Chunduru SK,
Condon SM, McKinlay M, Brink R, Leverkus M, Tergaonkar V, Schneider P, Callus BA,
Koentgen F, Vaux DL, Silke J. IAP antagonists target cIAP1 to induce TNFalpha-dependent
apoptosis. Cell. 2007; 131:682–693. [PubMed: 18022363]
38. Petersen SL, Wang L, Yalcin-Chin A, Li L, Peyton M, Minna J, Harran P, Wang X. Autocrine
TNFalpha signaling renders human cancer cells susceptible to Smac-mimetic-induced apoptosis.
Cancer Cell. 2007; 12:445–456. [PubMed: 17996648]
39. Lu J, Bai L, Sun H, Nikolovska-Coleska Z, McEachern D, Qiu S, Miller RS, Yi H, Shangary S,
Sun Y, Meagher JL, Stuckey JA, Wang S. SM-164: A novel, bivalent Smac mimetic that induces
apoptosis and tumor regression by concurrent removal of the blockade of cIAP-1/2 and XIAP.
Cancer Research. 2008; 68:9384–9393. [PubMed: 19010913]
40. Nikolovska-Coleska Z, Meagher JL, Jiang S, Yang CY, Qiu S, Roller PP, Stuckey JA, Wang S.
Interaction of a cyclic, bivalent smac mimetic with the x-linked inhibitor of apoptosis protein.
Biochemistry. 2008; 47:9811–9824. [PubMed: 18717598]
41. Nikolovska-Coleska Z, Wang R, Fang X, Pan H, Tomita Y, Li P, Roller PP, Krajewski K, Saito
NG, Stuckey JA, Wang S. Development and optimization of a binding assay for the XIAP BIR3
domain using fluorescence polarization. Analytical Biochemistry. 2004; 332:261–273. [PubMed:
15325294]
42. Nikolovska-Coleska Z, Meagher JL, Jiang S, Kawamoto SA, Gao W, Yi H, Qin D, Roller PP,
Stuckey JA, Wang S. Design and characterization of bivalent Smac-based peptides as antagonist
of XIAP and development and validation of a fluorescence polarization assay for XIAP containing
both BIR2 and BIR3 domains. Analytical Biochemistry. 2008; 374:87–98. [PubMed: 18023397]
J Med Chem. Author manuscript; available in PMC 2012 May 12.

Sun et al.
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Figure 1.
Chemical structures of Smac AVPI peptide and previously reported monovalent Smac
mimetics.
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Figure 2.
Chemical structures of previously reported bivalent Smac mimetics.
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Figure 3.
Chemical structures of previously reported monovalent Smac mimetic 15, bivalent Smac
mimetic 16 and an inactive analogue 17.
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Figure 4.
Chemical structures of new bivalent Smac mimetics.
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Figure 5.
Functional antagonism of Smac mimetics against XIAP linker-BIR2-BIR3 in a cell-free
caspase-9 functional assay. Data shown in the figure are averages and standard deviations of
duplicate wells in assay plates, and the figure is the representative of three independent
experiments.
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Figure 6.
Functional antagonism of Smac mimetics against XIAP linker-BIR2-BIR3 in a cell-free
caspase-3 functional assay. Data shown in the figure are averages and standard deviations of
duplicate wells in assay plates and the figure is representative of three independent
experiments.
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Figure 7.
Induction of cIAP-1, cIAP-2 and XIAP degradation, cleavage of PARP, and processing of
caspase-3 by compounds 16, 18, 19, 20 and 21 in the MDA-MB-231 cell line. Cells were
treated with different concentrations of Smac mimetics for 24 h and cIAP-1, cIAP-2, XIAP,
cleaved PARP (CL PARP), and cleaved caspase-3 (CL C3) were probed by Western blot
analysis.
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Figure 8.
(A) Concentrations of compounds 16 and 18 in 100 μL of cell lysates; and (B) amount of
compounds per μg of total proteins. 300nM of each compound was incubated with 10–
6
15×10 MDA-MB-231 cells for different times. After being washed by PBS, cells were
lysed and resuspended in 100 μL of deionized water. Compound concentrations and protein
concentrations were determined by LC-MS/MS and Micro BCA protein assay, respectively.
Data shown in the figure are averages and standard deviations of three independent
experiments.
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Figure 9.
Antitumor activity of compound 27 in the MDA-MB-231 xenograft model in SCID mice.
(A). Mean tumor volume. (B). Mean animal weight. Treatment started when the tumors
3
reached an average volume of 150mm on day 26. Treatment groups consisted of vehicle
control (9 mice per group), Taxotere 7.5mg/kg given intravenously on treatment days 2 and
9 with 8 mice per group, compound 27 at 1 mg/kg or 5mg/kg given intravenously on days
1–5 and 8–12, with 8 mice per group.
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Scheme 1.
Synthesis of bivalent Smac mimetics.
Reagents and conditions: (a) i. bis-azides, CuSO , (+)-sodium -L-ascorbate, tert-butanol-
4
H O 2:1; ii. 4 N HCL in 1,4-dioxane, MeOH; (b) 1,4-bis-(4-azido-butyl)-benzene (5 eq),
2
CuSO , (+)-sodium-L-ascorbate, tert-butanol-H O 2:1, 62%; (c) i. 33, CuSO , (+)-sodium -
4
2
4
L-ascorbate, tert-butanol-H O 2:1; ii 4 N HCL in 1,4-dioxane, MeOH
2
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