Structure-based design, synthesis, evaluation, and cristallographic studies of conformationally constrained Smac mimetics as inhibitors of the x-linked inhibitor of apoptosis protein (XIAP)

Article abstract of DOI:10.1021/jm8006849

Small molecules designed to mimic the binding of Smac protein to X-linked inhibitor of apoptosis protein (XIAP) are being pursued as a promising new class of anticancer drugs. Herein, we report the design, synthesis, and comprehensive structure-activity r

Full text of DOI:10.1021/jm8006849

J. Med. Chem. 2008, 51, 7169–7180
7169
Structure-Based Design, Synthesis, Evaluation, and Crystallographic Studies of
Conformationally Constrained Smac Mimetics as Inhibitors of the X-linked Inhibitor of
Apoptosis Protein (XIAP)†
Haiying Sun,‡, Jeanne A. Stuckey,§,O,2 Zaneta Nikolovska-Coleska,‡, Dongguang Qin,‡, Jennifer L. Meagher,O Su Qiu,‡,
,
Jianfeng Lu,‡, Chao-Yie Yang,‡, Naoyuki G. Saito,# and Shaomeng Wang*,‡,|,
Departments of Internal Medicine, Biological Chemistry, Pharmacology, Medicinal Chemistry, Radiation Oncology, ComprehensiVe Cancer
Center, Life Sciences Institute, Biophysics Research DiVision, UniVersity of Michigan, Ann Arbor, Michigan 48109
ReceiVed June 5, 2008
Small molecules designed to mimic the binding of Smac protein to X-linked inhibitor of apoptosis protein
(XIAP) are being pursued as a promising new class of anticancer drugs. Herein, we report the design,
synthesis, and comprehensive structure-activity relationship studies of a series of conformationally constrained
bicyclic Smac mimetics. Our studies led to the discovery of a number of highly potent and cell-permeable
Smac mimetics and yielded important new insights into their structure-activity relationship for their binding
to XIAP and for their activity in inhibition of cancer cell growth. Determination of the crystal structure of
one potent Smac mimetic, compound 21, in complex with XIAP BIR3 provides the structural basis for its
high-affinity binding to XIAP and for the design of highly potent Smac mimetics.
Introduction
suppressing function, XIAP is found to be highly expressed in
many human tumor cell lines and tumor samples from patients11
and plays an important role in conferring resistance on cancer
cells to a variety of anticancer drugs.8,9 Because XIAP blocks
apoptosis at the downstream effector phase, a point where
multiple signaling pathways converge, it represents a particularly
attractive molecular target for the design of new classes of
anticancer drugs aimed at overcoming the apoptosis resistance
of cancer cells.8,9,12
Apoptosis is a critical cell process in normal development
and homeostasis of multicellular organisms to eliminate un-
wanted or damaged cells. Inappropriate regulation of apoptosis
plays a major role in many human diseases, including cancer.1-4
Defects in the apoptosis machinery confers apoptosis resistance
on cancer cells to therapeutic agents, makes current anticancer
therapies less effective, and leads ultimately to their failure in
the clinic.2-4 Accordingly, targeting critical apoptosis regulators
aimed at overcoming apoptosis resistance of cancer cells is a
promising cancer therapeutic strategy.
The antiapoptotic function of XIAP is antagonized by second
mitochondria-derived activator of caspases or direct IAP binding
protein with low pI (Smac/DIABLO), a protein released from
mitochondria into the cytosol in response to apoptotic stimuli.13,14
Crystal and NMR structures15,16 show that Smac, through its
N-terminal AVPI (Ala1-Val2-Pro3-Ile4) motif, interacts with
the XIAP BIR3 domain at the same site where caspase-9 binds
and removes the inhibition of XIAP to caspase-9 by direct
competition.17,18 The mechanism by which Smac removes the
inhibition of XIAP to caspase-3/-7 is not entirely clear. It has
been proposed that Smac protein removes the inhibition of XIAP
to caspase-3/-7 by binding to the XIAP BIR2 domain through
its AVPI motif.19 Hence, the AVPI binding motif in Smac plays
a critical role for the interaction of Smac protein with XIAP
and its functional antagonism against XIAP.
The X-linked inhibitor of apoptosis protein (XIAPa) has been
identified as a key apoptosis inhibitor, although its role in cells
may not be limited to the regulation of apoptosis.5-10 XIAP
inhibits apoptosis through direct binding to and inhibition of
three cysteine proteases, an initiator caspase-9 and the two
effectors caspase-3 and -7.5-10 XIAP contains three baculoviral
IAP repeats (BIR) domains. While the third BIR domain (BIR3)
of XIAP selectively targets caspase-9, the BIR2 domain, together
with the immediate preceding linker, inhibits both caspase-3
and caspase-7. Because these caspases play a critical role in
the execution of apoptosis, XIAP functions as an efficient
inhibitor of apoptosis. Consistent with its potent apoptosis-
†
Coordinates of compound 21 in complex with XIAP BIR3 and structural
Earlier studies using Smac-based peptides containing the
AVPI binding motif, tethered to a carrier peptide for intracellular
delivery, have demonstrated that such compounds can enhance
the antitumor activity of chemotherapeutic agents and of TNF-
related apoptosis inducing ligand in vitro and in vivo.20-22
Although Smac-based peptides have served as useful tools for
important proof-of-concept studies, they are not suitable drug
candidates due to their very limited cellular activity and expected
poor in vivo stability. To overcome the limitations associated
with peptide-based Smac mimetics, a number of laboratories,
including ours, have pursued the design of peptidic and
nonpeptidic small-molecule Smac mimetics with a goal to obtain
more druglike compounds, which may be developed as a new
class of anticancer drugs.23-30
factors have been deposited in the RCSB Protein Data Bank (access code
2JK7).
* To whom correspondence should be addressed. Phone: 734-615-0362.
Fax: 734-647-9647. E-mail: shaomeng@umich.edu.
‡
Department of Internal Medicine, University of Michigan.
Department of Biological Chemistry, University of Michigan.
Department of Pharmacology, University of Michigan.
§
|
Department of Medicinal Chemistry, University of Michigan.
Department of Radiation Oncology, University of Michigan.
#
Comprehensive Cancer Center, University of Michigan.
Life Sciences Institute, University of Michigan.
O
2
Biophysics Research Division, University of Michigan.
a Abbreviations: IAP, inhibitor of apoptosis protein; XIAP, X-linked IAP;
Smac, second mitochondria-derived activator of caspases; DIABLO, direct
IAP binding protein with low pI; BIR, baculoviral IAP repeats (BIR)
domain; BIR2, the second BIR domain; BIR3, the third BIR domain; FP,
fluorescence polarization.
10.1021/jm8006849 CCC: $40.75
2008 American Chemical Society
Published on Web 10/28/2008

7170 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Sun et al.
Figure 1. Design and examples of conformationally constrained bicyclic Smac peptide mimetics.
Using a structure-based approach, our laboratory has reported
the design of a number of conformationally constrained, bicyclic
Smac mimetics.23,24,26,30 Our previous studies showed that these
designed Smac mimetics can achieve high binding affinities to
XIAP and are effective in inhibition of cell growth and induction
of apoptosis in cancer cells. For example, SM-131, which
contains a [7,5] bicyclic core structure, binds to XIAP BIR3
protein with a Ki of 61 nM in a competitive binding assay and
directly antagonizes the XIAP inhibition of caspase-9 activity
in a cell-free functional assay.26 This compound also potently
inhibits cancer cell growth and induces apoptosis in cancer cells
as a single agent.26
peptide inserts into a hydrophobic pocket in XIAP BIR3 and
the carbonyl group of Ile4 does not have specific interactions
with the protein.15 We thus replaced the Ile4 residue with a
simple benzylamine group, which led to compound 1.23,24
Compound 1 binds to XIAP BIR3 protein with a Ki of 0.29
µM in our fluorescence-polarization (FP) binding assay32 and
is 2 times more potent than the natural Smac AVPI peptide
(Ki ) 0.58 µM) under the same assay conditions. Compound 1
was thus used as a new lead in our subsequent design.
The isopropyl side chain in Val2 has no specific interactions
with the protein in the crystal structure, while the five-membered
ring of Pro3 has van der Waals contacts with the side chain of
Trp323 in the protein. Modeling suggested that the isopropyl
side chain in Val2 may be fused with the five-membered ring
of Pro3 to form a bicyclic lactam structure without causing steric
clashes with the protein.23,24 Cyclization of the side chain of
Val2 and the five-membered ring of Pro3 generates a new chiral
center. To probe the stereospecificity of this new chiral center
for binding to XIAP, we designed and synthesized stereoisomers
2 and 3, which contain a [6,5] bicyclic lactam core structure.24
Compound 2 binds to XIAP BIR3 with a Ki value of 4.47 µM,
but compound 3 shows no appreciable binding at concentrations
up to 100 µM. Hence, the binding data for compounds 2 and 3
established that the S-configuration (up-configuration) of the
chiral center in the bicyclic Smac mimetics is highly preferred
for their binding to XIAP BIR3. Accordingly, in our subsequent
design and synthesis, we have focused on compounds with the
S-configuration (up-configuration) for the chiral center.
Although compound 2 binds to XIAP BIR3 with a good
affinity, it is 8 times less potent than the Smac AVPI peptide
and 15 times less potent than compound 1. Modeling showed
that, although compound 2 largely mimics the bound conforma-
tion of the AVPI peptide to XIAP BIR3, its rigid [6,5] bicyclic
structure causes some deviations (Figure 2A) that may account
for its lower binding affinity to XIAP BIR3. Therefore, we
designed and synthesized compounds 423 and 5, in which the
six-membered ring in 2 was expanded to a seven- or eight-
membered ring, respectively. Modeling predicted that these two
compounds can nicely mimic the bound conformation of the
Smac AVPI peptide to XIAP BIR3 protein (Figure 2B,C) and
Although our previous studies23,24,26,30 have led to the
discovery of potent and cell-permeable Smac mimetics, our
understanding on their structure-activity relationship is still
limited. Furthermore, although molecular modeling was em-
ployed to predict the binding models of our designed Smac
mimetics to XIAP BIR3 protein in our previous studies, the
predicted binding models have not been experimentally confirmed.
To gain a more in-depth understanding of the structure-activity
relationship for our designed conformationally constrained Smac
mimetics for their binding to XIAP and for their cellular activity,
we have designed, synthesized, and evaluated a series of new
Smac mimetics. To obtain a solid structural basis for the
interaction of our designed Smac mimetics with XIAP BIR3,
we have determined a high-resolution crystal structure of a
potent Smac mimetic (compound 21) in complex with XIAP
BIR3. We report herein structure-based design, synthesis,
biochemical and biological evaluation, and crystallographic
studies of conformationally constrained Smac mimetics as
antagonists of XIAP.
Results and Discussion
Structure-Based Design of Conformationally Constrained
Smac Mimetics and Their Structure-Activity Relationship. We
have employed a structure-based strategy for the design of
conformationally constrained, bicyclic Smac mimetics (Fig-
ure 1).23,24
In the crystal structure of Smac protein in complex with XIAP
BIR3, the hydrophobic side chain of Ile4 in the Smac AVPI

Conformationally Constrained Smac Mimetics
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7171
Figure 2. Predicted binding models of compounds 2, 4, 5, 20, and 21 to XIAP BIR3 protein in comparison with that for Smac AVPI peptide in
the crystal structure. Carbon atoms in compounds 2, 4, 5, 20, and 21 and AVPI are shown in stick. The carbon atoms are colored in yellow in 2,
4, 5, 20, and 21 and in green in the AVPI peptide. The carbon atoms in several key residues in XIAP are colored in gray. Nitrogen and oxygen
atoms are colored in blue and red, respectively.
suggested that both compounds should bind to XIAP BIR3 with
a higher affinity than compound 2. Indeed, compounds 4 and 5
achieved Ki values of 0.15 and 0.10 µM, respectively, and are
30 or 45 times more potent than compound 2, confirming the
modeling prediction.
We carried out further modifications in both N-terminal
(region 1) and C-terminal (region 2) regions (Figure 1) in
compounds 2, 4, or 5 to further explore the structure-activity
relationships in these two regions. The results are summarized
in Table 1.
In the crystal structure of Smac protein in complex with XIAP
BIR3, the methyl group of Ala1 inserts into a small hydrophobic
pocket. Compounds 6, 7, and 8 were designed and synthesized
in which the methyl group in 2 was replaced with an ethyl,
n-propyl, or isopropyl group, respectively. While compound 6
with an ethyl group is 3 times more potent than 2, compound
8 with an isopropyl group is 10 times less potent than 2 and
compound 7 with an n-propyl group shows no appreciable
binding up to 100 µM to XIAP BIR3. These data demonstrated
that the small hydrophobic pocket in XIAP BIR3 occupied by
the methyl group in Ala1 in the Smac AVPI peptide can not
accommodate a group larger than an ethyl group for effective
binding. Therefore, only the methyl or ethyl group was
employed in this position in our subsequent design and synthesis.
The positively charged primary amino group in Ala1 in the
Smac AVPI has strong charge-charge interactions with the
negatively charged carboxylic group in the side chain of Glu314
of XIAP BIR3 and is critical for binding of Smac-based peptides
to XIAP BIR3. However, we found that Smac mimetics with
high binding affinities to XIAP BIR3 but containing this primary
amino group have very weak cellular activity.23,24,26 Therefore,
we replaced this primary amine with a secondary or tertiary
amine or a hydroxyl group, which can also form one or more
hydrogen bonds with the protein under physiological condi-
tions.26 Their binding data indicated that replacement of the
amino group with an N-methyl amino group in Smac mimetics
containing either a [7,5] or [8,5] bicyclic lactam leads to
reduction of the binding affinity by 2-3 times (13 vs 12, 20 vs
16, and 21 vs 17). However, replacement of the primary amino
group with an N,N-dimethyl amino (14) or a cyclic tertiary
amino (22 and 23) or a hydroxyl group (15 and 24) decreases
the binding affinities by 20-800 times. These data show that

7172 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Sun et al.
Table 1. Chemical Structures of Conformationally Constrained Bicyclic
Smac Mimetics and Their Binding Affinities to XIAP BIR3
the primary and secondary amino groups are highly preferred
at this site for Smac mimetics to achieve high binding affinities
to XIAP BIR3.
The crystal structure of Smac in complex with XIAP BIR3
showed that the carbonyl group of Ile4 is exposed to solvent
and has no specific interaction with the protein.15 However, we
hypothesized that this carbonyl group may contribute to the
binding affinity of Smac to XIAP by controlling the orientation
of the hydrophobic side chain of Ile4, thus reducing the entropic
loss upon binding. To test this hypothesis, we replaced the
benzyl group in 2, 4, and 5 with a diphenyl methyl group, which
led to compounds 9, 11, and 16, respectively. These three
compounds (9, 11, and 16) were found to be three times more
potent than their corresponding analogues 2, 4, and 5, supporting
our hypothesis.
Our modeling predicted that the pro R phenyl group enters
the hydrophobic pocket and the pro S phenyl group is exposed
to solvent. To test this modeling prediction, we have designed
compounds 18 and 19 by replacing either the pro R or pro S
phenyl group in 17 with a naphthyl group. It was found that
while the S form isomer 18 is as potent as 17, the R form isomer
19 is 35 times less potent than 17, thus confirming our modeling
prediction.
We next tested if other structures that can control and restrict
the orientation of the phenyl group can also improve the binding
affinity. To this end, we have designed and synthesized
compounds 25-27, in which the conformation of the phenyl
group was further constrained by a fused second ring and was
previously employed for the design of potent, Smac peptido-
mimetics.25 Compound 25, in which the chiral amine has the R
configuration, has a Ki value of 14 nM, whereas its stereoisomer
26 has a Ki value of 207 nM. Thus, compound 25 is 15 times
more potent than its isomer 26, indicating that the R form is
the desirable configuration for the chiral center. Compound 27,
in which the (R)-1,2,3,4-tetrahydro-naphthalenyl group in 25
was replaced by a (R)-2,3-dihydro-1H-indene group, also binds
to XIAP BIR3 with a high affinity and has a Ki value of 15
nM.
Functional Antagonism of Smac Mimetics against
XIAP BIR3 Protein in a Cell-Free Functional Assay. On the
basis of their expected mode of action, Smac mimetics should
antagonize the inhibition of XIAP BIR3 to caspase-9 by binding
to the same site where caspase-9 binds. We thus established a
cell-free functional assay using recombinant XIAP BIR3 protein
and evaluated the ability of our designed Smac mimetics to
antagonize the XIAP BIR3 protein.26 In this assay, addition of
dATP and cytochrome c into cell lysates activates caspase-9,
and recombinant XIAP BIR3 protein inhibits the activity of
caspase-9 in a dose-dependent manner. XIAP BIR3 protein at
0.5 µM is capable of completely inhibiting the activity of
caspase-9 under the assay conditions (Figure 3).
We evaluated two potent Smac mimetics, compounds 20 and
25, and the Smac AVPI peptide in this cell-free functional assay
for their ability to directly overcome the inhibitory effect of
XIAP BIR3 protein to caspase-9 activity (Figure 3). Compound
24, which is >500-times less potent than compounds 20 and
25 in its binding affinity to XIAP BIR3, was included as a
negative control. It was found that compounds 20, 25, and the
AVPI peptide all effectively and dose-dependently restore the
activity of caspase-9, whereas compound 24 has no significant
effect at concentrations as high as 100 µM. On the basis of the
kinetic data, the calculated EC50 values for compounds 20, 25,
and the AVPI peptide in restoration of the caspase-9 activity at
60 min are 0.78, 0.63, and 4.8 µM, respectively, consistent with

Conformationally Constrained Smac Mimetics
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7173
Figure 3. Functional antagonism of compounds 20, 24, 25, and the
AVPI peptide against XIAP BIR3 to promote the activity of caspase-9
in a cell-free functional assay.
Figure 4. Inhibition of cell growth by Smac mimetics in the MDA-
MB-231 breast cancer and SK-OV-3 ovarian cancer cell lines. Cells
were treated for 4 days, and cell growth was determined using WST-
based assay.
their binding data to XIAP BIR3. Hence, our functional assay
established that compounds 20 and 25 are potent antagonists
of XIAP BIR3 and both of them are also more potent than the
Smac AVPI peptide. Consistent with its weak binding affinity,
24 is inactive at concentrations as high as 100 µM in the
functional assay.
Evaluation of the Activity of Smac Mimetics in Cell
Growth Inhibition and Induction of Cell Death and Apo-
ptosis in Human Cancer Cell Lines. One major motivation for
the design and synthesis of nonpeptidic Smac mimetics is to
dramatically improve the cellular activity over Smac-based peptides.
Although Smac mimetics were initially thought to be primarily
useful for sensitizing cancer cells to other anticancer drugs,20,21,23,24,29
a number of studies have shown that properly designed Smac
mimetics are capable of inhibiting cancer cell growth and inducing
apoptosis in a subset of cancer cell lines25,30 such as the MDA-
MB-231 human breast cancer cell line and SK-OV-3 human
ovarian carcinoma cell line.
We have therefore tested compounds 20, 21, and 25 for their
activity in inhibition of cell growth and induction of cell death
and apoptosis as single agents in the MDA-MB-231 and SK-
OV-3 cancer cell lines. In our previous study,26 we showed that
methylation of the primary amine group in compound 12, which
resulted in compound 13, did not significantly alter the binding
affinity to XIAP BIR3 but led to dramatic improvement in the
cellular activity. To further confirm this observation in our Smac
mimetics containing the [8,5] bicyclic ring, we have included
compound 17, which contains a primary amine group and differs
from compound 21 by the methylation of the amine group.
Compound 24 was used as another control in these cellular
assays to demonstrate the specificity of compounds 20, 21, and
25. The results are shown in Figure 4.
Compounds 20, 21, and 25 potently inhibit cell growth in
the MDA-MB-231 breast cancer cell line in a dose-dependent
manner with IC50 values of 0.41, 0.41, and 0.1 µM, respectively.
Similarly, compounds 20, 21, and 25 also potently inhibit cell
growth in the SK-OV-3 ovarian cancer cell line with IC50 values
of 0.9, 1.0, and 0.18 µM, respectively. Consistent with its weak
binding affinity to XIAP BIR3, compound 24 has IC50 values
of 7.0 µM and >10 µM, respectively, being >30-times less
potent than compound 25 in both cancer cell lines. Consistent
with our previous observation, compound 17 has a weak cellular
activity in both cell lines, indicating that the methylation on
the primary amine group in 17 results in dramatic improvement
of the cellular activity.
We next evaluated if our designed Smac mimetics can
effectively induce apoptosis in the MDA-MB-231 cell line, and
the results are shown in Figure 5. As expected, compounds 20,
21, and 25 effectively induce apoptosis in a dose-dependent
manner. While all three compounds induce robust apoptosis at
100 nM, compound 25 is the most potent among them,
consistent with their potency in inhibition of cell growth. Similar
data were obtained using the SK-OV-3 ovarian cancer cell line
(data not shown). Our data thus indicate that potent Smac
mimetics not only potently inhibit cell growth but also ef-
fectively induce apoptosis in cancer cells.
Because XIAP potently inhibits caspase activity, we next
determined if compounds 20 and 25 induces apoptosis and cell
death in a caspase dependent manner. Using a trypan blue
exclusion assay, it was shown that both compounds effectively

7174 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Sun et al.
Similar to the Smac AVPI peptide, the first carbonyl group
in compound 21 forms a less than optimal hydrogen bond to
the indole NH group in Trp323. The second amino group in 21
has an optimal hydrogen bond with the backbone carbonyl group
of Thr308 and the carbonyl group in the [8,5] lactam ring forms
an optimal hydrogen bond with the backbone amino group of
Thr308. Another strong hydrogen bond is observed between
the third amino group and the backbone carbonyl group of
Gly306. For hydrophobic contacts, the ethyl group in 21 inserts
into a small hydrophobic pocket formed by the hydrophobic
side chains of Leu307 and Trp310, as well as the hydrophobic
portion of the side chain in Gln319, mimicking the methyl group
in alanine residues in the Smac AVPI peptide. The eight-
membered ring in 21 has extensive contacts with the side chain
of Trp323. While the five-membered ring in 21 still has contacts
with the side chain of Trp323, similar to that in Smac, but the
interaction appears to be weaker than that in Smac. However,
this five-membered ring in 21 gains additional new contacts
with Tyr324. As predicted by modeling, the pro R phenyl group
inserts into a hydrophobic pocket formed by the hydrophobic
side chains of Leu292 and the hydrophobic portion of the side
chains in Lys297 and Lys299.
Interestingly, the crystal structure also revealed a remarkable
dimer, which consists of two molecules of the protein and
compound 21 (Figure 7A). The dimeric interface created
between the two compound 21:XIAP BIR3 complexes in this
crystal structure is a unique dimer configuration when compared
to other BIR3 three-dimensional structures.33,34 This dimeric
interface is mainly stabilized by the presence of compound 21.
The pro S phenyl group in both molecules of compound 21
makes multiple hydrophobic interactions with Trp323 and
Tyr324 in the other protein molecule in the symmetric dimer.
In addition, the [8,5] bicyclic lactam ring from each molecule
of 21 stacks on the top of each other and has nice hydrophobic
contacts. Other protein-protein interactions that stabilize this
interface come from the N-terminal Phe250 from each molecule.
The hydrophobic side chain of each Phe250 residue in the
dimeric structure has extensive contacts with the diphenyl groups
in compound 21 from the opposite side. This dimeric structure
formed between 2 molecules of compound 21 and two XIAP
BIR3 molecules provides a structural basis for the design of
bivalent Smac mimetics to either induce dimerization for two
BIR domains or concurrent targeting of two different BIR
domains in the same IAP molecule.
Figure 5. Induction of apoptosis by Smac mimetics in the human breast
cancer MDA-MB-231 cell line. Cells were treated for 48 h, and
apoptosis was determined using Annexin V-propidium iodide (P.I.)
staining by flow cytometry. Annexin V (+) and P.I. (-) cells are labeled
as early apoptotic cells, whereas Annexin V (+) and P.I. (+) cells are
labeled as late apoptotic cells. Percentage of early and late apoptotic
cells are shown.
kills the MDA-MB-231 cancer cells in a dose-dependent manner
but 25 is more potent (Figure 6). Treatment with 25 at 10, 100,
and 1000 nM for 24 h kills 9%, 23%, and 65% of the MDA-
MB-231 cells, respectively. The cell killing by both 20 and 25
can be effectively attenuated by Z-DEVD-FMK, a cell-perme-
able caspase-3/7 inhibitor, indicating that cell death induction
by these Smac mimetics is caspase-3/-7 dependent (Figure 6).
Determination of the Crystal Structure of Compound
21 in Complex with XIAP BIR3 Domain. To firmly establish
the structural basis of the binding of our designed Smac
mimetics to XIAP BIR3, we have determined the crystal
structure of compound 21 in complex with XIAP BIR3 to 2.8
Å resolution (Figure 7).
The experimentally determined structure of compound 21 in
complex with one XIAP BIR3 molecule is in excellent agree-
ment with that predicted by computational modeling. The root-
mean-square-deviation (rmsd) between the non-hydrogen atoms
of compounds 21 in the crystal structure and the modeled
structure is 0.81 Å.
Synthesis of Designed Smac Mimetics. The synthesis for
Smac mimetics containing the [6,5] and [7,5] bicyclic lactam
systems have been reported previously by our group.23,24 The
general synthetic method for Smac mimetics containing the [8,5]
bicyclic lactam system is shown in Scheme 1.
The interactions of compound 21 with XIAP BIR3 closely
mimic those between the Smac AVPI peptide and the protein,
although some differences are observed. The methyl group in
Ala1 side chain in the Smac AVPI peptide is replaced with an
ethyl group in compound 21. This ethyl group makes more
extensive van der Waals interactions with Trp310 and Leu307
than the methyl group in Ala1 in Smac, which, in turn, produces
a minor displacement of the positively charged N-terminal amino
group. In Smac, this positively charged amino group in Ala1
has three hydrogen bonds to the side chains of Gln319 and
Glu314. In compound 21, the N-methylated amino group loses
contact with Gln319 but gains an additional hydrogen bonding
interaction with the backbone carbonyl group of Asp309.
The key Intermediate 28 was prepared according to a
previously reported method.30,31 Condensation of 28 with
corresponding amines gave a series of amides. After removal
of the Boc protecting group in these amides, the resulted
ammonium salts were condensed with corresponding N-Boc
amino acids, followed by deprotection of the Boc protecting
group to yield compounds 16-21 and 25-27. Refluxing of 17
with 1,4-dibromobutane or 2-bromoethyl ether under the exist-
ence of N,N-diisopropylethylamine in methanol furnished
compounds 22 and 23. Condensation of 28 and aminodiphe-
nylmethane furnished an amide. Removal of the Boc protecting

Conformationally Constrained Smac Mimetics
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7175
Figure 6. Induction of cell death by compounds 20 and 25 in the human breast cancer MDA-MB-231 cell line and dependence on caspase-3/7.
Cells were treated with different concentrations of Smac mimetic for 48 h and cell viability was determined using trypan blue exclusion assay. To
determine the dependence of cell death induction by Smac mimetics on caspase-3/-7, a cell-permeable caspase-3/-7 inhibitor, Z-DEVD-FMK, was
employed.
Figure 7. X-ray crystal structure of compound 21 in complex with human XIAP BIR3, determined to 2.8 Å resolution. (A) Overall representation
of compound 21 in complex with XIAP BIR3. Compound 21 is shown in green ball-and-stick. (B) Detailed interactions between compound 21 and
XIAP BIR3 residues shown as a 90° rotation of the yellow domain in A. Compound 21 is shown in yellow ball-and-stick. (C) Superposition of
modeled and crystal structures of compound 21 in complex with XIAP BIR3.
group in this amide followed by condensation of the resulted
ammonium salt with L-lactic acid yielded compound 24.
crystal structure of compound 21 in complex with XIAP BIR3
provides the structural basis for its high-affinity interaction with
XIAP BIR3 and for further design and optimization. These potent
and cell-permeable Smac mimetics serve as powerful pharmaco-
logical tools to further elucidate the role XIAP and other IAP
proteins in apoptosis regulation and promising lead compounds
for the development of a new class of anticancer drugs.
Summary
On the basis of the crystal structure of Smac in complex with
XIAP BIR3 domain, we have designed and synthesized a series
of conformationally constrained, bicyclic Smac mimetics. Our
studies have generated important structure-activity relationships
for their binding affinities to XIAP BIR3 and for their activity in
inhibition of cell growth in human cancer cells. Our efforts have
led to the design of a number of potent and cell-permeable Smac
mimetics such as 20 (SM-122), 21 (SM-130), and 25 (SM-230).
These compounds bind to XIAP BIR3 with high affinities and
antagonize XIAP BIR3 in cell-free functional assays. Importantly,
they are potent and effective in inhibition of cell growth and in
induction of apoptosis in cancer cells. Our determination of the
Experimental Section
1. Chemistry. General Methods. NMR spectra were acquired
1
at a proton frequency of 300 MHz. H chemical shifts are reported
with TMS (0.00 ppm) or DHO (4.70 ppm) as internal standards. 13
C
chemical shifts are reported with CDCl3 (77.00 ppm) or 1,4-dioxane
(67.16 ppm) as internal standards. The final products were purified by
HPLC on a Waters Sunfire C18 reverse phase semipreparative HPLC

7176 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Sun et al.
column (19 mm × 150 mm) using solvent A (0.1% of TFA in water)
and solvent B (0.1% of TFA in CH3CN) as eluents with a flow rate of
10 mL/min.
Scheme 1. General Synthesis of Conformationally Constrained
[8,5] Bicyclic Smac Mimeticsa
General Synthetic Method for [8,5] Ring Contained Smac
Mimetics. To a solution of compound 28 (1 mmol) in 10 mL of
CH2Cl2 were added corresponding amine (1.2 mmol), EDC (1.2
mmol), HOBt (1.2 mmol), and N,N-diisopropylethylamine (3 mmol)
at 0 °C. The mixture was stirred at room temperature overnight. After
condensation, the residue was purified by chromatography to give an
amide. To a solution of this amide in 5 mL of methanol was added 2
mL of HCl solution (4 N in 1,4-dioxane). The solution was stirred at
room temperature overnight and then concentrated to give an am-
monium salt. To a mixture of this salt in 10 mL of CH2Cl2 were added
a desired N-Boc-L-amino acid (1.2 mmol), EDC (1.2 mmol), HOBt
(1.2 mmol), and N,N-diisopropylethylamine (3 mmol) at 0 °C. The
mixture was stirred at room temperature overnight and then concen-
trated. The residue was purified by chromatography to give an amide.
To a solution of this amide in 5 mL of methanol was added 1 mL of
HCl solution (4 N in 1,4-dioxane). The solution was stirred at room
temperature overnight and then concentrated. The residue was purified
by C18 reverse phase semipreparative HPLC to give the expected Smac
mimetic.
(3S,6S,11S)-6-((S)-2-Amino-propionylamino)-5-oxo-decahydro-
pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide (16).
The gradient ran from 70% of solvent A and 30% of solvent B to
50% of solvent A and 50% of solvent B in 30 min. The purity was
checked by analytical HPLC to be over 98%. 1H NMR (300 M Hz,
D2O) δ 7.30-7.18 (m, 10H), 5.95 (s, 1H), 4.73 (m, 1H), 4.35 (m,
1H), 4.22 (m, 1H), 3.96 (m, 1H), 2.16 (m, 1H), 2.05 (m, 1H),
1.93-1.32 (m, 10 H), 1.39 (d, J ) 7.5 Hz, 3H). 13C NMR (75 MHz,
D2O) δ 173.67, 172.65, 169.93, 141.64, 141.45, 129.57, 129.45,
128.34, 128.09, 127.77, 62.50, 61.40, 58.16, 57.55, 51.42, 36.16, 33.30,
32.64, 28.07, 25.36, 22.14, 15.95. ESI MS: m/z 463.3 (M + H)+. HR
ESI MS for C27H35N4O3 required, 463.2709; found, 463.2699; Anal.
(C27H34N4O3 ·1.0HCl·1.0CF3COOH) C, H, N.
a Reagents and conditions: (a) (i) Amine, EDC, HOBt, N,N-diisopropy-
lethylamine, CH2Cl2, overnight; (ii) 4 N HCl in 1,4-dioxane, MeOH; (iii)
N-Boc-L-amino acid, EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2; (iv)
4 N HCl in 1,4-dioxane, MeOH. (b) 1,4-Dibromobutane or 2-bromoethyl
ether, N,N-diisopropylethylamine, MeOH, reflux, 67% for 22 and 54% for
23. (c) (i) Aminodiphenylmethane, EDC, HOBt, N,N-diisopropylethylamine,
CH2Cl2, overnight; (ii) 4 N HCl in 1,4-dioxane, MeOH; (iii) L-lactic acid,
EDC, HOBt, N,N-diisopropylethylamine, CH2Cl2, 71% over three steps.
172.16, 169.64, 141.38, 141.19, 139.58, 137.49, 134.18, 131.30,
129.21, 128.69, 128.18, 127.00, 125.97, 125.34, 124.57, 123.90,
62.01, 60.87, 54.62, 54.18, 51.13, 36.32, 33.58, 32.51, 27.74, 25.37,
25.04, 22.30, 9.10. ESI MS: m/z 527.3 (M + H)+. HR ESI MS for
C32H39N4O3 required, 527.3022; found, 527.3013; Anal. (C32H38N4-
O3 ·1.0HCl·1.0CF3COOH·0.8H2O) C, H, N.
(3S,6S,11S)-6-((S)-2-Methylamino-propionylamino)-5-oxo-decahy-
dro-pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide
(20). The gradient ran from 70% of solvent A and 30% of solvent
B to 50% of solvent A and 50% of solvent B in 30 min. The purity
1
was checked by analytical HPLC to be over 98%. H NMR (300
M Hz, D2O) δ 7.30-7.18 (m, 10H), 5.95 (s, 1H), 4.74 (m, 1H),
4.34 (m, 1H), 4.24 (m, 1H), 3.83 (m, 1H), 2.57 (s, 3H), 2.21-1.50
(m, 11H), 1.40 (d, J ) 7.0 Hz, 3H), 1.38 (m, 1H). 13C NMR (75
MHz, D2O) δ 173.77, 172.62, 169.95, 141.56, 141.28, 129.54,
129.44, 128.40, 128.35, 128.01, 127.82, 62.50, 61.40, 58.16, 57.49,
51.42, 36.16, 33.30, 32.64, 31.61, 28.07, 25.36, 22.14, 15.95. Anal.
(C28H36N4O3 ·1.0HCl·1.0CF3COOH) C, H, N.
(3S,6S,11S)-6-((S)-2-Methylamino-butyrylamino)-5-oxo-decahy-
dro-pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide
(21). The gradient ran from 70% of solvent A and 30% of solvent
B to 50% of solvent A and 50% of solvent B in 30 min. The
putridity was checked by analytical HPLC to be over 98%. 1H NMR
(300 M Hz, D2O) δ 7.32-7.19 (m, 10H), 5.95 (s, 1H), 4.75 (m,
1H), 4.36 (m, 1H), 4.25 (m, 1H), 3.71 (m, 1H), 2.56 (s, 3H),
2.18-1.40 (m, 14H), 0.87 (t, J ) 7.6 Hz, 3H). 13C NMR (75 MHz,
D2O) δ 173.76, 172.39, 168.73, 141.60, 141.27, 129.54, 129.44,
128.37, 128.05, 127.76, 62.96, 62.41, 61.44, 58.21, 51.51, 36.18,
33.40, 32.66, 32.04, 28.10, 25.38, 24.08, 22.11, 8.81. ESI MS: m/z
491.3 (M + H)+. HR ESI MS for C29H39N4O3 required, 491.3022;
found, 491.3026; Anal. (C29H38N4O3 · 1.0HCl · 1.0CF3COOH) C,
H, N.
(3S,6S,11S)-6-((S)-2-Amino-butyrylamino)-5-oxo-decahydro-
pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide (17).
The gradient ran from 70% of solvent A and 30% of solvent B to
50% of solvent A and 50% of solvent B in 30 min. The purity was
1
checked by analytical HPLC to be over 98%. H NMR (300 M
Hz, D2O) δ 7.30-7.18 (m, 10H), 5.95 (s, 1H), 4.71 (m, 1H), 4.34
(m, 1H), 4.22 (m, 1H), 3.83 (m, 1H), 2.14 (m, 1H), 2.03 (m, 1H),
1.93-1.20 (m, 12 H), 0.88 (t, J ) 7.5 Hz, 3H). 13C NMR (75
MHz, D2O) δ 173.67, 172.60, 169.96, 141.64, 141.34, 129.54,
129.45, 128.37, 128.05, 127.78, 62.41, 61.39, 58.18, 54.65, 51.41,
36.22, 33.43, 32.66, 28.09, 25.34, 25.04, 22.16, 9.07. ESI MS: m/z
477.3 (M + H)+. HR ESI MS for C28H37N4O3 required, 477.2866;
found, 477.2858. Anal. (C28H36N4O3 ·1.0HCl·2.0H2O) C, H, N.
(3S,6S,11S)-6-((S)-2-Amino-butyrylamino)-5-oxo-decahydro-pyr-
rolo[1,2-r]azocine-3-carboxylic Acid ((R)-naphthalen-1-yl-phenyl-
methyl)-amide (18). The gradient ran from 70% of solvent A and
30% of solvent B to 50% of solvent A and 50% of solvent B in 30
min. The purity was checked by analytical HPLC to be over 98%.
1H NMR (300 MHz, D2O) δ 7.75 (m, 1H), 7.65 (m, 1H), 7.58 (m,
1H), 7.32-7.10 (m, 4H), 7.10-6.92 (m, 5H), 6.58 (s, 1H), 4.63
(m, 1H), 4.27 (m, 1H), 3.80 (m, 1H), 2.12-1.65 (m, 6H), 1.65-1.20
(m, 8H), 0.86 (t, J ) 7.5 Hz, 3H). 13C NMR (75 MHz, D2O) δ
173.19, 172.59, 169.83, 141.15, 136.53, 134.23, 131.35, 131.11,
129.40, 128.32, 128.04, 127.37, 126.66, 126.08, 124.02, 62.20,
61.18, 54.96, 54.60, 51.33, 36.32, 33.32, 32.61, 27.92, 25.33, 25.01,
22.19, 9.08. ESI MS: m/z 527.3 (M + H)+. HR ESI MS for
C32H39N4O3 required, 527.3022; found, 527.3021. Anal. (C32H38N4-
O3 ·1.0HCl·1.0CF3COOH·0.6H2O) C, H, N.
(3S,6S,11S)-6-((S)-(2-pyrrolidin-1-yl-butyrylamino))-5-oxo-decahy-
dro-pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide
(22). To a solution of 17 (salt with HCl, 260 mg, 0.5 mmol) in 10
mL of methanol were added 1,4-dibromobutane (430 mg, 2 mmol)
and N,N-diisopropylethylamine (0.5 mL). The solution was refluxed
overnight and then condensed. The residue was dissolved in 5 mL
of methanol. To this solution was added 0.5 mL of TFA. After
condensation, the residue was purified by C18 reverse phase
semipreparative HPLC to give 22 (215 mg, 67%). The gradient
ran from 70% of solvent A and 30% of solvent B to 50% of solvent
A and 50% of solvent B in 40 min. The purity was checked by
(3S,6S,11S)-6-((S)-2-Amino-butyrylamino)-5-oxo-decahydro-pyr-
rolo[1,2-r]azocine-3-carboxylic Acid ((S)-naphthalen-1-yl-phenyl-
methyl)-amide (19). The gradient ran from 70% of solvent A and
30% of solvent B to 50% of solvent A and 50% of solvent B in 30
min. The purity was checked by analytical HPLC to be over 98%.
1H NMR (300 MHz, D2O) δ 7.58 (m, 1H), 7.20-6.50 (12H), 4.56
(m, 1H), 4.33 (m, 1H), 3.87 (m, 1H), 3.74 (m, 1H), 1.82-0.90 (m,
12H), 0.80 (t, J ) 7.4 Hz, 3H). 13C NMR (75 MHz, D2O) δ 172.22,
1
analytical HPLC to be over 98%. H NMR (300 M Hz, D2O) δ
7.30-7.19 (m, 10H), 5.95 (s, 1H), 4.77 (m, 1H), 4.36 (m, 1H),
4.25 (m, 1H), 3.68 (m, 1H), 3.50-2.70 (brs, 4H), 2.23-1.39 (m,
18H), 0.85 (t, J ) 7.2 Hz, 3H). 13C NMR (75 MHz, D2O) δ 173.78,
172.23, 168.82, 141.60, 141.26, 129.54, 129.45, 128.38, 128.06,
127.74, 69.12, 62.40, 61.45, 58.22, 51.52, 36.17, 33.45, 32.66,
28.12, 26.40, 25.38, 23.64, 23.23, 22.10, 8.98. ESI MS: m/z 531.3

Conformationally Constrained Smac Mimetics
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7177
(M + H)+. HR ESI MS for C32H43N4O3 required, 531.3335; found,
531.3334. Anal. (C32H42N4O3 ·1.0HCl·1.3H2O) C, H, N.
169.88, 138.67, 136.46, 129.89, 128.74, 128.13, 126.84, 63.01,
61.39, 57.48, 51.45, 48.44, 36.30, 33.30, 32.71, 31.64, 30.16, 29.10,
28.15, 25.43, 22.21, 20.22, 15.97. ESI MS: m/z 442.3 (M + H)+.
HR ESI MS for C25H37N4O3 required, 441.2866; found, 441.2864.
Anal. (C25H36N4O3 ·1.0HCl·1.0CF3COOH) C, H, N.
(3S,6S,11S)-6-((S)-2-Morpholin-4-yl-butyrylamino))-5-oxo-decahy-
dro-pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide
(23). To a solution of 17 (salt with HCl, 256 mg, 0.5 mmol) in 10
mL of methanol were added 2-bromoethyl ether (580 mg, 2.5 mmol)
and N,N-diisopropylethylamine (0.5 mL). The solution was refluxed
overnight and then condensed. The residue was dissolved in 5 mL
of methanol. To this solution was added 0.5 mL of TFA. After
condensation, the residue was purified by C18 reverse phase
semipreparative HPLC to give 23 (178 mg, 54%). The gradient
ran from 70% of solvent A and 30% of solvent B to 50% of solvent
(3S,6S,11S)-6-((S)-2-Methylamino-propionylamino)-5-oxo-decahy-
dro-pyrrolo[1,2-a]azocine-3-carboxylic Acid ((R)-Indan-1-ylamide)-
amide (27). The gradient ran from 70% of solvent A and 30% of
solvent B to 50% of solvent A and 50% of solvent B in 30 min.
1
The purity was checked by analytical HPLC to be over 98%. H
NMR (300 MHz, D2O) δ 7.23-7.06 (m, 4H), 5.19 (t, J ) 7.8 Hz,
1H), 4.75 (m, 1H), 4.32-4.18 (m, 2H), 3.83 (m, 1H), 2.98-2.69
(m, 2H), 2.56 (s, 3H), 2.36 (m, 1H), 2.22-1.50 (m, 12 H), 1.43
(m, 1H), 1.40 (d, J ) 7.0 Hz, 3H). 13CNMR (75 MHz, D2O) δ
174.18, 172.66, 169.87, 144.29, 143.14, 128.66, 127.35, 125.45,
124.66, 62.78, 61.45, 57.48, 55.33, 51.48, 36.25, 33.39, 33.30,
32.65, 31.60, 30.27, 28.20, 25.44, 22.17, 15.94. ESI MS: m/z 427.3
(M + H)+. HR ESI MS for C24H35N4O3 required, 427.2709; found,
427.2707. Anal. (C24H34N4O3 ·1.0HCl·1.0CF3COOH) C, H, N.
2. Molecular Modeling. The crystal structure of XIAP BIR3 in
a complex with the Smac protein15 (PDB reference: 1G73) was
used to predict the binding models of XIAP BIR3 bound to designed
compounds. We used the Amber program suite40 (version 7) to
perform all molecular dynamics (MD) simulations. A recent version
of the Amber force field (ff96)41 was used for the natural amino
acids in the complex and the TIP3P model42 was used for water
molecules. There is one Zn2+ ion covalently bound to C300, C303,
H320, and C327 in the XIAP BIR3 domain. This Zn2+ ion, while
important for structural integrity, has no direct interaction with the
ligands. We used parameters developed by Ryde43 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 algorithm44 was used to fix
the bonds involving hydrogen. The PME method45 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
neighboring pairs list was updated after every 20 steps. For the
refinement of the structure between compound 20 and XIAP BIR3,
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 conforma-
tions (typically 2000), evenly spaced in time, were collected for
structural analysis.
All the initial binding poses of designed ligands with XIAP
BIR3 were predicted using the GOLD program46 (version 2.2).
The center of the binding site for XIAP BIR3 was set at Thr308
and the radius of the binding site was defined as 13 Å, large
enough to cover the binding pocket. For each genetic algorithm
run, a maximum number of 200000 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 ligand. GoldScore implemented in Gold 2.2
was used as the fitness function to evaluate the docked
conformations. The 20 conformations ranked highest by each
fitness function were saved for analysis of the predicted docking
modes. For the docking poses reported, these were the highest
ranked conformations from the docking simulations.
1
A and 50% of solvent B in 40 min. H NMR (300 M Hz, D2O) δ
7.30-7.19 (m, 10H), 5.95 (s, 1H), 4.79 (m, 1H), 4.37 (m, 1H),
4.25 (m, 1H), 3.86 (brs, 4H), 3.67 (m, 1H), 3.31-3.20 (m, 4H),
2.18-1.39 (m, 14H), 0.85 (t, J ) 7.2 Hz, 3H). 13C NMR (75 MHz,
D2O) δ 173.77, 172.16, 167.95, 141.61, 141.26, 129.54, 129.45,
128.38, 128.06, 127.74, 70.21, 64.28, 62.40, 61.45, 58.22, 51.58,
51.02, 36.17, 33.44, 32.66, 28.12, 25.37, 22.09, 21.22, 9.14. ESI
MS: m/z 547.3 (M + H)+. HR ESI MS for C32H43N4O4 required,
547.3284; found, 547.3298. Anal. (C32H42N4O4 ·1.0HCl·2.5H2O)
C, H, N.
(3S,6S,11S)-6-((S)-2-Hydroxy-propionylamino))-5-oxo-decahy-
dro-pyrrolo[1,2-r]azocine-3-carboxylic Acid Benzhydryl-amide
(24). To a solution of 28 (320 mg, 1 mmol) in 10 mL of CH2Cl2
were added aminodiphenylmethane (220 mg, 1.2 mmol), EDC (230
mg, 1.2 mmol), HOBt (160 mg, 1.2 mmol), and N,N-diisopropy-
lethylamine (1.2 mL) at 0 °C. The mixture was stirred at room
temperature overnight. After condensation, the residue was purified
by chromatography to give an amide. To a solution of this amide
in 5 mL of methanol was added 2 mL of HCl solution (4 N in
1,4-dioxane). The solution was stirred at room temperature overnight
and then condensed to give an ammonium salt. To a mixture of
this salt in 10 mL of CH2Cl2 were added L-lactic acid (110 mg, 1.2
mmol), EDC (232 mg, 1.2 mmol), HOBt (164 mg, 1.2 mmol), and
N,N-diisopropylethylamine (1.2 mL) at 0 °C. The mixture was
stirred at room temperature overnight and then condensed. The
residue was purified by chromatography to give compound 24 (328
mg, 71% over three steps). 1H NMR (300 M Hz, CDCl3, TMS) δ
7.78 (brd, J ) 8.4 Hz, 1H), 7.37-7.20 (m, 11H), 6.22 (brd, J )
8.5 Hz, 1H), 4.88 (m, 1H), 4.64 (m, 1H), 4.23 (m, 2H), 3.80 (brs,
1H), 2.50 (m, 1H), 2.12 (m, 1H), 2.08-1.75 (m, 4H), 1.74-1.39
(m, 5H), 1.41 (d, J ) 7.2 Hz, 3H), 1.27 (m, 1H). 13C NMR (75
MHz, CDCl3) δ 174.17, 172.08, 169.77, 141.63, 141.47, 128.60,
128.52, 127.52, 127.36, 127.22, 68.27, 60.32, 59.48, 57.10, 49.55,
36.59, 35.36, 32.16, 25.03, 24.70, 22.94, 20.88. ESI MS: m/z 464.3
(M + H)+. HR ESI MS for C27H34N3O4 required, 464.2549; found,
464.2544. Anal. (C27H33N3O4 ·1.6H2O) C, H, N.
(3S,6S,11S)-6-((S)-2-Methylamino-propionylamino)-5-oxo-decahy-
dro-pyrrolo[1,2-a]azocine-3-carboxylic Acid ((R)-(1,2,3,4-Tetrahy-
dro-naphthalen-1-yl)-amide (25). The gradient ran from 70% of
solvent A and 30% of solvent B to 50% of solvent A and 50% of
solvent B in 30 min. The purity was checked by analytical HPLC
to be over 98%. 1H NMR (300 MHz, D2O) δ 7.22-7.04 (m, 4H),
4.87 (m, 1H), 4.70 (m, 1H), 4.32-4.16 (m, 2H), 3.90-3.78 (m,
1H), 2.68-2.60 (m, 2H), 2.56 (s, 3H), 2.16-1.42 (m, 16), 1.40 (d,
J ) 7.2 Hz, 3H). 13C NMR (75 MHz, D2O) δ 173.62, 172.70,
169.86, 138.61, 136.40, 129.76, 128.93, 128.06, 126.85, 62.76,
61.44, 57.49, 51.48, 48.62, 36.26, 33.30, 32.65, 31.61, 30.08, 29.19,
28.18, 25.44, 22.18, 20.47, 15.94. ESI MS: m/z 441.3 (M + H)+.
HR ESI MS for C25H37N4O3 required, 441.2866; found, 441.2862.
Anal. (C25H36N4O3 ·1.0HCl·1.0CF3COOH) C, H, N.
3. Protein Expression and Purification, Binding Assays to
XIAP BIR3, and Cell-Free Functional Assays for the Activity of
Caspase-9. Protein Expression and Purification. An N-terminal
6xHis-tagged BIR3 domain from human XIAP expressed in
abundance of 50 mg of protein per liter of culture in Escherichia
coli and was readily purified from soluble extracts. The sequence
encoding the BIR3 domain (residues 241-356) of the human XIAP
gene was cloned into the pET28a expression vector. High protein
expression levels were achieved by growing transformed BL21(DE3)
cells at 37 °C in 2xYT media containing 30 µg/mL kanamycin to
an optical density (600 nm) of 0.6 and inducing with 0.4 mM
(3S,6S,11S)-6-((S)-2-Methylamino-propionylamino)-5-oxo-decahy-
dro-pyrrolo[1,2-a]azocine-3-carboxylic Acid ((S)-(1,2,3,4-Tetrahy-
dro-naphthalen-1-yl)-amide (26). The gradient ran from 70% of
solvent A and 30% of solvent B to 50% of solvent A and 50% of
solvent B in 30 min. The purity was checked by analytical HPLC
to be over 98%. 1H NMR (300 MHz, D2O) δ 7.20-7.02 (m, 4H),
4.88 (m, 1H), 4.22 (m, 1H), 4.16 (m, 1H), 3.83 (m, 1H), 2.69-2.60
(m, 2H), 2.57 (s, 3H), 2.20-1.52 (m, 15 H), 1.44 (m, 1H), 1.40 (d,
J ) 7.2 Hz, 3H). 13C NMR (75 MHz, D2O) δ 173.65, 172.61,

7178 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22
Sun et al.
Table 2. Data Collection, Phasing, and Refinement Statistics of the
isopropyl-D-thiolgalactoside for 4 h at 25 °C. Cells pelleted at 5000g
were frozen at -80 °C and resuspended in lysis buffer containing
50 mM Tris pH 7.5, 200 mM NaCl, 50 mM Zn acetate, 3 mM
ꢀ-mercaptoethanol, and protease inhibitors. The cells were subjected
to sonication and the cellular debris pelleted by centrifugation at
30000g for 20 min. The soluble fraction was applied to Ni-NTA
agarose (Qiagen) pre-equilibrated with wash buffer containing 50
mM Tris pH 7.5, 200 mM NaCl, 50 mM Zn acetate, and 25 mM
imidazole. The BIR3 domain protein was then eluted from the
column using wash buffer containing 250 mM imidazole. To reduce
the number of protein aggregates, a final concentration of 1 mM
DTT was added to the elute before applying it to a Superdex-75
column (Pharmacia) equilibrated with 20 mM Tris pH 7.5, 200
mM NaCl, 50 mM Zn acetate, and 1 mM DTT. The resulting
protein was judged to be greater than 98% pure by the presence of
a single band on an SDS-PAGE gel.
Fluorescence-Polarization-Based Binding for XIAP BIR3 Pro-
tein. A sensitive in vitro binding assay using the fluorescence
polarization (FP)-based method32 was used to determine the binding
affinity of Smac mimetics to XIAP BIR3 protein. In this assay,
5-carboxyfluorescein was coupled to the lysine side chain of a
mutated Smac peptide with the sequence (AbuRPFK-Fam). This
fluorescently tagged peptide, named SM5F, was used as the
fluorescent tracer in the FP-based binding assay of different
compounds to the XIAP BIR3 protein (residues 241-356). The
Kd value of SM5F peptide to XIAP BIR3 protein was determined
to be 17.9 nM.32
Dose-dependent binding experiments were carried out with
serial dilutions of the tested compounds. An aliquot of the
samples and preincubated XIAP BIR3 protein (0.030 µM) and
SM5F peptide (5 nM) in the assay buffer (100 mM potassium
phosphate, pH 7.5; 100 µg/mL bovine gamma globulin; 0.02%
sodium azide, purchased from Invitrogen Life Technology), were
added to 96-well plates. For each assay, the controls included
XIAP BIR3 protein and SM5F (equivalent to 0% inhibition),
and SM5F only (equivalent to 100% inhibition). The polarization
values were measured after 3 h of incubation using an ULTRA
READER (Tecan U.S. Inc., Research Triangle Park, NC). IC50
values, the inhibitor concentration at which 50% of the bound
tracer is displaced, were determined from a plot using nonlinear
least-squares analysis. Curve fitting was performed using
GRAPHPAD PRISM software (GraphPad Software, Inc., San
Diego, CA).
Cell-Free Caspase-9 Functional Assay. MDA-MB-231 cell
lysates were prepared by solubilizing cells in ice cold buffer (pH
7.5), which contained 50 mM KCl, 5 mM EGTA, 2 mM MgCl2,
1 mM DTT, 0.2% CHAPS, 50 mM HEPES, and cocktail protease
inhibitors and incubated on ice for 10 min. Cytochrome c and dATP
were added into the cell lysates and incubated at 30 °C in a water
bath for 60 min to activate caspase-9. To inhibit caspase-9 activity,
recombinant human XIAP BIR3 protein (0.5 µM) was added in
the cell extract at the same time as Cytochrome c and dATP were
added and was found to dose-dependently inhibit the activity of
caspase-9. To determine caspase-9 activity, 25 µM of caspase-9
substrate (Z-LEHD-AFC) purchased from BioVision was added.
Fluorescence detection of substrate cleavage by caspase-9 was
carried out with an ULTRA READER using an excitation wave-
length of 400 nm and an emission wavelength of 505 nm, and the
reaction was monitored for 60-120 min. To determine the
antagonism of a Smac mimetic, different concentrations of a tested
Smac mimetic (from 100 µM to 1 nM) were added into the reaction
mixture together with XIAP BIR3 protein.
Crystal Structure of XIAP BIR3:Compound 21
data collection statistics
data set
XIAP BIR3:21
space group
unit cell (Å)
P6522
a ) b ) 115.76;
c ) 61.79
1.0000
2.8 (2.9 -2.8)
7.2 (30.8)
20 (3)
wavelength (Å)
resolution (Å)a
Rsym (%)b
c
I/σI
completeness (%)d
redundancy
99.3 (100)
>9
refinement statistics
resolution (Å)e
R-factor (%)f
Rfree (%)g
2.8
22.8 (37.5)
26.9 (46.3)
784
protein atomsh
water molecules
38
unique reflections
6181
rmsdi
bonds (Å)
angles (deg)
0.007
1.203
a Statistics for highest resolution bin of reflections in parentheses. b Rsym
) ∑h ∑j |Ihj- Ih |/∑h ∑j Ihj, where Ihj is the intensity of observation j of
reflection h and Ih is the mean intensity for multiply recorded reflections.
c Intensity signal-to-noise ratio. d Completeness of the unique diffraction
data. e Resolution cut-off used during heavy-atom refinement and phase
calculations. f R-factor ) ∑h ||Fo| - |Fc||/∑h |Fo|, where Fo and Fc are the
g
observed and calculated structure factor amplitudes for reflection h. Rfree
is calculated against a 10% random sampling of the reflections that were
removed before structure refinement. h Total number of protein atoms refined
in the asymmetric unit. i Root mean square deviation of bond lengths and
bond angles.
Cell Growth Assay. The effect of Smac mimetics on cell growth
was evaluated by a WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4-
nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium monosodium salt]
assay (Dojindo Molecular Technologies, Inc.). Cells (3000-4000
cells in each well) were cultured in 96-well tissue culture plates in
medium (200 µL) containing various concentrations of Smac
mimetics for the indicated time. At the end of incubation, WST-8
dye (20 µL) was added to each well and incubated for an additional
1-3 h, and then the absorbance was measured in a microplate reader
(Molecular Devices) at 450 nm. Cell growth inhibition was
evaluated as the ratio of the absorbance of the sample to that of
the control.
Apoptosis Assay. The apoptosis assay was performed with an
annexin-V/propidium iodide (PI) apoptosis detection kit (Roche)
according to the manufacturer’s instructions. Briefly, cells were
harvested, washed with ice-cold PBS, and then stained with
annexin-V-FITC and PI for 15 min at room temperature in the dark.
Stained cells were analyzed in a FACS calibur flow cytometer.
Annexin-V (-)/PI (-) cells were classified as live cells; Annexin-V
(+)/PI (-) cells were classified as early apoptotic cells; Annexin-
V (+)/PI (+) cells were classified as late apoptotic cells; Annexin
V (-) and P.I. (+) cells were classified as dead cells.
Cell Viability Assay. Cell viability was determined using a
trypan blue exclusion assay. Approximately 10000 MDA-MB-231
cells were seeded in 24-well plates and incubated at 37 °C in an
atmosphere of 95% air and 5% CO2 for 24 h before drug treatments.
Cells were treated with different concentrations of compounds 20
and 25 for 48 h. To examine the role of caspase-3/-7 in cell death
induction by Smac mimetics, a cell-permeable caspase-3/-7 inhibi-
tor, Z-DEVD-FMK (Calbiochem, San Diego, CA) was employed
and cells were treated with a combination of Z-DEVD-FMK with
compound 20 or 25 concurrently. To determine cell viability, 1:1
dilution of the suspension of 0.4% Trypan blue reagent (Invitrogen
Corporation) was used. Blue cells were counted as nonviable cells.
At least 100 cells were counted in the control well, and three
independent countings were performed.
IV. Assays for Cell Growth, Apoptosis, and Cell Viability.
Cell Lines. MDA-M-231 and SK-OV-3 cancer cell lines were
purchased from the American Type Culture Collection (Manassas,
VA), maintained in RPMI 1640 (Invitrogen), supplemented with
10% fetal bovine serum (Invitrogen) and 1% penicillin-streptomy-
cin, at 37 °C in 5% CO2. Both cell lines were cultured in RPMI
1640 supplemented with 10% fetal bovine serum (Invitrogen) and
1% penicillin-streptomycin in the presence of puromycin from
EMD Biosciences (2 µg/ml).

Conformationally Constrained Smac Mimetics
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 22 7179
genes in human cancers and myeloid leukemias. Clin. Cancer Res.
2000, 6, 1796–1803.
4. Crystallographic Studies. Crystallization of XIAP BIR3:
Compound 21 Complex. Bipyramidal crystals of the BIR3 domain
of human XIAP (residues 241-356) in complex with compound
21 were grown at 20 °C from hanging drop vapor diffusion
experiments by mixing 2 µL of protein (10 mg/ml preincubated
with 10 mM compound 21) in 20 mM Tris pH 7.5, 200 mM NaCl,
50 mM Zn acetate, and 1 mM DTT with 2 µL of precipitant (20%
polyethylene glycol 8000, 0.2 M MgCl2, and 0.1 M Tris-HCl pH
8.5). Crystals were cryoprotected with paratone-N and data collected
at a wavelength of 1.0 Å at the Advanced Photon Source on the
COMCAT 32-ID beam line equipped with a MAR165 CCD and
processed with DENZO/SCALEPACK.35 The complex crystallized
in space group P6522 with a solvent content of 66%,36 resulting in
one molecule per asymmetric unit. The data are 99.3% complete
to 2.8 Å resolution with a Rsym of 7.2%.
Structure Determination and Refinement. The BIR3:com-
pound 21 complex was solved to 2.8 Å resolution by molecular
replacement methods in CNS37 using the crystal structure of
XIAP BIR3 (residues 256-346) with another Smac mimetic
solved in our laboratory as the search model. Difference Fourier
maps (Fo - Fc) calculated after CNS rigid-body refinement of
the BIR3 domain against BIR3:compound 21 diffraction data
showed the presence of compound 21 and six additional residues
at the N-terminus (250-255). Multiple rounds of CNS refinement
and model building using O38 resulted in a BIR3:compound 21
structure refined to 2.8 Å resolution (Table 1). Simulated
annealing omit maps calculated in CNS were used to check for
the correct placement of atoms in the structure. The final
structure was analyzed in PROCHECK.39 All residues are in
the allowed regions of the Ramanchandran plot. The N-terminal
residues (241-249) and the C-terminal residues (346-356) were
disordered in the structure.
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Acknowledgment. The financial support from the Breast
Cancer Research Foundation, the National Cancer Institute, NIH
(R01CA109025), the Prostate Cancer Foundation, the Depart-
ment of Defense Prostate Cancer Program (W81XWH-04-1-
0213), and Ascenta Therapeutics are greatly appreciated. We
thank Dr. G. W. A. Milne and Karen Kreutzer for critical reading
of the manuscript.
Supporting Information Available: Elemental analysis data for
compounds 16-27. This material is available free of charge via
the Internet at http://pubs.acs.org.
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